Sirtuin Biology in Cancer and Metabolic Disease: Cellular Pathways for Clinical Discovery 0128224673, 9780128224670

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SIRTUIN BIOLOGY IN CANCER AND METABOLIC DISEASE

SIRTUIN BIOLOGY IN CANCER AND METABOLIC DISEASE Cellular Pathways for Clinical Discovery Edited By

KENNETH MAIESE Biotechnology and Venture Capital Development, Office of Translational Alliances and Coordination, National Heart, Lung, and Blood Institute; Cellular and Molecular Signaling, New York, NY, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-822467-0 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Ana Claudia A. Garcia Editorial Project Manager: Timothy Bennett Production Project Manager: Omer Mukthar Cover Designer: Christian J. Bilbow Typeset by MPS Limited, Chennai, India

Dedication

To all our readers and to the never-ending drive in all of us to reject the “status quo,” embrace innovation, and carry forward novel avenues of discovery that looks forward and inspires advanced treatments for cancer and metabolic diseases.

Contents

List of contributors About the editor Preface Acknowledgment

2.8 Concluding remarks and future perspectives References

xi xiii xv xvii

3. Sirtuins and metabolic regulation: food and supplementation SE´RGIO HENRIQUE SOUSA SANTOS, VICTOR HUGO DANTAS ˜ ES, JANAINA RIBEIRO OLIVEIRA AND GUIMARA

Section I

LUIZ FERNANDO REZENDE

Sirtuins and metabolic disease

3.1 Introduction 3.2 Tissue-specific sirtuin-modulated metabolic regulation 3.3 Nutrition as a therapeutic model for sirtuin regulation 3.4 Resveratrol 3.5 Gallic acid 3.6 Nonresveratrol related sirtuin activators 3.7 Food and sirtuins 3.8 Conclusion References

1. Sirtuins in metabolic disease: innovative therapeutic strategies with SIRT1, AMPK, mTOR, and nicotinamide KENNETH MAIESE

Abbreviations 1.1 Noncommunicable diseases 1.2 Metabolic disorders 1.3 Novel therapeutic strategies with sirtuins for metabolic disease 1.4 Silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) 1.5 SIRT1, metabolic function, and obesity 1.6 SIRT1 and AMP-activated protein kinase 1.7 SIRT1, mTOR, and metabolic disease 1.8 SIRT1, nicotinamide, and cellular metabolism 1.9 Future considerations Acknowledgments References

3 4 4 6

42 44 47 48 48 50 52 52

KULTIGIN TURKMEN

4.1 Introduction Part 1 4.2 Sirtuin 1 (SIRT1) in normal physiology Part 2 4.3 Diabetes mellitus and sirtuins 4.4 Hypertension and sirtuins 4.5 Novel treatment options in diabetes mellitus and diabetic kidney disease 4.6 Conclusion and future perspectives References

YI FANG AND XIAOLING LI

Introduction Stem cells and sirtuins SIRT1 in stem cell biology SIRT2 in stem cell biology SIRT3 in stem cell biology SIRT6 in stem cell biology SIRT7 in stem cell biology

39

4. Sirtuins in diabetes mellitus and diabetic kidney disease

6 7 8 9 10 11 13 13

2. Sirtuins in metabolic and epigenetic regulation of stem cells 2.1 2.2 2.3 2.4 2.5 2.6 2.7

34 35

25 26 26 31 32 32 33

61 62 62 65 65 71 71 72 73

5. Sirtuins and mitochondrial dysfunction JIAN-LI HE, TIAN-SHI WANG AND YI-PING WANG

5.1 Sirtuins are nutrient sensors 5.2 Sirtuins and mitochondrial biogenesis 5.3 Sirtuins and mitochondrial metabolism

vii

79 80 82

viii 5.4 Sirtuins and mitochondrial dysfunction in human diseases 5.5 Feasible clinical targets: posttranslational modifications of sirtuins regulate mitochondrial function Acknowledgments References

Contents

82 85 86 86

6. Sirtuins in immunometabolism HONGXIU YU

6.1 Brief introduction of immunometabolism 6.2 Role of sirtuins in immunometabolism 6.3 Conclusion and future considerations References

91 93 97 98

7. Mitochondrial sirtuins at the crossroads of energy metabolism and oncogenic transformation MAJA GRABACKA AND PRZEMYSLAW M. PLONKA

Abbreviations 7.1 Introduction—advantages of possessing mitochondria 7.2 Mitochondrial sirtuins 7.3 Lipoylation of multienzymatic complexes is essential for mitochondrial metabolism 7.4 Alternative lipoylation and its metabolic consequences 7.5 Regulation of pyruvate dehydrogenase complex by mitochondrial sirtuins 7.6 Alpha ketoglutarate dehydrogenase complex regulates gene expression 7.7 Fluctuations of the intracellular concentration of organic acids has far-reaching implications 7.8 Ketogenic enzymes ACAT1 and HMGCS2 as substrates for Sirt3 and Sirt5 7.9 Antagonistic roles of mitochondrial sirtuins in fed and fasted state 7.10 The interplay between Sirt3 and isocitrate dehydrogenase in cancer cells 7.11 Tumor-suppressing and tumor-promoting activities of sirtuins in the context of glutamine and glucose metabolism 7.12 Sirtuins regulate iron sulfur cluster assemblage 7.13 Mitochondrial fatty acid synthesis is linked to Fe S cluster assembly and protein lipoylation—implications for cancer cell metabolism 7.14 Consequences of Fe S cluster defects in cancer cells 7.15 Deoxyribonucleotide synthesis—toward the as yet unexplored areas of sirtuin research 7.16 Perspectives—evolutionary implications and new directions in cancer treatment References

104 105 106 106

Section II Sirtuins and cancer 8. Sirtuins and the hallmarks of cancer TALITA H.B. GOMIG, TAYANA S. JUCOSKI, ERIKA P. ZAMBALDE, ALEXANDRE L.K. AZEVEDO, DANIELA F. GRADIA AND ENILZE M.S.F. RIBEIRO

8.1 Introduction 8.2 Sirtuins in sustaining proliferative signaling and evading growth suppressors 8.3 Sirtuins and resisting cell death 8.4 Sirtuins in tumor-promoting inflammation and immune system function 8.5 Sirtuins in angiogenesis 8.6 Sirtuins in invasion and metastasis 8.7 Sirtuins in genome instability and replicative immortality 8.8 Sirtuins in reprogramming energy metabolism 8.9 Sirtuins and cancer therapy 8.10 Concluding remarks References

109

9. The bifunctional roles of sirtuins and their therapeutic potential in cancer

110

YEUAN TING LEE, YI JER TAN, PEI YI MOK, AYAPPA V. SUBRAMANIAM AND CHERN EIN OON

111 112 113

9.1 The mammalian sirtuins 9.2 Sirtuin modulators 9.3 Conclusion and future perspectives Acknowledgment References

129 132 135 136 137 137 138 139 141 142 142

153 160 168 169 169

114 115 115 116

116 118 119 120 121

10. Sirtuins and next generation hallmarks of cancer: cellular energetics and tumor promoting inflammation ROBERT KLESZCZ AND WANDA BAER-DUBOWSKA

10.1 Introduction: an overview of sirtuins involvement in inflammation and cancer metabolism 10.2 Nuclear and cytosolic sirtuins involvement in metabolism of cancer and inflammatory cells 10.3 Mitochondrial sirtuins 10.4 Sirtuins indeed link metabolism, inflammation, and cancer? 10.5 Conclusions and perspectives References

179 180 184 188 190 190

Contents

11. Sirtuins and cellular metabolism in cancers

13. Sirtuin signaling in hematologic malignancies

ZHEN DONG AND HONGJUAN CUI

RYAN A. DENU

11.1 The metabolic characteristics of cancers 11.2 The regulatory modes of sirtuins in controlling cellular metabolism 11.3 Direct epigenetic control of cellular metabolism by sirtuins 11.4 Direct posttranslational control of cellular metabolism by sirtuins 11.5 Indirect control of cellular metabolism by sirtuins 11.6 Conclusions References

195 197 198 200 206 211 211

12. Dual role of sirtuins in cancer

233 234 235 244 248 249 250

14. Impacts of sirtuin1 and sirtuin3 on oral carcinogenesis SHAJEDUL ISLAM, YOSHIHIRO ABIKO, OSAMU UEHARA, YASUHIRO KURAMITSU AND ITSUO CHIBA

MARGALIDA TORRENS-MAS AND PILAR ROCA

12.1 Introduction 12.2 Sirtuins and cancer metabolism 12.3 Sirtuins and oxidative damage 12.4 Sirtuins, genomic stability, and DNA repair 12.5 Sirtuins and metastasis 12.6 Sirtuins and cancer stem cells 12.7 Sirtuins and chemoresistance 12.8 Sirtuins: tumor suppressors or promoters? References

Abbreviations 13.1 Introduction 13.2 Hematologic malignancies 13.3 Sirtuins regulate pathways important for hematologic malignancies 13.4 Therapeutic opportunities 13.5 Conclusions References

ix

219 219 221 221 222 223 223 224 226

14.1 14.2 14.3 14.4

Introduction Overview of sirtuins Involvement of sirtuins in oral cancer Potential therapeutic implications of sirtuins in oral cancer 14.5 Concluding remarks References

Index

259 260 264 269 271 271

275

List of contributors

Yoshihiro Abiko Division of Oral Medicine and Pathology, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan

Hospital Affiliated, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China Shajedul Islam Division of Disease Control and Molecular Epidemiology, Department of Oral Growth and Development, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan; Division of Oral Medicine and Pathology, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan

Alexandre L.K. Azevedo Genetics Postgraduation Program, Genetics Department, Federal University of Parana´, Curitiba, Brazil Wanda Baer-Dubowska Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, ´ Poland Poznan,

Tayana S. Jucoski Genetics Postgraduation Program, Genetics Department, Federal University of Parana´, Curitiba, Brazil

Itsuo Chiba Division of Disease Control and Molecular Epidemiology, Department of Oral Growth and Development, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan

Robert Kleszcz Department of Pharmaceutical Biochemistry, ´ Poland Poznan University of Medical Sciences, Poznan, Yasuhiro Kuramitsu Advanced Research Promotion Centre, Health Sciences University of Hokkaido, Hokkaido, Japan

Hongjuan Cui State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, College of Sericulture and Textile and Biomass Science, Southwest University, Chongqing, P. R. China; Cancer Center, Medical Research Institute, Southwest University, Chongqing, P. R. China Ryan A. Denu University of Wisconsin-Madison, Madison, WI, United States

Yeuan Ting Lee Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia Xiaoling Li Signal Transduction Laboratory, Institute of Environmental Health Sciences, Triangle Park, NC, United States

National Research

Kenneth Maiese Biotechnology and Venture Capital Development, Office of Translational Alliances and Coordination, National Heart, Lung, and Blood Institute; Cellular and Molecular Signaling, New York, NY, United States

Zhen Dong State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, College of Sericulture and Textile and Biomass Science, Southwest University, Chongqing, P. R. China; Cancer Center, Medical Research Institute, Southwest University, Chongqing, P. R. China

Pei Yi Mok Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia

Yi Fang Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States

Janaina Ribeiro Oliveira Laboratory of Health Science, Postgraduate Program in Health Sciences; State University of Montes Claros (UNIMONTES), Montes Claros, Brazil

Talita H.B. Gomig Genetics Postgraduation Program, Genetics Department, Federal University of Parana´, Curitiba, Brazil

Chern Ein Oon Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia Przemyslaw M. Plonka The Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, The Jagiellonian University in Krako´w, Krako´w, Poland

Maja Grabacka The Department of Biotechnology and General Technology of Foods, Faculty of Food Technology, University of Agriculture in Krako´w, Krako´w, Poland

Luiz Fernando Rezende Laboratory of Health Science, Postgraduate Program in Health Sciences; State University of Montes Claros (UNIMONTES), Montes Claros, Brazil

Daniela F. Gradia Genetics Postgraduation Program, Genetics Department, Federal University of Parana´, Curitiba, Brazil

Enilze M.S.F. Ribeiro Genetics Postgraduation Program, Genetics Department, Federal University of Parana´, Curitiba, Brazil

Victor Hugo Dantas Guimara˜es Laboratory of Health Science, Postgraduate Program in Health Sciences; State University of Montes Claros (UNIMONTES), Montes Claros, Brazil

Pilar Roca Translational Oncology Multidisciplinary Group, Research Institute of Health Sciences (IUNICS), University of Balearic Islands, Health Research Institute of the Balearic Islands (IdISBa), E-07122 Balearic Islands, Palma, Spain; Ciber Obesity and Nutrition Physiopathology (CIBEROBN, CB06/03), Health Institute Carlos III, Madrid, Spain

Jian-Li He Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China; State Key Laboratory of Oncogenes and Related Genes, Renji

xi

xii

List of contributors

Se´rgio Henrique Sousa Santos Postgraduate Program in Food and Health; Food Engineering School, Agricultural Sciences Institute (ICA), Federal University of Minas Gerais (UFMG), Montes Claros, Brazil; Laboratory of Health Science, Postgraduate Program in Health Sciences; State University of Montes Claros (UNIMONTES), Montes Claros, Brazil Ayappa V. Subramaniam Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia Yi Jer Tan Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia Margalida Torrens-Mas Translational Oncology Multidisciplinary Group, Research Institute of Health Sciences (IUNICS), University of Balearic Islands, Health Research Institute of the Balearic Islands (IdISBa), E-07122 Balearic Islands, Palma, Spain Kultigin Turkmen Division of Nephrology, Department of Internal Medicine, Meram School of Medicine, Necmettin Erbakan University, Konya, Turkey Osamu Uehara Division of Disease Control and Molecular Epidemiology, Department of Oral Growth and

Development, School of Dentistry, Health University of Hokkaido, Hokkaido, Japan

Sciences

Tian-Shi Wang Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China; State Key Laboratory of Oncogenes and Related Genes, Renji Hospital Affiliated, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China Yi-Ping Wang Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Key Laboratory of Breast Cancer in Shanghai, Cancer Institute, and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College, Fudan University, Shanghai, P. R. China; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, P. R. China Hongxiu Yu Institutes of Biomedical University, Shanghai, P. R. China

Sciences,

Fudan

Erika P. Zambalde Genetics Postgraduation Program, Genetics Department, Federal University of Parana´, Curitiba, Brazil

About the editor

Kenneth Maiese is an internationally recognized physician-scientist, healthcare executive, and editor with broad research, clinical, and leadership experience in academia, the federal government, and industry. He was born and raised in New Jersey and was the Valedictorian of his high school class at Pennsauken High School. He graduated from the University of Pennsylvania Summa cum Laude with Distinction and was a Teagle Scholar, Grupe Scholar, and Joseph Collins Scholar at Weill Medical College of Cornell University. Dr. Maiese was subsequently trained as a physician-scientist at Cornell, the National Institutes of Health, and as a senior executive at the Harvard T.H. Chan School of Public Health. He has extensive experience in academic medicine, healthcare delivery, business development, managed care, biotechnology, and drug development, holding positions as member and advisor for the National Institutes of Health Biotechnology and Venture Capital Development, National Institutes of Health Innovation Network, Chief Medical Officer, tenured Professor and Chair and Chief of Service of the Department of Neurology and Neurosciences of Rutgers University, Global Head of Translational Medicine and External Innovation, Board Member of the Cancer Institute of New Jersey, Steering Committee Member for the Foundation for the National Institutes of Health, tenured Professor in Neurology, Anatomy & Cell Biology, Molecular Medicine, the Barbara Ann Karmanos Cancer Institute, and the National Institute of Health Center at Wayne State University. He is the Founding Editor and Editor-in-Chief of multiple highly successful international journals. Dr. Maiese maintains therapeutic and scientific expertise in multiple medical disciplines, regulatory policy, and drug commercialization. His work has to elucidate a number of new avenues for the fruitful discovery of innovative strategies to treat neurodegenerative diseases, cardiovascular disorders, metabolic dysfunction, and cancer and he has led the development of first-in-class pharmaceuticals. Early in his career, Dr. Maiese received outstanding investigator awards, received the Hoechst Award for exceptional basic science work, was named a Johnson & Johnson Distinguished Investigator, was chosen as a Henrietta B. and Frederick H. Bugher Foundation Investigator, received the Albrecht Fleckenstein Memorial Award for Distinguished Achievement in Basic Research, was elected to America’s Top Physicians and The Best of US Physicians, was recipient of Albert Nelson Marquis Lifetime Achievement Award, and was elected as an America’s Health Insurance Plans (AHIP) Executive Leadership Fellow. His highly cited work has received the distinction of “High Impact Research and Potential Public Health Benefit” by the National Institutes of Health. He has been fortunate to have benefited from continuous funding from sources that include the American Diabetes Association, the American Heart Association, the Bugher Foundation, the Johnson & Johnson Focused Giving Award, and the National Institutes of Health. His service on Executive Councils for Graduate Schools has fostered innovative graduate student training programs and he was elected as board and advisor member to develop and execute inaugural MD/PhD Degree Programs and new Translational Medicine Programs. He chairs national grant committees, formulates regulatory and utilization management policies for national clinical care guidelines, and is a chartered panel member or consultant for numerous national and international foundations as well as multiple study sections and special emphasis panels for the National Institutes of Health. Dr. Maiese is frequently honored as the chairperson and/or the plenary and keynote speaker for a number of international symposiums, organizations, and presentations to federal policy makers.

xiii

Preface

believed that at least another 400 million individuals either suffer from metabolic disease or are at risk for developing DM. These estimates for the number of individuals who may be undiagnosed or at risk to develop DM are expected to increase to 700 million by the year 2045. Furthermore, at least 7 million individuals over the age of 18 remain undiagnosed with DM. Prevalence of DM also has increased from 9.5% during the period of 1999 2002 to 12% during the period of 2013 16. Risk factors for developing complications of DM include tobacco consumption, lack of physical activity, hypertension, and elevated serum cholesterol. Obesity is considered to be an additional risk factor for the development of DM. Obesity and excess body fat can increase the risk of developing DM and can affect stem cell proliferation, agingrelated illnesses, inflammation, oxidative stress injury, and mitochondrial function. In regards to financial impact, the cost for the care of patients with DM is estimated to be at least US$ 760 billion, and these costs consume more than 17% of the Gross Domestic Product in the United States. Loss of function and disability as a result of DM leads to another estimated US$ 69 billion. Interestingly, cancer and DM share not only common risk factors but also biological mechanisms that may influence each of these diseases. Cancer and DM share the risk factors of obesity, poor nutrition, aging, and inactivity. The mechanisms that link cancer and DM may involve hyperglycemia, hyperinsulinemia, and inflammation. As a result, promotion of proper nutrition, exercise, and weight management can improve outcomes for individuals with cancer or DM. DM, primarily type 2, is associated with increased risk for the development of cancer in the liver, pancreas, colon, rectum, breast, bladder, and endometrial. Individuals with DM also have an increased risk for the development of non-Hodgkin’s lymphoma. In general, with some exceptions, women with DM are approximately 27% more likely to develop cancer when compared to women without DM, and men with DM are 19% more likely to develop cancer when compared to men without DM. In addition, some therapies for DM, such as metformin, may be associated with a lower risk for the development of cancer.

Life expectancy continues to increase throughout the world and is approaching 80 years of age for individuals. The rise in life span across the globe also has paralleled the increase observed with noncommunicable diseases (NCDs). NCDs are increasing in incidence throughout the world. At least 70% of deaths that are documented each year are the result of NCDs. Interestingly, NCDs affect a significant proportion of the population in low- and middle-income countries with at least one-third of the population under the age of 60 suffering from NCDs. Of the greater than 40 million individuals that die each year from NCDs, at least 15 million individuals are between 30 and 69 years old. The 10 leading causes of death in the world are cardiac disease, cancer, trauma, respiratory disease, stroke, Alzheimer’s disease, diabetes mellitus (DM), influenza and pneumonia, kidney disease, and suicide. Of these NCDs, cancer is a significant concern since it is the second leading cause of death throughout the world. It is estimated that approximately one in six deaths result from cancer, and at least 70% of cancer deaths are in low- and middle-income countries. The most significant risk factor for cancer is tobacco use which represents more than 20% of cancer deaths. High body mass index, lack of physical activity, low fruit and vegetable intake, alcohol consumption, and tobacco use together lead to greater than 33% of cancer-associated deaths. Furthermore, cancer also has a significant effect on world economies and creates a large financial drain on resources. It is estimated that only 20% 30% of low- and middle-income countries have the appropriate capability to treat cancer-related illnesses. In contrast, over 90% of high-income countries have effective treatment services for cancer. Recent estimates report that in the United States, the cost for cancer therapy is almost US$ 150 billion, and the estimated lost earnings for individuals in the United States as a result of cancer-related illnesses exceed another US$ 100 billion per year. In addition to cancer, metabolic disorders that include DM also represent a significant component of NCDs. At present, at least 500 million individuals have DM and approximately 80% of adults with DM are living in low- and middle-income countries. It is

xv

xvi

Preface

Current treatments for both cancer and DM remain limited and, in many cases, cannot block disease progression. For example, early diagnosis and rapid treatment of DM can provide some degree of protection and may partially limit the progression of DM. Yet, even under the best of circumstances, tight serum glucose control does not blunt the complications that can arise during DM or completely prevent the risk of tumorigenesis. As a result, novel avenues for therapeutic strategies to address cancer and metabolic disorders are extremely warranted. An exciting prospect for the development of innovative therapeutics involves sirtuins. Sirtuins are histone deacetylases that have an intricate role in the onset and development of cancer and metabolic disease. Sirtuins are expressed throughout the body, have wideranging biological effects, and significantly affect both cellular survival and longevity during acute and longterm illnesses. In general, sirtuins have an intricate and complex role in the pathology, progression, and treatment of multiple disease entities that involve cancer, metabolic disease and DM, neurodegenerative disorders, cognitive loss, behavior disorders, cardiovascular disease, atherosclerosis, immune system dysfunction, reproductive disease, endocrine disorders, gastrointestinal disease, and aging-related disorders. In particular, sirtuins can oversee critical pathways that involve stem cell maintenance, cellular proliferation, metabolic homeostasis, apoptosis, and autophagy that can impact cellular dysfunction and unchecked cellular growth that can occur during cancer and metabolic disease. Sirtuin Biology in Cancer and Metabolic Disease: Cellular Pathways for Clinical Discovery is a unique resource for detailing the basic and clinical roles of sirtuins, recent discoveries on the understanding of sirtuin biology, and the broad applicability for sirtuin pathways to be targeted for the clinical care of cancer and metabolic disorders. With chapters authored by internationally recognized experts, this book fills a significant void to provide a foundation for elucidating the intimate relationship between cancer and metabolic disease that intersects with sirtuin pathways. Each chapter offers an intuitive perspective of advances on the application of sirtuin pathways for cancer and

metabolic disease that will be become a “go-to” resource for a broad audience of scientists, physicians, pharmaceutical industry experts, nutritionists, and students. Sirtuin Biology in Cancer and Metabolic Disease: Cellular Pathways for Clinical Discovery begins with Section I, Sirtuins and Metabolic Disease. In this section, the role of sirtuins in metabolic disease, relation to other integral biologic pathways of sirtuins, regulation of stem cells, supplementation of nutritional requirements, immune function modulation, and maintenance of mitochondrial function are presented. Additionally, a discussion is expanded on sirtuin pathways that affect glucose sensitivity and tolerance, obesity, the nervous system, renal disease, and the link between energy metabolism and transformation into cancer. Section II, Sirtuins and Cancer, provides a critical perspective for the role of sirtuins as an agent that oversees tumorigenesis and metabolic pathways that influence cancer cells. Sirtuins are discussed as both tumor-promoting and suppressor agents that can change depending upon environmental stimuli: signal transduction pathways of sirtuins are highlighted that involve inflammatory modulation, invasion and migration capabilities of sirtuins are dissected, the impact of changing cellular metabolism dynamics is highlighted, and the development of sirtuin modulators as chemotherapeutic agents is presented. In addition, the role of sirtuins is examined for multiple cancer cell growths, such as oral carcinogenesis and hematologic malignancies, and the sirtuin mechanisms that lead to cellular inflammation and the development of metastatic disease are presented. Sirtuin Biology in Cancer and Metabolic Disease: Cellular Pathways for Clinical Discovery offers a compelling and thought-provoking perspective of the examination of the intriguing biology of sirtuins that ties cancer and metabolic disease together and provides a critical platform for the development of sirtuin-based novel therapeutic strategies to effectively treat cancer and metabolic disorders with precision in order to minimize any potentially detrimental clinical outcomes. Kenneth Maiese Editor

Acknowledgment

My sincere appreciation goes to the extraordinary devotion and expertise of our contributors. The ongoing support, time, and undertakings that I have received from the Elsevier team, especially, Tari Broderick and Timothy Bennett, have made this book a reality.

xvii

C H A P T E R

1 Sirtuins in metabolic disease: innovative therapeutic strategies with SIRT1, AMPK, mTOR, and nicotinamide Kenneth Maiese1,2 1

Biotechnology and Venture Capital Development, Office of Translational Alliances and Coordination, National Heart, Lung, and Blood Institute; 2Cellular and Molecular Signaling, New York, NY, United States O U T L I N E Abbreviations

3

1.6 SIRT1 and AMP-activated protein kinase

8

1.1 Noncommunicable diseases

4

1.7 SIRT1, mTOR, and metabolic disease

9

1.2 Metabolic disorders

4

1.8 SIRT1, nicotinamide, and cellular metabolism

10

1.3 Novel therapeutic strategies with sirtuins for metabolic disease

1.9 Future considerations

11

6

Acknowledgments

13

1.4 Silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae)

6

References

13

1.5 SIRT1, metabolic function, and obesity

7

Abbreviations AGEs AgRP AD AMPK Aβ Bad Deptor DNA DM EPO 4EBP1 FRAP SGK1 HbA1c IGF-1 IRS-1 FoxOs mLST8

advanced glycation end products agouti-related peptide Alzheimer’s disease AMP-activated protein kinase β-amyloid BCL2-associated agonist of cell death DEP domain-containing mTOR interacting protein deoxyribonucleic acid diabetes mellitus erythropoietin eukaryotic initiation factor 4E (eIF4E)-binding protein 1 FKBP12-rapamycin-associated protein glucocorticoid induced protein kinase 1 hemoglobin A1c insulin growth factor-1 insulin receptor substrate 1 mammalian forkhead transcription factors mammalian lethal with Sec13 protein 8, termed mLST8

Sirtuin Biology in Cancer and Metabolic Disease. DOI: https://doi.org/10.1016/B978-0-12-822467-0.00006-1

3

© 2021 Elsevier Inc. All rights reserved.

4 mSIN1 mTOR mRNA mTORC1 mTORC2 NAD1 NADP1 NMN NCDs NF-κB p70S6K PPAR PGC PS PDK1 PI 3-K PRAS40 POMC Akt AGC PKCα PTP LKB1 SIRT1 TNF-α UCP

1. Sirtuins in metabolic disease: innovative therapeutic strategies with SIRT1, AMPK, mTOR, and nicotinamide

mammalian stress-activated protein kinase interacting protein mechanistic target of rapamycin messenger ribonucleic acid mTOR Complex 1 mTOR Complex 2 nicotinamide adenine dinucleotide nicotinamide adenine dinucleotide phosphate nicotinamide mononucleotide noncommunicable diseases nuclear factor-κB p70 ribosomal S6 kinase peroxisome proliferator-activated receptor peroxisome proliferator-activated receptor gamma coactivator phosphatidylserine phosphoinositide-dependent kinase 1 phosphotidylinositide 3-kinase proline rich Akt substrate 40 kDa proopiomelanocortin protein kinase B protein kinase A/protein kinase G/protein kinase C protein kinase C-α protein tyrosine phosphatase serine-threonine liver kinase B1 silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) tumor necrosis factor-α uncoupling protein

1.1 Noncommunicable diseases Metabolic disorders, that include diabetes mellitus (DM), are considered to be a leading cause of death in the world as part of noncommunicable diseases (NCDs) [1
5]. NCDs are increasing in incidence throughout the world [2,6
8] and according to the World Health Organization, almost 70% of deaths that are documented each year are the result of NCDs [9,10]. Both wealthy and low-income countries are affected by NCDs. Greater than 10% of the population less than 60 years of age is affected in high-income countries [9]. NCDs affect a greater proportion of the population in low- and middle-income countries with at least one-third of the population under the age of 60 suffering from NCDs. Of the over 40 million individuals that die each year from NCDs, 15 million individuals are younger, with ages between 30 and 69 years old. The rise in NCDs follows an observed increase in life expectancy of the world’s population [11,12]. After a recent reduction that has been reported in deaths from opioid overdoses, life expectancy is increasing again in the United States [13]. The age of the world’s population continues to increase with new estimates of life expectancy approaching 80 years of age [14]. With life expectancy marked by a 1% decrease in the age-adjusted death rate from the years 2000 through 2011 [15], the number of individuals over the age of 65 has been observed to double during the prior 50 years [16]. In consideration of less developed nations, the number of older individuals in large developing countries such as India and China also will increase from 5% to 10% over the next several decades [17,18]. Although many factors may account for the observed increased in life span for the world’s population, the 10 leading causes of death, which are cardiac disease, cancer, trauma, respiratory disease, stroke, Alzheimer’s disease (AD), DM, influenza and pneumonia, kidney disease, and suicide, have remained the same [13]. Yet improvements in preventive medical care, more focused public health guidelines and sanitation measures, and new treatments for multiple disorders that involve endocrine disease, metabolic disorders, and nutrition have assisted with improvements in life span longevity [3,19
26].

1.2 Metabolic disorders As a significant component of NCDs, DM is a disorder that is increasingly being targeted for the development of new treatment strategies to reduce death and disability for the world’s population [27
29] (Table 1.1). Approximately 80% of adults with DM are living in low- and middle-income countries [4]. Currently, almost

I. Sirtuins and metabolic disease

1.2 Metabolic disorders

5

TABLE 1.1 B-cell function (Mitochondrial D). Innovative treatment strategies with SIRT1 for metabolic disease • Metabolic disorders, such as diabetes mellitus (DM), are a significant component of the increased prevalence of noncommunicable diseases (NCDs) and affect all organs and systems of the body. • Given the challenges with limited therapeutic options for metabolic disease, the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1) and the pathways with AMP-activated protein kinase (AMPK), the mechanistic target of rapamycin (mTOR), and nicotinamide offer exciting avenues for innovative treatment strategies for metabolic disease. • SIRT1 can increase life span in higher organisms and limit oxidative stress injury, block β-amyloid (Aβ) and tau toxicity that can affect cognition, prevent apoptotic cell death, limit the development of obesity, regulate nutritional intake, maintain mitochondrial function, and oversee insulin sensitivity. • AMPK through autophagy activation can reduce insulin resistance, improve cognition in models of Alzheimer’s disease and DM, promote clearance of Aβ and tau, preserve β-cell function, and block hyperglycemic cell death. • mTOR can improve insulin secretion, decrease insulin resistance, limit apoptotic cell death, foster the differentiation of adipocytes, block Aβ toxicity, and maintain mitochondrial function and neurovascular cell survival independently and through the trophic factor erythropoietin (EPO). • Nicotinamide, a product and inhibitor of SIRT1, can decrease insulin resistance, prevent muscle atrophy during DM, reduce mitochondrial stress through AMPK, reduce hemoglobin A1c (HbA1c) levels, and prevent palmitate-induced hepatotoxicity via SIRT1-dependent induction of autophagy. • SIRT1 has an intimate but complex relationship with mTOR, AMPK, and nicotinamide such that biological feedback pathways exist that require a fine balance in modulation in order to achieve optimal clinical efficacy.

500 million individuals have DM [5,17,30
32]. At least another 400 million individuals are believed to either suffer from metabolic disease or be at risk for developing DM [4,33
35]. In addition, according to the International Diabetes Federation the number of individuals with DM is expected to rise to 700 million individuals by the year 2045 [4]. In the United States, almost 35 million individuals, which represents approximately 10% of the population, are diagnosed with DM [1]. At least seven million individuals over the age of 18 remain undiagnosed with DM and in the year 2018 it was estimated that almost 35% of adults in the United States had prediabetes based on their fasting glucose and hemoglobin A1c (HbA1c) levels [36]. Prevalence of DM also has increased from 9.5% during the period of 1999
2002 to 12% during the period of 2013
16. In the adult population, it was noted that prevalence varied by indicators of socioeconomic status, such as education level. At least 13% of adults with less than a high school education had DM compared to almost 10% of individuals with a high school education and DM and 7.5% of individuals with greater than a high school education and DM. Risk factors for developing complications of DM included tobacco consumption, physical inactivity, hypertension, and elevated serum cholesterol [2]. Obesity is considered to be another risk factor for the development of DM. Obesity results in impaired glucose tolerance that leads to DM progression [24,37
43]. Obesity and excess body fat can increase the risk of developing DM in young individuals [44] and can affect stem cell proliferation, aging, inflammation, oxidative stress injury, and mitochondrial function [39,45
51]. In regard to the financial considerations for DM, at least US$ 20,000 are required to care for each individual with DM per year. The care for patients with DM equals approximately US$ 760 billion [4]. This care consumes more than 17% of the Gross Domestic Product in the United States, per the Centers for Medicare and Medicaid Services (CMS) [52]. When considering the loss of function and disability that result from DM individuals, an estimated US$ 69 billion are consumed from reduced productivity linked to DM. The toxic effects of DM do not exclude any organs of the body and can affect all cellular systems [53]. In the peripheral nervous system, at least 70% of individuals with DM can develop diabetic peripheral neuropathy. DM can result in autonomic neuropathy [54] and peripheral nerve disease [55
58]. Assessments of peripheral neuropathies can be challenging, since the disorder is chronic in nature, may be subclinical, and prior deficits may go undetected even after improved control over glucose homeostasis has been initiated. In the central nervous system, DM can cause insulin resistance and dementia in patients with AD [2,6,11,29,53,59
61]. DM can affect multiple cellular pathways that lead to the progression of cognitive loss [17,62
67]. DM also has been tied to mental illness [68,69], cerebral vascular injury [17,34,70
73], impairment of microglial activity [29,59
61], and can impact stem cell proliferation [17,41,62
66]. DM also can result in endothelial dysfunction [11,17,74
76], cardiovascular disease [35,37,75,77
83], retinal disease [84
86], and immune and infectious disorders [87
93].

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1. Sirtuins in metabolic disease: innovative therapeutic strategies with SIRT1, AMPK, mTOR, and nicotinamide

1.3 Novel therapeutic strategies with sirtuins for metabolic disease Given the growing prevalence of DM, the significant number of individuals that remain undiagnosed with DM, and the severe financial impact on global economies, new and innovative therapeutic strategies are vital to be developed for the treatment of metabolic disorders, such as DM. With conventional therapies, early diagnosis of DM and rapid treatment can offer some degree of protection and may inhibit the progression of DM [5,11,94
98]. However, even under the best of circumstances, tight serum glucose control does not blunt the complications that can arise during DM [2,99]. Careful nutritional and exercise management also is considered to be important for DM care, but in some cases these strategies may be less than beneficial dependent upon the degree of reduced oral intake and a decrease in organ mass through processes that involve autophagy [100]. DM also has additional risk factors when one considers the development of neurodegenerative disorders and cognitive loss that can be compounded by hypertension, low education in early life, and tobacco use [2,29,53,101]. For example, vascular disease as a result of DM may lead to dementia [2,6,8,11,53,102
104]. As a result, new avenues for therapeutic strategies to address metabolic disorders are urgently needed. One innovative strategy that offers exciting prospects involves the silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1) and its associated pathways with AMP-activated protein kinase (AMPK), the mechanistic target of rapamycin (mTOR), and the vitamin nicotinamide (Table 1.1).

1.4 Silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) SIRT1 is a member of the sirtuin family (sirtuin 1) and a histone deacetylase [11,17,22,105
114] that can transfer acetyl groups from ε-N-acetyl lysine amino acids to the histones of deoxyribonucleic acid (DNA) to control transcription (Table 1.1). Seven mammalian homologues of Sir2 that include SIRT1 through SIRT7 have been identified. These histone deacetylases can control cellular metabolism, cell senescence, cell function, cell injury, and posttranslation modifications of proteins [2,8,115
118]. SIRT1 depends upon the substrate nicotinamide adenine dinucleotide (NAD1) [90,109,119
121]. Histone deacetylases are enzymes that transfer acetyl groups from ε-N-acetyl lysine amino acids that exist on the histones of DNA to regulate transcription. Although histone deacetylases primarily oversee DNA transcription, they may be involved with posttranslational changes of proteins as well such as the ability of SIRT1 to control the posttranslational phosphorylation of mammalian forkhead transcription factors [122,123]. During deacetylase reactions, sirtuins, that include SIRT1, transfer the acetyl residue from the acetyllysine residue of histones to the ADP-ribose moiety of NAD1, resulting in the production of nicotinamide, 20 -O-acetyl ADP ribose, and deacetylated proteins. SIRT1 expression is present in the brain, heart, liver, pancreas, skeletal muscle, spleen, and adipose tissues. In the cell, SIRT1 is present in the nucleus and cytoplasm with dominant expression in the nucleus [11,85,90,106,112,118,124
126]. In disorders that can lead to oxidative stress, such as DM [5,11,44,94,98,127,128], SIRT1 activation can decrease oxidative stress, prevent diabetic retinal injury, and block cell injury that would lead to memory loss [2,85,112,129
135]. Activation of SIRT1 has been associated with reduction in oxidative stress and the protection of cognition [136,137]. Oxidative stress may lead to aberrant cell cycle reentry that can lead to neuronal death during AD [138,139]. Limiting the generation of reactive oxygen species in models of AD has led to reduced toxicity of Aβ, suggesting that oxidative stress is a critical component in the pathology of AD [126]. SIRT1 may directly block Aβ and tau toxicity and preserve mitochondrial function. SIRT1 may limit the toxicity of Aβ [140
143]. SIRT1 also can oversee neurovascular protection in the brain that would be relevant for ischemicmediated dementia that can occur during DM [17,89,90,105,112,119,120,130,144
147]. SIRT1 can increase life span in higher organisms and protect against oxidative stress in neuronal cells [129]. Pancreatic SIRT1 activation through exercise also has been shown to potentially reduce oxidative stress complications during diabetes [148]. SIRT1 can control pathways of apoptosis and autophagy [113,114] (Table 1.1). In regard to autophagy, activation of autophagy in human lung cancer cells leads to increased cell survival. SIRT1 inhibition leads to prosurvival autophagy activity and inhibition of apoptosis in these cells [149]. SIRT1 also can upregulate autophagy and mitochondria function in embryonic stem cells during periods of oxidative stress [150]. Through SIRT1 activity and autophagic flux, senescence is inhibited in vascular endothelial cells [151]. SIRT1 is also tied to apoptosis. SIRT1 activation blocks external membrane phosphatidylserine (PS) exposure during the early phases of apoptosis in mature cells [123,152
154]. SIRT1 can limit apoptosis initiated by tumor necrosis factor-α (TNF-α) in endothelial progenitor cells [155]. Loss of SIRT1 expression in endothelial progenitor

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1.5 SIRT1, metabolic function, and obesity

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cells leads to apoptotic cell death that can occur in smokers and chronic obstructive disease patients [156]. Apoptotic cell death during oxidative stress in cardiac cells is minimized through the activation of SIRT1 [157].

1.5 SIRT1, metabolic function, and obesity SIRT1 plays a critical role in cellular metabolism [28,43,106,119,158,159] (Table 1.1). For example, SIRT1 can oversee the development of fat progression and obesity. SIRT1 in association with peroxisome proliferatoractivated receptor-γ (PPAR-γ) can control adipogenesis. SIRT1 protein can repress PPAR-γ to result in the mobilization of fatty acids from white adipocytes upon fasting and lead to the loss of fat through lipolysis [160]. PPAR-γ also can interact with SIRT1 and form a negative feedback to regulate SIRT1 expression and activity [161]. SIRT1 expression has been found to be depressed in adipose tissue in obese rodents. Loss of SIRT1 in white adipose cells results in the impairment of fatty acid mobilization. Treatment with resveratrol, an activator of SIRT1, can lead to calorie restriction to prevent obesity as a result of a high calorie diet in mice [162]. SIRT1 also has been shown to be expressed in anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus and regulate food intake and cellular metabolism [163]. Overexpression of SIRT1 in the hypothalamus prevents mammalian forkhead transcription factor 1 (FoxO1), a mammalian forkhead transcription family member [8,110,164], from promoting hyperphagia and weight gain [163]. Yet lack of SIRT1 in POMC neurons leads to obesity due to reduced energy expenditure [165]. Additional studies have shown that melatonin can reduce inflammation in adipocytes that can limit obesity through pathways that involve SIRT1 [48] and that decreased expression of SIRT1 during hyperglycemia and obesity can increase stroke infarct size [72]. In relation to cellular metabolic regulation, SIRT1 regulates the activity of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) through deacetylation. PGC-1α is a member of a family of transcriptional coactivators that includes PGC-1α, PGC-1β, and PGC-1 related coactivator (PRC). PGC-1α leads to gene transcription and increases the expression of genes that regulate mitochondrial functions and fatty acid oxidation [166]. Increased PGC-1α activity can function to protect against some metabolic diseases and improve mitochondrial biogenesis. SIRT1 can oversee PGC-1α activity in the liver to lead to gluconeogenic genes and hepatic glucose output. SIRT1 can control the ability of PGC-1α to repress glycolytic genes in response to fasting and pyruvate [167]. Hepatic SIRT1 with PPAR-α, a receptor that can increase free fatty acid uptake and decrease lipolysis, activates PGC-1α to lead to lipid homeostasis. SIRT1 deletion in the liver results in the loss of PGC-1α activity and the subsequent impairment of fatty acid oxidation, leading to the develop of hepatic steatosis during high-fat diets [168]. SIRT1 has an important role in the management of insulin sensitivity. During high-fat diets, increased SIRT1 activity can regulate glucose and hepatic lipid homeostasis and protects against metabolic syndrome [169]. SIRT1 activation also limits diabetic myocardial injury by inhibiting high mobility group box 1/nuclear factor-κB (NF-κB) pathway-associated proteins [170]. During the administration of high-fat diets, SIRT1 expression is decreased in the pancreas and liver and may be associated with insulin resistance [171]. SIRT1 has been shown to be markedly decreased in insulin-resistant cells and reduction of SIRT1 levels in gastrocnemius muscle leads to insulin resistance [172]. In addition, knockdown or blockade of SIRT1 activity impairs insulin signaling by interfering with insulin-stimulated insulin receptor phosphorylation and glycogen synthase [172]. On the other hand, overexpression of SIRT1 in the liver can attenuate hepatic steatosis and lead to improved glucose homeostasis [173]. Insulin sensitivity is improved in diabetic rats during exercise training through the activation of SIRT1 [148]. The ability of SIRT1 to improve insulin sensitivity may occur through a number of mechanisms that include modulation of fat mobilization [160], control of gluconeogenesis [167], and limiting the onset of inflammation [168]. SIRT1 also serves as a positive modulator of insulin signaling in insulin-sensitive organs and activates the insulin downstream target protein kinase B (Akt) through phosphotidylinositide 3-kinase (PI 3-K) [174]. In addition, SIRT1 can stimulate glucose-dependent insulin secretion from pancreatic β cells by repressing the uncoupling protein (UCP) gene UCP2 [175]. Resveratrol, an activator of SIRT1, can promote glucose-stimulated insulin secretion in insolinoma INS-1E cells and human islets that is dependent on the activity of SIRT1 [176]. SIRT1 also oversees insulin sensitivity through protein tyrosine phosphatase (PTP). In the PTP family [177
182], PTP1B has been identified to negatively regulate insulin signal transduction by targeting the insulin receptor. PTP1B deficiency or inhibition leads to improved insulin sensitivity and glycemic control. SIRT1 overexpression or SIRT1 activation can reduce both the PTP1B messenger ribonucleic acid (mRNA) and protein levels during insulin resistance for improved glucose homeostasis. An increase in PTP1B expression prevents

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1. Sirtuins in metabolic disease: innovative therapeutic strategies with SIRT1, AMPK, mTOR, and nicotinamide

SIRT1-mediated glucose uptake and insulin receptor phosphorylation in response to insulin stimulation, illustrating that SIRT1 improves insulin sensitivity through the repression of PTP1B [172].

1.6 SIRT1 and AMP-activated protein kinase SIRT1 may regulate insulin sensitivity and metabolism through the phosphorylation of AMPK and protect against metabolic syndrome [43,53,112,183,184] (Table 1.1). Activation of AMPK through phosphorylation functions to promote insulin sensitivity, fatty acid oxidation, and mitochondrial biogenesis. This leads to the generation of ATP and reduction in oxidative stress [2,12]. AMPK has been shown to limit the disability and hyperalgesia from diabetic neuropathy in animal models [57]. Diets associated with fish oil consumption can result in increased AMPK activity and block endothelial progenitor cell dysfunction and ischemic injuries [78]. AMPK can reduce insulin resistance, since the loss of AMPK results in reduced tolerance to the development of insulin resistance [185]. During periods of dietary restriction that may increase life span [129], AMPK can be one of several pathways to shift to beneficial oxidative metabolism [186]. AMPK can reduce ischemic brain damage in diabetic animal models [187], improve memory retention in models of AD and DM [188], offer elimination of ß-amyloid (Aß) in the brain [189], facilitate tau clearance [190], modulate chronic inflammation in neurodegenerative disorders [22,184,191], and prevent Aß neurotoxicity [192]. The AMPK pathway is relevant with current treatments used for DM and SIRT1. Biguanides and metformin rely upon AMPK and autophagy to restore cellular function. Metformin blocks mTOR activity, promotes autophagy, and can function at times in an AMPK-independent manner [193]. Through metformin, AMPK can become activated, leading to autophagy induction, and protecting against diabetic apoptotic cardiac cell loss [194]. Metformin also has been shown to prevent lipid peroxidation in the brain and spinal cord and to decrease caspase activity during toxic insults [195]. These observations of metformin to offer protection during DM may be associated with the ability of autophagic pathways to limit oxidative stress under some circumstances [38,196]. AMPK can oversee metabolic pathways through both apoptosis and autophagy. During metabolic disease, autophagy can remove misfolded proteins and eliminate nonfunctioning mitochondria to maintain β-cell function and prevent the onset of DM [197]. Exercise in mice has been demonstrated to promote the induction of autophagy and regulate glucose homeostasis [198]. Autophagy can improve insulin sensitivity during the administration of high-fat diets in mice [185] and may offer protection to microglia during acute glucose fluctuations [61]. During periods of hyperglycemia, AMPK increases basal autophagy activity [110,199] and maintains endothelial cell survival [76,200]. AMPK can modulate apoptosis and autophagy during coronary artery disease [201], cholesterol efflux [202], endothelial dysfunction during hyperglycemia [76], and oxidative stress [159,203]. Antisenescence activity also can be promoted by AMPK activation and the increase of autophagic flux [151]. Activation of autophagy pathways is not always beneficial and may require careful modulation during metabolic disease [42,53,59,61,89,93,204]. Increased activity of autophagy can lead to the loss of cardiac and liver tissue in diabetic rats during attempts to achieve glycemic control through diet modification [100]. During elevated glucose exposure, advanced glycation end products (AGEs), agents that can result in DM complications, have been shown to lead to autophagy activation and vascular smooth muscle proliferation that can result in atherosclerosis [205], as well as cardiomyopathy [206]. During elevated glucose exposure, autophagy can impair endothelial progenitor cells, lead to mitochondrial oxidative stress [207], and prevent angiogenesis [208]. Chronic inflammatory conditions such as lichen planus also have been tied to mTOR inhibition and autophagy activation [209]. In regard to SIRT1, SIRT1 can control AMPK through the AMPK kinase, serine-threonine liver kinase B1 (LKB1). Overexpression of SIRT1 results in the deacetylation of LKB1, leading to its translocation from the nucleus to the cytoplasm, where LKB1 activates AMPK [210]. AMPK, in turn, can the lead to the activation of SIRT1. AMPK cannot directly activate SIRT1, but may enhance SIRT1 activity. However, AMPK-mediated impairment of muscle differentiation during glucose restriction and PGC-1α-mediated gene expression is dependent upon SIRT1. AMPK activation enhances SIRT1 activity either by increasing cellular NAD1/NADH ratio, resulting in the deacetylation and modulation of the activity of downstream SIRT1 targets that include the PGC-1α, FoxO1, and FoxO3a [211], or by upregulating nicotinamide phosphoribosyltransferase (Nampt) during glucose restriction, leading to increased NAD1 and decreased nicotinamide, an inhibitor of SIRT1 [212]. The SIRT1 activator resveratrol also has been demonstrated to increase AMPK activity through SIRT1-dependent or -independent mechanisms [211,213]. Resveratrol increases AMPK phosphorylation to protect cells against elevated glucose concentration, improve insulin sensitivity, and stimulate glucose transport. SIRT1 and AMPK can function to prevent hyperglycemic cell death in endothelial cells [76] and maintain mitochondrial homeostasis [43]. Increased

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AMPK activation limits myocardial infarct size in both nondiabetic and diabetic rat hearts following ischemia/ reperfusion, which may be mediated through the inhibition of mitochondrial permeability transition pore opening in cardiomyocytes [214]. AMPK signaling has been shown to reverse hyperalgesia during diabetic neuropathy in animal models [57].

1.7 SIRT1, mTOR, and metabolic disease A significant pathway for SIRT1 to impact metabolic disease and cellular survival involves mTOR [5,28,43,115,151,215
217] (Table 1.1). mTOR is a 289-kDa serine/threonine protein kinase that is encoded by the single gene FRAP1 [2,218
220]. mTOR can be referred to by other terms such as the mammalian target of rapamycin and the FK506-binding protein 12-rapamycin complex-associated protein 1 [113,221]. The target of rapamycin (TOR) was first described in Saccharomyces cerevisiae with the genes TOR1 and TOR2 [221]. Through the use of rapamycin-resistant TOR mutants, TOR1 and TOR2 were found to encode the Tor1 and Tor2 isoforms in yeast [222]. Rapamycin is a macrolide antibiotic in Streptomyces hygroscopicus that blocks TOR and mTOR activity [34]. mTOR serves as the principal component of the protein complexes mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2) [223
225] (Table 1.1). mTORC1 is composed of Raptor, Deptor (DEP domain-containing mTOR interacting protein), the proline rich Akt substrate 40 kDa (PRAS40), and mammalian lethal with Sec13 protein 8, termed mLST8 (mLST8) [2,113]. Rapamycin inhibits the activity of mTORC1 by binding to immunophilin FK-506-binding protein 12 (FKBP12) that attaches to the FKBP12-rapamycin-binding domain (FRB) at the carboxy (C)-terminal of mTOR to interfere with the FRB domain of mTORC1 [113]. mTORC1 is more sensitive to inhibition by rapamycin than mTORC2, but chronic administration of rapamycin can inhibit mTORC2 activity as a result of the disruption of the assembly of mTORC2 [184,220]. mTORC1 binds to its constituents through the protein Ras homologue enriched in brain (Rheb) that phosphorylates the Raptor residue serine863 and other residues that include serine859, serine855, serine877, serine696, and threonine706 [226]. The inability to phosphorylate serine863 limits mTORC1 activity, as observed through the use of site-direct mutation of serine863 [227]. mTOR can control Raptor activity and this activity can be blocked by rapamycin [227]. Deptor, also an inhibitor of mTORC1, prevents mTORC1 activity by binding to the FAT (FKBP12-rapamycin-associated protein (FRAP), ataxia-telangiectasia (ATM), and the transactivation/transformation domain-associated protein) domain of mTOR. If the activity of Deptor is lessened, Akt, mTORC1, and mTORC2 activities are increased [228]. PRAS40 blocks mTORC1 activity by preventing the association of p70 ribosomal S6 kinase (p70S6K) and the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) with Raptor [2,113,229,230]. Akt also is important in this pathway since mTORC1 activity is increased once phosphorylation of PRAS40 occurs by Akt. This releases the binding of PRAS40 and Raptor to sequester PRAS40 in the cell cytoplasm with the docking protein 14-3-3 [231
234]. In contrast to Deptor and PRAS40, mLST8 promotes the activity of mTOR. This requires the binding of p70S6K and 4EBP1 to Raptor [235]. mLST8 also has a number of other functions. It can oversee insulin signaling through the mammalian transcription factor FoxO3 [89,236], is necessary for Akt and protein kinase C-α (PKCα) phosphorylation, and is required for Rictor to associate with mTOR [236]. In contrast to mTORC1, mTORC2 is composed of Rictor, Deptor, the mammalian stress-activated protein kinase interacting protein (mSIN1), mLST8, and the protein observed with Rictor-1 (Protor-1) [2,8,113]. mTORC2 controls cytoskeleton remodeling through PKCα and cell migration through the Rac guanine nucleotide exchange factors P-Rex1 and P-Rex2 and through Rho signaling [237]. mTORC2 fosters activity of protein kinases that include glucocorticoid induced protein kinase 1 (SGK1), a member of the AGC family of protein kinases. Protor-1, a Rictor-binding subunit of mTORC2, leads to SGK1 activity [238,239]. mSin1 is vital for the assembly of mTORC2 and for mTORC2 to phosphorylate Akt [240]. Rictor and mSIN1 phosphorylate Akt at serine473 and promote threonine308 phosphorylation through phosphoinositide-dependent kinase 1 (PDK1) to be protective for cellular survival. mTOR is intimately tied to cellular metabolic function and disease [2,34,57,241
244]. Activation of mTOR may limit cognitive loss that can be a result of DM [2,28,113,245
247]. For example, mTOR activation can prevent microglial injury during oxidative stress and limit Aß toxicity in neurons [232,246,248,249]. In addition, mTOR activation can prevent diabetic neuropathy [58] and reduce ischemic stroke injury in conjunction with circadian clock genes [2,22,250
252].

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1. Sirtuins in metabolic disease: innovative therapeutic strategies with SIRT1, AMPK, mTOR, and nicotinamide

Decreased activity of mTOR has been shown to increase mortality in murine models of DM [253]. During mTOR inhibition with rapamycin, reduced β-cell function, insulin resistance, and decreased insulin secretion can promote the progression of DM [254]. Proper translocation of glucose transporters to the plasma membrane in skeletal muscle are also affected during loss of mTOR activity [255]. mTOR activation has been found to be diminished and this loss of mTOR may possibly be responsible for insulin resistance and the increased risk of vascular thrombosis in patients with metabolic syndrome [256]. Activation of mTOR pathways that involve p70S6K and 4EBP1 can improve insulin secretion in pancreatic β-cells and increase resistance to β-cell streptozotocin toxicity and obesity in mice [257]. Loss of p70S6K activity leads to hypoinsulinemia and glucose intolerance with decreased pancreatic β-cell size [258]. mTOR activity can protect pancreatic β-cells against cholesterolinduced apoptosis [259], lead to enhanced neuronal cell survival in cell models of DM [260], and prevent glucolipotoxicity [261]. mTOR activity can allow for the differentiation of adipocytes [262], prevent endothelial cell dysfunction during hyperglycemia [76], and preserve glucose homeostasis [263]. mTOR provides protection as part of the Mediterranean diet to reduce obesity in the population. The diet may reduce Aβ toxicity in astrocytes through enhanced Akt activity by consumption of polyphenol of olives and olive oil that ultimately could prevent the onset or progression of AD [242]. In addition, growth factors that offer cellular protection against oxidative stress, such as insulin-like growth factor-1 (IGF-1) [264
267] and erythropoietin (EPO) [2,38,84,97,128,268
272] use SIRT1 and mTOR pathways (Table 1.1). EPO utilizes components of the mTOR pathway, such as PRAS40 and Akt, to enhance cell survival [231,273
275] and limit toxic cellular environments [249,276
278]. EPO also plays a significant role as a potential cellular protectant during aging [279] and DM with the modulation of mTOR pathways, in part, to maintain cellular survival [34,84,96,97,268,270,280
284]. In regard to SIRT1, EPO protects cells against toxic environments through SIRT1 [90,96,224,285]. EPO increases metabolic activity and maintains adipose energy homeostasis in adipocytes to protect against metabolic dysfunction through the combined activation of PPAR-α and SIRT1 [286]. EPO uses SIRT1 to modulate skeletal myogenic differentiation [287]. In central nervous system endothelial cells, EPO fosters the subcellular trafficking of SIRT1 to the nucleus to promote vascular cell protection and to prevent mitochondrial depolarization, cytochrome c release, BCL2-associated agonist of cell death (Bad) activity, and caspase activation [153]. EPO can protect human cardiomyocytes against mitochondrial dysfunction through the activation of SIRT1 during chemotherapy toxicity [107]. EPO also blocks the loss of neuronal cells in the brain through the upregulation of SIRT1 [288]. It is important to note that SIRT1 has an inverse relationship with mTOR [2,76,113,126,149,151,184,215,216]. SIRT1 activity results in neurite outgrowth and increased neuronal survival during nutrient limiting conditions with the inhibition of mTOR [289]. SIRT1 can promote tumor cell growth with autophagy activity that requires mTOR inhibition, illustrating that SIRT1 and autophagy pathways can be targets to control tumor cell growth [149]. During oxidative stress, SIRT1 can promote autophagy induction and the inhibition of mTOR to prevent mitochondrial dysfunction in embryonic stem cells [150] and can influence mitochondrial turnover through mitophagy [43]. During periods of hyperglycemia, SIRT1 blocks vascular cell injury during inhibition of mTOR activity [76]. Inhibition of mTOR with SIRT1 activation can increase cell survival for photoreceptor cells [115] and prevent cell senescence [151,290]. However, under some conditions that may involve dopaminergic neuronal cell loss, a balance in activities of SIRT1, mTOR, and forkhead transcription factors is necessary for neuroprotection [147]. This work suggests that there may be biological feedback pathways with mTOR, such as through AMPK inhibition, to prevent excessive mTOR activity. For example, if mTOR activity is unchecked during the inhibition of AMPK activity, mTOR and p70S6K can lead to glucose intolerance by inhibiting the insulin receptor substrate 1 (IRS-1) [291]. In addition, mTOR inhibition may reduce stroke infarct size during models of DM [187] and also can be necessary for maintaining a balance between pancreatic β-cell proliferation and cell size [292].

1.8 SIRT1, nicotinamide, and cellular metabolism As previously mentioned, the sirtuin deacetylation reaction generates nicotinamide as a product from the transfer of the acetyl residue from the acetyllysine residue of histones to the ADP-ribose moiety of NAD1 that leads to the generation of nicotinamide (Table 1.1). The agent and vitamin nicotinamide has a vital role during metabolic dysfunction and DM [12,44,95,96,293
295]. Nicotinamide is the amide form of vitamin B3 (niacin) and it is obtained through synthesis in the body or as a dietary source and supplement, such as from animal sources or plants [295]. Nicotinic acid is the other form of the water-soluble vitamin B3 [296]. The primary form of niacin in dietary plant sources is nicotinic acid that is rapidly absorbed through the gastrointestinal epithelium [297].

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1.9 Future considerations

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Nicotinamide is generated through the conversion of nicotinic acid in the liver or through the hydrolysis of the coenzyme ß-nicotinamide adenine dinucleotide (NAD1). Once nicotinamide is obtained in the body, it functions as the precursor for NAD1 [294,298]. It is also required for the synthesis of nicotinamide adenine dinucleotide phosphate (NADP1) [299]. Nicotinamide is changed to its mononucleotide form (NMN) with the enzyme nicotinic acid/nicotinamide adenylyltransferase yielding the dinucleotides NAAD1 and NAD1. NAAD1 converts to NAD1 through NAD1 synthase [300] or NAD1 can be synthesized through nicotinamide riboside kinase that phosphorylates nicotinamide riboside to NMN [301,302]. Nicotinamide through NAD1 can be directly utilized by cells to synthesize NAD1 [12,293
295,303]. Nicotinamide participates in energy metabolism through the tricarboxylic acid cycle by utilizing NAD1 in the mitochondrial respiratory electron transport chain for the production of ATP, DNA synthesis, and DNA repair [304
306]. Specific levels of NAD1 may be a critical factor for cell survival [303,307,308]. Nicotinamide offers protection usually in a specific concentration range [298]. Administration of nicotinamide in a range of 5.0
25.0 mmol/L can significantly protect neurons during oxidative stress injuries. This concentration range is similar to other injury paradigms in both animal models [309] and in cell culture models [294,310,311]. Elevated concentrations of nicotinamide in some experimental models may not offer protection and can be detrimental [312,313]. Yet increased administration of nicotinamide may be useful against tumorigenesis [314] and lead to apoptotic cell death in cancer cells [315,316]. In relation to cellular metabolism and DM, nicotinamide can lower insulin resistance and glucose release in combination with other factors to prevent the onset and progression of DM [317
319] (Table 1.1). Nicotinamide may prevent skeletal muscle atrophy during DM [320], reduce mitochondrial stress through AMPK activation [321], and reduce inflammation of the brain during DM with the administration of niacin [322]. In animal models, nicotinamide can maintain normal fasting blood glucose with streptozotocin-induced DM [323,324] and block oxidative stress pathways that lead to cell death and apoptosis [311,325
328]. In addition, nicotinamide can significantly improve glucose utilization, prevent excessive lactate production, and improve electrophysiologic capacity in ischemic animal models [329]. Oral nicotinamide administration (1200 mg/m2/day) has been shown to protect pancreatic β-cell function and prevents clinical disease in islet-cell antibody-positive first-degree relatives of type-1 DM [330]. Nicotinamide administration (25 mg/kg) in patients with recent onset type-1 DM combined with intensive insulin therapy for up to 2 years after diagnosis experienced significantly reduce HbA1c levels [331]. However, prolonged exposure of nicotinamide has been reported to result in impaired pancreatic β-cell function and cell growth [332,333]. Nicotinamide also may inhibit cytochromes P450 and hepatic metabolism [334]. As a result, the duration of nicotinamide administration may influence the efficacy of this agent since long-term administration also has been reported to support glucose intolerance in some animal models [307]. Since SIRT1 through the transfer of the acetyl residue from the acetyllysine residue of histones to the ADP-ribose moiety of NAD1 can lead to the production of nicotinamide, it is of interest to also note that nicotinamide is intimately linked to SIRT1 through autophagic pathways. Nicotinamide can promote the delayed induction of autophagy and subsequently decreased survival in cancer cells [335]. Mitochondrial autophagy (mitophagy) can result in an increased NAD1/NADH ratio during nicotinamide administration [132,307,336,337]. In addition, chronic administration of nicotinamide can lead to skeletal muscle lipotoxicity and glucose intolerance during autophagy activation [307]. Nicotinamide has been shown to prevent palmitate-induced hepatotoxicity via SIRT1-dependent induction of autophagy [338]. As an inhibitor of SIRT1, nicotinamide can limit cancer cell growth and in combination with chemotherapeutic agents lead to apoptotic cell death [154,339,340]. Furthermore, through SIRT1 inhibition, nicotinamide may exert antiinflammatory properties and affect the transcriptional regulation of inflammatory genes [341]. Additional studies suggest the nicotinamide, although an initial inhibitor of SIRT1, may subsequently promote SIRT activity as a result of the cellular conversion of nicotinamide to NAD1 [109].

1.9 Future considerations NCDs continue to impact a greater proportion of individuals throughout the world and affect both wealthy and low-income nations. Metabolic disorders, including DM, are a significant component of NCDs and currently lead to disability and death for an estimated 500 million individuals throughout the globe (Table 1.1). Metabolic disorders, such as DM, have marked deleterious effects since they can affect all organs of the body. Possibly more important is the observation that another 400 million individuals are believed to suffer from metabolic disease or are at risk for developing DM. In addition, obesity in the population represents an important risk factor for developing DM and can impair stem cell proliferation, aging, inflammation, and mitochondrial function through the generation of oxidative stress. In regard to financial costs to provide care for metabolic disorders, DM

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consumes more than 17% of the Gross Domestic Product in the United States and an estimated US$ 69 billion are consumed from reduced productivity as a result of DM. Metabolic disorders are complex in nature and present therapies only offer a minimal degree of protection since even under the best of circumstances, tight serum glucose control does not blunt the complications that can arise during DM. DM also has additional risk factors that include the development of neurodegenerative disorders and cognitive loss that can be compounded by hypertension, low education in early life, and tobacco use. The development of innovative and effective strategies is vital for the successful treatment of metabolic disorders. SIRT1 and its associated pathways with AMPK, mTOR, and the vitamin nicotinamide fill this essential need to offer exciting possibilities to address metabolic disease (Fig. 1.1). SIRT1, a histone deacetylase, is expressed in the brain, heart, liver, pancreas, skeletal muscle, spleen, and adipose tissues and during deacetylase reactions leads to the production of nicotinamide. SIRT1 activation can function to limit oxidative stress to potentially increase life span and limit disorders such as diabetic retinal injury and cognitive loss. SIRT1 activation blocks apoptotic cell death and, at times, can foster autophagy activation to protect embryonic stem cells during oxidative stress. In conjunction with pathways that involve PPAR-γ, PPAR-α, PGC-1α, PTP, and Akt, SIRT1 activity can improve insulin sensitivity, regulate glucose homeostasis, and limit the development of obesity. SIRT1 also has been shown to be present in POMC neurons and AgRP neurons in the arcuate nucleus of the hypothalamus to regulate food intake. SIRT1 relies upon AMPK to reduce insulin resistance and promote fatty acid oxidation and mitochondrial function. In turn, AMPK can promote the activation of SIRT1. SIRT1 and AMPK can work in association to prevent hyperglycemic cell death, maintain mitochondrial homeostasis, and reverse diabetic neuropathy symptoms. Through AMPK, induction of autophagy occurs that can offer cellular protection during elevated glucose and maintain glucose homeostasis. However, this upregulation of autophagy requires a fine balance during metabolic disorders. During periods of autophagy and metabolic dysfunction, loss of cardiac and liver tissue can occur in animal models, progenitor cell survival can become impaired, chronic inflammation can occur, and progression of atherosclerosis can ensue. SIRT1 also relies upon the mTOR pathway to oversee cellular metabolism. mTOR activation can limit cognitive loss during DM, prevent central nervous system microglial injury during oxidative stress and Aß toxicity, lessen diabetic neuropathy, and reduce ischemic stroke injury in conjunction with circadian clock genes. Loss of mTOR activity can impair β-cell function, insulin sensitivity, adipocyte differentiation, cellular survival, and insulin secretion. Furthermore, tropic factors, such as EPO, use mTOR to maintain adipose energy homeostasis and protect neuronal, vascular, and cardiac cells against mitochondrial dysfunction. Under several scenarios, SIRT1 holds an inverse relationship with mTOR to limit mTOR activity in order to foster neuronal and vascular survival, prevent cell senescence, and promote autophagy to block mitochondrial dysfunction and limit mitophagy. Given these observations with SIRT1 and mTOR, a biological feedback pathway between SIRT1 and mTOR may be FIGURE 1.1 Novel therapeutic pathways of SIRT1 for metabolic disease. Metabolic disorders, that include diabetes mellitus (DM), are an important component of noncommunicable diseases (NCDs) and can affect all organs and systems of the body. The silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1) and its associated pathways with AMP-activated protein kinase (AMPK), the mechanistic target of rapamycin (mTOR), and nicotinamide offer new prospects for the development of treatments for metabolic disease. SIRT1 oversees metabolic function though a variety of mechanisms that involve insulin sensitivity and obesity, AMPK with preservation of β-cell function, mTOR with its subcomponents of mTORC1 and mTORC2 that can impact adipocyte differentiation and function through erythropoietin (EPO) to protect neuronal and vascular cell survival, and nicotinamide that can limit mitochondrial stress and preserve mitochondrial function. These pathways are closely tied to autophagy induction and apoptosis that ultimately can determine metabolic homeostasis.

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necessary to achieve a high level of protection in the body and prevent excessive mTOR activity that may result in glucose intolerance, increased stroke size during DM, and loss of pancreatic β-cell proliferation. As a product of the SIRT1 deacetylation reaction, nicotinamide participates in energy metabolism through the tricarboxylic acid cycle by utilizing NAD1 in the mitochondrial respiratory electron transport chain and has a prominent role in overseeing cellular metabolism. Nicotinamide can reduce insulin resistance, maintain normal fasting blood glucose levels, and protect pancreatic β-cell function. Nicotinamide has been shown to block palmitate-induced hepatotoxicity through SIRT1-dependent induction of autophagy and may limit tumorigenesis through its inhibition of SIRT1. Yet the concentration of nicotinamide may be critical during metabolic disease, since prolonged or increased levels of nicotinamide administration may result in glucose intolerance and be toxic to cells. Furthermore, nicotinamide may also foster SIRT activity as a result of the cellular conversion of nicotinamide to NAD1. These studies highlight the importance of maintaining the proper balance of SIRT1 activity and nicotinamide levels to achieve an effective outcome for the treatment of metabolic disorders. It is clear that SIRT1 has a vital role in the maintenance of glucose homeostasis in the body and holds an intricate and complex relationship with AMPK, mTOR, and nicotinamide to maintain cellular metabolism and protect against toxic environments. In light of the fact that SIRT1 and its association with AMPK, mTOR, and nicotinamide can affect a number of organs throughout the body and oversee multiple biological process, additional studies should provide further insight for the development of new and robust strategies for the treatment of metabolic diseases that limit any unwarranted clinical outcomes.

Acknowledgments This research was supported by the following grants to Kenneth Maiese: American Diabetes Association, American Heart Association, NIH NIEHS, NIH NIA, NIH NINDS, and NIH ARRA.

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2 Sirtuins in metabolic and epigenetic regulation of stem cells Yi Fang and Xiaoling Li Signal Transduction Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, NC, United States O U T L I N E 2.1 Introduction

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2.2 Stem cells and sirtuins

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2.3 SIRT1 in stem cell biology 2.3.1 SIRT1 is important for normal embryogenesis and animal development 2.3.2 SIRT1 maintains pluripotent ESCs through multilevel mechanisms 2.3.3 SIRT1 is important for the maintenance of diverse ASC pools 2.3.4 SIRT1 is important in maintaining/promoting stemness and survival of CSCs

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2.4 SIRT2 in stem cell biology 2.4.1 SIRT2 promotes differentiation of ESCs in vitro 2.4.2 SIRT2 promotes survival of CSCs

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2.5 SIRT3 in stem cell biology

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2.5.1 SIRT3 maintains the pool and regenerative capacity of HSCs during aging

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2.6 SIRT6 in stem cell biology 32 2.6.1 SIRT6 epigenetically promotes proper lineage commitment of ESCs and animal development 32 2.6.2 SIRT6 controls regeneration and stress resistance in HSCs and mesenchymal stem cells 33 2.6.3 SIRT6 suppresses stemness of CSCs 33

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2.7 SIRT7 in stem cell biology 2.7.1 SIRT7 regulates embryogenesis and life span through maintenance of genome stability 2.7.2 SIRT7 regulates quiescence and regenerative capacity of HSCs

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2.8 Concluding remarks and future perspectives

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References

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33 34

2.1 Introduction Sirtuins are highly conserved NAD1-dependent protein deacylases, which function to transfer a variety of lipid acyl-groups, including acetyl, succinyl, malonyl, glutaryl, or long-chain acyl-groups, from the substrate proteins to the adenosine diphosphate ribose (ADP)-ribose moiety of NAD1 [1
4]. Seven members of sirtuins have been identified in mammals (SIRT1 to SIRT7) [5], and these seven proteins are localized in distinct subcellular localizations and have different tissue expression patterns with differential substrate specificities [6
10]. Since NAD1 is required for sirtuins’ enzymatic activity and the concentration of NAD1 is determined by cellular metabolic state, sirtuins are able to sense cellular energy status and then modulate the functions of a wide array of protein substrates, ranging from histones and transcription factors to metabolic enzymes and cell membrane proteins. Consistently, a wealth of studies demonstrate that sirtuins play critical roles in modulating metabolism homeostasis, nutrient sensing, aging, and age-associated conditions/diseases [11,12]. Sirtuin Biology in Cancer and Metabolic Disease. DOI: https://doi.org/10.1016/B978-0-12-822467-0.00004-8

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2. Sirtuins in metabolic and epigenetic regulation of stem cells

Emerging evidence has revealed that members of the Sirtuin family of unique enzymes also function as pivotal regulators of stem cell biology. Stem cells, including pluripotent stem cells (PSCs) and adult stem cells (ASCs), are characterized by their ability to self-renew while maintaining their pluri/multipotency. This ability is supported by unique sets of metabolism programs and specific epigenetic status in stem cells. For instance, the rapidly proliferating embryonic stem cells (ESCs) demand a high glycolytic flux under aerobic conditions (“Warburg effect”), and are also dependent on exogenous glutamine and one-carbon catabolism [13
19]. These metabolic programs not only provide anabolic precursors for rapid cell proliferation, but also produce many intermediate metabolites, including acetyl-CoA, NAD1, α-ketoglutarate, and S-adenosylmethionine (SAM), which function as substrates or cofactors for enzymes that regulate chromatin modification and gene expression [19
24]. Thus the unique metabolic state of stem cells directly links to distinctive epigenetics and gene expression profiles, greatly influencing their self-renewal and pluri-/multipotency [15,25]. Recent literature shows that sirtuins are important in maintaining stem cell pluri-/multipotency, promoting cell survival, and regulating differentiation by modulating metabolisms and epigenetics. In this chapter, we summarize the involvement of individual sirtuins in metabolic and epigenetic regulation of stem cells, including PSCs and embryogenesis, ASCs and tissue regeneration, as well as cancer stem cells (CSCs) and cancers.

2.2 Stem cells and sirtuins PSCs are stem cells that give rise to all cells involved in tissue development in the body. There are two types of PSCs, ESCs and induced pluripotent stem cells (iPSCs). ESCs are derived from the inner cell mass (ICM) of preimplantation embryos. iPSCs are reprogrammed from adult somatic cells in vitro through simultaneous overexpression of four core pluripotent factors, Oct4, Sox2, Klf4, and cMyc (OSKM). Both ESCs and iPSCs can be indefinitely maintained and expanded in the pluripotent state in vitro, and are capable of differentiating into all the derivatives of the three germ layers. Among the seven sirtuin members, SIRT1 has the highest expression in mouse ESCs (mESCs), followed by SIRT6 and SIRT7, while the expression of other sirtuins, including mitochondrial sirtuins, is low in mESCs [26]. Furthermore, germline knocking out SIRT1, SIRT6, or SIRT7, but not other sirtuins, causes significant developmental defects [7,27
33], demonstrating that those three nuclear sirtuins are important in the regulation of ESC biology and animal development. ASCs are stem cells that persist throughout the life to replace cells lost due to homeostatic turnover, disease, and injury [34,35]. They are characterized by their ability to self-renew and differentiate to generate all the cell types in a tissue. ASC populations have varying proliferative plasticity and distinct turnover patterns in different tissues. Based on their turnover rates in various tissues/organs, ASCs can be categorized into three types: ASCs that continuously cycle themselves in high-turnover tissues, like intestinal stem cells (ISCs) and the short-term hematopoietic stem cells (ST-HSCs); ASCs that are maintained in quiescent state, and whose proliferation is strongly induced by injury or other environmental/developmental cues, such as muscle satellite cells (SCs), adult neural stem cells (NSCs), and long-term hematopoietic stem cells (LT-HSCs); and ASCs that periodically alternate between quiescent and proliferative states, like hair follicle stem cells. In a given tissue, ASCs are all delicately balanced between quiescence, proliferation/self-renewal, and differentiation. Disruption of ASC homeostasis, either by a decline in ASC number and/or functions or by aberrant activation of ASCs, has been linked to a number of human disorders, including degenerative disease, aging, and cancer. Recent evidence indicates that sirtuins, including SIRT1, SIRT3, SIRT6, and SIRT7 are critical in maintaining ASC (particularly HSC) homeostasis, tissue regeneration, and aging through diverse mechanisms. CSCs, similar to normal stem cells, possess the ability to self-renew and differentiate to give rise to all the cell types in a cancer [36]. Although they are a small population of a tumor, CSCs are able to generate a new tumor, resulting in tumor relapse and metastasis [37,38]. As conventional chemotherapy and radiotherapy designed to reduce cancer mass primarily target the differentiated or differentiating cancer cells but fail to kill CSCs, development of therapy against CSCs would greatly improve the survival and life quality of certain cancer patients, particularly for patients with metastatic tumors. Given the importance of sirtuins in PSCs and ASCs, it is not surprising that several members of this family, including SIRT1, SIRT2, and SIRT6, are also key regulators of CSC functions [39].

2.3 SIRT1 in stem cell biology 2.3.1 SIRT1 is important for normal embryogenesis and animal development SIRT1 is highly expressed in preimplantation embryos, ESCs, and male germ cells, but has relatively low expression in differentiated cells and tissues, as well as most adult tissues [27,40,41]. This tissue expression

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2.3 SIRT1 in stem cell biology

pattern suggests a fundamentally important role of SIRT1 in ESCs, embryogenesis and development. In particular, SIRT1 is most highly expressed at the 2-cell stage when the embryos begin to activate its own genome [41], implying that SIRT1 is one of the first activated genes during embryogenesis. Consistently, SIRT1 null zygotes generated through in vitro fertilization of SIRT1 deficient sperm and oocytes display a substantially compromised efficiency of developing to the two-cell stage compared to control zygotes [42]. Importantly, germline deletion of SIRT1 in mice results in severe developmental defects on various genetic backgrounds, including intrauterine growth retardation, developmental defects of the retina and heart, defective germ cell differentiation, bone developmental delay, and neonatal lethality [27,28,43
45]. Additionally, impaired SIRT1 stability induced by loss of USP22, a deubiquitinating enzyme that stabilizes SIRT1, is also linked to defective embryogenesis in USP22 deficiency mice [46]. Please note that depending on different genetic backgrounds, there are some variations in the penetrance and severity of SIRT1 deletion-induced developmental phenotypes. Mice lacking SIRT1 on the FVB/N genetic background [47] or a few percentages of SIRT1 null mice on some mixed backgrounds [27,28] can survive into adulthood. However, despite these variations, growth retardation and sterility with developmental defects are all observed in these adult mice. Specifically, spermatogenic stem cell number in E15.5 embryos is severely reduced, and spermatogenesis but not oogenesis is abrogated in adult SIRT1 knockout animals [42]. Moreover, germ cell-specific deletion of SIRT1 still disrupts spermatogenesis, indicating that the action of SIRT1 in germ cells is cell-autonomous [44,48]. All these observations strongly indicate that SIRT1 is a key regulator of normal embryogenesis and animal development.

2.3.2 SIRT1 maintains pluripotent ESCs through multilevel mechanisms SIRT1 deficiency leads to loss of pluripotency and reduced survival of ESCs. Consistent with its high expression in ESCs, SIRT1 maintains pluripotency by interacting with key pluripotency genes, such as Oct4 and Myc, modulating DNA methylation, and meanwhile repressing differentiation signals such as cellular retinoic acid (RA) signaling. SIRT1 also promotes ESC survival by regulating cellular endogenous reactive oxygen species (ROS) responses and suppressing p53 activation-induced DNA damage and apoptosis. So SIRT1 is multifaceted in pluripotency maintenance (Fig. 2.1). In hESCs, SIRT1 is one of the downstream factors mediating Oct4-dependent pluripotency maintenance [49]. Oct4 transcriptionally activates the expression of SIRT1, which in turn deacetylates and destabilizes p53, a well-known SIRT1 deacetylation substrate [49], to promote pluripotency. Consistently, silencing Oct4 leads to a reduction of SIRT1 expression and activation of p53, which induces hESC differentiation [49]. In mESCs, SIRT1 maintains the naı¨ve state through direct deacetylation of Oct4 [50]. During the naive-to-primed transition, the activity of SIRT1 is reduced and Oct4 becomes hyperacetylated. Acetylated Oct4 binds to an enhancer to induce the expression of Otx2, which interacts with acetylated Oct4 to induce the primed pluripotency gene network [50]. In addition to Oct4, SIRT1 also deacetylates Myc to metabolically and epigenetically maintain pluripotent mESCs [41]. Deacetylation of Myc, including both N-Myc and c-Myc, stabilizes Myc and/or enhances its recruitment to a promoter of methionine adenosyltransferase 2a (Mat2a), thereby promoting Mat2a expression. Mat2a is critical to the conversion of methionine to SAM and the maintenance of appropriate methylation levels of FIGURE 2.1 SIRT1 is a multifaceted regulator

Oct4

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in the pluripotent ESCs. SIRT1 is critically involved in the maintenance of pluripotent ESCs and animal development through multilevel mechanisms. The expression of SIRT1 in ESCs is under transcriptional control of Oct4, and the interaction between SIRT1, Oct4, and Myc are important to metabolically and epigenetically maintain pluripotent ESCs. SIRT1 also actively represses the expression of developmental and differentiation genes through deacetylation of histones and inhibition of cellular RA signaling. Finally, SIRT1 enhances stress response of ESCs by repressing p53 and promoting DNA repair.

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histones, especially H3K4me3. Therefore deletion of SIRT1 reduces Mat2a expression and H3K4me3 level, altering gene expression profiles, compromising the pluripotency, and sensitizing mESCs to methionine restriction-induced differentiation and apoptosis [41]. This SIRT1-Myc regulated metabolic and epigenetic regulation is also important to animal development, as SIRT1 KO embryos are sensitive to maternal methionine restriction-induced lethality, whereas maternal methionine supplementation increases the survival of SIRT1 KO newborn mice [41]. SIRT1 also actively maintains pluripotency by selectively preventing abnormal DNA methylation of some developmental genes in mESCs through antagonizing Dnmt3l [51]. Dnmt3l is a catalytically inactive DNA methyltransferase that interacts with Dnmt3a and Dnmt3b to stimulate de novo methylation [52
54]. SIRT1 transcriptionally suppresses Dnmt3l expression and physically deacetylates Dnmt3l, thereby destabilizing Dnmt3l protein [51]. SIRT1 deficiency leads to a high level of Dnmt3l, abnormal DNA methylation, repressed expression of imprinted and germline genes, and delayed neurogenesis and spermatogenesis [51]. Therefore, SIRT1-mediated DNA methylation status is important for the maintenance of differentiation potential or pluripotency in ESCs. Additionally, SIRT1 directly represses differentiation of ESCs by regulating histone acetylation and RA signaling. Highly expressed SIRT1 in ESCs deacetylates histones and represses the transcription of some developmental genes, while reduction of SIRT1 during differentiation reactivates expression of these key developmental genes [55]. During differentiation, SIRT1 level is reduced either by microRNAs-mediated inhibition of SIRT1 protein translation [40], or by CARM1-HuR-dependent destabilization of SIRT1 mRNA [55,56]. SIRT1 also represses the differentiation of mESCs through inhibiting RA signaling [45]. CRABPII, a cellular RA binding protein that is translocated into the nucleus to activate retinoic acid receptor (RAR) signaling upon RA treatment, becomes acetylated in the nucleus. SIRT1 interacts with and deacetylates CRABPII, recycling CRABPII back to the cytosol, thereby inactivating the RAR-mediated activation of differentiation genes. Notably, SIRT1 deficiency-induced development defects in mice, particularly bone development delay, are associated with increased RA signaling [45]. Finally, SIRT1 is important in regulating ESC survival in response to various stresses. SIRT1 strongly enhances the survival of hESCs by promoting DNA repair and inhibiting p53 [57], and promotes telomere elongation and genome stability during reprogramming in iPSCs [58]. SIRT1 also protects ESCs against different degrees of oxidative stress via different mechanisms. In response to endogenous ROS, SIRT1 functions to maintain heathy pluripotent ESCs by sensitizing mESCs to mitochondrial p53-induced apoptosis while inhibiting nuclear p53-mediated suppression of Nanog expression [59]. In contrast, SIRT1 protects ESCs from apoptosis induced by high concentrations of exogenous ROS (e.g., 1 mM H2O2) in part through the class III PI3K/Beclin 1 and mTOR-mediated autophagy [60]. Collectively, SIRT1 is critically involved in the maintenance of pluripotent ESCs and animal development through regulation of pluripotency factors, metabolism, epigenetics, redox homeostasis, and cellular stress response.

2.3.3 SIRT1 is important for the maintenance of diverse ASC pools SIRT1 is an important factor for preserving ASC pools in multiple tissues through diverse mechanisms (Fig. 2.2). SIRT1 interacts with Lamin A protein to prevent progeroid syndrome, a group of rare genetic disorders characterized with severe early-onset premature aging [61]. Progeroid syndrome is caused by progerin, a truncated Lamin A protein in human. Knockout of Zmpste24, a metalloproteinase responsible for prelamin A maturation, recapitulates the phenotypes of progerin, and results in quick depletion of multiple ASC pools including mesenchymal stem cells (MSCs), hair follicle progenitor cells, and hematopoietic stem cells (HSCs) in mice [61]. Liu et al showed that both progerin and unprocessed prelamin A protein are defective in binding and activation of SIRT1, while promoting interaction between SIRT1 and Lamin A by resveratrol activates SIRT1 and rescues ASC decline in Zmpste242/2 mice [61]. In addition, long-term culture with resveratrol retains MSCs’ proliferative and differentiation potential, and resveratrol treatment significantly improves bone regeneration after MSC transplantation in rats [62]. These findings indicate that maintaining ASC pools could be one of the mechanisms through which SIRT1 promotes longevity. In ASCs that continuously divide asymmetrically in high-turnover tissues, such as ISCs, SIRT1 is necessary and sufficient to promote their self-renewal and expansion in response to caloric restriction (CR) [63]. CR activates SIRT1 in ISCs, which then deacetylates S6K1, facilitating mTORC1 to phosphorylate S6K1 and stimulating protein synthesis [63]. To activate SIRT1, CR promotes production of a paracrine factor cyclic ADP ribose (cADPR) in Paneth cells, a key constituent of the mammalian ISC niche, but not in ISCs [64]. The released cADPR

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FIGURE 2.2

SIRT1 is important for the maintenance of diverse ASC pools. SIRT1 is critical to maintain diverse ASC pools through maintenance of self-renewal or quiescence and regeneration capacity, particularly in response to environmental stress and injury. In the intestine, caloric restriction promotes production of cADPR in Paneth cells, which function in ISCs to promote NAD1 synthesis and SIRT1 activity. Activated SIRT1 then deacetylates S6K1, promoting its phosphorylation by mTORC1 and subsequent protein synthesis. This action of SIRT1 is important for ISCs self-renewal. In muscle stem cells SCs, SIRT1 represses muscle specific gene expression to maintain their quiescence and regeneration capacity. Injury induces a metabolic switch from oxidative phosphorylation to glycolysis, which reduces cellular NAD1 levels and SIRT1 activity, leading to desuppression of muscle genes and SC differentiation. In HSCs, SIRT1 represses the activity of p53 and transcription of Hox9 to maintain their genome stability and proliferation in response to environmental stress.

then acts on ISCs to activate CaMKK and AMPK, thereby stimulating the transcription of Nampt, the ratelimiting enzyme in a NAD1 salvage pathway, and enhancing the activity of SIRT1 [63,64]. This data also suggests that SIRT1-mediated preservation of the ASC pool could be a part of the longevity response to CR. In ASCs that are maintained in quiescent state but strongly induced by injury, such as the skeletal muscle stem cells SCs and adult NSCs, SIRT1 is required for the maintenance of their quiescence and regeneration capacity [65
67]. When quiescent SCs are induced to proliferate upon injury, SIRT1 activity is decreased by reduced intracellular NAD1 levels due to a metabolic switch from fatty acid oxidation to glycolysis [65]. As a result, H4K16ac level is elevated, and muscle gene transcription is enhanced [65]. When the deacetylase domain of SIRT1 is specifically ablated in skeletal muscle, myogenic genes are expressed in SCs, leading to premature differentiation, and impairing muscle growth during development and regeneration upon injury [65]. In adult NSCs, SIRT1 also maintains their quiescence, as specific deletion of SIRT1 in adult NSCs enhances their self-renewal [66,67] and promotes lineage specification to oligodendrocyte progenitor cells/oligodendrocytes [68]. This impact of SIRT1 is also sensitive to metabolic switch/redox stress and involves inhibition of Hes1 transcription factor [66], Notch signaling [67], or cell metabolism and growth factor signaling [68]. It would be interesting to determine whether SIRT1 similarly impacts long-term maintenance of the NSC pool and neurogenesis in response to injury as in SCs and skeletal muscle in the future. SIRT1 is also important for the maintenance of quiescence and regeneration capacity of HSCs in response to environmental stress and aging [69]. SIRT1 KO mice do not exhibit any abnormalities in their hematopoietic compartment under normal conditions [70]. However, SIRT1 deficient mESCs are compromised in their hematopoietic commitment in vitro and SIRT1 KO mice have decreased survival of hematopoietic progenitors ex vivo, particularly under hypoxic conditions or under conditions of delayed growth factor addition [71]. Moreover,

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conditional ablation of SIRT1 in adult hematopoietic stem/progenitor cells (HSPCs) cell-autonomously induces HSPC expansion and loss of long-term repopulation capacity under stress [72]. This stress-induced loss of HSC function is associated with genomic instability, p53 activation, and increased DNA damage in SIRT1-deficient HSPCs [72]. SIRT1 loss also causes a significant increase of H4K16ac and upregulates the expression of Hoxa9, a key regulator of HSPC function and proliferation [72]. Collectively, SIRT1 is critical to maintain diverse ASC pools through maintenance of self-renewal or quiescence and regeneration capacity, particularly in response to stress and injury (Fig. 2.2).

2.3.4 SIRT1 is important in maintaining/promoting stemness and survival of CSCs Consistent with its role in maintenance of pluripotent ESCs and functional pools of ASCs, SIRT1 is critical for stemness and survival of CSCs (Fig. 2.3). For example, SIRT1 is highly expressed in a number of CSCs, such as glioma stem cells (GSCs), breast cancer stem cells (BCSCs), colorectal CSC-like cells, CSC-like ovarian cancer cell lines, and liver CSCs. In GSCs, SIRT1 is required not only for the maintenance of stem cells, but also for oncogenic transformation through suppressing p53-dependent tumor surveillance [73]. Silencing of SIRT1 significantly enhances the sensitivity of CD1331 GSCs to radiation and increases the effectiveness of radiotherapy in the inhibition of xenografted CD1331 GSCs [74]. In BCSCs, SIRT1 is a direct target of miR-34a via its highly conserved 30 -UTR [75]. Low levels of miR-34a and high levels of SIRT1 are important for the expression of BCSC markers, maintenance of BCSC pool, and formation of mammosphere in vitro, as well as tumor burden in xenografts [75]. In colorectal CD1331 CSC-like cells, SIRT1 promotes colony and sphere formation in vitro, and enhances tumorigenesis of colorectal cancer cells in vivo by stimulating the expression of several stemness-associated genes [76]. In addition, SIRT1 promotes the hypoxia-induced CSC-like properties in human ovarian cancer cell lines [77]. SIRT1 is also highly expressed in liver CSCs and is important to maintain stemness properties or self-renewal and tumorigenic potential of liver CSCs [78,79]. Mechanistically, SIRT1 enhances SOX2 expression by reducing c-Myc USP22 SRT1720 TV-6 Ac Ac

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FIGURE 2.3 SIRT1 regulates the stemness and self-renewal of CSCs. In BCSCs, SIRT1 is under negative control of miR-34a and is important to promote the expression of stem cell markers and maintenance. In liver CSCs, SIRT1 functions to promote Sox2 expression and mitochondrial respiration, which are important for liver CSCs self-renewal, tumorigenic potential, and stemness. Finally, SIRT1 is highly expressed in LSCs, either by increase in expression or by c-Myc/USP22-mediated stabilization, to inhibit p53 signaling and enhance mitochondrial gene expression and mitochondrial respiration via PGC-1α, which are important for LSC survival and proliferation. Inhibition of SIRT1 activity by TV-6 suppresses tumorigenic activity of LSCs in vitro and in vivo. In contrast, in MDS HSPCs, SIRT1 is repressed by miR-9 and miR-34a. Expression of SIRT1 or activation of SIRT1 by SRT1720 deacetylates TET2 and restores its function. This action increases 5hmC on the enhancers of a number of genes involved in proliferation, adhesion, and TGF-β signaling and represses their expression, thereby inhibiting MDS HSPC proliferation and functions.

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the DNA methylation level in the CpG islands of SOX2 promoter [78], and increases mitochondrial respiratory capacity by promoting the translocation of mitochondrial ribosomal protein S5 (MRPS5) to mitochondria through deacetylation [79]. Increased mitochondrial respiratory capacity is required for liver CSC stemness, and MRPS5 is critical to promoting mitochondrial respiratory capacity via promoting the function of Complex-I and generation of NAD1 [79]. Several studies from the McCormack group indicate that SIRT1 is important for the survival and proliferation of leukemic stem cells (LSCs). Both chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) are organized as a hierarchy, with small populations of self-renewing LSCs that give rise to the bulk of leukemic cells [80
82]. The McCormack group found that SIRT1 protein level is highly expressed in LSCs from both CML and AML [81,82]. The mRNA level of SIRT1 is increased in CML, whereas in AML, SIRT1 protein stability is enhanced due to c-MYC induced overexpression of USP22, a protein deubiquitinase that stabilizes SIRT1 [46]. This increase in SIRT1 protein inhibits p53 signaling and enhances mitochondrial gene expression and mitochondrial respiration via PGC-1α, which are important for LSC survival and proliferation [81
83]. Conversely, knocking down/out SIRT1 expression or inhibiting SIRT1 activity by Tenovin-6 (TV6), a small molecule inhibitor of SIRT1 [84], decreases LSC proliferation, enhances apoptosis, and impairs their colony-forming ability [81,82]. More interestingly, the combined use of TV6 with kinase inhibitors (imatinib for BCR-ABL tyrosine kinase, or AC220 for FLT3, a kinase that commonly duplicated in AML) [82,85], leads to an enhanced inhibition of LSCs and improved survival of animals receiving CML LSCs transplantation or xenografted with imatinib-resistant blast crisis CML patient sample [81,82]. Given that inhibition of SIRT1 has only minimal impact on normal human CD341 HSCs, SIRT1 provides a good candidate for targeting LSCs, and the synergetic treatment could potentially cure leukemia patients with normal p53 function. However, in contrast to its pro-proliferation and self-renewal function in LSCs, SIRT1 activation is important to disrupt maintenance of myelodysplastic syndrome (MDS) stem and progenitor cells (HSPC) [86]. MDS is a group of clonal hematopoietic disorders, characterized by morphological dysplasia and ineffective hematopoiesis, leading to cytopenias and a 30% risk of transformation to AML [87]. Sun et al. found that SIRT1 protein levels are kept low by miR-9 and miR-34a in MDS HSPCs. Genetic or pharmacological activation of SIRT1 by a SIRT1 agonist SRT1720 leads to deacetylation and activation of TET2, a safeguard against HSPC transformation, which increases 5-hydroxymethylcytosine (5hmC) on the enhancers of a number of genes involved in proliferation, adhesion, and TGF-β signaling and repress their expression. This action of SIRT1 inhibits MDS HSPC proliferation and functions [86]. Therefore, depending on its deacetylation targets, SIRT1 exerts distinct functions in different CSCs.

2.4 SIRT2 in stem cell biology 2.4.1 SIRT2 promotes differentiation of ESCs in vitro In contrast to SIRT1, SIRT2, a predominately cytosolic sirtuin, is highly expressed in differentiated somatic cells but not in PSCs [88], and is not essential for embryonic viability and postnatal development in mice [32]. SIRT2 is induced during RA-induced differentiation of mESCS, and represses GSK3β to promote differentiation [89], implying that SIRT2 is important for differentiation of ESCs. Cha et al. found that SIRT2 is the primary deacetylase that deacetylates and inhibits glycolytic enzymes in hESCs [88]. So, SIRT2 expression in hESCS is repressed by miR200c-5p to allow high flux of glycolysis [88], which is important for ESC growth and pluripotency maintenance [16,90
92]. Induction/overexpression of SIRT2 in ESCs represses glycolysis and promotes differentiation, and inhibition of SIRT2 in fibroblasts promotes the reprogramming of human fibroblasts to iPSCs [88]. However, complete depletion of SIRT2 prevents generation of pluripotent stem cells from mouse embryonic fibroblasts (MEFs) [93]. Collectively, SIRT2 is critical in controlling metabolic switch between PSCs and differentiated cells in vitro.

2.4.2 SIRT2 promotes survival of CSCs SIRT2 has been shown to promote CSC function and survival. For example, in contrast to SIRT1 and SIRT3, which are expressed in both glioblastoma stem cells (GSCs) and NSCs, SIRT2 is exclusively restricted to GSCs [94]. In BCSCs, the activity of aldehyde dehydrogenase 1A1 (ALDH1A1), a marker commonly used to isolate stem cells, is a direct deacetylation substrate of SIRT2 [95]. Zhao et al. showed that ALDH1A1 low in acetylation

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displays a high ALDH1 activity and is capable of promoting self-renewal of BCSCs. They further found that Notch signaling activated ALDH1A1 through the induction of SIRT2, leading to ALDH1A1 deacetylation and enzymatic activation to promote proliferation BCSCs. In breast cancer xenograft models, replacement of endogenous ALDH1A1 with an acetylation mimetic mutant inhibited tumorigenesis and tumor growth [95].

2.5 SIRT3 in stem cell biology 2.5.1 SIRT3 maintains the pool and regenerative capacity of HSCs during aging SIRT3 is a mitochondrial sirtuin which deacetylates mitochondrial proteins and reduces oxidative stress [96]. SIRT3 is highly enriched in HSCs and is reduced with age, indicating an important role in aging [96]. Consistently, Brown et al. showed that SIRT3 is not required for normal HSC homeostasis in young mice, but is essential in aged mice [96]. Deletion of SIRT3 reduces the HSC pool in aged mice and compromises HSC self-renewal upon serial transplantation stress, which is in part due to hyperacetylation of SOD2 and subsequent increase in oxidative stress [96]. Overexpression of SIRT3 in aged HSCs reduces oxidative stress and rescues their reconstitution capacity, highlighting the link between mitochondrial function and HSCs, and the role of this surtuin in regulating the aging-associated degeneration [96].

2.6 SIRT6 in stem cell biology 2.6.1 SIRT6 epigenetically promotes proper lineage commitment of ESCs and animal development

Ln cP R ES S1

SIRT6 is a nuclear sirtuin evolutionally important for proper differentiation of ESCs (Fig. 2.4). SIRT6-deficient ESCs are defective in differentiation, forming small embryoid bodies (EBs) and exhibiting skewed differentiation toward neuroectoderm when induced to differentiation [97]. However, although SIRT6 represses glycolysis that is important for ESC self-renewal [16,90
92] through deacetylation of histones [98], elevated glycolysis is not a causation for the observed developmental phenotypes in SIRT6 KO ESCs [97]. Instead, SIRT6 deacetylates

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FIGURE 2.4 SIRT6 epigenetically regulates proper lineage commitment of ESCs and animal development, and inhibits stemness and proliferation of HSCs and CSCs. In mouse, monkey, and human, SIRT6 represses expression of metabolic genes (particularly glycolytic genes), core pluripotent genes, and maternal imprinted lncRNA H19 through deacetylation of H3K9ac and K3K56ac. These actions of SIRT6 metabolically and epigenetically safeguard the balance between pluripotency and differentiation. In hESCs, a p53-targeted lncRNA, lncPRESS1, physically interacts with SIRT6 and prevents its chromatin localization, which in turn maintains high levels of H3K56ac and H3K9ac at promoters of pluripotency genes. Therefore, in contrast to SIRT1, SIRT6 is an evolutionally conserved regulator of ESC differentiation and animal development both in vitro and in vivo. Consistently, SIRT6 also functions to inhibit self-renewal of HSCs and to suppress cancer stem-like capacity in CSCs.

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H3K56ac and H3K9ac, thereby repressing expression of the core pluripotency genes (OCT4, SOX2, and NANOG) and promoting differentiation. In hESCs, SIRT6 is inhibited by a p53-targeted lncRNA, lncPRESS1 [99]. LncPRESS1 physically interacts with SIRT6 and prevents its chromatin localization, maintaining high levels of H3K56ac and H3K9ac at promoters of pluripotency genes [99]. Deletion of SIRT6 leads to high levels of H3K56ac and H3K9ac, and persistent expression of OCT4, SOX2, and NANOG in ESCs and EBs, thereby preventing differentiation [97]. Moreover, OCT4 and SOX2 enhance the expression of TET1 and TET2, which increase 5-hydroxymethylcytosine (5hmC), particularly on neural genes, and shift differentiation toward neuroectoderm [97]. Besides epigenetic regulation, SIRT6 also deacetylates p53 at lysine 381 to reduce the stability and activity of p53 [100]. Consistent with its role in ESC differentiation, SIRT6 is important for embryogenesis and animal development. SIRT6 knockout mice show developmental delay and premature death [97], and haploinsufficiency of Trp53 dramatically extends the life span of these mice [100]. Deletion of SIRT6 in cynomolgus monkey leads to hyperacetylation of histone, activating the long noncoding RNA H19 and resulting in prenatal developmental retardation and neonatal death [101]. A homozygous SIRT6 inactive mutation in human also leads to hyperacetylation of H3K9 and H3K56, dramatically elevating pluripotent genes and causing severe congenital anomalies and perinatal lethality [102]. Collectively, SIRT6 is critical in safeguarding the balance between pluripotency and differentiation [97].

2.6.2 SIRT6 controls regeneration and stress resistance in HSCs and mesenchymal stem cells SIRT6 is also important in maintaining HSC quiescence and long-term repopulation capacity by inhibiting HSC proliferation. Specific depletion of SIRT6 in adult mouse HSCs leads to enhanced H3K56ac level and increases the expression of transcription factors in the Wnt pathway, which results in a twofold expansion of HSPC [103]. Enhanced proliferation makes HSCs more prone to depletion, and impairs their long-term repopulation capacity [103]. In addition, SIRT6 helps to maintain the functionality of MSCs through ameliorating stress in vitro [104]. SIRT6-deficient human MSCs have increased ROS levels and elevated vulnerability to oxidative injury, and exhibit accelerated cell attrition after implantation to the muscles of immunodeficient mice [104]. This action of SIRT6, again, has been attributed to its deacetylation activity on H3K56ac, which directly upregulates a number of NRF2-regulated antioxidant genes by recruiting RNAP II to their promoters [104]. Although it remains unclear how a deacetylase that usually represses gene expression through histone deacetylation acts as a coactivator to promote expression of antioxidant genes in MSCs, this study indicates that SIRT6 is important for MSC stress resistance and delay of aging.

2.6.3 SIRT6 suppresses stemness of CSCs In contrast to the prostemness function of SIRT1 and SIRT2 in CSCs, SIRT6 appears to suppress cancer stemlike capacity in breast, lung, and colorectal cancer cells [105] and inhibit colorectal CSC proliferation [106], which is consistent with its reported tumor-suppressing activity [107]. Ioris et al. reported that overexpression of SIRT6 reduces growth, progression, and grade of breast tumors in a mouse model with PI3K activation, by suppressing the PI3K-boosted glycolysis/pentose phosphate pathway and hindering their stemness. This anticancer stemness action of SIRT6 requires the activation of PI3K, as ablation of a PI3K-activating mutation in otherwise isogenic cancer cells is sufficient to convert SIRT6-sensitive into SIRT6-insensitive cells [105]. However, intriguingly, although the anticancer stemness action of SIRT6 is in line with its pluripotency suppressing activity in ESCs [97], SIRT6 overexpression suppresses PI3K signaling at the transcriptional level and antagonizes tumor sphere formation independent of its histone deacetylase activity in the PI3K activation cancer model [105]. Additional studies are required to dissect the activity dependent and independent mechanisms of SIRT6 in repressing stemness of different types of stem cells.

2.7 SIRT7 in stem cell biology 2.7.1 SIRT7 regulates embryogenesis and life span through maintenance of genome stability SIRT7, a nuclear sirtuin enriched in the nucleolus, also plays a role in animal development [33]. SIRT7 is important in maintaining genome stability. When recruited to sites of DNA damage, SIRT7 deacetylates

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H3K18ac, helping recruitment of damage response factors to DNA double-strand breaks and promoting DNA nonhomologous end joining repair [33]. SIRT7-deficient pups display defective embryogenesis and growth retardation, and are born at sub-Mendelian ratios. More than 20% of SIRT7 null mice die within the first month of life, and the remaining knockouts show signs of accelerated aging with dramatically reduced life span [33].

2.7.2 SIRT7 regulates quiescence and regenerative capacity of HSCs SIRT7 is also important in modulating HSC quiescence and regenerative capacity through regulating metabolic homeostasis [108]. The expression pattern of SIRT7 in HSCs is similar to that of SIRT3, with high expression in HSCs and reduction with age [108], which indicates a critical role in regulating HSC aging. Quiescent HSCs are metabolically inactive. When induced to proliferate, they readily turn into a metabolically active state associated with a dramatic increase of mitochondrial mass. Mohrin et al., found that SIRT7, which is induced by mitochondrial protein folding stress (PFSmt), interacts with nuclear respiratory factor 1 (NRF1), a master regulator of mitochondria, to inhibit the expression of mitochondrial ribosomal proteins and mitochondrial translation factors [108]. SIRT7 knockdown cells increase mitochondria mass and proliferation, demonstrating that SIRT7 is important for preserving HSC quiescence [108]. Consistently, SIRT7 KO HSCs exhibit phenotypes of aging in vivo, including increased PFSmt and apoptosis, loss of quiescence, reduced repopulation capacity, and myeloid-biased differentiation. Conversely, upregulation of SIRT7 improves the regenerative capacity of aged HSCs [108].

2.8 Concluding remarks and future perspectives Emerging evidence reveals important functions of the sirtuin family in regulation of self-renewal, stemness maintenance, pluri-/multipotency, and differentiation of both PSCs and ASCs. Dysfunction and dysregulation of sirtuins in stem cells are linked to a number of human diseases, including developmental defects, degenerative diseases, and cancer. Therefore, it is clear that stem cell sirtuins are at the crossroads of development, aging, and cancer. Given the essential role of NAD1 in diverse cellular processes, including energy metabolism, redox sensing, DNA repair, and genome stability [109], it is not surprising that stem cells harness the sirtuin family of NAD1-dependent enzymes to tightly link their metabolic plasticity to epigenetics, transcription, genome stability, and stress resistance in response to developmental and environmental cues. However, despite their common dependence on cellular NAD1, different sirtuins display cell type-specific and/or stage-dependent impacts on stem cell biology. On the one hand, different sirtuins often have distinct, even contrasting, functions in the same stem cell type. For example, as nuclear sirtuins in ESCs, SIRT1 maintains pluripotency and represses differentiation genes, whereas SIRT6 suppresses pluripotency genes and is critical for their proper lineage commitment upon differentiation. This phenomenon is likely more consistent with their expression dynamics and/or substrate specificities than NAD1 sensing. On the other hand, the same sirtuin may elicit differential impacts in different stem cells. For instance, SIRT6 maintains the quiescent state and long-term regeneration capacity of ASCs, but suppresses the stemness of ESCs. Much work is still needed to comprehend how different members of sirtuins integrate distinct environmental cues via their differential dosages, distinct sensitivities to NAD1, diverse substrates, and interacting partners at various subcellular compartments to coordinate environmental influence on stem cell activities. It is also worth noting that mitochondrial sirtuins, SIRT3 in particular, are crucial defenders from oxidative stress in various cell types, including HSCs, during the process of aging, yet evidence for their contribution to the maintenance of PSCs is still lacking. This is likely due to the fact that PSCs have evolved a variety of mechanisms to protect themselves from ROS, including their preferential use of glycolysis instead of oxidative phosphorylation to reduce ROS production [25,110] and their employment of a number of alterative mechanisms to protect against oxidative stress [111]. Consistently, PSCs such as ESCs have relatively low expression of mitochondrial sirtuins [26]. Nevertheless, given the prevalence of obesity and metabolic syndrome in the reproductive population as well as the increasing parental age in modern society, it will be interesting to study whether mitochondrial sirtuins can regulate the maintenance and survival of PSCs, particularly iPSCs and germline stem cells, under these oxidative stress-associated conditions. Recent advancements in the field have shown that lysine acetylation is just one type of a widespread protein modification known as lipid lysine acylation. These short-chain and long-chain lipids are generated

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by completely different metabolic pathways, and distinct protein acylations are associated with differential regulation of gene transcription, particularly during germ cell development [112,113]. Intriguingly, in addition to deacetylation, sirtuins have been shown to have activities to remove lipid acyl-groups from protein lysine side chains [112,114
116]. Further studies on sirtuin-mediated protein deacylation in stem cells will shed light on the functions of these newly discovered protein modifications in regulating stem cell biology.

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55 e15.

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C H A P T E R

3 Sirtuins and metabolic regulation: food and supplementation Se´rgio Henrique Sousa Santos1,2, Victor Hugo Dantas Guimara˜es2, Janaina Ribeiro Oliveira2 and Luiz Fernando Rezende2 1

Postgraduate Program in Food and Health; Food Engineering School, Agricultural Sciences Institute (ICA), Federal University of Minas Gerais (UFMG), Montes Claros, Brazil 2Laboratory of Health Science, Postgraduate Program in Health Sciences; State University of Montes Claros (UNIMONTES), Montes Claros, Brazil O U T L I N E 3.1 Introduction

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3.2 Tissue-specific sirtuin-modulated metabolic regulation 3.2.1 Liver 3.2.2 Adipose tissue 3.2.3 Heart and skeletal muscle 3.2.4 Kidneys 3.2.5 Pancreas 3.2.6 Brain

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3.3 Nutrition as a therapeutic model for sirtuin regulation 3.3.1 Polyphenols

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3.4 Resveratrol

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3.5 Gallic acid

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3.6 Nonresveratrol related sirtuin activators

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3.7 Food and sirtuins 3.7.1 Mediterranean diet 3.7.2 Berberin 3.7.3 Green cardamom 3.7.4 Cocoa 3.7.5 Indole-3-carbinol 3.7.6 Xanthigen

50 50 51 51 51 51 52

3.8 Conclusion

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3.1 Introduction Economic and cultural changes observed in the last decade have substantially modified people’s way of life. Several factors, such as lifestyle, eating habits, and stress, have been indicated as prevalent issues in the increased levels of nontransmissible chronic diseases, including obesity [1]. Obesity is a major health issue worldwide with a current high occurrence and an estimated 2.3 billion people will be affected worldwide by 2025 [2]. The condition is characterized by excessive body fat deposits associated with reduced energy expenditure and/or food intake imbalance [3]. Excessive body fat itself constitutes an important metabolic syndrome (MS) component, a condition associated with serious complications such as type 2 diabetes (DM2), hypertension, hypertriglyceridemia, reduced glucose tolerance, increased cholesterol, impaired insulin sensitivity, nonalcoholic fatty liver disease (NAFLD), and cardiovascular diseases [4,5].

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3. Sirtuins and metabolic regulation: food and supplementation

As previously stated, obesity plays a central role in MS onset. Increased caloric intake promotes adipocytes hypertrophy and abnormal adipokines secretion [6]. Those changes can be observed in the white adipose tissue (WAT) via promotion of the secretion of nonesterified fatty acids (NEFAs) in the circulatory system, impairing peripheral tissues’ insulin signaling and reducing its sensitivity [7
9]. Moreover, elevated serum NEFAs increase expression of several proinflammatory cytokines, such as tumoral necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) [10] (Fig. 3.1). Those changes may compromise the function of several organs, lower the individual life quality and modulate many enzymes, amongst which we highlight the sirtuins (Sirt). Sirtuins have recently become important targets in the field of health research as modulators of several metabolic diseases, like obesity and its complications [11,12]. The discovery of sirtuin’s chemical structure as well as its regulators (either inhibitors or activators) made possible the identification of sirtuin’s actions over many diseases and its possible therapeutic role [13]. Many authors suggest sirtuin modulation as a possible pathway for the control and management of several age-related diseases, such as diabetes, cancer, cardiovascular and neurodegenerative disorders [14], each somehow associated with pathological inflammatory conditions [15]. Sirtuins are found everywhere in humans (Fig. 3.2), that is, SIRT1 is expressed in brain, liver, pancreas, adipose tissues, skeletal muscle, and heart [16]. Sirtuins may function as deacetylases and/or ADP-ribosyltransferases. Deacetylases-activated sirtuins remove acetylated lysine residues acetyl groups from several target proteins, including histones and transcription factors. Sirtuin 1 (SIRT1) and Sirtuin 3 (SIRT3) are considered crucial to the control of metabolic processes. They are located in the nucleus and mitochondria, respectively, and are expressed in a wide variety of tissues with numerous target proteins [17].

FIGURE 3.1 Obesity development and metabolic disorders: Obesity is characterized by excessive body fat. Increased adiposity leads to adipocyte hypertrophy, which in turn starts to secrete more adipokines. Among the adipokines we highlight MCP-1, which promotes the recruitment of monocytes (Mφ) from the bloodstream, which are released from adipose tissue and may be differentiated into the Mφ1 lineage, causing inflammation, through the interleukins secretion associated with tumor necrosis factors (TNF). Adipokine secretion deregulation, as well as inflammatory factors, may lead to organ dysfunction, compromising their tasks and leading to the development of type 2 diabetes mellitus (DM2), cardiovascular diseases (CVDs), cancer, nonalcoholic fatty liver disease (NAFLD), insulin resistance, and other coexisting metabolic changes in the metabolic syndrome.

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FIGURE 3.2 Sirtuin expression in many organs and its role over metabolic physiological regulation.

SIRT1 and SIRT3 coordinately increase cellular energy reserves, thus maintaining cell energy homeostasis. Any defects on SIRT-controlled pathways result in many metabolic disturbances. Therefore genetic or pharmacological sirtuin activations might lead to metabolic benefits ultimately protecting mice from diet-induced obesity, DM2, and NAFLD [17]. Mammal SIRT1 deacetylation is involved in a series of functions, such as apoptosis, cell cycle, circadian rhythm, mitochondrial function, and metabolism-related target proteins. Currently, researchers are focused on SIRT1’s impact on glucose homeostasis and energy balance [18]. While SIRT1 plays an important role in metabolic function, SIRTs 3 and 5 are located in the mitochondria, thus controlling mitochondrial energy metabolism itself. As the most well-known and studied of the mitochondrial sirtuins, SIRT3 deacetylates several mitochondrial proteins, thus also playing a role in ATP production. Mammal sirtuins are regulated not only by the NAD1/NADH ratio or cellular stressors, but also by transcription/translation-related endogenous proteins as well as many microRNAs [16]. SIRT1 interacts with and regulates a number of histones and nonhistones protein substrates. The huge variety of endogenous targets is associated with the diverse array of biological functions controlled by SIRT1, being involved in age and development, energetic metabolism, inflammation, and DNA double-strand repair, amongst others [18]. Several mice models were used to elucidate and characterize sirtuin’s metabolic functions. Whole body SIRT1-KO mice present low birth weight and don’t live past the initial postnatal stage, while race-specific SIRT-KO mice exhibit developmental and metabolic abnormalities, such as cardiac defects and reduced motor activity, but also improved glucose homeostasis. Some studies opted for a tissue-specific knockout model, such as liver-specific SIRT1-KO mice, although many of the results are often contradictory. Moreover, not only SIRT1 knockdown but also overexpression have been used as a model of investigation, such as the whole body SIRT1 overexpression mice that showed a resistance to high-fat diet (HFD)-induced metabolic dysfunction as well as studies on other sirtuins, specially their importance in mitochondrial metabolic regulation [17,18].

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3. Sirtuins and metabolic regulation: food and supplementation

3.2 Tissue-specific sirtuin-modulated metabolic regulation 3.2.1 Liver In the liver, SIRT1 has several deacetylation targets affecting gluconeogenesis and fat homeostasis. Many of those deacetylation events present opposite effects, thus highlighting the complex nature of SIRT1 function in this organ. For example, SIRT1 deacetylates two distinct gluconeogenesis coactivators with opposite functions, while PGC-1α is an activator [19], CRTC2 acts as an ubiquination and degradation factor [20]. It is postulated then that SIRT1 enacts temporal changes during fasting periods, alternating between an initial CRTC2-mediated mechanism to a posterior PGC-1a induced at a later stage. SIRT1 is reduced at the right moment because the SIRT1 promoter itself is regulated by CRTC2 mediated by the CREB transcription factor [21]. The lower CRTC2 and subsequent SIRT1 thus establish a lower activity level suitable for SIRT1 substrate and PGC-1α at later fasting periods. As for the fat homeostasis, SIRT1 deacetylates LXR nuclear receptor [22] to activate SREBP1 gene for synthesis. However, SIRT1 deacetylates SREBP1 itself to inhibit its activity [23]. Other SIRT1 substrates include FXR nuclear receptor for biliary synthesis [24] and LKB1 linking SIRT1 and AMPK [25,26], as previously described. In vitro studies showed that SIRT1 increases fatty acids oxidation through PPARα receptor activation [27]. Hepatocytesspecific SIRT1-KO mice showed glucose overproduction, chronic hyperglycemia, and increased ROS production, thus resulting in mTORC2/AKT-mediated oxidative stress and consequently insulin resistance [28]. Moreover, numerous studies examined the role of hepatic SIRT1 on many alimentary conditions, and found that liverspecific SIRT1-KO leads to physiological hypersensitivity to a fat-rich diet [29
32], with the exception being that overexpression protects against hepatic steatosis [33,34]. Accordingly, fat-rich diet-induced inflammation leads to JNK1 kinase-induced SIRT1 phosphorylation and its subsequent degradation, as observed in WAT [35]. SIRT3 has several targets in hepatocytes. In fasting conditions, SIRT3 is increased and promotes fatty acids oxidation [36], while also activating the urea cycle [22] and liver ketogenesis [37]. SIRT3 inhibition in a fatty diet-fed mice results in earlier obesity, hyperlipidemia, insulin resistance, and hepatic steatosis. It has also been shown that both obesity as well as a chronic HFD reduced SIRT3 activity, induced hyperacetylation of several mitochondrial proteins, and impaired mitochondrial function [38
40]. SIRT3-KO mice fed a methionine/choline deficient (MCD) diet showed elevated ALT serum levels, increased hepatic content, and increased expression of inflammatory genes as well as reduced fibrogenic activity (SOD2). However, SIRT3 overexpression resulted in the opposite effects, suggesting that SIRT3-KO aggravates MCD-induced NASH, while SIRT3 overexpression attenuates the MCD-induced phenotype [41,42]. Liver SIRT4 shows an antagonistic role to SIRT1 given that its Knockout protects against HFD-induced hepatic steatosis [43], possibly because SIRT4 deacetylates and inhibits malonyl-CoA decarboxylase (MCD1), converting malonyl-CoA to acetyl-CoA [44], thus inhibiting the accumulation of malonyl-CoA, the main precursor for fat synthesis, as well as reducing SIRT4 levels in RC. Finally, hepatic SIRT6, like SIRT1, seems protective, given that two studies shows that SIRT6-KOs are more prone to hepatic steatosis [42,45,46].

3.2.2 Adipose tissue Adipose tissue has been increasingly implicated in energy homeostasis, with SIRTs 1, 2, and 6 directly involved in adipocytes function; SIRT1 has been the most explored. SIRT1 increases adiponectin expression, an important adipokine for energy metabolism, at least in part through FOXO1 protein deacetylation [47], which lowers susceptibility to obesity and diabetes, increases insulin sensitivity, and promotes glucose homeostasis. SIRT1 also inhibits white adipocytes formation and activates lipolysis through PPAR-γ suppression, an important regulator in adipocytes [48]. While SIRT1 and NAD1 are both induced in CR mice WAT [33], SIRT1 is reduced in obese mice [49] and human WATs [50]. A possible mechanism for SIRT1 inhibition in obese animals is suggested by the findings that HFD triggers SIRT1 cleavage in WAT by Caspase 1 [49]. WAT-specific SIRT1-KO mice are prone to diabetes, possibly because the “first blow” in that direction has already been taken. Interestingly, SIRT2 also plays a functional role in adipocytes biology through preadipocytes differentiation [51]. More data corroborate the hypothesis that SIRT1 in the WAT promotes metabolic health through reduced fat deposits. SIRT1 promotes fat mobilization from the WAT to bloodstream during fasting periods in order to facilitate its oxidation in liver and muscle [48]. SIRT1 also promotes WAT browning, which might lead to in situ fat oxidation [52]. Consistently, SIRT1 inhibition in WAT is associated with inflammatory processes and macrophage recruitment [53]. A deacetylation target from SIRT1 in WAT is the nuclear receptor PPARγ [54]. SIRT1 affects

I. Sirtuins and metabolic disease

3.2 Tissue-specific sirtuin-modulated metabolic regulation

43

gene expression from a PPARγ target genes subset restricting differentiation of preadipocytes precursor cells [48]. Finally, SIRT1 transgenic mice show higher insulin sensitizing adipokines [55]. Further roles for sirtuins in adipokines production and endocrine signaling between WAT and hypothalamus remain a fascinating study subject. SIRT2 may also inhibit adipocytes differentiation, at least in part through FOXO1 protein deacetylation [56]. Moreover, SIRT2 activates FOXO3 in adipocytes, thus reducing oxidative stress [57]. SIT6 has been found to be a negative regulator for DGAT1 gene expression, which reduces triglycerides biosynthesis [58].

3.2.3 Heart and skeletal muscle Studies suggest that calorie restriction (CR); physical exercise, and resveratrol may induce mitochondrial biosynthesis and increase mice and human skeletal muscle stress tolerance [59
61], with at least three pathways being implicated in those effects. Firstly, skeletal muscle endothelial nitric oxide synthase (eNOS) activity is induced by CR in mice and humans [62,63]. Given that SIRT1 is known to deacetylate and activate eNOS [64], SIRT1 induction in skeletal muscle might help to explain the whole chain of events. eNOS2/2 mice show no CR-induced mitochondrial activity [62], proving the involvement of eNOS. Secondly, PGC-1α deacetylation is also induced by CR or resveratrol and jumpstarts mitochondrial biogenesis [19,65]. Thirdly, CR-induced adipokine binds to its skeletal muscle receptor and stimulates SIRT1/AMPK axis causing Ca21 release and subsequent activation of AMPKkinase calmodulin-dependent protein kinase, a pathway that can also be activated by physical exercise. Glucose effects over cultivated skeletal muscle cells are dependent on SIRT1
AMPK
FOXO1 interaction, AMPK and FOXO1 [66,67], and, unsurprisingly, skeletal muscle tissues-specific SIRT1-KO impairs CR-induced physiological changes [68]. As for the heart, studies in mice showed that SIRT1’s gain or loss of function protects against oxidative stress such as in ischemia
reperfusion injuries (IRI) [69,70]. SIRT1 may also protect against moderate levels of specific cardiac overexpression but be harmful at higher levels [71]. Protection against hypertrophy might involve a pathway including PPARα and fat oxidation [72]. Although, surprisingly, a study has shown that SIRT1 or PPARα haplosufficiency protected against pressure overload
induced hypertrophy [73]. Notably, CR induced SIRT1 nuclear location through a mechanism requiring eNOS, which was associated with ischemia tolerance [74]. SIRT1 and eNOS might comprise a self-reinforcing activity loop, similar to SIRT1 and AMPK. As for other sirtuins, it has been shown that SIRT6 attenuates AKT signaling in heart, protecting against hypertrophy [75], in which case SIRT6 acts as an IFG-activated gene corepressor by binding to c-jun transcription factor. Finally, a recent study showed that mice knockout for a complex 1 subunit in the heart had increased NADH and SIRT3 inhibition [76], rendering those rats more susceptible to cardiac insufficiency, thus reinforcing SIRT3’s importance for proper cardiac function [77].

3.2.4 Kidneys Many studies have indicated SIRT1-mediated renal function protection, starting with the reported oxidative stress mitigation by SIRT2 in HK-2 cells [78]. SIRT1 genetic activation, either through transgenic mice or STACs administration, protects those animals against several renal injuries models [79
82]. SIRT1 also mitigates fibrosis subsequent to acute renal failure by Smad4 deacetylation and TGF-β signaling suppression [83]. Moreover, SIRT1 is required for renal protection against RC-induced hypoxia, in this case by FOXO3 deacetylation and autophagy activation [84]. Finally, CR is also a renal protector in a rat model of diabetes, associated with SIRT1 activation and NF-κB deacetylation [85]. A recent study suggests a novel communication scheme between renal compartments mediated by SIRT1. In a compartment consisting of proximal tubule cells, SIRT1 establishes nicotinamide mononucleotide (NMN) precursor levels. Released NMN activates SIRT1 in a different renal compartment, constituted by podocytes, to activate stress resistance. This circuit net effect is kidney protection and diabetic albuminuria inhibition [86]. Conversely, SIRT1 might contribute to autosomic-dominant polycystic kidney disease (ADPKD), and hereditary diseases that affects up to 1 in 400 individuals as a consequence of PKD1 or PKD2 genetic defects and lead to renal insufficiency by the fourth to sixth age decade. A recent study shows that c-MYC-induced SIRT1 upregulation is present in an ADPKD murine model [87] in which genetic or pharmacological SIRT1 inhibition suppresses cysts growth, indicating that SIRT1 might induce cyst formation through cell survival mechanisms such as P53 deacetylation and inhibition. Although unusual for SIRT1 to have a protective effect in a normal physiological environment, SIRT1 inhibition might prove effective in a pathological scenario.

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3. Sirtuins and metabolic regulation: food and supplementation

Besides the functions previously described, the kidneys also play a major role in the renin
angiotensin system (RAS), given that the justaglomerular cells from kidney afferent arterioles are the major renin production site [88]. RAS peptide cascade regulates metabolism at several key points. Both RAS peptide cascade and sirtuins are modulated by diverse diet composition, suggesting a similar effect from these systems. Those findings led to other studies that confirmed this interaction both in vivo and ex vivo with cultured cells in which the activation or inhibition of RAS/Ang-(1
7) axis modulates sirtuins expression [89].

3.2.5 Pancreas Both SIRT1 and SIRT4 have been described as playing a major role in pancreatic cells regulation. SIRT1 increases insulin gene transcription and improves glucose-stimulated insulin secretion (GSIS) through UCP2 gene expression reduction [90], given that mice overexpressing SIRT1 showed increased GSIS [91], while also protecting pancreatic β-cells from inflammation-induced apoptosis [92]. Conversely, SIRT4 inhibits GDH-mediated amino acids-induced insulin secretion [93].

3.2.6 Brain The hypothalamus is a major brain region that is fundamental for the control of human physiology. SIRT1 levels in the hypothalamus are affected by diet and seem to mediate many aspects in this structure control [94]. In the absence of SIRT1, the somatotropic axis is inhibited and during caloric restriction SIRT1 levels are increased in both the lateral (LHA) and dorsomedial hypothalamic (DHA) axes. SIRT1 promotes through physical activity an increase in body temperature by supraregulation of orexin receptor 2, which is involved in appetite-regulating feedback mechanisms. In anorexigenic pro-opiomelanocortin (POMC) neurons SIRT1 is key for the diet-induced energy expenditure [95], and SIRT1-KO POMC neurons are more susceptible to obesity development [96]. SIRT1 levels are reduced with aging in the central nervous system (CNS) and, indeed, SIRT1 overexpression can delay the agerelated decay in circadian functions [97], while also delaying aging and prolonging mice life expectancy [98].

3.3 Nutrition as a therapeutic model for sirtuin regulation Metabolic changes are directly related to sirtuin level and activity, thus being able to contribute to obesityrelated comorbidities. Currently, several medicines with a therapeutic approach to treat obesity, such as Orlistat, present serious side effects including increased blood pressure, dry mouth, headache, and insomnia [99]. Sibutramin, a weight control drug, was removed from circulation by the FDA in 2010 because of increased cardiovascular risks. In this sense, there has been observed an increase in the search for alternative and natural antiobesity products. Natural products capable of regulating PPARγ expression, transcriptional activity, and/or sirtuin-mediation have been highlighted as having potential antiadipogenesis action and therefore targets for antiobesity agents [100]. Nowadays, most natural products investigated are phytochemicals obtained from plants used as food and/or medicine, which are both relatively safe and of easy access [100]. Diet-obtained phytochemicals, mainly from fruits and vegetables, may be employed as antiobesity agents given that evidence suggests they may reduce adipose tissue mass, through adipogenesis inhibition, lipolysis stimulation, and apoptosis of existing adipocytes. Diet-obtained phytochemicals include terpenoids, polyphenols, phytosterols and alkaloids, which are amongst the most common components with antiobesity properties [101,102]. Moreover, therapeutic measures have been focused on sirtuin activity modulation through stimulus or inhibition via synthetic or natural active principles (Fig. 3.3), particularly food and supplements. Based on these findings, nutritional studies have focused on the use of food for the modulation of sirtuins, using fruits, vegetables, good red wine, and dark chocolate. Nutritional trends have adopted the “sirtfood diet,” developed by nutritionists Aidan Goggins and Glen Matten, a nutritional method based on the use of diet polyphenols, imitating the effects of caloric restriction, fasting, and exercise. Sirtuins-selecting activators and inhibitors are still in their initial stages, but some natural compounds are already commercially available [103]. The interest for new sirtuin-modulating molecules is based on potential health benefits found in caloric restrictive diets; these not only improve mammal metabolism but also prolong their life expectancy through SIRT1 induction [104], although its role in this regard is still debatable [105]. Fig. 3.3 shows some of the better-known compounds and food that act upon sirtuin modulation to improve the metabolic overall outcome.

I. Sirtuins and metabolic disease

3.3 Nutrition as a therapeutic model for sirtuin regulation

45

FIGURE 3.3 Natural and synthetic molecules applied on sirtuins metabolic activation or inhibition.

3.3.1 Polyphenols Flavonoids are metabolic polyphenols with pharmacological properties synthesized by plants and fungi, and due to their extensive biological activities, they have been studied for medicine development. Flavonoids have been shown to modulate NAD1-dependent histones deacetylase activity, like SIRT1 (resveratrol, piceatannol, butein, isoliquiritigenin, fisetin, quercetin) [106] and SIRT6 (catechin, luteolin, quercetin, kaempferol, myricetin, delphinidin, cynidin) [107] (Table 3.1). SIRT6 targets lysines 9 (H3K9) and 56 (H3K56) on the H3 sequence and has been shown to efficiently deacetylatethese sites. Those SIRT6 functions are involved in the regulation of several genes, including stress-response. SIRT6-deficient cells presented oxidative stress sensitivity and a reduced DNA repair capability. SIRT6 is also involved in glucose and lipids metabolism, regulating the expression of multiple glycolytic and lipidic genes related to cell response [108,109]. Those SIRT6 functions highlight its importance to aging processes and the protection of many cell functions. Therefore SIRT6-regulating compounds are promising therapeutic targets for age-related diseases, including but not limited to cancer, diabetes, neurodegenerative disorders, and metabolic disturbances [107].

I. Sirtuins and metabolic disease

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3. Sirtuins and metabolic regulation: food and supplementation

TABLE 3.1 Polyphenols activity on activation and(or) inhibition of Sirt 1 and Sirt 6. Inhibition (mean) Compound

Sirtuin

10 µM

100 µM

Activation

EC50

References

Butein

1

Na

8.53

Na

Na

[106]

Resveratrol

1

Na

13.4

Na

Na

Piceatannol

1

Na

7.90

Na

Na

Isoliquiritigenin

1

Na

7.57

Na

Na

Fisetin

1

Na

6.58

Na

Na

Quercetin

1

Na

4.59

Na

Na

10 µM(%)

100 µM(%)

(1)-Catechin

6

17.0

26.0

0.8

Na

(2)-Catechin

6

25.0

54.0

0.4

Na

(2)-Gallocatechin

6

17.0

23.0

0.2

Na

(2)-Catechin gallate

6

62.0

88.0

0.1

2.5

(2)-Gallocatechin gallate

6

79.0

84.0

0.01

5.4

(2)-Epicatechin

6

Nd

10.0

Nd

Na

(2)-Epigallocatechin

6

Nd

4.1

Nd

Na

(2)-Epicatechin gallate

6

15.0

60.0

0.1

Na

(2)-Epigallocatechin gallate

6

10.0

42.0

0.8

Na

Naringenin

6

8.3

23.0

1.1

Na

Eriodictoyl

6

19.0

27.0

1.1

Na

Apigenin

6

Nd

1.0

0.9

Na

Luteolin

6

24.0

29.0

1.2

270

Kaempferol

6

40.0

24.0

2.2

Nd

Quercetin

6

27

40.0

1.5

990

Myricetin

6

Nd

0.0

2.3

404

Cyanidin

6

Nd

0.0

2.6

460

Delphinidin

6

Nd

0.0

2.2

760

Genistein

6

1.8

9.6

0.7

Na

Biochanin A

6

13

24.0

Nd

Na

Nicotinamide

6

Na

58.0

Nd

Na

Gallic Acid

6

Na

14.0

1.4

Na

Hydroferulic acid

6

Na

212.0

1.8

Na

p-Coumaric acid

6

Na

8.5

0.6

Na

Caffeic Acid

6

Na

25.9

1.0

Na

trans-Ferulic acid

6

Na

28.6

0.9

Na

Sinapic Acid

6

Na

8.0

0.6

Na

EC50, Half maximal effective concentration; Na, not available; Nd, not determined.

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[107]

47

3.4 Resveratrol

3.4 Resveratrol O-3,5,40 -Trihydroxystilbene, commonly known as resveratrol, is a polyphenol whose molecule contains two phenyl rings separated by a methylene bridge, and was the first compound known to simulate the effects of CR as a sirtuin-stimulator [106,110]. Resveratrol can be found in several foods (Table 3.2), like berries, peanut, red grape, and red wine (0.1 a 14.3 mg/L concentrations), which might help to explain the lower rates of cardiac diseases in France despite its saturated fat
rich diet [141
143]. Resveratrol can induce CR-like genetic expression TABLE 3.2 Composition of resveratrol in food using the Phenol-explore database [140]. Categories of food

Food

Mean content

Min

Max

SD

n

N

References

Fox grape, red wine

0.25 mg/100 mL

0.01

0.67

0.30

4

4

[111,112]

Alcoholic beverages Wines—Berry wines

Wines—Grape wines

Wines—Sparkling wines

23

Fox grape, white wine

0.01 mg/100 mL

1.00

0.02

0.01

2

2

[112]

Muscadine grape, red wine

3.02 mg/100 mL

1.41

4.41

1.13

5

5

[111]

Wine (red)

0.27 mg/100 mL

0.00

2.78

0.31

478

956

[112
122]

23

Wine (rose´)

0.12 mg/100 mL

5.00

0.29

0.08

18

36

[112,113,123,124]

Wine (white)

0.04 mg/100 mL

0.00

0.17

0.03

101

270

[116,118,125,126]

23

23

23

Champagne

9.00

mg/100 mL

8.00

0.01

1.15

4

4

[127]

Chocolate, dark

0.04 mg/100 g FW

0.04

0.04

0.00

1

1

[128]

Bilberry, raw

0.67 mg/100 g FW

0.67

0.67

0.00

1

1

[129]

European cranberry

1.92 mg/100 g FW

1.92

1.92

0.00

1

1

[129]

Grape (black)

0.15 mg/100 g FW

0.02

0.58

0.20

7

7

[121,130]

Grape (green)

0.02 mg/100 g FW

0.00

0.04

0.02

5

5

[121,131]

Lingonberry, raw

3.00 mg/100 g FW

3.00

3.00

0.00

1

1

[129]

Redcurrant, raw

1.57 mg/100 g FW

1.57

1.57

0.00

1

1

[129]

Strawberry, raw

0.35 mg/100 g FW

0.35

0.35

0.00

1

1

[129]

Grape (green), pure juice

5.0823 mg/100 mL

0.00

0.01

2.3123

12

12

[132]

Peanut, butter

0.04 mg/100 g FW

0.01

0.07

0.02

35

35

[133
135]

Vinegar

4.5723 mg/100 mL

0.00

0.01

4.08e-03

6

92

[136]

Peanut

0.08 mg/100 g FW

9.0023

1.12

0.28

16

16

[134,137]

0.19

0.08

12

12

[138,139]

2.20

0.17

0.05

11

11

[139]

0.00

0.01

3.49e-03

12

12

[134,135]

0.16

0.05

7

7

[138]

Coffee and cocoa Cocoa—Chocolate

Fruits and fruit products Fruits—Berries

Nonalcoholic beverages Fruit juices—Berry juices

Oils Oils—Nut oils

Seasonings Other seasonings

Seeds Nuts

Peanut, dehulled Peanut, dehulled, roasted Peanut, roasted Pistachio, dehulled

0.07 mg/100 g FW 0.02 mg/100 g FW 23

5.37

mg/100 g FW

0.11 mg/100 g FW

23

3.00

23

23

9.00

FW, Fresh weight; Max, maximum; Min, minimum; n, number of original data points used to calculate the mean content value; N, total number of original individual samples analyzed; SD, standard deviation. Composic¸a˜o de resveratrol em alimento utilizando-se o banco de dados Phenol-explore [140].

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3. Sirtuins and metabolic regulation: food and supplementation

patterns in many tissues, while also reducing aging signs without affecting any sirtuin gene expression [144,145]. In vivo studies have shown that resveratrol improves the overall health of hypercaloric diet
fed mice [59]. Moreover, resveratrol-fed elderly mice show a marked reduction in signs of aging, including reduced albuminuria, decreased inflammation and apoptosis in the vascular endothelium, increased aortic elasticity, greater motor coordination, reduced cataract formation, and preserved bone mineral density [145]. Due to its low bioavailability, reworked resveratrol versions with improved bioavailability were developed, with one such formulation containing 150 mg/day resveratrol (resVida) having demonstrated beneficial effects in healthy obese males, lowering liver lipidic content, blood glucose, triglycerides, alanine-aminotransferase, and inflammatory markers, thus emulating CR-diet effects [146]. Moreover, oral resveratrol supplementation acutely improved endothelial-dependent vasodilatation in a dose-related manner, which was correlated with the plasma resveratrol content [147]. Resveratrol administered as Longevinex formulation improved flow-mediated dilation after 3 months of treatment, with the parameter returning to the baseline 3 months after treatment interruption, without affecting blood pressure, insulin resistance, lipid profile, or inflammatory markers [148]. SRT501, another commercial resveratrol formulation, reworked the molecular signature that overlaps with resveratrol, like improved mitochondrial biogenesis, metabolic signaling pathways, and impaired proinflammatory pathways in hypercaloric diet-fed mice. SRT501 generated a genetic profile mirroring that of CR [149]. It has also been demonstrated that those formulations reduced blood glucose and improved insulin sensitivity in Phase II type 2 diabetes patients, highlighting its potential clinical utility [150]. Furthermore, 30 mg/kg/day resveratrol administration for 60 days to HFD-fed mice reduced these animals’ body fat, total cholesterol, triacylglycerol, transaminases, and insulin plasma levels, results that were accompanied by reduced liver TNF-α, IL-6, and NF-κB mRNA levels. Analysis of liver adipogenesis-related genes indicated reduced ACC, PPAR-γ, and SREBP-1 mRNA levels in obese mice treated with the polyphenol through increased SIRT1 expression, resulting in reduced hepatic steatosis [151].

3.5 Gallic acid Gallic acid (3,4,5-trihidroxybenzoic acid) (GA) is a natural polyphenol found in microalgae, some bacteria and vegetables (Table 3.3) [197]. In vivo studies have shown that GA has antiobesity, hypolipidemic and antidiabetogenic effects [198
200]. Amongst the many mechanisms through which GA may regulate the metabolic functions is sirtuin modulation. A 9-week 10 mg/kg/day GA treatment of HFD-fed mice improved blood glucose levels and insulin homeostasis, and delayed body weight gain without affecting food intake, results that might be, at least in part, attributed to the AMPK/Sirt1/PGC1α pathways modulation, as confirmed by the SIRT1-KO mice which showed lower expression of mitochondrial function-related genes and reduced PGC1α deacetylated form. Moreover, it also promotes WAT tissue browning through Uncoupling Proteins (UCPs) 1 and 3 [201]. Another study found similar results in standard chow and HFD-fed mice treated for 60 days with 100 mg/kg GA, resulting in SIRT1 increased expression in WAT [202].

3.6 Nonresveratrol related sirtuin activators Given the aforementioned importance of the sirtuins, there has been a great interest in discovering a more potent activator than resveratrol. Milne et al. identified nonresveratrol-related SIRT1 activators with significantly increased action. Amongst those, SRT1720 compound, which shares the same molecular binding and activation site with resveratrol, is the most effective so far, with stimulatory activity at 750% on 10 μM dose with substrate deacetylation both in vitro and in vivo [149]. Furthermore, SIRT1720’s effectiveness was observed for in vivo type 2 diabetes models: genetically obese mice (Lep ob/ob), DIO mice, and fa/fa Zucker rats. SRT1720 to DIO mice not only reduced postprandial blood glucose, partially restored insulin levels, and lowered fast blood glucose, but also improved oxidative metabolism in skeletal muscle, brown adipose tissue, and liver through a global metabolic adaptation that mimics lower energy levels [61,149,203]. Other SRT1720-treatment benefits included lower liver triglycerides and aminotransferases as well as lipogenic genes expression [204]; increased average and maximum life expectancy of HFD-fed mice, increased insulin sensitivity, and normalized the genetic profile for apoptosis and inflammation markers [205]. In a preclinical cancer treatment evaluation, SRT1720 induced apoptosis in vitro in multiple cells myeloma and reduced xenoinserted growth tumor through a mechanism involving the ATM-CHK2/Caspase8
9 signaling pathway [206].

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3.6 Nonresveratrol related sirtuin activators

TABLE 3.3 Composition of Gallic acid in food using the Phenol-explore database [140]. Categories of food

Food

Mean content

Min

Max

SD

n

N

References

Beer (ale)

0.11 mg/100 mL

0.03

0.18

0.08

3

3

[152,153]

Beer (dark)

0.03 mg/100 mL

0.03

0.03

0.00

1

1

[152,153]

Beer (regular)

0.07 mg/100 mL

0.00

0.70

0.17

25 47

[153
158]

Liquors—Nut liquors

Walnut, liquor

15.15 mg/100 mL

11.20

21.70

4.81

4

4

[159]

Spirits—Brandy

Cognac

0.23 mg/100 mL

0.10

0.47

0.19

3

36

[160]

Spirits—Rum

Rum

0.06 mg/100 mL

0.06

0.06

0.00

1

21

[160]

Spirits—Whisky

Scotch whisky

0.09 mg/100 mL

0.05

0.12

0.03

4

66

[160]

Wines—Fortified Wines

Sherry

0.60 mg/100 mL

0.60

0.60

0.00

1

1

[161]

Wines—Grape wines

Wine (red)

3.59 mg/100 mL

0.00

12.60

2.71

99 514 [117,119,162]

Wine (rose´)

1.04 mg/100 mL

0.94

1.43

0.35

2

Wine (white)

0.22 mg/100 mL

0.00

1.10

0.32

22 105 [116,162,164]

Champagne

0.05 mg/100 mL

0.01

0.08

0.03

4

4

[127]

Dried fruits—Dried other fruits

Date, dried

1.56 mg/100 g FW

0.00

3.09

1.55

3

3

[165]

Fruits—Berries

Blackberry, raw

4.67 mg/100 g FW

2.00

9.00

3.79

3

3

[166]

Cloudberry

4.20 mg/100 g FW

4.20

4.20

0.00

1

1

[167]

6.00

0.00

1

1

[168]

Alcoholic beverages Beers

Wines—Sparkling wines

5

[113,163]

Fruits and fruit products

23

23

mg/100 g FW 6.00

23

Fruits—Citrus

Grapefruit

6.00

Fruits—Other fruits

Date, fresh

0.16 mg/100 g FW

0.00

0.48

0.28

3

3

[165]

Fruits—Tropical fruits

Banana, raw

1.00 mg/100 g FW

0.87

1.08

0.12

3

3

[169]

Jams—Pome jams

Quince, jam

0.67 mg/100 g FW

0.00

6.05

1.63

20 20

[170]

Grape (green), pure juice

0.10 mg/100 mL

0.10

0.10

0.00

1

18

[171]

Sea-buckthornberry, pure juice

0.20 mg/100 mL

0.15

0.26

0.08

2

2

[172]

Apple (cider), juice from concentrate 0.04 mg/100 mL

0.00

0.12

0.06

6

6

[173]

Nonalcoholic beverages Fruit juices—Berry juices

Fruit juices—Pome juices

Apple (cider), pure juice

0.18 mg/100 mL

0.14

0.22

0.03

4

4

[173]

Apple (dessert), pure juice

0.66 mg/100 mL

0.00

1.36

0.77

4

4

[174
176]

Fruit juices—Tropical fruit juices Pomegranate, pure juice

0.45 mg/100 mL

0.03

3.08

0.86

13 13

[177]

Tea infusions

Tea (black), infusion

4.63 mg/100 mL

0.07

14.58

4.38

39 53

[178
181]

Tea (green), infusion

0.49 mg/100 mL

4.0023 3.33

0.89

44 44

[178
182]

Tea (oolong), infusion

0.68 mg/100 mL

0.02

3.50

1.20

14 14

[178,179,183]

Common sage, dried

5.25 mg/100 g FW

0.00

10.50

7.42

2

2

[184,185]

Marjoram, dried

1.60 mg/100 g FW

1.60

1.60

0.00

1

1

[186]

Oregano, dried (wild marjoram)

5.15 mg/100 g FW

0.00

10.30

7.28

2

2

[184,185]

Other seasonings

Vinegar

2.59 mg/100 mL

0.00

9.50

2.36

27 187 [136,187,188]

Spices

Cloves

458.19 mg/100 g FW 17.47

Seasonings Herbs

783.50 298.99 5

5

[189
191] (Continued)

I. Sirtuins and metabolic disease

50

3. Sirtuins and metabolic regulation: food and supplementation

TABLE 3.3 (Continued) Categories of food

Food

Mean content

Nuts

Chestnut, raw

Soy and soy products

Min

Max

SD

n

N

References

479.78 mg/100 g FW 276.00 907.00 182.55 9

9

[192]

Soybean, sprout, raw

0.70 mg/100 g FW

0.70

0.70

0.00

1

1

[193]

Cabbages

Cauliflower, raw

0.69 mg/100 g FW

0.68

0.70

0.01

2

2

[193]

Fruit vegetables

Eggplant (purple), whole, raw

0.14 mg/100 g FW

0.11

0.20

0.05

3

3

[194]

Olive (black), raw

0.02 mg/100 g FW

0.01

0.04

0.02

3

3

[195]

Chicory (green), raw

25.84 mg/100 g FW

12.75

38.93

18.51

2

2

[196]

Chicory (red), raw

14.56 mg/100 g FW

7.99

30.94

8.57

6

6

[196]

Seeds

Vegetables

Leaf vegetables

FW, Fresh weight; Max, maximum; Min, minimum; n, number of original data points used to calculate the mean content value; N, total number of original individual samples analyzed; SD, standard deviation. Composic¸a˜o de resveratrol em alimento utilizando-se o banco de dados Phenol-explore [140].

Besides SRT1720, new compounds were identified, like oxazolo [4,5-b] pyridine [207] and imidazo (1,2-b) pyridazine, thiazole derivatives containing oxazolopyridine nuclei with potential new therapeutic targets for treatment of several disturbances. The most potent analog from this series, compound 29, showed significant oral bioavailability in mice and rats in three different models, without affecting body weight, as well as general chemical clinics and hematology [208]. Another recently developed new compound is SRT2104, capable of attenuating inflammation in a dextran sulfate sodium-induced (DSS) colitis model, with a lower colitis score compared to prednisone as well as improving weight loss. SRT2104 has been also tested in a Phase II patient clinical study with individuals suffering from metabolic, inflammatory, and cardiovascular diseases, after it was concluded that it was safe and well-tolerated in Phase I trials with healthy subjects [209]. In addition, pyrrolo [3,2-b] quinoxaline has been described as potent lipolytic agents with in vitro antiinflammatory properties, although its possible effect on animal models remains untested [210]. Other compounds, prepared by Mai et al., are 1,4-dihydropyridine (DHP) derivatives exhibiting a benzyl group in the N1 position, which have been shown to act as potent human SIRT1, SIRT2, and SIRT3 activators, with increased mitochondrial activity and reduced murin C2C12 myoblasts senescence [211]. In conclusion, so far no sirtuin activator has been shown to extend standard diet-fed mice life expectancy, but their effects over general health and age-related diseases might be just as interesting.

3.7 Food and sirtuins 3.7.1 Mediterranean diet Diet is a modifiable environmental factor, being one of the cornerstones for obesity management, which might also modify sirtuins activity as previously described. The Mediterranean diet (MD) is a healthy eating habit based on a diet common in Mediterranean countries [212]. MD is characterized by very low NEFAs ingestion and high intake of polyunsaturated fatty acids (PUFA) and micronutrients, including vitamins and minerals known to improve plasmatic antioxidative capabilities [213]. Moreover, both the quality and quantity of ingested lipids affect lipids and triglycerides stocks in adipose tissue and even other organs, such as the liver [214], resulting in a deleterious effect. A study conduct with obese patients in Naples, Italy showed that adhesion to MD for 7 days was associated with increased SIRT4 levels, lower ectopic fat deposition, and adipocytes dysfunction when compared to patients with lower adhesion to MD, regardless of BMI. Circulating SIRT4 levels varied not only according to energy intake but due to intake of antioxidants-containing foods, such as fruits, olive oil, vegetables, and fish [215]. This positive association between MD and circulating SIRT4 levels suggests that a SIRT4 increase might represent yet another antioxidant metabolic advantage to obese individuals and reinforces the importance of the role of nutrition professionals in multidisciplinary teams for obesity control.

I. Sirtuins and metabolic disease

3.7 Food and sirtuins

51

3.7.2 Berberin Berberin, a commonly used medicine in traditional Chinese medicine, has attracted increasingly more attention for its potential therapeutic qualities on hepatic steatosis, diabetes, and dislipidemia treatment [216]. It has been shown that berberin and its derivatives reduced HepG2 cells hepatic steatosis [217] as well as HFD-fed mice hepatic steatosis [218
221]. In a double-blind study, it also reduced NAFLD [222] and serum cholesterol in hypercholesterolemic patients [223]. Although berberin’s beneficial effects seem to be, at least in part, AMPK-mediated [224,225] and LDLR increased expression [223], one of the mechanisms for the hepatocytes’ lipids metabolism improvement is due to SIRT1-mediated autophagy induction and FGF21 expression. Berberin stimulates SIRT1 deacetylation activity and induces autophagy through autophagy-protein 5, while also promoting hepatic gene expression and circulating levels of FGF21 and chthonic bodies in a SIRT1-dependent manner in mice [226].

3.7.3 Green cardamom Green cardamom (GC) (Elettaria cardamomum) is an aromatic plant traditionally used as a culinary ingredient, commonly cultivated in Sri Lanka, Thailand, and Guatemala. It is an important source of flavonoids, alkaloids, terpenoids, anthocyanins, and phenolic compounds [227,228]. GC has been used for the treatment of several disturbances, including asthma, indigestion, and congestive hepatopathy [229], mainly because of its antioxidant, antiinflammatory, antitumor, and antimicrobial activities [230,231]. Furthermore, it can also be used in the treatment of obesity and its associated comorbidities, such as NAFLD; 3 g/day for 3 months GC administration reduced liver fat, reactive C-protein, and antiinflammatory factors through increased SIRT1 in plasma [232]. Those effects might be associated with its active principles: 1,8-cineol [eucaliptol], beta-pinenol, and geraniol, polyphenols and known sirtuin modulators [233
235]. The mechanisms for GC’s antiinflammatory effects include reduced inflammatory cells infiltration, lipidic peroxidation, and advanced protein oxidation products (APOP), as well as an increase in antioxidants enzymes [236,237], reduced inflammatory mediators, such as COX-2, iNOS and NF-κB [238
240], lower hemolysis by vitamin E deficiency [241], and increased activity of adipogenesis and NBK cells [230].

3.7.4 Cocoa Cocoa (Theobroma cacao) is a flavonoids-rich fruit, with potent antioxidant effects and well-established benefits for cardiovascular health, due to improved endothelial function and reduced cellular neurodegeneration [242,243]. A study using pholyphenol-enriched cocoa evaluated its potential benefits through the sirtuins pathway in a diabetic retinopathy model. It was found that SIRT1 activity was reduced in retinal tissue of diabetic mice, NAD1 intracellular levels were increased, and Lys310-p65 acetylation was increased in the retinal tissue. 0.12
22.9 mg/kg/day Cacao administration by Gavage for 16 weeks reduced SIRT1 inhibition and increased NAD1, leading to lLys310-p65 deacetylation, which then regulated GFAP expression in diabetic rats retinal tissue; all those effects allowed for SIRT1-mediated neuroprotection in diabetic retinopathy-presenting tissues [244].

3.7.5 Indole-3-carbinol Indol-3-carbinol (I3C) is a natural product found in cabbage and broccoli with chemotherapeutic properties, including antiproliferative and proapoptotic properties against several types of cancer [245]. In vitro, I3C targets a wide array of cell cycle and survival-related signaling pathways, such as AKT, NF-κB, leukemia/lymphoma B2 cells, mitogen-activated kinases, cyclin-dependent kinases, and cyclin D1 [246]. Recently, I3C has been highlighted as a possible antiobesity agent, being able to reduce blood glucose and insulin in HDF-fed mice [247] as well as reducing inflammatory cytokines such as IL-6 expression in adipocytes and macrophage [248]. In vivo it was shown that 100 mg/kg/day I3C administration reduced body weight and visceral fat weight gain in HFD-fed mice [249]. I3C binds to SIRT1 and activates 3T3-L1 cell deacetylation and reduced adipogenic C/EBPα, PPARγ2, FAS, and aP2 mRNA levels in 3T3-L1 cells but not in SIRT1 knockdown cells, thus it was concluded that I3C specifically inhibits adipocytes differentiation through SIRT1 binding and activity regulation, suggesting an important route to SIRT1-direct drugs [250].

I. Sirtuins and metabolic disease

52

3. Sirtuins and metabolic regulation: food and supplementation

3.7.6 Xanthigen Xanthigen is a source of punic acid and fucoxantin, derivatives from pomegranate seed oil and brown seaweed, respectively, both with antitumor, antioxidant, and antiinflammatory properties [100,251]. A study found that xanthigen interfered with adipocytes suppression, as seen on 3T3-L1 pre-adipocytes. Also, Western blot analysis showed increased SIRT levels, indicating that xanthigen is somehow involved in the suppression of adipocytes differentiation through C/EBPβ, C/EBPδ, and PPARγ inhibition coupled to SIRT1 pathway stimulation and FOXOs and AMPK signaling pathways modulation [252].

3.8 Conclusion In conclusion, we observed that sirtuins are enzymes with great relevance as potential therapeutic targets for improvements in life quality, life expectancy, metabolic disorders, and age-related diseases, given their capability of acting as metabolism modulators. The use of sirtuins activators, as functional diet supplements or even as medicine, has proven to be an effective strategy in the amelioration of obesity-related comorbidities and metabolic syndrome.

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101.

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C H A P T E R

4 Sirtuins in diabetes mellitus and diabetic kidney disease Kultigin Turkmen Division of Nephrology, Department of Internal Medicine, Meram School of Medicine, Necmettin Erbakan University, Konya, Turkey O U T L I N E 4.1 Introduction

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Part 1 4.2 Sirtuin 1 (SIRT1) in normal physiology

62 62

Part 2 4.3 Diabetes mellitus and sirtuins

65 65

4.4. Hypertension and sirtuins 4.5. Novel treatment options in diabetes mellitus and diabetic kidney disease

71 71

4.6. Conclusion and future perspectives

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References

73

4.1 Introduction Sirtuins are a group of NAD-dependent enzymes that consist of seven different structural subtypes, SIRT1 to SIRT7 (Table 4.1). Mainly they remove the acetyl groups from lysine residues of various enzymes, transcription factors, and also histones of DNA. There are two possibilities for the effects of deacetylation of the proteins mentioned above. The first possibility is the activation of transcription factors. In this case, the system can be activated or inhibited according to transcription factor activity. The second possibility is the inhibition of the target factors and in this case again the system can be activated or inhibited. However, on the contrary, deactylated histones cause downregulation of their target proteins. Sir 2 was the first sirtuin identified in Saccharomyces cerevisae. After the discovery of Sir 2 in yeast, researchers showed that these enzymes could prolong life. After the discovery of the role of prolonging life, studies were initiated to identify the members of the sirtuin family in mammals and many articles have been published on this subject up to the present. From a search in Pubmed, we can see that the current number of articles on sirtuins is around 12,000 and this number is increasing day by day. In my opinion, the main reason why the Sirtuin family is very popular among researchers is that these enzymes control life-leading mechanisms and pathways. In particular, calorie restriction (CR)-induced effects activate sirtuins and eventually induce organ protection and increase cell survival (Fig. 4.1). Diabetes mellitus (DM) and its complications are worldwide problems. The prevalence of DM is growing, probably due to increased consumption of food and decreased physical activity. According to the American Diabetes Association (ADA) data, in 2017 an estimated 24.7 million people in the United States were diagnosed with DM, representing 7.6% of the total population (and 9.7% of the adult population). The estimated national cost of diabetes in 2017 is US$ 327 billion, of which US$ 237 billion (73%) represents direct health care expenditures attributed to diabetes and US$ 90 billion (27%) represents lost productivity from work-related absenteeism, reduced productivity at work and at home, unemployment from chronic disability, and premature mortality in Sirtuin Biology in Cancer and Metabolic Disease. DOI: https://doi.org/10.1016/B978-0-12-822467-0.00014-0

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© 2021 Elsevier Inc. All rights reserved.

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4. Sirtuins in diabetes mellitus and diabetic kidney disease

TABLE 4.1 The locations and functions of sirtuin family members. Sirtuin group

Enzyme localization

Enzyme activity

SIRT1

Cytoplasm and nucleus

Deacetylase

SIRT2

Cytoplasm and nucleus

Deacetylase

SIRT3

Cytoplasm, mitochondrion and nucleus

Deacetylase

SIRT4

Mitochondrion

ADP-ribosyl transferase

SIRT5

Mitochondrion

Deacetylase

SIRT6

Nucleus

Deacetylase and ADP-ribosyl transferase

SIRT7

Nucleus

Deacetylase

FIGURE 4.1 The relation between calorie restriction, NAD, and sirtuins.

the United States [1]. Hence, we should initiate new strategies and develop new treatment modalities to overcome these problems. In this chapter, first of all, I would like to focus on the physiological functions of sirtuins, and thereafter I will comprehensively discuss the pathophysiological roles of sirtuins in DM and related diseases, including hypertension and diabetic nephropathy.

Part 1 4.2 Sirtuin 1 (SIRT1) in normal physiology In the cell, the localization of sirtuins is very important for their functions. Sirtuin 1 (SIRT1) is found both in the nucleus and cytoplasm. Because of its widespread localization, SIRT1 might have various physiological and maybe pathological metabolic effects under certain stress conditions. The main functions of this molecule include deacetylating nuclear histone proteins as well as deacetylation of nonhistone proteins including peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α), insulin receptor substrate-1 (IRS-2), peroxisome proliferator-activated receptor-alpha (PPAR-α), PPAR-γ, mitochondrial uncoupling protein 2 (UCP-2), liver X receptor (LXR), farnesoid X receptor (FXR) and sterol-regulatory-element binding protein (SREBP) [2 6]. By deacetylating histone proteins in the nucleus by SIRT1, repression of gene transcription

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occurs [7]. On the other hand, the metabolic effects of SIRT1 regulate insulin secretion, adipogenesis, and myogenesis. Hence, SIRT1 is the most common member of sirtuins, probably due to the generalized effects on the cell cycle, mitochondria metabolism, energy homeostasis, inflammation, oxidative stress, and apoptosis [8]. However, other sirtuins might play a role regarding the functions of mitochondria. For instance, SIRT5 regulates the urea cycle through the direct activation of carbamoyl phosphate synthetase [9]. Subsequent studies have revealed that SIRT5 also exhibits demalonylase and desuccinylase activities, through which it controls ketogenesis [10]. An important effect of SIRT5 as an inducer of the energetic flux via glycolysis has also been shown [11]. After deactylation of transcription factors or histones the second important step is conversion of nicotinamide adenine dinucleotide (NAD) to nicotinamide (NAM). We understood that without NAD, SIRT1 cannot exert its functions. On the other hand, NAD is reduced to NADH in mitochondria under certain conditions. Therefore NAD should be supplied via salvage and de novo pathways. In the salvage pathway, NAM can be converted to nicotinamide mononucleotide (NMN) by the enzyme “intracellular NAM phosphoribosyl transferase” (iNampt) and thereafter NMN is converted to NAD via the enzyme named “nicotinamide mononucleotide adenyl transferases2” (Nmnat). On the contrary, in the de novo pathway, NAD is obtained from tryptophan and nicotinic acid by various enzymes [12]. In this regard, I would like to continue with the metabolic effects of SIRT1, 3, and 4 in mammals.

4.2.1 Major roles of SIRT1 in glucose metabolism SIRT1 can influence many steps of glucose metabolism in liver, pancreas, muscle, and adipose tissue (Fig. 4.2). Mainly SIRT1 affects cell metabolism by deacetylating and activating PGC-1α [13]. During the fasting state, the balance between insulin and glucagon (decreased insulin versus increased glucagon) stimulates gluconeogenesis. Modulation of gluconeogenesis occurs via cAMP-response element binding protein (CREB), regulated transcription coactivator 2 (CRTC2), and Forkhead box 1 (FOXO1) (Fig. 4.3) [14,15]. The latter is one of the most important transcriptional factors, and has been found to be very effective in terms of sensing nutrient deprivation and promoting cellular homeostasis [16]. The link between FOXO proteins, signal transducer and activator of transcription 3 (STAT3), and SIRT1 regarding hepatic glucose metabolism has also been identified. FOXO1, -3a, and -4 were found to be closely associated with increased expression of gluconeogenesis genes and decreased expression of glucokinase [17,18]. SIRT1 also regulates gluconeogenesis via deacetylation, thereby deactivating STAT3 which can inhibit the transcription of gluconeogenic genes in normal conditions [19]. In this context, FOXO proteins along with STAT3 and SIRT1 regulate glucose metabolism [20] and feeding behaviors [21]. Insulin sensitivity has also been considered to be an important part of the glucose metabolism. Protein tyrosine phosphatase 1B (PTP1B) is a tyrosine phosphatase for the insulin receptor that has been found to have an important role in insulin sensitivity and diet-induced obesity [22]. PTP1B can be repressed via deacetylation. Activation

FIGURE 4.2 Metabolic effects of SIRT1 in peripheral organs.

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4. Sirtuins in diabetes mellitus and diabetic kidney disease

FIGURE 4.3 The enzymes involved in glucose and lipid metabolism.

of SIRT1 inhibits PTP1B. In this regard, SIRT1 might improve insulin sensitivity in insulin-resistant conditions via reducing PTPB1B activity [23]. The role of SIRT1 in the pancreas was also demonstrated. Experimental data of SIRT1 overexpression suggested that serum insulin and cholesterol were diminished along with the diminution of the adipose tissue volume and decreased obesity-induced insulin resistance [24,25]. Interestingly, SIRT1-deficient mice also exhibit low levels of serum glucose and insulin [26]. Despite the repetitive results of the studies regarding the CR-induced SIRT1 expression, Moynihan et al. [6] demonstrated that β-cell-specific SIRT1 transgenic mice exhibit insulin secretion ex vivo in pancreatic β-islet cells in response to high glucose. This result was confirmed by Bordone et al. [26] who also pointed out that insulin secretion was reduced in SIRT1 knockout mice and in pancreatic β-islet cell lines in which SIRT1 had been knocked down by RNA interference. UCP-2 is a mitochondrial inner membrane protein that regulates mitochondrial ATP synthesis. The effects mentioned above partially depend on the SIRT1-mediated inhibition of UCP-2 protein in pancreatic islet β-cells [6]. In accordance, SIRT1 knockout mice exhibit increased UCP-2 in β-cells along with low levels of serum insulin [26]. Higher pancreatic secretion of insulin and ATP were demonstrated in UCP-2 knockout mice [27]. According to these study results, SIRT1 might be a positive regulator rather than a suppressor of insulin in the postprandial fed state.

4.2.2 Major roles of SIRT1 in lipid metabolism In the fasting state, free fatty acids (FFA) are one of the main energy sources of our body. PPAR-α increases mitochondrial free fatty acid β oxidation via SIRT1-induced PGC1-α deactylation in the liver and striated and smooth muscles (Figs. 4.2 and 4.3) [3]. In addition, SIRT1 knockout mice exhibit hepatosteatosis due to accumulation of fats in the liver [28]. However, PPAR-γ, which is another important regulator of adipogenesis from the PPAR family, is inactivated by SIRT1. In this regard, inhibition of PPAR-γ causes diminished adipogenesis, therefore obesity, and increases free fatty acid release in white adipose tissue [29]. Liver kinase B1 (LKB1) is a primary upstream kinase of adenosine monophosphate-activated protein kinase (AMPK), a necessary element in cell metabolism that is required for maintaining energy homeostasis. SIRT1 stimulates LKB1 and AMPK that enhance fatty acid β-oxidation in the liver [30]. In contrast, SIRT1 inhibits SREBP 1 and this inhibition results in increased lipolysis in the liver [2]. Liver X receptor-alpha (LXR-α) and beta (LXR-β) are nuclear receptor proteins in humans which are encoded by the NR1H3 and NR1H2 genes (nuclear receptor subfamily 1, group H3 and H2), respectively. Another regulatory function of SIRT1 is cholesterol biosynthesis. LXR-α and LXR-β are two important nuclear proteins that maintain and control synthesis of cholesterol. These proteins stimulate the activation of ABCA-1 that results in high-density lipoprotein (HDL) synthesis. In this context, Li et al. [31] showed that deacetylation of LXR proteins might be associated with the pathogenesis of atherosclerosis, especially in chronic metabolic disorders including diabetes. Farnesoid X receptor (FXR), also known as bile acid receptor, another member of the nuclear receptor family, was found to be closely related to glucose and lipid metabolism. Deacetylation of FXR by SIRT1 represses the activation of this protein, and therefore the deleterious metabolic effects were inhibited [32]. In the following section, I will continue with the roles of sirtuins in terms of the pathogenesis of DM.

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4.2.3 Major roles of SIRT3 in glucose metabolism and lipid metabolism SIRT 3, a mitochondria localized sirtuin, also has beneficial effects on glucose metabolism by increasing insulin sensitivity and decreasing serum glucose. Hirschey et al. recently demonstrated that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. According to the results of this study, increased obesity, insulin resistance, hyperlipidemia, and steatohepatitis are prominent in mice lacking SIRT3 compared to wild-type mice [33]. The same group also identified a single nucleotide polymorphism which encodes a point mutation in the SIRT3 protein. As a result, mitochondrial protein acetylation is impaired and polymorphism of SIRT3 has been shown to be associated with metabolic syndrome [33]. SIRT3 is also involved in the pathogenesis of hepatic lipid metabolism by deacetylating and activating longchain acyl CoA dehydrogenase [34].

4.2.4 Major roles of SIRT4 in glucose and lipid metabolism Another important sirtuin that takes part in glucose metabolism is SIRT4. In contrast to SIRT1, both the localization and the function of SIRT4 are different. SIRT4 is located in mitochondria and transfers ADP-ribose to the substrates. One of the target enzymes of SIRT4 is glutamate dehydrogenase (GDH) which converts glutamate to α-ketoglutarate in the mitochondrion [35]. SIRT4 inhibits amino acid-induced insulin secretion via repression of GDH [36]. During fasting, SIRT4 is found to be inhibited in liver to induce glucogenesis from amino acids and fats and in the meantime inhibition of SIRT4 allows insulin secretion from β-cells. However, SIRT4 is activated and the reactions mentioned above are reversed in fed states [35]. In contrast, SIRT4 has opposite effects on free fatty acid metabolism and induction of SIRT4 might increase hepatosteatosis [37].

Part 2 4.3 Diabetes mellitus and sirtuins DM and its complications have been increasing worldwide during the last decades despite the emergence of diagnostic tools and therapeutic applications. Hence, DM has been accepted as an epidemic worldwide, except in Africa. The prevalence of DM is growing probably due to increased consumption of food and decreased physical activity. According to the ADA data, in 2017 an estimated 24.7 million people were diagnosed with DM in the United States [1]. Besides the increase in prevalence and incidence of DM, morbidity and mortality are also increasing in this population. In this regard, diabetes was both found to be closely associated with cardiovascular morbidity and mortality [38]. Traditional risk factors including hyperglycemia, insulin resistance hyperlipidemia, and so forth can be recognized and treated, however, today we can not achieve the desired results. The most important questions are what are the main struggles faced by clinicians and how can they deal with these problems in diabetic patients? wFirst of all, internists, endocrinologist, nephrologists, and cardiologists encounter difficulties regarding diabetic macro- and microvascular complications even in diabetic patients with strict blood glucose level and blood pressure control [39,40]. Additionally, the entire pathophysiology of this chronic metabolic disorder remains mysterious despite the advances in diagnostic and therapeutic strategies. To date, novel risk factors, including increased inflammatory cytokines due to low-grade persistent inflammation [41], adipose tissue hormones (adipokines), including obstatin, leptin, resistin, and the renin angiotensin aldosterone system are determined as the most detrimental factors attributed to this heightened CV morbidity and mortality in diabetic and obese patients [42 44]. There are some treatment options to overcome novel risk factors [45,46], however, it is illogical to treat these various entities separately. Therefore the main source of the detrimental pathogenetic mechanisms should be determined and new therapeutic molecules should be identified to accomplish the treatment of diabetes. In this context, three main questions should be answered. First, what are the main attributed pathways to overcome these undesirable pathophysiologic events in normal physiology? Second, why do these unwanted

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events occur despite defensive mechanisms? And third, are there any novel treatment modalities to treat diabetes and its complications? To answer these questions we have to highlight additional pathophysiological mechanisms. In the following part of this chapter we aimed to bring out these novel pathways regarding sirtuins.

4.3.1 The roles of sirtuins in the pathogenesis of diabetes mellitus As mentioned above, DM and its complications are worldwide problems that we should overcome. Insulin resistance is the prominent risk factor and as a result hyperinsulinemia occurs in the early stages of type 2 DM. Impaired glucose uptake and utilization follow this stage and hyperglycemia and hyperinsulinemia contribute to the pancreatic β-islet cell destruction in the following stages of diabetes [44,47]. At the end, hyperglycemia and related metabolic alterations, including advanced glycation end products (AGEs), polyol, hexosamine, and protein kinase C pathways, collectively contribute to the classical pathogenesis of DM. The main reasons for the undesirable complications of DM might be due to unknown pathophysiological mechanisms. In this context, as mentioned above, SIRT1 induces gluconeogenesis and inhibits glycolysis in liver during fasting via deacetylating FOXO1 and PGC1α. One of the most important question is how do these changes regarding gluconeogenesis and glycolysis occur? Rodgers et al. [48] demonstrated that hepatic PGC1α is upregulated and gluconeogenesis is increased, which can further increase hyperglycemia in diabetic mice. Yechoor et al. [49] showed that SIRT3 mRNA is downregulated in the muscle insulin receptor knockout mice. Another study demonstrated that SIRT3 induces the ketogenesis by activating acetyl Co-A synthetase in the mammalian cells [50]. Hence, one might expect that SIRT3 might play an important role in the increased ketogenesis observed in DM.

4.3.2 Sirtuins and diabetic kidney disease Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease and an independent risk factor for cardiovascular diseases [51]. All cell types of kidney including glomerular podocytes, mesangial, tubular, endothelial, and interstitial cells can be affected by hyperglycemic injury. Glomerular basement membrane thickening, mesangial expansion, and glomerular podocyte loss are the main features involved in DKD [52]. Regardless of the cause, glomerular sclerosis, interstitial fibrosis, and tubular atrophy are the final consequences. In normal physiology, SIRT1 is extensively expressed from proximal and distal tubular cells as well as podocytes [53]. SIRT1 is also found to be involved in water and sodium physiology. In this regard, SIRT1 decreases the transcription of alpha subunits of eNaC channels and therefore decreases sodium and water reabsorption in the distal tubules [54]. Another system which has very active players in the progression of DKD is the renin angiotensin aldosterone system (RAAS). Among them, angiotensin II (ANG II), is the most studied molecule and plays a key role in the development and progression of diabetic nephropathy. Angiotensin 1 7, a metabolic by-product ameliorates the effects of ANG II. Previous studies showed that ANG 1 7 can activate ERK1/2 and cAMP/protein kinase A-dependent pathways in glomerular mesangial and proximal tubular cells (PTCs) [55,56] and was found to be protective in terms of DKD in Zucker diabetic fatty rats [57]. Recently, Mori et al. reported that ANG 1 7 ameliorates DKD and reduces tubulointerstitial fibrosis (TIF) and inflammation in the kidney via the SIRT1-FOXO1 pathway [58]. In addition, decreased expression of SIRT1 is found to be closely associated with increased transcription of angiotensin II type 1 receptor (AT1R) in podocytes [59]. However, increased expression of SIRT1 downregulates AT1R in vascular smooth muscle cells [60]. Collectively, in the pathologic conditions, SIRT1 is found to be protective against various injury to tubules and glomerulus [61 63]. Like SIRT1, SIRT3 also has a role in the kidney [64]. As we know very well, the functions of SIRT3 are closely related to mitochondrial energy homeostasis and antioxidant defense in proximal and distal tubules which have a very high number of mitochondria [65,66] In this regard, Perico et al. [67] demonstrated that SIRT3 can mediate microtubule network-dependent trafficking of functional mitochondria between renal tubular epithelial cells. This process preserves the proper cellular bioenergetic profile and antioxidant defense in PTCs. 4.3.2.1 What are the effects of SIRT6 and SIRT7 in kidney? SIRT6 has an important role in maintaining glomerular selectivity to plasma proteins. We got these data from the studies done by Huang et al. [68]. Their group clearly showed that deletion of SIRT6 in mice resulted in podocyte injury and decreased slit diaphragm proteins foot process effacement and eventually increased proteinuria. They also concluded that Sirt6 deletion can accelerate renal hypertrophy.

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Although there have been not many studies on SIRT7 in the literature, a few studies have shown that SIRT7 also has an effect on renal functions. The studies regarding SIRT7 demonstrated that this subtype has an ability to deacetylate KCC4 transport protein which is expressed at the basolateral membrane of the α-intercalated cells. This channel can transport potassium with chloride and also has an important role in maintaining pH in the collecting ducts especially during metabolic acidosis [69]. Hence it is wise to consider that SIRT7 can control renal acid base and electrolyte balance. Additionally Miyasato and colleagues [70] recently found that Sirt7 knockout mice were resistant to cisplatin-induced acute kidney injury (AKI). Furthermore, they showed that loss of SIRT7 decreases the expression of tumor necrosis factor-α (TNF-α) by regulating the nuclear expression of the transcription factor nuclear factor kappa B. According to the results of this study, SIRT7 might play an important role in cisplatin-induced AKI and suggest the possibility of SIRT7 as a novel therapeutic target for cisplatin-induced nephrotoxicity. What are the protective effects of SIRT1 in different parts of the kidney? In the following part, I would like to answer this question using data from literature [71].

4.3.3 The roles of SIRT1 in the glomerulus in diabetic kidney disease 4.3.3.1 Results from animal models of diabetes mellitus In the literature, there is an accumulation of data regarding the pathogenetic role of decreased expression of SIRT1 in the DKD (Table 4.2). In this context, Koya and colleagues clearly demonstrated that SIRT1 activity is diminished in type 2 diabetic rats, and these changes were ameliorated by CR [72 75]. Recent studies revealed that mice with 80% knockdown of renal SIRT1 expression have normal glomerular function under the basal condition. However, nephron- and tubulotoxic agents easily compromise the glomerular and tubular functions and lead to end-stage renal failure, especially in patients with DKD. Results obtained from the mice model of diabetes have shown the dark side of sirtuin-related pathways in DKD. For instance, data obtained from diabetic mice model of doxorubicin-induced nephropathy revealed that proteinuria, glomerulosclerosis, mitochondrial injury, and impaired autophagy of damaged mitochondria can occur, especially in SIRT1 deficiency [76]. Replacement of SIRT1 in the early phase of doxorubicin-induced nephropathy prevented the progression of glomerular injury and reduced the accumulation of dysmorphic mitochondria in podocytes but TABLE 4.2 The effects of SIRT1 and associated pathways in cell culture and animal models of diabetes mellitus. Tissue/cell type

Animal model/cell culture

Target protein/ enzyme/ pathway

Definition of SIRT1 effect

References

Glomerulus STZ-diabetic mice

Histone H3, Activation of SIRT1 by resveratrol treatment involves change in MAP kinase p38, phosphorylation of histone H3, expression of Sir-2, p53, and p38 in p53 DKD.

[75]

Glomerulus Diabetic WFR

NF-kβ

Increased inflammation through increased NF-kβ secondary to decreased SIRT1.

[73]

Proteinuria, glomerulosclerosis, mitochondrial injury and impaired autophagy of damaged mitochondria can occur especially in SIRT1 deficiency

[76]

Glomerulus Doxurubicin-induced nephropathy in diabetic rat Tubulus

Cell culture

Foxo3a

SIRT1 protects against oxidative stress-induced proximal tubule cell apoptosis by acetylation of FOXO3a

[77]

Tubulus

Cell culture

Foxo3a, Bnip3

SIRT1 can induce Bnip3 associated mitochondrial autophagy through FOXO3 pathway.

[72]

Mesangial cells

Cell culture

AMPK, mTOR

SIRT 1 augments AMPK and inhibits mTOR pathways

[78]

Mesangial cells

Cell culture

Smad7

Inhibition of Smad7 by SIRT1 decreases TGFβ-induced apoptosis

[79]

Mesangial cells

Cell culture

p53

Inhibition of p53 by SIRT1 decreases oxidative stress-induced apoptosis

[80]

Podocytes

Cell culture

Foxo4

Inhibition of Foxo4 by SIRT1 decreases AGE-induced apoptosis

[81]

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did not reverse the progression of glomerulosclerosis and proteinuria. Chuang et al. [76] also demonstrated that SIRT1 knockdown mice with DM developed more albuminuria and mitochondrial dysfunction compared with diabetic mice without SIRT1 knockdown. Recently, Hasegawa et al. [82] showed the close relation between PTC and podocytes. In this regard, they demonstrated that metabolic effects mediated by glucose in PTC affected podocytes and eventually increased albuminuria. In detail, high glucose in PTC decreased SIRT1 levels and thereafter decreased NMN levels. This can cause a vicious cycle because decreased NMN levels further decrease SIRT1 in podocytes. The decrease in SIRT1 in podocytes induces many reactions. Among these reactions, deacetylation of H3 and H4 histones via SIRT1 inducing DNA methylation of the CpG islands around the claudin-1 gene is the most important step. This reaction is activated by DNA methyl transferase (DNMT). After DNA methylation, the claudin-1 gene is activated and increases claudin-1 expression which interacts with the β-catenin/snail pathway. Eventually, activation of the latter pathway induces decreased podocin and snaptopodin protein and at the end of these alterations foot process effacement and proteinuria occurs. The importance of this study is that researchers clearly showed proximal tubule podocyte interaction occurred before well-known mechanisms including tubuloglomerular feedback and glomerular tubular balance. In this context, altogether the results mentioned above clearly show the beneficial effects of SIRT1 in terms of homeostatic maintenance of podocytes under stress conditions. 4.3.3.2 Results from cell culture studies Cell culture is an indispensable method for understanding the exact pathological pathways. In this regard, the exact roles of SIRT1 in the glomerular cells has been clarified by the results of cultured mesangial cells and podocytes (Table 4.2). In cultured mesangial cells, oxidative stress-related apoptosis was suppressed by SIRT1 via deacetylation of p53 [83] and TGF β-related apoptosis was also found to be inhibited by SIRT1 via deacetylation of Smad7 [79]. Recently, Zhua et al. demonstrated that NAD concentration and SIRT 1 activity of the mesangial cell were reduced when these cells were exposed to high glucose [84]. Additionally, advanced glycated end-product was found to be an inducer of apoptosis of podocytes via increasing Foxo4. Activation of SIRT1 deacetylates and inhibits Foxo4, therefore protecting podocytes from AGE-induced apoptotic cell death in diabetes [81]. In this regard, SIRT1 may act as a guardian against apoptotic cell death or hypertrophy of the mesangial cells and podocytes in DKD. Hence, it might be wise to consider the therapeutic approaches activating SIRT1 in the early phase of DKD before the onset of sclerotic lesions.

4.3.4 The roles of SIRT1 in the tubulointerstitium in diabetic kidney disease Oxidative stress is enormously high in the renal medulla, especially due to hypoxia and hypertonicity. In recent studies, SIRT1 was excessively detected in the interstitial cells of the renal medulla, when compared with the renal cortex. It is wise to hypothesize that excessive SIRT1 activity can fight with excessive oxidative stress in this part of the kidney. Some in vitro studies support this theory. In this regard, He et al. [77] showed the beneficial effects of SIRT1 activation in terms of oxidative stress in the interstitium of renal medulla. 4.3.4.1 Results from animal models of diabetes mellitus In this part of the review, we would like to describe the role of SIRT1 in the pathogenesis of diabetic TIF. In the progression of DKD, tubular atrophy and interstitial fibrosis eventually occur. Hence, one of the aims of the therapy of DKD should include the prevention of diabetic TIF. Previous studies demonstrated that SIRT1 can act as an antifibrotic molecule through Smad3 and TGF-β pathways which were mentioned above as common players in the pathogenesis of DKD [77,85]. Another system which has very active players in the progression of DKD is the RAAS. Among them, angiotensin II (ANG II), is the most studied molecule and plays a key role in the development and progression of diabetic nephropathy. Angiotensin 1 7 is a metabolic by-product that ameliorates the effects of ANG II. Previous studies showed that ANG 1 7 can activate ERK1/2 and cAMP/protein kinase A-dependent pathways in glomerular mesangial and PTCs [55,56] and was found to be protective in terms of DKD in Zucker diabetic fatty rats [57]. Recently, Mori et al. [58] reported that ANG 1 7 ameliorates DKD and reduces TIF and inflammation in the kidney via the SIRT1-FOXO1 pathway. In light of these data, we should consider renin angiotensin aldosterone system blockers in the treatment of DKD.

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4.3.4.2 Results from cell culture studies From the results of cell culture studies, we have learned that increased activity of renal tubular SIRT1 might attenuate diabetic albuminuria by epigenetically suppressing claudin-1 overexpression in podocytes [82]. Additionally, direct renoprotective effects of SIRT1 overexpression in proximal tubular epithelial cells were also shown. In this regard, SIRT1 was shown to be a defender against renal tubular cell apoptosis by inducing catalase through the deacetylation of FOXO3 in vivo [62]. Furthermore, Hasegawa et al. [61] also demonstrated that renal proximal tubular cell-specific SIRT1 transgenic mice were resistant against cisplatin-induced apoptosis and tubulopathy by enhancing medium-chain acyl-CoA dehydrogenase, which is a rate-limiting enzyme of mitochondrial β oxidation. In another study, SIRT1 activated PGC-1α and activation of this enzyme is found to induce PTC repair via activating mitochondrial functions [86] and oxidative phosphorylation [87]. Kim and collegues also reported that the activation of SIRT1 by resveratrol diminished cisplatin-induced proximal tubular cell apoptosis through the deacetylation of p53 [80]. In the following section, I would like to mention the novel pathways and sirtuins involved in the pathogenesis of DM and DKD. Among them, I will start with the relation between autophagy and SIRT1. Thereafter, I will continue with AMPK pathway, which is the most important pathway of energy sensation in diabetes. Eventually I will get onto the mTOR pathway and discuss the relationship with sirtuins regarding diabetes and DKD.

4.3.5 The roles of SIRT1 and autophagy in diabetes mellitus and diabetic kidney disease Autophagy is a tightly regulated degradation process that involve many complicated reactions and ultimately ends with lysosomal degradation of nonuseful proteins and organelles [88]. This lysosomal degradation pathway is considered essential for homeostasis, development, differentiation, and survival of the organisms [89]. The role of autophagy in metabolic disorders is currently under intense investigation and it has been revealed that autophagy has a protective role in pancreatic β-cells against various deleterious stresses [90,91]. Physiological situations, such as nutrient starvation, antiaging, cell growth control, tumor suppression, and inherent immunity, and pathological insults, such as hypoxia, energy depletion, and drugs (mTOR inhibitors such as sirolimus), may induce autophagy. Many nutrient-sensing pathways, including AMPK, are involved in autophagy, especially in starvation [92]. Alteration of these pathways under hyperglycemic conditions may impair the autophagic response stimulated by miscellaneous stresses. One of the important studies in the literature showed that autophagy-deficient mice had pancreatic β-cell degeneration and impaired glucose tolerance with reduced insulin secretion. The results of this study clearly suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of pancreatic β-cells [90]. Among other, especially SIRT1 was found to be one of the positive regulators of autophagy. In this regard, the decrease in SIRT1 expression observed in the diabetic kidney may lead to the dysregulation of autophagy. Recent studies clearly showed that in proximal tubule cells SIRT1 decreased albuminuria in DM through maintaining NMN concentrations around glomeruli and controlling podocyte function [82,93]. In addition, SIRT1 was found to be closely associated with the survival of cells in an affected kidney by modulating their responses to various stress stimuli, taking part in arterial blood pressure control, protecting against cellular apoptosis in renal tubules by catalase induction, and triggering autophagy. The phosphorylation of AMPK positively regulated SIRT1 [94] and thereafter autophagy [95], was also decreased in the DKD. Hence, activation of SIRT1 may become a novel target in the treatment of diabetic nephropathy [44,96]. The role of autophagy in diabetes was also shown in mice deficient of an autophagy related protein named sequestosome 1 (Sqstm1/p62). The Sqstm1/p62-deficient mice exhibit severe hyperglycemia and eventually diabetes [97]. Kitada et al. [73] demonstrated accumulation of Sqstm1/p62 in the PTCs of diabetic Wistar Fatty Rats (WFRs) kidneys, which means that an impairment of the autophagy system occurred in DKD. They also showed that restoration of SIRT1 expression and AMPK activation via CR improved the function of the autophagy system, which resulted in normalization of mitochondrial morphological changes and Sqstm1/p62 accumulation in the kidney of WFRs. According to the results of this study, the existence of autophagosomes in the PTCs of the kidney in WFRs treated with CR was consistent with the existence of normal morphological mitochondria. What effects can be seen in podocytes regarding the relation between sirtuins and autophagy? In this context, Hartleben et al. [98] gave careful attention to podocytes as sites of “an unusually high level of constitutive autophagy.” They advocated that autophagic pathways can be activated against podocytopathy to diminish proteinuria in mice. Inhibition of autophagy by lacking one of the autophagosomal genes, Atg5, was found to be closely associated with proteinuria. In this regard, autophagy might play a protective role against podocyte injury.

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4.3.6 The roles of SIRT1 and adenosine monophosphate-activated protein kinase pathway in diabetic kidney disease In normal physiology, AMPK may activate the phosphorylation of acetyl-CoA-carboxylase, which results in decreased lipogenesis and enhanced lipolysis. So, AMPK is activated under energy-depleted conditions and is likely to be suppressed in diabetic nephropathy. Hence, suppression in AMPK activity in DKD may trigger lipogenesis and thereafter lipotoxicity-associated kidney injury may occur in susceptible kidneys. Decreased phosphorylation of AMPK which in turn inactivated AMPK was reported in the glomerulus and tubules in animal models of type 1 and type 2 DM [78,99,100]. In addition, glomerular and tubular lesions are reversed when AMPK activators such as 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside (AICAR) and metformin are administered in streptozosin (STZ)-induced DKD [101]. AMPK activity and plasma adiponectin levels were also found to be significantly lower in db/db diabetic mice when compared with control mice [78]. Additionally, Cammisotto et al. [102] demonstrated that adiponectin might stimulate AMPK activity in podocytes, mesangial cells, and glomerular endothelial cells. Sharma and colleagues also showed decreased podocyte AMPK activity along with albuminuria in mice with DKD [103]. In a study with diabetic akita mice the results clearly demonstrated that activation of AMPK by AICAR significantly improved kidney injury including albuminuria [104]. Many nutrient-sensing pathways including AMPK, mTOR, and sirtuins may also be involved in autophagy especially in starvation [92]. Alteration of these pathways under hyperglycemic conditions may impair the autophagic response stimulated by miscellaneous stresses. Recent research demonstrated that positive effects of resveratrol on glucose metabolism and insulin sensitivity are closely associated with AMPK subunit α activation of this agent rather than the stimulatory effect on SIRT1.

4.3.7 The roles of SIRT1 and mTOR pathway in diabetic kidney disease The third and the last pathway is the mammalian target of rapamycin (mTOR) pathway, which is a serine/ threonine kinase that regulates both cell growth and cell cycle progression through the phosphatidyl 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway. Phosphatidylinositol-3 kinase (PI3K) converts the lipid phosphatidylinositol biphosphate (PIP2) into phosphatidylinositol triphosphate (PIP3), which localizes protein kinase B (Akt) to the membrane. mTOR phosphorylates both ribosomal protein S6 kinases (p70S6K1) and eukaryotic initiation factor 4E-binding proteins (4E-BP1) via independent pathways, resulting in activation of S6K1 and inactivation of 4E-BP1 [105]. Increased p70S6K1 and eIF4E act independently to promote cell growth and cell-cycle progression. Therefore mTOR plays a critical role in transducing proliferative signals mediated through the PI3K/Akt signaling pathway, principally by activating downstream protein kinases that are required for both ribosomal biosynthesis and translation of key mRNAs of proteins required for progression of G1 to S phase [106]. This means that mTOR regulates both cell cycle progression and cell growth. DKD is found to be closely associated with dysregulated cell growth and cell-cycle progression, exaggerated proliferative responses, and increased apoptosis in mesangial and PTCs [107]. In this regard, researchers hypothesized that there might be a link between DKD and the mTOR pathway. Thereafter, many studies showed that there has been a significant early activation of mTOR-dependent pathways that induce podocyte injury, mesangial expansion, glomerular basement membrane thickening, glomerular hypertrophy, and tubular dearrangements in animal models of diabetes [108,109]. One of the attributed mechanisms of the mTOR pathway that leads to interstitial fibrosis is the increased expression of profibrotic cytokines including TGF-β and connective tissue growth factor in DKD [108]. Moreover, administration of rapamycin, an inhibitor of mTOR, to diabetic mice reversed these pathological depositions mentioned above and diminished proteinuria in DKD [110]. Recently, mTOR Complex 1 (mTORC1) activity was found to be essential to maintain podocyte homeostasis, however hyperactivation of mTORC1 may cause glomerular lesions commonly seen in DKD [109,110]. Additionally, podocyte-specific repression of raptor, a component of mTORC1 that inhibits mTOR pathway have demonstrated beneficial effects on mesangial matrix expansion, glomerulosclerosis and eventually proteinuria diabetic mice [111]. In this regard, researchers decided to try rapamycin, an mTOR inhibitor, as a treatment agent to attenuate glomerular and mesangial lesions commonly seen in DKD. Rapamycin was found to be beneficial in terms of mesangial expansion and glomerular basement membrane thickness in rats with DKD [112]. The first reports are promising and in the near future rapamycin might be a therapeutic option in DKD.

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4.4 Hypertension and sirtuins Most diabetic patients have hypertension. The pathogeneses of both diabetes and hypertension have similarities. So we can say that diabetes and hypertension are twins. As mentioned, a close relation has been revealed between sirtuins and diabetes. The main question is whether or not sirtuins are also associated with blood pressure control. In this context, recent studies have attempted to find a link between hypertension (HT) and SIRT1. The plausible explanation regarding the relation between HT and SIRT1 came from the study done by Miyazaki and colleagues. Their group have demonstrated that SIRT1 overexpression reduces the expression of the angiotensin-1 (AT1) receptor of angiotensin II (AT1R) in vivo, while nicotinamide, a SIRT1 inhibitor, increases this expression [60]. Additionally, they reported that the SIRT 1 activator, resveratrol, negatively regulated AT1R expression via reducing specificity protein 1 (Sp1) in cultured vascular smooth muscle cells and suppressed AT1R in the aorta and reversed HT in mice [60]. SIRT1 also promotes vasodilation by deacetylating nitric oxide (NO) synthetase in the endothelial cells [8]. In a recent study, endothelial NO was found to be crucial in SIRT1associated CR-induced cardioprotection and blood pressure control [113]. The role of aldosterone in the pathogenesis of HT has been well-known for years. Renal tubular sodium reabsorption via epithelial sodium channels (eNac) in the apical membranes of collecting duct principal cells is the primary target of aldosterone. Increased levels of serum aldosterone induce eNac expression of collecting tubular cells which contribute HT [114]. In this regard, Zhang et al. [54] demonstrated that SIRT1 can diminish the expression of eNac alpha subunits via hypermethylation of a histone H3K79 methyltransferase (also named disruptor of telomeric silencing 1). This effect was found to be independent of both deacetylase activity of SIRT1 and the mineralocorticoid signaling pathway. In addition to the experimental studies investigating the relation between SIRT1 and HT, a recent clinical study revealed that SIRT1 gene polymorphism (rs2273773) was significantly associated with ambulatory blood pressure levels in Han Chinese patients with hypertension [115]. This experimental and clinical evidence collectively demonstrated that in the near future SIRT1 might be a candidate target molecule in the treatment of HT.

4.5 Novel treatment options in diabetes mellitus and diabetic kidney disease Because much research has demonstrated various positive effects of sirtuins on glucose, lipid, and carbohydrate metabolism, scientists also attempted to translate these data into pharmaceuticals. In this context, resveratol, is a plant polyphenol, was the first molecule introduced as a SIRT1 inducer [116]. Much experimental data showed the beneficial effects of resveratrol in animals. Yamazaki et al. [117] showed that treatment of mice with nonalcoholic fatty liver disease with a synthetic SIRT1 activator, SRT1720, might decrease the serum lipid levels, oxidative stress, and inflammation. In addition, Feig et al. [118] showed that activation of SIRT1 by SRT1720 protected the organism from diet-induced insulin resistance and obesity via increasing oxidation of fat in liver, adipose tissue, and skeletal muscle. Also SRT3025 which is another SIRT1 activator is found to improve glycemic control with greatly reduced islet α-cell mass and lower plasma glucagon concentrations [119]. However, in the following years, it was demonstrated that positive effects of resveratol on glucose metabolism and insulin sensitivity are closely associated with AMPK subunit α activation of this agent rather than the stimulatory effect on SIRT1. In this regard, Um et al. [120], for the first time, demonstrated that resveratrol could not improve glucose tolerance and insulin sensitivity in AMPK α knockout mice. In humans, Timmers et al. [121] also showed the beneficial effects of resveratrol in obese patients in terms of lowering systolic blood pressure, serum lipid and glucose levels, and inflammation parameters. Beside resveratrol and SIRT1 synthetic activators, NMN, a NAD1 intermediate, is another molecule that has been demonstrated to have beneficial effects by improving glucose and lipid levels in aging-induced diabetes [122]. Additionally, niacin, vitamin B3, is also an important intermediate for the biosynthesis of NAD1 that can used for the activation of SIRT1 [123]. Another molecule that has been tried to increase sirtuin activity is curcumin. Curcumin is a polyphenol extracted from the rhizome of Curcuma longa. In a study done by Ortega-Dominguez et al. [124] curcumin was found to prevent the increase of mitochondrial fission 1 protein, and the decrease of NAD-dependent deacetylase SIRT3 in cisplatin-induced AKI. They also showed that the mitophagy associated proteins parkin and phosphatase and tensin homolog (PTEN)-induced putative kinase protein 1 (PINK1) were found to be decreased. According to the results of this study, curcumin might ameliorate the tubular damage and renal complications by preserving redox balance and mitochondrial bioenergetics via SIRT3 activation.

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There is also SIRT3 activators. One of them is honokiol which is an active compound of Magnolia officinalis. M. officinalis is a well-known antiinflammatory and antioxidant herbal medicine which has been widely used in China [125]. In this context, honokial is found to decrease insulin resistance by targeting PTP1B and decrease glucose levels in type 2 diabetic mice [126]. Recently, Li et al. [127] demonstrated that honokiol might be beneficial via SIRT3-induced deacetylation of a transcription factor named KLF15 and thereafter decreases angiotensin-2 induced fibrosis in hypertension-induced chronic kidney disease. In addition, another study also showed that activation of SIRT3 via honokiol treatment might have beneficial effects on unilateral ureteral obstructioninduced renal fibrosis through SIRT3-dependent regulation of mitochondrial functions and the NF-κB/TGF-β1/ Smad signaling pathway [128]. Slybin (also known as silibinin) is a major active constituent of silymarin, a standardized extract of the milk thistle seeds. Wang et al. [129] studied the effects of SIRT1 and autophagy in streptozotocin-induced DM and assessed the protective mechanism of silibinin in terms of relationship between autophagy and SIRT1. They concluded that systemic administration of silibinin reversed streptozotocin-induced downregulation of SIRT1 expression and SIRT1 might play a role in regulating the physiological level of autophagy. Hence, silibinin may reverse hyperglycemia and repair damaged pancreatic β-cells via the promotion of SIRT1 expression and recovering autophagy. Stanniocalcin-1 has antioxidant and antiapoptotic activities, which play a role in kidney protection, including DKD. A recent study suggested that stanniocalcin-1 ameliorates oxidative stress and cell apoptosis in the kidneys of db/db mice and high glucose-treated cells by inhibiting Bnip3 expression through AMPK/Sirt3 pathway activation [130]. Thus the authors concluded that stanniocalcin-1 might become a potential drug for the treatment of DKD patients. 5-Aminoimidazole-4-carboximide riboside (AICAR) is also tested in diabetic mice regarding the energy sensing pathway including AMPK [131]. Data are limited regarding the relation between SIRT and AICAR in DKD, however, in a mice model of AKI, Morigi et al. [132] showed that treatment with the AMPK agonist AICAR or the antioxidant agent acetyl-L-carnitine (ALCAR) restored SIRT3 expression and activity, improved renal function, and decreased tubular injury in wild-type animals, but had no effect in Sirt32/2 mice. Moreover, they demonstrated that Sirt3-deficient mice given cisplatin experienced more severe AKI than wild-type animals and died, and neither AICAR nor ALCAR treatment prevented death in Sirt32/2 AKI mice. However, there are also conflicting results regarding the effects of novel synthetic SIRT1 activators on glucose and lipid metabolism. SIRT1 activators might induce insulin secretion and sensitivity, and reduce adipogenesis, but also induce gluconeogenesis in the liver, that may worsen hyperglycemia in diabetic. Additionally, nowadays neither resveratrol nor the other agents mentioned above are approved by Food and Drug Administration (FDA) and European Medicines Agency (EMA). So how can clinicians use these data regarding sirtuins in their daily practice? In this context, CR provides a desirable metabolic profile and improvement of the mitochondrial functions in humans via activating several genes including SIRT1 [133]. Hence, CR with increased physical activity should be encouraged, especially in obese diabetic patients. The second option in our hand is metformin, which is a commonly used antidiabetic agent. Metformin decreases insulin resistance and hyperglycemia via inhibiting gluconeogenesis and hepatic glucose output and activation of free fatty acid oxidation in skeletal muscle [134]. Some of these beneficial effects of metformin were attributed to SIRT1 activation via the AMPK pathway [135]. Hence, among the mentioned treatment options, except for metformin and CR, none of them as been used in a large series of clinical trials. To date, therefore, it is wise to use only metformin along with CR in obese type 2 diabetic patients to get beneficial metabolic effects. Lastly, in this section, I would like to mention mesenchymal stromal cell (MSC) transplantation which is found to restore kidney impairment and provide renoprotection in a mice model of cisplatin-induced kidney injury. In this context, a recent study revealed that MSCs-transplanted mice with cisplatin-induced AKI showed improved mitochondrial biogenesis in proximal tubules by enhancing PGC1α expression, NAD1 biosynthesis, and SIRT3 activity. However, in MSC-treated SIRT3-deficient mice, these beneficial effects could not be seen. Hence, the authors concluded that MSCs transplantation into mice with AKI might stimulate renal tubular cell growth and enhance mitochondrial function via SIRT3 [67].

4.6 Conclusion and future perspectives Inflammation, oxidative stress, apoptosis, autophagy, and various endogenous pathways are involved in the pathogenesis and progression of DM. Sirtuins are key molecules that might exert renoprotective effects by modulating these pathways. Hence, activation of sirtuins, especially SIRT1 and SIRT3, might result in various beneficial

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metabolic effects, making these proteins a target for new drugs, especially for the future treatment of DM and DKD. However, there are many missing pieces in the puzzle and further experiments and clinical studies are needed to highlight the exact roles of sirtuins in DM.

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C H A P T E R

5 Sirtuins and mitochondrial dysfunction Jian-Li He1,2, Tian-Shi Wang1,2 and Yi-Ping Wang3,4 1

Department of Biochemistry and Molecular Cell Biology, Shanghai Key Laboratory for Tumor Microenvironment and Inflammation, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China 2State Key Laboratory of Oncogenes and Related Genes, Renji Hospital Affiliated, Shanghai Jiao Tong University School of Medicine, Shanghai, P. R. China 3Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Key Laboratory of Breast Cancer in Shanghai, Cancer Institute, and Shanghai Key Laboratory of Medical Epigenetics, Shanghai Medical College, Fudan University, Shanghai, P. R. China 4Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, P. R. China O U T L I N E 5.1 Sirtuins are nutrient sensors

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5.2 Sirtuins and mitochondrial biogenesis

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5.3 Sirtuins and mitochondrial metabolism

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5.4.4 Neurodegeneration 5.4.5 Aging 5.4.6 Tumorigenesis

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5.5 Feasible clinical targets: posttranslational modifications of sirtuins regulate mitochondrial function 85

5.4 Sirtuins and mitochondrial dysfunction in human diseases 82 5.4.1 Diabetes and obesity 83 5.4.2 Cardiovascular diseases 84 5.4.3 Renal disease 84

Acknowledgments

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5.1 Sirtuins are nutrient sensors Sirtuins are nicotinamide adenine dinucleotide (NAD1)-dependent deacylases. They are evolutionarily conserved from yeast to mammals. The importance of sirtuins is largely based on their ability to sense and respond to nutrients, and to maintain metabolic homeostasis [1]. Sirtuins were initially discovered in the budding yeast (Saccharomyces cerevisiae) contributing to the replicative life span [2]. The mammalian genome encodes seven different paralogues of sirtuins. Mammalian sirtuins show diverse spatial distribution in cells. SIRT1, SIRT6, and SIRT7 localize primarily in the nucleus, while SIRT2 functions in the cytoplasm. SIRT3, SIRT4, and SIRT5 reside within mitochondria [3]. Mitochondria are metabolic organelles serving as cellular powerhouses. Sirtuins have been shown to regulate mitochondrial function with their diverse deacylase activities. Among mitochondrial sirtuins, SIRT3 is the major deacetylase with a broad range of substrates. SIRT3 modulates oxidative phosphorylation, fatty acid oxidation (FAO), reactive oxygen species (ROS) response, and even genomic stability [4 7]. SIRT4 is one of the least wellcharacterized members of the sirtuin family, with deacetylase, lipoamidase, and ADP-ribosylase activities, and which controls multiple metabolic processes, including glutamate and leucine metabolism, FAO, pyruvate oxidation, and mitochondrial dynamics [8 11]. SIRT5 regulates metabolic enzymes involved in the tricarboxylic acid cycle, FAO, and adenosine triphosphate (ATP) synthesis by modulating the acylation status of lysine residues,

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FIGURE 5.1 Sirtuins are nutrient sensors.

such as glutarylation, succinylation, and malonylation [12 14]. How cells modulate the specificity of sirtuins toward different acylation modifications remains poorly understood. The activity of sirtuins is tightly controlled by nutrients, linking the nutrient supply with the acylation status of sirtuins’ substrate. Therefore, sirtuins serve as nutrients sensors within cells. Similarly, many nutrientresponsive signaling proteins, such as insulin-like growth factor 1 (IGF1), the target of rapamycin (mTOR), and AMP-activated protein kinase (AMPK), sense nutrients and regulate cellular metabolic activity correspondingly [15]. These nutrient-sensing pathways often collaborate to sense specific nutrient signals and modulate cellular metabolism and maintain mitochondrial homeostasis [16,17] (Fig. 5.1). Sirtuins balance anabolism and catabolism in response to the sufficiency and scarcity of nutrients such as glucose, lipids, and glutamine. The dysfunction of sirtuins is linked to a deficient response to metabolic stress. Podocyte injury, a contributor to diabetic nephropathy, is at least in part caused by high glucose-induced mitochondria dysfunction. Interestingly, SIRT6 and AMPK were reported to transduce the stress signal of high glucose. SIRT6 was downregulated by high glucose and potentially contributed to podocyte apoptosis [18]. Sirtuins are also involved in the pathogenesis of obesity. Nutrient-rich diet decreased SIRT1 activity to induce SIRT3 hyperacetylation in a model of diet-induced obesity [19]. On the contrary, calorie restriction upregulated the expression of most sirtuins to reprogram mitochondrial metabolism [6,20]. Unlike other sirtuins, SIRT4 was induced in a fed state and is involved in mammalian target of rapamycin complex 1 (mTORC1) activation to trigger anabolic signaling [21]. In addition, sirtuins modulate hepatic cellular metabolism in response to different lipid species. Low-level palmitate treatment showed a beneficial effect by enhancing SIRT3 activity and consequent upregulation of mitochondrial metabolism [22]. Lipid droplet-derived monounsaturated fatty acids were sensed by SIRT1, which further promoted peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α)/peroxisome proliferator-activated receptor α (PPARα)-dependent mitochondrial biogenesis and oxidative metabolism [23]. Interestingly, SIRT7-deficient mice were resistant to high-fat diet-induced fatty liver, obesity, and glucose intolerance [24]. Amino acids are also regulators of sirtuin activity. Glutamine is a major source of carbon and nitrogen for proliferating cells. Amino acid sensor mTORC1 downregulated the activity of SIRT4 to enhance glutamine metabolism during transformation and cancer development [25].

5.2 Sirtuins and mitochondrial biogenesis Mitochondria are responsible for not only energy production, but also biomass production. Mitochondria are believed to have an endosymbiotic origin and have maintained a genome outside the nucleus during evolution. Mitochondria mass is regulated on at least three different levels: (1) biomass production: new mitochondria are built by biomolecules encoded by both nuclear and mitochondrial genome (nDNA and mtDNA) [26]; (2) mitochondria quality control: cellular stress, such as ROS and depolarization agents [27,28], causes damage to mitochondria, necessitating a process termed mitophagy to remove defective mitochondria [29]; and (3) mitochondria dynamics: mitochondria form dynamic tubular networks by fission and fusion in response to cellular metabolic requirements [30]. Sirtuins are involved in regulating the transcription of genes encoding mitochondrial proteins. Sirtuins bind to various transcription regulators of mitobiogenesis to modulate target gene expression. For example, SIRT1 mRNA expression was increased by calorie restriction [31], which further activated PGC-1α to enhance mitochondrial biogenesis [32,33]. SIRT1 also bound to transcription factor v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) and enhanced mitobiogenesis in immune cells to maintain energy production in monocytes [34]. Interestingly, SIRT1 and PGC-1α were suggested to regulate mitobiogenesis through directly

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localizing into mitochondria. SIRT1 and PGC-1α formed a complex with the mitochondrial transcription factor A (TFAM) [35], which potentially regulated mtDNA replication and transcription (Fig. 5.2A). Activating transcription factor 2 (ATF2) is an important regulator of mitochondrial function. SIRT6-activated PGC-1α gene expression by recruiting phosphorylated ATF2 to PGC1-α and increasing General control non-derepressible 5 (GCN5)dependent PGC-1α acetylation [36,37] (Fig. 5.2A). Another nuclear sirtuin, SIRT7, physically interacted with nuclear respiratory factor 1 (NRF1) and inhibited the transcription of genes encoding mitochondrial ribosomal proteins, resulting in the reversal of stem cell aging [38]. SIRT7 was also shown to regulate the expression of nuclear-encoded mitochondrial proteins by deacetylating transcription factor GA binding protein β1 (GABPβ1) [39] (Fig. 5.2A). In hepatic cells, SIRT7 was reported to be modified by protein arginine methylation, linking glucose availability to the transcription of nuclear-encoded mitoribosomal genes [40]. Sirtuins are regulators of protein translation in mitochondria. The proteins encoded by mtDNA are synthesized by mitochondrial ribosomes. Mitoribosomal proteins are encoded in the nuclear genome, synthesized in the cytoplasm, and transported into mitochondria. Together with mitochondrial rRNAs and tRNAs, which are transcribed from mtDNA, mitoribosomal proteins assemble into functional ribosomes to synthesize mtDNA-encoded proteins [41]. SIRT3 was shown to interact with and deacetylate mitochondrial ribosomal protein L10 (MRPL10). In mouse models and cultured cell lines, MRPL10 hyperacetylation enhanced the translational efficiency of mitoribosomes [42]. Other than the mitoribosome, mitochondrial tRNA is potentially regulated by SIRT3 as well. tRNA methylation is an indispensable process for tRNA maturation and proper function in protein translation. Notably, the methylation step is dependent on a key metabolite, 5,10-methylenetetrahydrofolate, which is generated by a mitochondrial enzyme serine hydroxymethyltransferase 2 (SHMT2) [43]. SIRT3 was shown to deacetylate SHMT2 and increase its catalytic activity [44]. Therefore SIRT3 may control protein synthesis by regulating tRNA methylation and maturation (Fig. 5.2B). Sirtuins are involved in the clearance of damaged mitochondria. Mitophagy is critical for cells to degrade deficient or unnecessary mitochondria. BCL2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) is a key determinant for mitophagy. SIRT1 deacetylated forkhead box O3 (Foxo3) to enhance the expression of Bnip3. Importantly, Bnip3 deacetylation was necessary for preventing kidney damage caused by aging or hypoxia [45] (Fig. 5.2C). It is noteworthy that mitochondrial dynamics is also under the control of sirtuins. Mitochondria undergo elongation and division in response to metabolic signals. Mitofusin 1 (MFN1) and optic atrophy 1 (OPA1), two proteins responsible for mitochondria fusion, turned out to be the substrate for SIRT1 and SIRT3, respectively

FIGURE 5.2 Sirtuins regulate mitochondria biogenesis. (A) Sirtuins control the transcription of mitochondria genes through interacting with transcription factors. (B). SIRT3 regulates mitochondrial protein translation by modulating the MRPL10 acetylation and tRNA methylation. (C) SIRT3 deacetylates FOXO3 to enhance BNIP3 expression and consequent mitophagy. (D) Sirtuins are involved in the regulation of mitochondria dynamics by modulating the activity of MFN1, OPA1, and DRP1.

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(Fig. 5.2D). On one hand, SIRT1 expression was increased in hypoxic cells, leading to deacetylation and stabilization of MFN1. The consequent mitochondria elongation was vital for cells to adapt to low oxygen conditions [46]. On the other hand, SIRT3 deacetylated OPA1 and increased its GTPase activity to protect the mitochondrial network from genotoxic agents in cardiomyocytes [47]. Mitochondria fission counteracts with mitochondria fusion to modulate mitochondrial structure. One of the key drivers of mitochondrial fission is dynamin-related protein 1 (Drp1). SIRT4 suppressed Drp1 phosphorylation, a key step in mitochondrial recruitment of Drp1 (Fig. 5.2D). The interaction between SIRT4 and Drp1 was linked to the invasion and migration of cancer cells [11].

5.3 Sirtuins and mitochondrial metabolism The majority of mitochondrial metabolic enzymes are modified by acylation, and therefore substrates for sirtuins. Recent studies have connected mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5) with various metabolic pathways. Mitochondrial sirtuins control the acetylation of key enzymes in specific pathways and regulate FAO, tricarboxylic acid cycle (TCA cycle), urea cycle, and other metabolic pathways. For example, SIRT3 controlled the acetylation and activity of coenzyme A synthetase 2 (ACS2), long-chain acyl-coenzyme A dehydrogenase (LCAD), isocitrate dehydrogenase 2 (IDH2), glutamate dehydrogenase (GDH), and superoxide dismutase 2 (SOD2) [5,6,48 50]. SIRT4 seems to be a versatile regulator of mitochondrial metabolism. SIRT4 used NAD1 to ADP-ribosylate and downregulate GDH activity [51]. In addition, SIRT4 functions as a lipoamidase to regulate pyruvate oxidation. Pyruvate is an important substrate for mitochondria. Dihydrolipoyllysine acetyltransferase (DLAT) is an essential subunit of mitochondrial pyruvate dehydrogenase complex (PDH). Interestingly, glutamine enhanced the lipoamidase activity of SIRT4 to inhibit the lipoyl levels of DLAT, thereby suppressing pyruvate oxidation [8]. SIRT5 has been shown to remove multiple types of acylation modifications. SIRT5 was able to deacetylate, desuccinylate, and deglutarylate carbamoyl phosphate synthetase 1 (CPS1). The deacylase activity of SIRT5 toward CPS1 is essential to upregulate the urea cycle for ammonia detoxification [52 54]. Notably, recent protein succinylome analysis revealed that 29% of mitochondrial proteins were lysine-succinylated. Protein succinylation of mitochondrial proteins also plays a regulatory role in cellular metabolism, such that SIRT5 desuccinylated HMGCS2 to promote ketogenesis in mouse liver [14,55].

5.4 Sirtuins and mitochondrial dysfunction in human diseases Mitochondria are hubs in the metabolic network. Therefore mitochondrial dysfunction disorders cell metabolism and contributes to the development of various human diseases. Because sirtuins are key regulators of mitochondrial function, deregulation of sirtuins leads to aberrant acetylation, or more broadly, acylation of their substrates. Recent investigations have demonstrated that deregulation of sirtuins and mitochondrial function is implicated in multiple human diseases, such as diabetes, heart disease, kidney disease, neurodegeneration, and cancer (Figs. 5.3 and 5.4).

FIGURE 5.3 Sirtuins regulate mitochondrial metabolism and homeostasis by modulating various metabolic enzymes.

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FIGURE 5.4 Schematic overview of the deregulation of sirtuins links to mitochondrial dysfunction and human diseases.

5.4.1 Diabetes and obesity Aberrant mitochondrial function is a significant character of diabetes and obesity. Sirtuins are intensively involved in metabolic disorders of mitochondria. Adiponectin, an antidiabetic adipokine, and its receptor AdipoR1 were shown to be induced by extracellular Ca21 influx to activate CaMKKβ-AMPK-SIRT1-PGC-1α axis, resulting in an increase of mitochondrial mass in myocytes. Therefore disruption of SIRT1 caused mitochondrial dysfunction and insulin resistance in obese animals [56]. SIRT2 is potentially protective for obesity and diabetes. SIRT2 attenuated oxidative stress and mitochondrial dysfunction by increasing the expression of mitochondrial fusion protein Mfn2 and by decreasing the expression of mitochondria-fission protein Drp1 in HepG2 cells. SIRT2 mRNA levels were inversely correlated with indexes of obesity and insulin resistance in peripheral blood mononuclear cells (PBMCs) of human subjects [57]. SIRT3 is a key modulator of mitochondrial function in skeletal muscle and adipose tissue. SIRT3 expression was found to be decreased in the skeletal muscle of type 1 and type 2 diabetic models, while Sirt3-deletion mice exhibited reduced mitochondrial oxidation and impaired insulin signaling in skeletal muscle [58]. Of note, SIRT3 activity and mitochondrial antioxidant defense were decreased in skeletal muscle during human pregnancy and gestational diabetes mellitus [59]. Recent reports demonstrated that genetic ablation of Sirt3 and carnitine acetyltransferase, which disrupted acetyl-lysine turnover in the mitochondria of muscle, promoted insulin resistance and redox stress [60]. SIRT3 activation was also necessary to reduce oxidative stress and prevent mitochondrial dysfunction after intracerebral hemorrhage in diabetic rats [61]. Calcium overload is an important factor causing mitochondrial malfunction. SIRT3 was able to inhibit mitochondrial calcium overload and prevent the loss of brown adipose tissue during obesity and metabolic disorders [62]. SIRT4 regulates mitochondrial metabolism in pancreatic β cells and hepatic cells. SIRT4 was able to ADP-ribosylate GDH and inhibit glutamate oxidation in pancreatic β cells [51]. SIRT4 knockdown increased mitochondrial and FAO gene expression in liver and muscle cells, which might provide therapeutic benefits for type 2 diabetes [9]. SIRT5 plays an important role in regulating the metabolic activities of hepatic cells and pancreatic β cells. Overexpression of hepatic SIRT5 by CRISPR/ Cas9 gene-editing ameliorated the metabolic abnormalities in an obesity mouse model [63] SIRT5 overexpression not only improved the secretory capacity of β cells but also reduced glucolipotoxicity-induced apoptosis when cells were stressed with excess palmitate and glucose [40]. SIRT6 and SIRT7 are irreplaceable in maintaining metabolic homeostasis. The deletion of Sirt6 resulted in severe hypoglycemia, liver steatosis, and promoted obesity and diabetes in mice [64,65]. Activation of hepatic SIRT6 suppressed hepatic gluconeogenesis by increasing GCN5-dependent PGC-1α acetylation [37]. SIRT7 was evidenced to deacetylate NRF1 and modulate the expression of nuclear-encoded mitochondrial genes. Sirt7 deficiency induced multisystemic mitochondrial dysfunction and metabolic disorders in mice [39].

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5.4.2 Cardiovascular diseases Aberrant expression of sirtuins tends to deregulate mitochondrial metabolism, mitochondrial biogenesis, and mitophagy in the cardiovascular system. Cardiac deletion of Sirt1 caused cardiomyopathy, while the activation of SIRT1 by resveratrol reversed the cardiac injuries through PGC-1α-dependent mitochondrial biogenesis [10]. SIRT2 promoted AMPK activation and cardiac metabolism to ameliorate angiotensin II-induced cardiac hypertrophy [66]. Besides, SIRT3 was able to decrease the susceptibility of cardiac-derived cells and adult heart to ischemia reperfusion (IR) injury [67]. Sirt3-knockout mice showed high cardiac lipotoxicity and cardiac dysfunction [68]. SIRT3 worked together with FOXO3A and Parkin to promote mitophagy in cardiomyocytes. However, suppression of SIRT3-FOXO3A-Parkin signaling exacerbated the development of diabetic cardiomyopathy [69]. In addition, SIRT3 was reported to deacetylate SOD2 and promote mitochondrial ROS scavenging. By decreasing ROS level, SIRT3 reduced the vascular inflammation induced by the activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome [70,71]. In cardiomyocytes, SIRT4 was decreased after myocardial ischemia reperfusion injury, while overexpression of SIRT4 restored mitochondrial function and ameliorated myocardial injury [72]. Sirt5 ablation was accompanied by a strong increase of protein succinylation in mouse heart. This observation suggests that the desuccinylase activity of SIRT5 plays a significant role in cardiac function. In support of this notion, cardiac deletion of Sirt5 decreased FAO, glucose oxidation, and mitochondrial NAD1/NADH in a model of heart muscle hypertrophy [73]. More importantly, loss of Sirt5 led to hypertrophic cardiomyopathy and increased cardiac ischemia reperfusion injury [74,75].

5.4.3 Renal disease In both human patients and mouse models of severe diabetic kidney disease, expression of SIRT1 was decreased in kidney glomeruli, proximal tubules, and podocytes [76,77]. On the contrary, upregulation of SIRT1 by calorie restriction enhanced Bnip3-dependent mitochondrial autophagy and attenuated hypoxia-associated mitochondrial damage, which is protective in the aged kidney [45]. Similarly, activation of the SIRT1/PGC-1α pathway by SIRT1 activator SRT1720 induced mitochondrial biogenesis and accelerated the recovery of renal function after kidney ischemia reperfusion [78]. These observations indicate that activating SIRT1 is a beneficial therapeutic strategy for kidney disease. Renal SIRT3 level was reported to be associated with oxidative stress and mitochondrial damage. Specifically, cisplatin reduced SIRT3 expression in cultured human tubular cells and caused mitochondrial fragmentation [79]. SIRT3 is potentially linked to renal inflammation. TFAM, a substrate for SIRT3, was shown to mediate the intracellular release of mtDNA and increase renal inflammation and fibrosis [80].

5.4.4 Neurodegeneration Neurodegenerative diseases are tightly connected with mitochondrial dysfunction. Extensive evidence from human patients and mouse models of Huntington’s disease (HD) revealed abnormal mitochondrial permeability transition, defective mitochondrial calcium dynamics, and decreased mitochondrial respiration in HD cerebral cortex and basal ganglia [81 83]. In transgenic and chemical-induced mouse models of HD, SIRT1 activation is critical to protect against motor impairment through multiple targets including the TORC1 and cyclic AMPresponsive element-binding protein (CREB) transcriptional pathway [84,85]. Overactivation of SIRT2 potentially contributed to Alzheimer’s disease (AD) by downregulating the acetylation of tubulin, as SIRT2 inhibition improved microtubule assembly, autophagic vesicle traffic, and mitochondria degradation [86]. Additionally, the neuronal function is affected by mitochondrial sirtuins. SIRT3 deacetylated SOD2 and cyclophilin D in neuronal mitochondria to prevent neuronal death in mouse models of epilepsy and HD [87]. Neuronal SIRT3 was shown to be activated by physiological stress to counteract the degeneration [87]. Sirt4 deletion reduced the expression of glutamate transporter in mouse brain, which was linked to seizure phenotypes [88]. SIRT5 was reported to maintain the expression of SOD2 and mitochondrial antioxidant capacity, explaining its role in ameliorating nigrostriatal dopaminergic degeneration in a mouse model of Parkinson’s disease (PD) [89].

5.4.5 Aging Mutations, deletions, and release of mtDNA contribute to aging [90,91]. Transcriptional factor TFAM is responsible for mtDNA replication and transcription, which are essential for mitochondrial homeostasis. SIRT3

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was shown to deacetylate TFAM and maintain mtDNA integrity [7,92]. Calorie restriction prevents aging with its mechanism not fully understood. Upon calorie restriction, Sirt3 activated Idh2 to increase mitochondrial NADPH level, providing reducing power to support ROS clearance and prevent the age-related hearing loss [49]. Furthermore, SIRT4 was reported to control leucine catabolism and regulate leucine-induced insulin secretion, preventing age-related insulin resistance [10].

5.4.6 Tumorigenesis Mounting evidence suggests that sirtuins play a complex role in tumorigenesis with both tumor-promoting and tumor-suppressing functions. Here we provide an overview of current knowledge of sirtuins in mitochondrial dysfunction and tumorigenesis. SIRT2’s effect on tumor development is probably dependent on the tissue of origin. In human breast cancers and hepatocellular carcinoma, SIRT2 was suggested to be a tumor suppressor through its role in regulating mitosis and genome integrity [90]. However, inactivation of p73 by SIRT2 was critical for the proliferation and tumorigenicity of glioblastoma cells [93]. Moreover, SIRT2 inhibition by a selective inhibitor promoted c-Myc ubiquitination and degradation. SIRT2 inhibitor displayed a broad anticancer effect in various cancer cells [94]. In line with this finding, genetic depletion and pharmacological inactivation of SIRT2 antagonizes aggressive traits and tumorigenesis in basal-like breast cancer [95]. A series of studies have identified SIRT3 as a mitochondria-localized tumor suppressor [96,97]. In human breast tumors, SIRT3 loss resulted in mitochondrial metabolism remodeling and genomic instability [97]. SIRT3 could also destabilize HIF-1α to reduce ROS production and reprogram mitochondrial metabolism in breast cancer cells [98]. In addition, mice with Sirt3 deficiency showed higher expression of iron and transferrin receptor (TfR1) in the pancreas, which contributed to the tumor-suppressive activity of SIRT3 in pancreatic cancers [99]. Interestingly, some studies described SIRT3 as a tumor promoter. SIRT3 knockdown significantly inhibited tumorigenesis in a xenograft model of human melanoma [100]. The expression of SIRT4 is decreased in several types of human tumors, and its lower expression in mitochondria resulted in stress-induced genomic instability and glutamine-dependent proliferation [101]. Due to the intimidate relationship between sirtuins and aging, sirtuins are believed to be promising targets in cancer treatment. A more comprehensive understanding of sirtuins in the malignant transformation and progression of different cancers would aid in target selection and drug screening.

5.5 Feasible clinical targets: posttranslational modifications of sirtuins regulate mitochondrial function The close link between sirtuins and mitochondrial function paves a way to control mitochondria activity by modulating the activity of sirtuin proteins. Similar to their substrates, sirtuins are modified at posttranslational levels as well. The posttranslational modifications (PTM) of sirtuins provide an opportunity for therapeutic intervention of sirtuins. Specifically, the effect of mammalian sirtuins on mitochondrial metabolism has been reported to be regulated by various PTM. For example, phosphorylation of SIRT1, SIRT3, and SIRT6 was demonstrated to control their deacylase activity. SIRT1 is modified by serine phosphorylation. The cAMP/PKA pathway was responsible for SIRT1 phosphorylation, which increased the intrinsic deacetylase activity of SIRT1 to promote PGC-1α deacetylation and FAO [102] (Fig. 5.5A). It is noteworthy that SIRT3, as a vital regulator of mitochondrial stress response in cancer cells, is also modified by phosphorylation. Radiotherapy induced cyclin B1-CDK1 to activate SIRT3 via threonine/serine phosphorylation [103]. In addition, PKCζ phosphorylated SIRT6 to promote its chromatin enrichment and the expression of fatty acid β-oxidation-related genes [104]. Sirtuins are also modified by acetylation, methylation, and SUMOylation. Downregulation of SIRT1 induced SIRT3 hyperacetylation and consequent mitochondrial dysfunction in aging- and obesity-related diseases [19] (Fig. 5.5B). Protein arginine methylation has emerged to modulate the deacetylase activity of sirtuins. SIRT7 was methylated by arginine methyltransferase 6 (PRMT6), which inhibited its histone H3K18 deacetylase activity to promote mitochondrial biogenesis and respiration (Fig. 5.5C) [40]. Protein SUMOylation, referring to the conjugation of small ubiquitinlike protein (SUMO) onto lysine residues of target protein, also modulates mitochondrial activity. During fasting, SUMO-specific protease SENP1 accumulated in the mitochondrial matrix to deSUMOylate and activate SIRT3. This process was indispensable for SIRT3 to promote FAO and maintain energy homeostasis. Importantly, the suppression of SIRT3 deSUMOylation has been demonstrated to prevent HFD-induced obesity [105, 106] (Fig. 5.5D).

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FIGURE 5.5 PTM of sirtuins regulate mitochondrial function. (A) Phosphorylation of SIRT1, SIRT3, and SIRT6 promotes mitochondrial biogenesis and metabolism. (B) SIRT1 downregulation causes SIRT3 hyperacetylation and mitochondrial dysfunction. (C) Methylation of SIRT7 by PRMT6 inhibits its H3K18 deacetylase activity and promotes mitochondrial biogenesis. (D) DeSUMOylation of SIRT3 by SENP1 upregulates its deacetylase activity and mitochondrial metabolism.

Based on their important role in regulating mitochondrial function and cellular metabolism, sirtuins hold great promise to be therapeutic targets in the treatment of human diseases as mentioned above. Further insights into the catalytic and regulatory mechanism of sirtuins will expedite the development of promising inhibitors.

Acknowledgments This work was supported by the Natural Science Foundation of China (Nos. 81790251 and 81772946 to Y.-P.W.), the Young Elite Scientist Sponsorship Program by CAST (2018QNRC001 to Y.-P.W.), High-Level Local University Construction Project of Shanghai Jiao Tong University School of Medicine (18zxy004 to T.-S.W.), Shanghai Committee of Science and Technology (15ZR1424500 to T.-S.W.), and the China Postdoctoral Science Foundation (2019M661548 to J.-L. H.).

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Skeletal muscle MnSOD, mitochondrial complex II, and SIRT3 enzyme activities are decreased in maternal obesity during human pregnancy and gestational diabetes mellitus. J Clin Endocrinol Metab 2013;98 (10):E1601 9. [60] Williams AS, Koves TR, Davidson MT, Crown SB, Fisher-Wellman KH, Torres MJ, et al. Disruption of acetyl-lysine turnover in muscle mitochondria promotes insulin resistance and redox stress without overt respiratory dysfunction. Cell Metab 2020;31(1):131 47. [61] Zheng J, Shi L, Liang F, Xu W, Li T, Gao L, et al. Sirt3 ameliorates oxidative stress and mitochondrial dysfunction after intracerebral hemorrhage in diabetic rats. Front Neurosci 2018;12:414. [62] Gao P, Jiang Y, Wu H, Sun F, Li Y, He H, et al. Inhibition of mitochondrial calcium overload by SIRT3 prevents obesity or age-related whitening of brown adipose tissue. Diabetes 2020;69(2):165 80. [63] Du Y, Hu H, Qu S, Wang J, Hua C, Zhang J, et al. SIRT5 deacylates metabolism-related proteins and attenuates hepatic steatosis in ob/ob mice. EBioMedicine 2018;36:347 57. [64] Kuang J, Chen L, Tang Q, Zhang J, Li Y, He J. The role of Sirt6 in obesity and diabetes. Front Physiol 2018;9:135. [65] Zhong L, D’Urso A, Toiber D, Sebastian C, Henry RE, Vadysirisack DD, et al. The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Cell 2010;140(2):280 93. [66] Tang X, Chen XF, Wang NY, Wang XM, Liang ST, Zheng W, et al. SIRT2 acts as a cardioprotective deacetylase in pathological cardiac hypertrophy. Circulation 2017;136(21):2051 67. [67] Parodi-Rullan RM, Chapa-Dubocq X, Rullan PJ, Jang S, Javadov S. High sensitivity of SIRT3 deficient hearts to ischemia-reperfusion is associated with mitochondrial abnormalities. Front Pharmacol 2017;8:275. [68] Zeng H, Vaka VR, He X, Booz GW, Chen JX. High-fat diet induces cardiac remodelling and dysfunction: assessment of the role played by SIRT3 loss. J Cell Mol Med 2015;19(8):1847 56. [69] Yu W, Gao B, Li N, Wang J, Qiu C, Zhang G, et al. Sirt3 deficiency exacerbates diabetic cardiac dysfunction: role of Foxo3A-Parkinmediated mitophagy. Biochim Biophys Acta Molecular Basis Disease 2017;1863(8):1973 83. [70] Traba J, Geiger SS, Kwarteng-Siaw M, Han K, Ra OH, Siegel RM, et al. Prolonged fasting suppresses mitochondrial NLRP3 inflammasome assembly and activation via SIRT3-mediated activation of superoxide dismutase 2. J Biol Chem 2017;292(29):12153 64. [71] Chen ML, Zhu XH, Ran L, Lang HD, Yi L, Mi MT. Trimethylamine-N-oxide induces vascular inflammation by activating the NLRP3 inflammasome through the SIRT3-SOD2-mtROS signaling pathway. J Am Heart Assoc 2017;6(9):e006347. [72] Zeng G, Liu H, Wang H. Amelioration of myocardial ischemia-reperfusion injury by SIRT4 involves mitochondrial protection and reduced apoptosis. Biochem Biophys Res Commun 2018;502(1):15 21. [73] Hershberger KA, Abraham DM, Martin AS, Mao L, Liu J, Gu H, et al. Sirtuin 5 is required for mouse survival in response to cardiac pressure overload. J Biol Chem 2017;292(48):19767 81. [74] Sadhukhan S, Liu X, Ryu D, Nelson OD, Stupinski JA, Li Z, et al. Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function. Proc Natl Acad Sci U S A 2016;113(16):4320 5. [75] Boylston JA, Sun J, Chen Y, Gucek M, Sack MN, Murphy E. Characterization of the cardiac succinylome and its role in ischemiareperfusion injury. J Mol Cell Cardiol 2015;88:73 81. [76] Hasegawa K, Wakino S, Simic P, Sakamaki Y, Minakuchi H, Fujimura K, et al. Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing Claudin-1 overexpression in podocytes. Nat Med 2013;19(11):1496 504.

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6 Sirtuins in immunometabolism Hongxiu Yu Institutes of Biomedical Sciences, Fudan University, Shanghai, P. R. China O U T L I N E 6.1 Brief introduction of immunometabolism

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6.2 Role of sirtuins in immunometabolism 6.2.1 SIRT1 6.2.2 SIRT2 6.2.3 SIRT3, SIRT4, and SIRT5

93 93 96 96

6.2.4 SIRT6 6.2.5 SIRT7

97 97

6.3 Conclusion and future considerations

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References

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6.1 Brief introduction of immunometabolism Metabolism fuels all cellular processes, ranging from development, proliferation, differentiation, and the effector functions [1]. First, uptake and utilization of nutrients provides the substrates for adenosine triphosphate (ATP) synthesis to sustain various cellular programs. Second, it provides the building blocks for the synthesis of macromolecules, such as RNA, DNA, proteins, and membranes, that are necessary for the proliferation and function of cells. Besides maintaining homeostasis, the immune system senses and defends against pathogens for the life of the organism. These diverse functions are energetically expensive [2]. For instance, the sensing of a pathogen or environmental threats results in the secretion of inflammatory cytokines and chemokines by the innate immune cells and the clonal expansion of adaptive immune cells. Because immune cells lack significant stores of nutrients, they will dramatically increase the uptake of glucose, amino acids, and fatty acids from their microenvironment [3]. The processes require precise control of cellular metabolic pathways [4,5]. Following the entry of glucose into cells via glucose transporters, glucose is metabolized by the interconnected pathways of glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS). The glycolysis occurs in the cytoplasm, whereas the latter two are restricted to the mitochondria. Glucose is firstly phosphorylated to glucose 6-phosphate (G-6-P) by hexokinases. During glycolysis, G-6-P is metabolized to pyruvate, reducing nicotinamide adenine dinucleotide (NAD1) to NADH and generating ATP. In hypoxia, pyruvate is reduced to lactate, restoring NAD1 levels in the cell. In normoxia, pyruvate is metabolized to acetyl-coenzyme A (acetyl-CoA), which is oxidized in the TCA cycle to generate NADH. In the redox reactions of OXPHOS, electrons are sequentially transferred to generate a H1 gradient across the inner mitochondrial membrane and to generate ATP. In contrast to glycolysis, mitochondrial OXPHOS is a highly efficient form of generating ATP. Besides glucose metabolism, immune cells utilize three additional pathways—the pentose phosphate pathway (PPP), glutaminolysis, and fatty acid oxidation (FAO)—to meet their functional demands [6,7]. Recently, amino acid metabolism has also been proved to have an important role in controlling immune cells function [8
11]. The metabolism is integrated to determine the immune cells’ ability to divide, differentiate, and carry out effector functions in immune response [12]. Cellular metabolism plays important roles in regulating acute and chronic inflammation, which involves both innate and adaptive immune cells [13,14]. During the early stage of acute inflammation, such as sepsis, a high

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amount of energy is required and provided by glycolysis. Increased glucose uptake, PPP activation, and glycolysis lead to lactic acid accumulation. Moreover, this initial defensive pathway rapidly becomes toxic to cells by generating excessive reactive oxygen species (ROS) and prompting cell death pathways. Septic patients are likely to spend less than a day, or only a few hours, in the cytokine storm of hyperinflammation. Then the acute systemic inflammation with sepsis will switch to hypoinflammation or adaptation phase [15]. The later stage of acute inflammation is a low-energy response. Fatty acid enters mitochondria and generates acetyl-CoA, which undergoes the TCA cycle to provide NADH as a reducing agent for oxygen support of ATP generation. An increase in ATP production by mitochondrial OXPHOS occurs. Chronic inflammation such as hyperlipidemia, diabetes, obesity, and nonalcoholic fatty liver disease (NAFLD) are associated with altered immune cell metabolism and ineffective immunity [16]. For instance, lipid accumulation in adipose tissue promotes the proinflammatory state of macrophages, further exacerbating obesity-related adipose tissue inflammation [17]. Insulin itself might promote T cell activation through its ability to enhance glucose uptake, amino acid transport, lipid metabolism, and protein synthesis [18]. Different immune cell functions are associated with distinct metabolic configurations [19,20]. Generally, quiescent immune cells rely primarily on FAO and the TCA cycle coupled with OXPHOS to generate ATP. Activated immune cells use glycolysis to generate biomass and ATP [21]. Macrophages are the first line of defense against pathogens and injury and are necessary for the maintenance of tissue homeostasis [22]. Macrophages have been classified into classically activated macrophages (M1) and alternatively activated macrophages (M2). Whereas lipopolysaccharide (LPS) and interferon-γ (IFN-γ) drive M1 macrophage activation, interleukin-4 (IL-4) and IL-13 polarize macrophages to the alternative M2 state [23]. M2 macrophages use fatty acid primarily derived from acquired triacyglycerols to support OXPHOS, and have an intact TCA cycle. M1 macrophages involve an increase in glycolysis, while the TCA cycle is fragmented in cells. Dendritic cells (DCs) are antigen-presenting cells (APC) that critically influence decisions about immune activation or tolerance [24]. Impaired DC function is at the core of common chronic disorders. Knowledge of the mechanisms regulating DC function is necessary to understand the immune system and to prevent disease and immunosenescence. There are four major subsets of DCs: conventional DCs (cDCs), Langerhans cells, monocyte-derived DCs, and plasmacytoid DCs (pDCs). These cells are related to each other as they have a common myeloid progenitor. The development of DCs from progenitor cells is associated with mitochondrial biogenesis. Immature DCs use FAO as a core metabolic process. Activation of DCs leads to a rapid increase in flux through glycolysis and the associated PPP, with an accompanying increase in fatty acid synthesis [25,26]. Once triggered to secrete antibodies, activated B cells are glycolytic. However, the detailed metabolic profiles of differentiated and memory B cells remain to be explored. T cells are the central players in the adaptive immune response. They have evolved to rapidly respond to invading pathogens. The T cell metabolic machinery is regulated for coordination of the transitions in different phases from initial cell growth, massive clonal expansion and differentiation, a contraction or death phase, to the maintenance of immune memory [27,28]. During the initial growth phase, T cells switch from the FAO in naive T cells to the glycolytic, pentose phosphate and glutaminolytic pathways in activated T cells [29]. Furthermore, activated T cells can differentiate into different functional T cell subsets depending on the cytokines and other extracellular signals. These subsets determine the nature of the immune response. Whereas CD81 T cells differentiate into cytotoxic T lymphocytes that kill host cells infected with pathogens, CD41 T cells can differentiate into an effector T cell (Th1, Th2, Th9, or Th17) that mediate appropriate immune responses or induced regulatory T cells (iTreg cells) that suppress uncontrolled immune responses [30,31]. At the end of an immune response, the majority of effector T cells undergo apoptosis, whereas a small percentage persist as memory T cells (Tmem cells), which are responsible for enhanced immunity after reexposure to the pathogen. Of these various T cell subsets, the Tmem cells and iTreg cells rely on oxidizing fatty acid to support OXPHOS, whereas cytotoxic T lymphocytes and effector T cells sustain high glycolytic activity and glutaminolytic activity [32,33]. Glycolysis allows the efficient translation of mRNAs encoding effector cytokines, such as IFN-γ. In terms of metabolic requirements, effector T cells are similar to oncogenically transformed cells [34]. The metabolic signals including extracellular nutrient environment or intracellular metabolic status of immune cells are sensed and transduced to affect cell cycle, differentiation, cell death, and immunological functions. Such signals include metabolites involved in cell metabolic pathways or metabolic products, by-products, and cofactors such as ATP, NAD1-NADH, nicotinamide adenine dinucleotide phosphate (NADP1)-NADPH, acetyl-CoA, and ROS [29,35]. The sensors are cell-surface receptors or transporters that physically interact with and respond to metabolic signals by changes in their biological status. The physical interactions among metabolic signals and sensors trigger a series of intracellular signaling events that result in immune cell differentiation and activation [36].

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Sirtuins, prolyl-4-hydroxylase (PHD) proteins, adenosine 50 -monophosphate (AMP)-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), pyruvate kinase M2 (PKM2), and hypoxia-induced factor 1α (HIF-1α) are central players in the coordinated sensing of cellular metabolic signal and dictation of cell fate [37
41]. Furthermore, every step of metabolic signal transduction in immune cells can be modulated, which in turn result in changes in their functional properties [42]. For instance, either pharmacological activation of AMPK or T cell-specific deletion of mTOR is sufficient to drive T cell differentiation toward iTreg cells after antigen stimulation [43,44]. When glycolysis is inhibited by modulation of PKM2 [45], macrophages undergo a shift toward a more M2 macrophage-like state. A similar effect is observed in Th17 cells, which become more like Treg cells if glycolysis is inhibited [40]. This plasticity of immune cells is important. For example, the metabolic reprogramming of immune cells from an inflammatory to an antiinflammatory phenotype or vice versa can be used as a strategy to boost antitumor immunity or inhibit hyperimmune response. Immune cell metabolism has therefore become an attractive target area for therapeutic purposes [46,47].

6.2 Role of sirtuins in immunometabolism The mammalian sirtuins are a family of NAD1-dependent deacetylases and consist of seven members (SIRT1
SIRT7) [48]. Sirtuins display a cell type-dependent manner of subcellular localization and biological functions [49]. SIRT1 and SIRT2 shuttle between the nucleus and cytoplasm. SIRT3
5 mainly localize in mitochondria, while SIRT6 is found exclusively in the nucleus. SIRT7 is found in both the nucleus and cytoplasm. Substrates of sirtuins include histones and nonhistone proteins such as nuclear transcription factors and the key enzymes involved in glycolysis, FAO, the TCA cycle, and other oxidative and metabolic pathways [50]. Although deacetylase activity was initially reported to be conserved in mammalian sirtuins, different sirtuins exhibit different acyl group preferences [51]. Among the seven members, SIRT4
7 exhibit weak or undetectable deacetylation activity. SIRT2 has efficient demyristoylase activity [52]. SIRT5 is lysine desuccinylase, deglutarylase, and demalonylase [53,54], while SIRT4 and SIRT6 possess ADP-ribosyltransferase activity [55,56]. Because sirtuins are expressed throughout the body, NAD1 is rate-limiting for sirtuins enzymatic reactions, and as a result, sirtuins serve as metabolic sensors of intracellular NAD1 and the redox state [57,58]. Although much are known about the sirtuins in metabolism, the effect of sirtuins on immune cell function and metabolic configuration have not been systematically explored. In this chapter, sirtuins’ functions in immune response and immunometabolism will be discussed. The main findings discussed in the present chapter are summarized in Table 6.1.

6.2.1 SIRT1 6.2.1.1 SIRT1 in macrophage SIRT1 is the most studied family member among sirtuins. Accumulated evidence shows the important role of SIRT1 in immune cell function and immune response. Yoshizaki et al. showed the antiinflammatory role of SIRT1 in macrophages [59]. Knockdown of SIRT1 in the mouse macrophage RAW264.7 cell line and in intraperitoneal macrophages increases intracellular inflammatory c-Jun N-terminal kinase (JNK) and IκB kinase (IKK) pathways, and inflammatory gene tumor necrosis factor alpha (TNF-α) expression. Activated SIRT1 in macrophages restrains proinflammatory responses. SIRT1 activator treatment of Zucker fatty rats leads to glucose tolerance, insulin sensitivity, and reduced tissue inflammatory responses. These in vitro and in vivo results raise the possibility that macrophage SIRT1 may play a beneficial role in regulating the inflammatory component of metabolic diseases. Consistently, Schug et al. have demonstrated that SIRT1 plays a key role in regulating the inflammatory and immune responses in mice [60]. Using a myeloid cell-specific Sirt1 knockout (Mac-Sirt1 KO) mouse model and bone marrow-derived macrophages (BMDMs), they have showed that deletion of SIRT1 in macrophages leads to the components of the nuclear factor kappa B (NF-κB) pathway being hyperacetylated, resulting in increased expression of IL-6 and IL-1β genes. Mac-Sirt1 KO mice challenged with a high-fat diet (HFD) display high levels of activated macrophages in liver and adipose tissue, predisposing mice to the development of insulin resistance and metabolic disorders. This work provides both in vitro and in vivo evidence as to how SIRT1 functions in macrophages or systemically. Furthermore, they also demonstrated that the SIRT1 homolog SIRT6 has a compensatory effect on NF-κB activation in Mac-Sirt1 KO BMDMs.

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TABLE 6.1 Sirtuins function in immnue cells. Gene Immunue cells

Model

Target

Function

References

SIRT1 Macrophage

SIRT1 knockdown RAW264.7 cell

JNK, IKK pathway

Antiinflammation

[59]

Macrophage

Myeloid-specific-Sirt1 KO mouse

NF-κB

Antiinflammation

[60]

Macrophage

Phagocytosis

AP-1

Antiinflammation

[61]

Macrophage

Myeloid-specific-Sirt1 KO mouse

Proinflammation

[62]

Macrophages and adipocyte

Myeloid-specific-Sirt1 KO mouse and Adipocyte-specific-Sirt1 KO mouse

Antiinflammation

[63]

THP-1 promonocyte

Sepsis

PGC-1α

Supports FAO

Inflammation homestasis

[64]

Myeloid-derived suppressor cell

Myeloid-specific-Sirt1 KO mouse

mTOR, HIF-1α

Inhibits glycolysis

Inhibits tumor growth

[65]

Dendric cell

DC-specific-Sirt1 KO mouse

PPAR-γ

Antiinflammation

[66]

Dendric cell

DC-specific-Sirt1 KO mouse

HIF-1α

T cell differention

[67]

CD4 T cell

Sirt1 KO mouse

AP-1

Inhibits autoimmunity

[68]

Th9 cell

CD4-specific-Sirt1 KO mouse

mTOR, HIF-1α

1

CD81 memory T cell

Inhibits glycolysis

Antiallergic [69] airwayInflammation, promotes tumor growth

Inhibits glycolysis

T cell differention

[70]

Inhibits glycolysis

Inhibits phagocytosis by macrophages

[71]

SIRT2 Macrophage

Sirt2 KO mouse

Macrophage

Sirt2 KO mouse

NF-κB

Proinflammation

[72]

Macrophage

Sirt2 KO mouse

NF-κB

Antiinflammation

[73]

SIRT5 Macrophage

Sirt5 KO mouse

NF-κB

Proinflammation

[74]

No effect on infection

[75]

Macrophages

Sirt5 KO mouse

Macrophage

Sirt5 KO mouse

PKM2

Inhibits glycolysis

Antiinflammation

[76]

Sepsis

HIF-1α

Represses glucose metabolism

Inflammation homestasis

[64]

SIRT6 THP-1 promonocyte CD41 T cells, macrophage

Sirt6 KO mouse, T cell-specific and myeloid-specific Sirt6 KO mouse

[77]

Dendric cell

Sirt6 KO mouse

[78]

In addition, SIRT1 deacetylates activator protein-1 (AP-1) to improve macrophage function [61]. AP-1 is a transcription factor, binding to the promoters of the target gene to modulate their expression, which is in turn involved in cell proliferation, differentiation, and inflammation. Cyclooxygenase-2 (COX-2) is the rate-limiting enzyme for prostaglandin (PG) production. Macrophage-derived excessive PGE2 inhibits the phagocytosis activity of macrophages. Zhang et al. have proved SIRT1 deacetylates AP-1 to repress its transcriptional activity and inhibit COX-2 expression and PGE2 production in macrophages, resulting in improved macrophage phagocytosis. In 2015 Ka et al. provided in vitro evidence that myeloid SIRT1 plays a crucial role in macrophage polarization and chemotaxis [62]. Pancreatic islets, liver, and adipose tissues from Sirt1 KO HFD mice show increased macrophages infiltration and tissue inflammation. Sirt1 KO mice fed a HFD exhibit glucose intolerance, reduced insulin secretion, and insulin sensitivity with a slight decrease in body weight. The metabolic phenotype is in contrast to those from the study by Schug et al. [60], in which Sirt1 KO mice exhibit greater weight gain and adipose tissue mass. The author said these opposite results may result from differences in the diet composition.

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Considering the dynamic interplay between adipocytes and macrophages within adipose tissue during the pathogenesis of obesity, Hui et al. have generated adipocyte and myeloid cell-specific Sirt1 KO mice and compared the metabolic phenotypes of them [63]. Compared to myeloid-selective Sirt1 depletion, mice with adipocyte-specific deletion of Sirt1 are more susceptible to diet-induced insulin resistance. Mechanistically, the phenotype in adipocyte specific Sirt1 KO mice is associated with an increased macrophage infiltration into adipose tissues and their polarization toward M1 macrophage. In 2012 Liu et al. provide evidence that SIRT1 and SIRT6 can directly regulate immune cell metabolism [64]. In acute immune responses, the THP-1 promonocytes prefer to use glycolysis to provide high energy, which is sustained by the HIF-1α and NF-κB pathway. As the early inflammatory response changes to late adaptation, glucose metabolism switches to FAO. SIRT1 and SIRT6 integrate sequential reprogramming of metabolic and acute inflammatory responses. SIRT1 supports FAO by activating the peroxisome proliferator-activated receptor-coactivator 1-α (PGC-1α) pathway; SIRT6 represses glucose metabolism by epigenetically silencing the HIF-1α pathway. 6.2.1.2 SIRT1 in myeloid-derived suppressor cells In other innate immune cells, SIRT1 also plays important roles. For example, SIRT1 directs the differentiation of myeloid-derived suppressor cells (MDSCs) [65]. MDSCs are one of the major components of the immunosuppressive network responsible for immune cell tolerance in cancer [79]. Polarized MDSCs lineages can be distinguished as M1 and M2 cells. Liu et al. found that SIRT1 deficiency in MDSCs directs a specific switch to M1 lineage, in favor of tumor cell attack. mTOR- and HIF-1α-induced glycolytic activity is required for differentiation determined by SIRT1. 6.2.1.3 SIRT1 in dendritic cells SIRT1 plays an essential role in mediating proinflammatory and metabolic signaling in DCs. DCs are professional APCs required for the initiation of immune responses [80]. In addition to presenting antigens, DC-derived cytokines and chemokines can result in either a proinflammatory or antiinflammatory environment, engaging distinct T-cell differentiation programs on naive CD41 T cells [81]. As shown previously, in response to antigen stimulation, naive CD41 T cells may differentiate into at least five types of effector Th cells, namely Th1, Th2, Th9, Th17, and iTreg cells, which differ from each other in their cytokine secretion profile and function [82]. Th1 induction by DCs is well documented, and rapid progress is being made on Th17 and Treg stimulation. For example, DC-producing IL-12 can support Th1 development, whereas DC-producing IL-10 or transforming growth factor-β (TGF-β1) can support Treg development [83]. However, the molecular mechanisms by which DCs direct Th2 responses remain more elusive. It has been demonstrated that inappropriate Th2 responses are responsible for the development of allergic diseases [84]. Legutko et al. revealed the function of SIRT1 in the regulation of DCs function and Th2 responses. Firstly, they proved that pharmacological inhibition of SIRT1 dampens Th2 responses and subsequent allergic inflammation by interfering with lung DC function in a murine model of antigen-induced airway allergy. Then, by using DCs cell-specific Sirt1 deletion mouse model, they demonstrated that SIRT1 represses the activity of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ) in DCs, thereby favoring their maturation toward a pro-Th2 phenotype. Lack of SIRT1 expression in DCs reduces macrophage and eosinophilic lung infiltration in the murine model [66]. Moreover, by using DCs-specific Sirt1-deficient mouse, Liu et al. indicated that SIRT1 is a metabolic checkpoint regulating the differentiation of Th1 and Treg cells through a HIF-1α-dependent and mTOR-independant pathway [67]. By using DCs-specific Sirt1 deletion mice and microbial-induced inflammation model, Liu and colleagues proved that SIRT1 is required for the reciprocal production of IL-12 and TGF-β1 production in DCs as well as the expression of their receptor in responding T cells, resulting in a differential lineage engagement of Th1 and Treg. The study further implicated a metabolic checkpoint in DCs requiring the interplay of SIRT1 and HIF-1α, two metabolic sensors of redox and oxygen states, respectively. 6.2.1.4 SIRT1 in T cells SIRT1 is expressed in all tissues but is abundant in the thymus, particularly in CD41CD81 thymocytes, suggesting an involvement of SIRT1 in T cell development and function. It has been reported that SIRT1 is involved in maintaining T cell tolerance and its expression is induced considerably in anergic T cells [68,85,86]. CD41CD81 thymocytes from Sirt12/2 mice exhibit increased sensitivity to irradiation
induced apoptosis [87]. T cell-specific deletion of Sirt1 substantially promotes the generation and function of Treg cells in vitro and in vivo [88].

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However, neither the metabolic signals upstream of SIRT1 nor the molecular mechanism that mediates the downstream effects of SIRT1 in these contexts are clear. Moreover, Yu et al. have suggested that SIRT1-dependent glycolytic activity is critical for the differentiation of Th9 cells [69]. The authors have checked the expression of SIRT1 in various T cell subsets Th1, Th2, Th17, Treg, and have found that Th9 cells exhibit a lower expression than other cells. Moreover, Th9 cells exhibit more glycolytic activity than other cells. Deficiency of Sirt1 in CD41 T cells increase IL-9 production and glycolysis. Accordingly, ectopic SIRT1 expression in CD41 T cells inhibits IL-9 production and glycolysis. The work further demonstrates that SIRT1-dependent Th9 cells are important in tumor development and allergic airway inflammation by inhibiting the differentiation of Th9 cells in an mTOR-HIF-1α-dependent glycolytic pathway. This work also indicated metabolic reprogramming to be an immunotherapeutic approach in inflammation and cancer. In 2018 Jeng et al. revealed the role of SIRT1 in regulating CD81 memory T cells [70]. Human CD81CD282 T cells are very cytotoxic, producing greater levels of effector molecules, such as granzyme B (GZMB). Jeng et al. found that CD81CD282 T cells have a decreased expression of SIRT1 and an enhanced capacity of glycolysis. Loss of SIRT1 enhances glycolytic capacity and produces much more GZMB. These data suggest SIRT1 is a regulator of human CD81 memory T cell metabolism and activity. The SIRT1 may provide a potential therapeutic intervention to reprogram terminally differentiated memory T cells. In addition to these works, there are several reviews summarizing the role of SIRT1 in immune cells, inflammation, and cancer [15,89].

6.2.2 SIRT2 SIRT2 is involved with numerous processes such as DNA damage, infection, and carcinogenesis. However, the role of SIRT2 in the inflammatory process remains largely unknown. Ciarlo et al. showed that SIRT2 is the most highly expressed sirtuins in myeloid cells, especially macrophages [71]. By comparing Sirt2 KO mice with the control mice, Ciarlo et al. have suggested that Sirt2 deficiency has no major impact on the development of immune cells, including T cells (double negative T cells, single positive T cells, naı¨ve T cells, and memory T cells), B cells (immature and mature B cells), DCs, and Treg cells. Sirt2 deficiency increases phagocytosis by macrophages and protects from chronic staphylococcal infection. Interestingly, the glycolytic activity was higher in Sirt2 KO than wild-type (WT) BMDMs exposed to Staphylococcus aureus AW7. Moreover, when glycolysis BMDMs was inhibited, it significantly reduced the phagocytosis of Staphylococcus aureus AW7. These results have suggested that the metabolic capacity of macrophages may provide a mechanism by which SIRT2 inhibits phagocytosis. Lee et al. have evaluated the effects of SIRT2 in macrophages [72]. They isolated BMDMs from Sirt2 KO and control mice or RAW264.7 macrophage cells. The authors have demonstrated that the deficiency of Sirt2 results in the inhibition of NF-κB activation through reducing the phosphorylation and degradation of IκBa, then exerting an apparently inhibitory effect on the expression of M1-macrophage-related proinflammatory genes. While Sirt2 deficiency has not shown any significant inhibitory effect on M2-macrophage-related genes. Except for phosphorylation, posttranslational modification of the p65 subunit by acetylation is a major aspect of the regulation of NF-κB activity [73]. It has been demonstrated that SIRT2 regulates NF-κB by binding to NF-κB and mediating the deacetylation of NF-κB. In contrast to Lee’s report, Lo Sasso et al. have reported that Sirt2 deficiency promotes M1-macrophage and inhibits M2- macrophage polarization, mimicking a proinflammatory milieu [90]. More specifically, Sirt2 KO mice develop more severe colitis when exposed to the chemical colitis inducer, dextran sulfate sodium (DSS).

6.2.3 SIRT3, SIRT4, and SIRT5 SIRT3, SIRT4, and SIRT5 are mainly localized in mitochondria. They are well described in the literature to coordinate metabolic pathways involved in stress responses, aging, and others. It is noteworthy that whereas mitochondria play a key role in energetic metabolism, except for SIRT5, no direct evidence has been reported for a role of mitochondrial SIRT3 and SIRT4 during inflammation. SIRT5 is one of the least characterized sirtuins. SIRT5 also localizes in the cytoplasm and is ubiquitously expressed in organs. SIRT5 is a weak deacetylase and recent data suggest that SIRT5 primarily is a lysine demalonylase, desuccinylase, and deglutarylase [53,91,92]. SIRT5 desuccinylates and deglutarylates carbamoyl phosphate synthetase 1 (CSP1) to increase ammonia detoxificationm and desuccinylates succinate dehydrogenase (SDH)

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to repress cellular respiration [93,94]. Overall, SIRT5 is emerging as a key regulator of metabolism. Emerging evidence has revealed that SIRT5 have a pivotal role in immunometabolism. Interestingly, the role of SIRT5 in inflammation is controversial. Qin and colleagues have shown that Sirt5 deficiency decreases LPS-triggered inflammation in both acute and immunosuppressive phases of sepsis. Function screening have revealed that SIRT5 and SIRT1/2 have opposite expression patterns and functions in macrophages [74]. In response to stimulation with LPS, Sirt5 KO peritoneal macrophages produce less proinflammatory cytokines (IL-6 and TNF-α) than control macrophages. Sirt5 deficiency protects mice from endotoxin tolerance. Moreover, SIRT5 counteracts the inhibitory effects of SIRT2 and enhances the inflammatory responses in macrophages by activation of the NF-κB pathway. By using a different genomic background Sirt5 KO mice, Heinonen et al. have showed that Sirt5 deficiency does not affect IL-6 and TNF-α production and proliferation by macrophages [75]. Sirt5 deficiency does not affect the major T cells, B cells, and DCs subsets development. Moreover, Sirt5 deficiency does not worsen endotoxemia and bacterial infection. Wang and colleagues provided evidence that SIRT5 targets PKM2 to protect mice from DSS-induced colitis [76]. PKM2, which catalyzes the rate-limiting step of glycolysis [95], can regulate HIF-1α activity and IL-1β induction and is a critical determinant of the Warburg effect in LPS-activated macrophages [41]. The tetramer of PKM2 has a high pyruvate kinase activity, and localizes mainly in the cytosol, whereas dimeric PKM2 preferentially localizes in the nucleus and has a high protein kinase activity. In this work, Sirt5 deficiency leads to increased glycolysis, hypersuccinylation, and dimerization of PKM2 in BMDMs. The dimeric PKM2 in the nucleus directly interacts with HIF-1α and promotes the HIF1α-dependent gene expression, notably IL-1β. This will have an important consequence for the DSS-induced colitis in mice. The study therefore identifies SIRT5 as an important negative regulator of glycolysis in macrophages.

6.2.4 SIRT6 Consistent with previously mentioned works [64], SIRT6 is emerging as an important regulator of glucose metabolism [96]. Sirt6 knockout mice die by severe hypoglycemia and persistent inflammation. SIRT6 has been further proved to have a role in chronic liver inflammation and dendritic cells development. Xiao et al. have provided evidence to show that compared to global Sirt6 KO mice, deletion of Sirt6 in T cells or myeloid-derived cells is sufficient to induce liver inflammation and fibrosis [77]. This result suggests that Sirt6 deficiency in the immune cells is the cause. Consistently, macrophages derived from the bone marrow of Sirt6 KO mice show increased IL-6 and TNF-α expression levels and are hypersensitive to LPS activation. By inhibiting c-JUN-dependent expression of proinflammatory genes, SIRT6 has been shown to play an antiinflammatory role in mice. Lasiglie shows that SIRT6 stimulates the development of myeloid, conventional DCs (cDCs) [78]. Sirt6 KO mice exhibit low frequencies of bone marrow cDCs precursors and low yields of bone marrow-derived cDCs compared to WT animals. Moreover, Sirt6 KO cDCs show a decrease of IL-12, TNF-α, and IL-6 production, which means reduced SIRT6 activity may contribute to immunosenescence.

6.2.5 SIRT7 SIRT7 is the last studied among the seven mammalian sirtuins. SIRT7 is an important regulator of rRNA and protein synthesis for maintenance of normal cellular homeostasis. Vakhrusheva et al. showed that in Sirt7 KO mice, the SIRT7 disruption predisposes the mice to heart hypertrophy together with increasing cardiac inflammation [97]. These authors observed an increased infiltration of immune cells, with higher levels of pro- and antiinflammatory cytokine production. These results suggest that SIRT7 is also involved in inflammation. Information regarding the possible involvement of SIRT7 in inflammatory signaling pathways and mechanisms are still needed.

6.3 Conclusion and future considerations The sirtuins have been implicated in a variety of biological processes including cell cycle progression, apoptosis, cellular senescence, stress response, neuronal protection, adaptation to caloric restriction, metabolism, and tumorigenesis. In addition, accumulated evidence has shown that sirtuins integrate metabolism, bioenergetics, and immunity during inflammation. Moreover, many independent studies regarding the same sirtuin could exert

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Fatty acid

Glucose

FIGURE 6.1 Metabolic control of sirtuins in immune cells. Metabolic signals are sensed by sirtuins, resulting in the activation of distinct signaling pathways to initiate the adaptations of immune cells.

Insulin

SIRT1,2,5,6 mTOR

IKK NFκB JNK

HIF-1α PKM2

AP-1

PGC-1α

c-JUN

PPAR-γ

IL-1β,TNF-α,IL-6,IL-9 Glucose metabolism Fatty acid oxidation

Macrophage

DC

MDSC

T-cell

proinflammation or antiinflammation, indicating that regulation of the inflammatory response by sirtuins is subtler than previously thought. Furthermore, sirtuins’ effects on inflammation can be a double-edged sword, for instance, low levels accentuate early acute inflammation-related autotoxicity by increasing NF-κB activity, and prolonged increases in SIRT1 during late inflammation are associated with immunosuppression and increased mortality [21]. Better understanding of sirtuins’ role in regulation of inflammation and immumetabolism is in its infancy. Except for SIRT1, other sirtuin family members’ roles in immunometabolic reprogramming are largely unknown. SIRT7 in particular is abundant in immune organs. Many works focus on the glucose and fatty acid metabolism regulated by sirtuins. Amino acid abundance and composition may serve as a quality control checkpoint in immune cells [8,37], and more attention should be paid to amino acid metabolism. The architectural principles for the metabolic control of sirtuins in immune cells are shown in Fig. 6.1.

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70. [94] Zhou L, Wang F, Sun R, et al. SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense. EMBO Rep 2016;17(6):811
22. [95] Christofk HR, Vander Heiden MG, Harris MH, et al. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 2008;452(7184):230
3. [96] Zhong L, D’Urso A, Toiber D, et al. The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha. Cell 2010;140(2):280
93. [97] Vakhrusheva O, Smolka C, Gajawada P, et al. Sirt7 increases stress resistance of cardiomyocytes and prevents apoptosis and inflammatory cardiomyopathy in mice. Circ Res 2008;102(6):703
10.

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C H A P T E R

7 Mitochondrial sirtuins at the crossroads of energy metabolism and oncogenic transformation Maja Grabacka1 and Przemyslaw M. Plonka2 1

The Department of Biotechnology and General Technology of Foods, Faculty of Food Technology, University of Agriculture in Krako´w, Krako´w, Poland 2The Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, The Jagiellonian University in Krako´w, Krako´w, Poland O U T L I N E Abbreviations

104

7.10 The interplay between Sirt3 and isocitrate dehydrogenase in cancer cells

115

7.11 Tumor-suppressing and tumor-promoting activities of sirtuins in the context of glutamine and glucose metabolism

115

7.12 Sirtuins regulate iron
sulfur cluster assemblage

116

116

7.1 Introduction—advantages of possessing mitochondria

105

7.2 Mitochondrial sirtuins

106

7.3 Lipoylation of multienzymatic complexes is essential for mitochondrial metabolism

106

7.4 Alternative lipoylation and its metabolic consequences

109

7.5 Regulation of pyruvate dehydrogenase complex by mitochondrial sirtuins

110

7.13 Mitochondrial fatty acid synthesis is linked to Fe
S cluster assembly and protein lipoylation—implications for cancer cell metabolism

7.6 Alpha ketoglutarate dehydrogenase complex regulates gene expression

111

7.14 Consequences of Fe
S cluster defects in cancer cells

118

7.7 Fluctuations of the intracellular concentration of organic acids has far-reaching implications

112

7.15 Deoxyribonucleotide synthesis—toward the as yet unexplored areas of sirtuin research

119

7.8 Ketogenic enzymes ACAT1 and HMGCS2 as substrates for Sirt3 and Sirt5

113

7.16 Perspectives—evolutionary implications and new directions in cancer treatment

120

7.9 Antagonistic roles of mitochondrial sirtuins in fed and fasted state

References

121

114

Sirtuin Biology in Cancer and Metabolic Disease. DOI: https://doi.org/10.1016/B978-0-12-822467-0.00001-2

103

© 2021 Elsevier Inc. All rights reserved.

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7. Mitochondrial sirtuins at the crossroads of energy metabolism and oncogenic transformation

Abbreviations 2-HG ACADSB ACAT1 ACSM1 aKG AML BCKDC CIA Clp CPT1 D2HGDH DLAT DLD DLST EGF ETC FRDA FXN GAPDH GCS GDH GLS1 GSH GSSG HCC HIF-1α HMGCL HMGCS2 IDH IRP1, IRP2 ISC JmjC KAR1 KAT2A KDM KGDC KGDH L2HGDH LCAD LDH LECA LIAS LIPT1 LIPT2 LYRM mACO MBP MCAT MCCC MCD MDH MECR MEFs MoaA mtACP mtFAS NIF NMP NSCLC OADC OXPHOS OXSM PDC

2-hydroxyglutarate short-branched-chain acyl-CoA dehydrogenase acetyl-CoA acetyltransferase 1 acyl-CoA synthetase medium-chain family member 1 alpha ketoglutarate (2-oxoglutarate) acute myeloid leukemia branched chain ketoacyl-CoA dehydrogenase complex cytosolic iron
sulfur cluster assembly caseinolytic peptidase carnitine palmitoyltransferase D-2-hydroxyglutarate dehydrogenase dihydrolipoyl acetyltransferase dihydrolipoyl dehydrogenase dihydrolipoyl succinyltransferase epidermal growth factor electron transfer chain Friedereich ataxia frataxin glyceraldehyde-3-phosphate dehydrogenase glycine cleavage system glutamate dehydrogenase glutaminase 1 glutathione (reduced) glutathione (oxidized) hepatocellular carcinoma hypoxia inducible factor 1 alpha 3-hydroxy-3-methylglutaryl-CoA lyase 3-hydroxy-3-methylglutaryl-CoA synthase 2 isocitrate dehydrogenase iron regulatory proteins 1, 2 mitochondrial iron
sulfur cluster assembly Jumonji C domain 3-ketoacyl-ACP reductase lysine acetyltransferase 2A lysine demethylase alpha ketoglutarate (2-oxoglutarate) dehydrogenase complex alpha ketoglutarate (2-oxoglutarate) dehydrogenase L-2-hydroxyglutarate dehydrogenase long chain acyl-CoA dehydrogenase lactate dehydrogenase last eukaryotic common ancestor lipoic acid synthase lipoyltransferase 1 lipoyltransferase 2 Leucine-tyrosine-arginine (LYR) motif containing proteins mitochondrial aconitase methyl-binding domain protein malonyl-CoA:ACP transferase methylcrotonyl-CoA carboxylase complex malonyl-CoA decarboxylase malate dehydrogenase 2-enoly-ACP reductase mouse embryonic fibroblasts molybdenum cofactor biosynthesis protein A mitochondrial acyl-carrier protein mitochondrial fatty acid synthesis nitrogen fixation system nucleotide monophosphate nonsmall cell lung cancer oxoadipate dehydrogenase complex oxidative phosphorylation ketoacyl-ACP synthase pyruvate dehydrogenase complex

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7.1 Introduction—advantages of possessing mitochondria

PDH PDP1 PEP PHD PDK1
4 PKL PKM PKR PPP Ppt2 RIC RNR ROS SAM SDH SDHA, SDHB SUF TET TfR1 THF VLCAD

105

pyruvate dehydrogenase pyruvate dehydrogenase phosphatase 1 phosphoenolpyruvate prolyl hydroxylase pyruvate dehydrogenase kinases 1
4 pyruvate kinase L isoenzyme pyruvate kinase M isoenzyme pyruvate kinase R isoenzyme pentose phosphate pathway phosphopantetheine transferase 2 repair of iron clusters ribonucleotide reductase reactive oxygen species S-adenosylmethionine succinate dehydrogenase succinate dehydrogenase subunits A, B sulfur mobilization system ten-eleven translocation transferrin receptor 1 tetrahydrofolate very-long-chain acyl-CoA dehydrogenase

7.1 Introduction—advantages of possessing mitochondria Mitochondria are the organelles of vital importance for the synthesis of key cellular components and the place that integrate various metabolic processes. They carry out respiration, intertwine biosynthesis and degradation of major nutrients, and provide indispensable intermediates for other organelles. The acquisition of mitochondria was undoubtedly one of the most important steps in the eukaryotic evolution, but the exact chronology of the gradual, mutual adaptations of host and endosymbiont, as well as the calculations of the initial costs and benefits of this partnership are a subject of vivid discussions [1
3]. Nevertheless, the acquisition of mitochondria was crucial, primarily not because of energy generating advantage, but supposedly due to the provision of efficient iron
sulfur (Fe
S) cluster assembly machinery, the mitochondrial ISC (iron sulfur cluster) system. Fe
S clusters are believed to be one of the most ancient inorganic catalysts, indispensable for life in any form. This notion has been recently challenged by creating a genetically manipulated Escherichia coli strain devoid of ISC and sulfur mobilization (SUF) systems (ultimately lethal), that was able to survive, when the components of the eukaryotic mevalonate pathway synthesizing isoprenoid units were introduced [4]. Nevertheless, it is quite clear that Fe
S lacking organisms could not compete with optimally equipped counterparts and would be most likely eliminated by natural selection in a real environment. Living organisms developed three enzymatic systems for Fe
S assembly, namely prokaryotic SUF, nitrogen fixation (NIF), and ISC systems, as well as the eukaryotic cytoplasmic iron
sulfur cluster (CIA) system. Mitochondrial ISC machinery is inherited from an alpha proteobacterial endosymbiont. Although it is accepted that the last eukaryotic common ancestor (LECA) surely possessed Fe
S assembly machinery, it is not yet exactly clear which of the prokaryotic systems supported CIA in LECA [5]. It must be also noted that the first reductases utilizing Fe
S clusters must emerge as early as in the so-called “RNA World,” because it is hardly possible to imagine deoxyribonucleotide (therefore, DNA) synthesis from ribonucleotides without the participation of such enzymes as ribonucleotide reductases (RNR) [6
8]. In mammalian cells, Fe
S clusters are indispensable prosthetic groups for a huge variety of enzymes including respiratory complexes (I, II, III), Krebs cycle enzymes (aconitase, succinate dehydrogenase, fumarase), the enzymes involved in tRNA maturation (TYW1, CDKAL1, CDK5RAP1, ELP3), and DNA replication and repair (RNR, polymerases δ, ε, ζ, primase, helicases, nucleases, DNA glycosylases) [9,10]. The proper assembly of Fe
S clusters and the transfer to apoproteins is carried out by mitochondrial ISC and cytoplasmic CIA systems. Apart from being the main source of ATP, mitochondria harbor enzymatic machinery for intermediate metabolism and coenzyme biosynthesis (such as lipoic acid or heme, just to mention a few), as well as take part in the maintenance of cellular homeostasis and act as sensors of nutritional status. As a fulfillment of these roles, two main signaling routes between the mitochondrion and the nucleus have evolved: retrograde and anterograde signaling. Anterograde signaling (nucleus to mitochondria) is a consequence of progressive loss of transcriptional and translational autonomy and the increasing number of nuclear-encoded proteins with a final destination in mitochondria. Retrograde signaling (mitochondria to nucleus) provide an important feedback that can prompt

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7. Mitochondrial sirtuins at the crossroads of energy metabolism and oncogenic transformation

with rescue and repair actions in the case of defects in mitochondrial function. Efficient nuclear
mitochondrial cooperation is a matter of cell survival and in the situations of severe imbalance, mitochondria initiate the pathway to apoptosis. All these necessary functions of mitochondria are performed by a network of proteins, whose activities are finely tuned by numerous posttranslational modifications and allosteric regulation. Mitochondrial sirtuins (Sirt3, Sirt4, and Sirt5) play a crucial role in these processes directly due to their deacylation or ADP-ribosylation activities, or indirectly by regulating the concentration of important metabolites, such as acetyl-CoA, succinyl-CoA, etc.

7.2 Mitochondrial sirtuins Sirtuins have gained much attention in the last two decades, due to their apparent engagement in diverse phenomena, beginning from life span and cell survival, metabolism, and the response to nutritional status. Based on phylogenetic conservation of the core domain, mammalian sirtuins are divided into four subclasses: I—Sirt1, 2, 3 with deacetylase activity; II—Sirt4 with ADP-ribosyltransferase and lipoamidase activity; III—Sirt5 with weak deacetylase and strong desuccinylase, demalonylase and deglutarylase activities; and IV—Sirt6, 7 with both deacetylase and ADP-ribosyltransferase [11]. All sirtuins are NAD-dependent enzymes that cleave this coenzyme producing nicotinamide and 2-O-acyl-ADP ribose derivative. They possess deacylase and/or ADPribosyltransferase activities. In the last 20 years, rapidly emerging discoveries have revealed that acetylation, succinylation, and the other acyl PTMs are much more frequent in the mitochondrial proteome than was previously believed [12
14]. The constant progress in sophisticated proteomic analyses and surveys has led to the identification of new amino acid sites, which contribute to enzymatic activity or protein
protein interactions and can be regulated through acylation. Although protein acylation (especially acetylation and succinylation) is mostly a nonenzymatic process [15], the cleavage of these modifications is entirely enzymatic, executed just by sirtuins. Recently, the mitochondrial sirtuins (Sirt3, 4, 5) have emerged as the key regulators of a global enzymatic activity and metabolic fluxes through various intertwingled pathways carried out in the mitochondria, such as fatty acid oxidation, ketogenesis, the Krebs cycle, respiration, amino acid and pyruvate metabolism. The clear distinction of mitochondrial sirtuins based on their enzymatic activities is somewhat difficult, as some of the catalytic activities overlap (Table 7.1), but generally Sirt3 is regarded as deacetylase that recognizes acylated lysines of neutral charge [39], Sirt4 cleaves negatively charged moieties with branched acyl chain (methylglutaconyl, methylglutaryl, and 3-hydroxy-3-methylglutaryl), as well as biotin and lipoamide [27,29], whereas Sirt5 prefers shorter, unbranched carboxylic acid moieties (succinyl, malonyl, glutaryl) [33,36,37,39]. Multiple mitochondrial proteins are modified by acylation, which has a strong impact on their enzymatic activity. This is a part of feedback response in tuning the enzyme activity. The occupation of acetylation, succinylation, malonylation, and glutarylation sites frequently overlap (Table 7.2), creating an “acylation code” [38]. These modifications executed on lysine residues are mutually exclusive, but the degree of the overlap among the acetylome, succinylome, malonylome, and glutarylome varies depending on the tissue/ cell compartment (e.g., hepatocytes, as compared to embryonic fibroblasts and mitochondria to cytoplasm) and the nutritional status (fed or fasted) [29,38,42]. For example, in mouse embryonic fibroblasts (MEFs), 997 succinylated and 2240 acetylated sites were identified, 282 of which overlap [31]. The majority of modifications are highly prevalent in mitochondria, but some, such as malonylation, are much more frequent in cytoplasmic proteins [38]. Among mitochondrial proteins the degree of overlap between acetylation and succinylation sites reaches 80% [39], nevertheless there are some succinylated proteins present exclusively in cytoplasm or nucleus [31]. Reactive thioesters are the intermediates of all major catabolic pathways, that is, degradation of pyruvate, fatty acids, amino acids, and Krebs cycle components and the diversity in their relative abundance in various tissues might be the main reason for the different patterns in acylome in these tissues [42].

7.3 Lipoylation of multienzymatic complexes is essential for mitochondrial metabolism Lipoylation is a special kind of posttranslational modification that engages lipoic acid attachment to multimeric enzyme complexes of 2-ketoacid dehydrogenases: pyruvate dehydrogenase (PDC), alpha-ketoglutarate dehydrogenase (KGDC), branched chain ketoacyl-CoA dehydrogenase (BCKDC), oxoadipate dehydrogenase (OADC), as well as the glycine cleavage system (GCS). Lipoic acid is a sulfur derivative of octanoic acid

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7.3 Lipoylation of multienzymatic complexes is essential for mitochondrial metabolism

107

TABLE 7.1 The enzymatic activities of mitochondrial sirtuins and their selected important target proteins. Mitochondrial sirtuins

EC number

Sirt3

2.3.1.286

Enzymatic activities Deacetylase (Protein N-acetyltransferase)

Decrotonylase Sirt4

2.4.2.30

Selected substrates (References)

Regulated processes/pathways

PDH [16], mACO [17]

TCA

ACS2 [18]

Fatty acid synthesis

ACLY [19]

Fatty acid synthesis

ACAT1 [16,20]

Ketogenesis

HMGCS2 [21]

Ketogenesis

LCAD, VLCAD [22,23]

Fatty acid oxidation

ACADSB [24]

Branched chain amino acid metabolism

OTC [24]

Urea cycle

Histone H3 [25]

Transcription

ADP-ribosyltransferase GDH [26] PDH [27,28]

Lipoamidase

Demethylglutarylase MCCC [29] Demethylglutaconylase De-HMG-ase Deacetylase MCD [30] Sirt5

2.3.1.B43

Glutamine/glutamate metabolism, anaplerosis Acetyl-CoA synthesis Branched chain amino acid catabolism

Fatty acid synthesis /fatty acid oxidation balance

PDH, SDH, IDH [31]

Acetyl-CoA synthesis, TCA

HMGCS2, ACAT1 [32]

Ketogenesis

VLCAD [23], HADHA [31]

Fatty acid oxidation

CPS1 [33]

Urea cycle

GLS1 [34]

Glutaminolysis

PKM2 [35]

Glycolysis

Deglutarylase

CPS1, HADHA [36]

Urea cycle

Demalonylase

CPS1 [37,38]

Urea cycle

Aldolase, GAPDH and other glycolytic enzymes [38]

Glycolysis

Desuccinylase Deacetylase (weak)

The abbreviations of enzyme names are explained in the abbreviations section.

(Fig. 7.1A), synthesized in the FASII mitochondrial fatty acid pathway (mtFAS) and covalently linked to a lysine residue in the evolutionary conservative lipoyl-binding domain, through an amide bond [28]. Lipoamide cofactor is crucial for the redox reactions as it may cyclically form reduced thiol groups (dihydrolipoamide), oxidized disulfide, or a S-adduct with a ketoacid substrate. Lipoamide forms a “swinging arm,” which facilitates the substrate shuttle within the multiprotein complexes and the transfer to coenzyme A (Fig. 7.1B) [43]. These enzymatic complexes have a similar structure that consists of three main components, E1, E2, and E3 (Fig. 7.1B). The E2 component (dihydrolipoyl acetyltransferase, DLAT, in PDC; dihydrolipoyl succinyltransferase, DLST, in KGDC; lipoamide acyltransferase in BCKDC, and protein H in GCS, oxoadipate dehydrogenase complex shares E2 and E3 components with KGDC) possesses lipoamide. E1 components catalyze substrate decarboxylation using thiamine pyrophosphate, E2 components generate acyl-CoA by means of lipoamide reduction to dihydrolipoamide, and the third component E3 regenerates lipoamide by the redox reactions that utilize FAD and NADH coenzymes. Interestingly, the components of all five lipoyl-modified enzyme complexes possess acetylation and succinylation sites targeted by Sirt3 and Sirt5 [31,39,40]. All these enzymatic complexes have a role in the mitochondrial metabolism and are the source of numerous reactive acyl-CoA thioesters: PDC produces acetyl-CoA, a two-carbon unit for sustaining the Krebs cycle; KGDC

I. Sirtuins and metabolic disease

TABLE 7.2 The selected examples of lysine acylome overlapping in the mitochondrial proteins involved in various metabolic pathways. Potential number and type of acylated sites

The particularly important modified lysine residues

Protein

Ac

Suc

Mal

Glu

Carbamoyl phosphate synthase 1 (CPS1)

55

41




43

K55, K219, K412, K892, K915, K1360, K1486

Urea cycle

Ornithine carbamoyltransferase (OTC)

9

11




11

K88

Urea cycle

3-Hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2)

12

15







K83, K310, K447, K473

Ketogenesis

Acetyl-CoA acetyltransferase (ACAT1)

12

12

6

11

K174, K181, K251, K263, K268, K273

Ketogenesis

3-Hydroxyacyl-CoA dehydrogenase (HADH)

11

11




11

K81, K185

Beta oxidation

Trifunctional enzyme, A subunit (HADHA)

25

25




15

K295, K406, K505, K540, K644

Beta oxidation

Very long chain acyl-CoA dehydrogenase (ACADVL)

10

10







K298, K299, K482, K492, K507

Beta oxidation

Succinate dehydrogenase subunit A (SDHA)

18

10

1

13

K179

Krebs cycle, respiration

Malate dehydrogenase 2 (MDH2)

14

14

3

13

K185, K301, K307, K314

Krebs cycle

Malonyl-CoA decarboxylase (MCD)

6

2







K471

Acetyl-CoA synthesis

PDHA (E1 component of Pyruvate dehydrogenase complex (PDC))

5

5







K321

Acetyl-CoA synthesis

Dihydrolipoyl acetyltransferase (DLAT, E2 component of PDC)

1

2







K132, K259—Lipoylated

Acetyl-CoA synthesis

Dihydrolipoyl dehydrogenase (DLD, E3 component of PDC, KGDC, BCKDH)

10

12







K143, K410, K417

Acetyl-CoA synthesis; Krebs cycle, Amino acid metabolism

PDHX (PDC component X)

1

1







K97 lipoylated

Acetyl-CoA synthesis

ATP synthase subunit O

9

6

2

K90, K162

Respiration

Glutamate dehydrogenase (GDH)

24

19

3




Amino acid metabolism

Methylcrotonyl-CoA carboxylase (MCCA)

13

6




4a

K84, K480, K503 C172—ADP-ribosylated K66, K655, K683, K667—biotinylated active site

a Glutaryl moiety derivatives: methylglutaryl-, 3-hydroxy-3-methylglutaryl-, methylglutaconyl- modifications at K66, K432, K490, K501, K677, K691, K692. Yet the significance of PTMs at a particular site and stoichiometry of a given modification, is still to be evaluated (based on [16,20
24,29,31,32,37,38,40,41]). Ac, Acetylation; Glu, glutarylation; Mal, malonylation; Suc, succinylation.

Pathway and action

Amino acid metabolism

7.4 Alternative lipoylation and its metabolic consequences

109

FIGURE 7.1 Lipoic acid is a crucial cofactor in the 2-oxoacid dehydrogenase complexes. Pyruvate dehydrogenase serves as an example of such a lipoylated enzyme complex. (A) synthesis of lipoic acid moiety from octanoyl acid, a product of the mitochondrial fatty acid synthesis pathway (mtFAS). Mitochondrial acyl-carrier protein (ACP) provides octanoyl residue, which is transferred to glycine cleavage system component, protein H, by octanoyltransferase (LIPT2). Lipoic acid synthase (LIAS) inserts sulfur atoms from Fe
S cluster and S-adenosylmetionine (SAM) to form lipoic acid. Lipoate is then transferred by lipoyltransferase LIPT1 from H protein to the lysine residue on the apo-pyruvate dehydrogenase E2 component. (B) Pyruvate dehydrogenase complex consists of three types of subunits: pyruvate dehydrogenase (PDH) E1 that decarboxylates pyruvate to acetyl moiety using thiamine pyrophosphate prosthetic group (TPP); dihydrolipoyl acetyltransferase (DLAT) is a E2 component that employs lipoamide to transfer the acetyl group from TPP to CoA-SH, forming acetyl-CoA; finally dihydrolipoate is oxidized back by dihydrolipoyl dehydrogenase (DLD) E3 component, which is a flavoprotein that uses FAD and NAD as electron acceptors.

produces succinyl-CoA in the Krebs cycle as well; the BCKDC and branched chain amino acid degradation pathway is a source of several acyl-CoAs (isovaleryl, methylbutyryl, isobutyryl), which ultimately generate acetylCoA and succinyl-CoA. The GCS decarboxylates glycine and participates in the methylation of tetrahydrofolate (THF) to form 2,10-methylene-THF, which is an intermediate in the formation of methyl group donors (such as S-adenosylmethionine, SAM). These compounds are necessary for protein and DNA methylation and epigenetic regulation [44]. Importantly, the SAM radical family of enzymes comprises tRNA nucleoside thiolating enzymes (TYW1, ELP3, CDK5RAP1), lipoic acid synthase (LIAS), or molybdenum cofactor biosynthesis protein A (MoaA) [9]. The common feature of these enzymes is the usage of [4Fe
4S] clusters during catalysis and cleavage of SAM to 5-adenosyl radical (5-dA*) and methionine [43]. Mammalian lipoic acid biosynthesis and protein lipoylation is linked to mitochondrial fatty acid synthesis (mtFAS). Mitochondrial acyl-carrier protein (mtACP) is especially important in this pathway. mtACP is a donor of the octanoyl chain to octanoyltransferase (LIPT2), which moves the octanoate from ACP to the protein H of GCS. Then LIAS uses its two [4Fe
4S] clusters, SAM, and an additional Fe
S containing scaffold protein to insert two sulfur atoms into the acyl chain and to form lipoic acid moiety [45] (Fig. 7.1A). One of its Fe
S clusters serves as a sulfur donor and is destroyed during catalysis, but then is reassembled with the help of a Fe
S donating scaffold protein (see also Section 7.13). Methionine and 5-dA* are released in the reaction, too [43]. The formed lipoic acid is transferred finally from H protein to E2 subunits of 2-ketoacid dehydrogenase complexes by lipoyltransferase LIPT1.

7.4 Alternative lipoylation and its metabolic consequences Recently, a novel mechanism that controls protein lipoylation has been discovered [46]. The polymerase δ-interacting protein 2 (Poldip2) has been shown to regulate lipoylation of PDC and KGDC taking part in the, so-called, salvage pathway. This is a process of exogenous lipoic acid utilization, an alternative to lipoic acid de novo synthesis. The acyl-CoA synthetase medium-chain family member 1 (ACSM1), which participates in this process, activates the absorbed lipoic acid to a nucleotide conjugate, lipoyl-NMP. This enzyme possesses multiple acetylation and succinylation sites recognized by Sirt3 and Sirt5 [31,39,40].

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In mitochondria, Poldip2 binds to CLPX protein, which is a subunit of proteasome-like caseinolytic peptidase (Clp) complex. CLPX acts as an ATP-dependent unfoldase that controls the entry of proteins intended for degradation into the Clp proteolytic cavity. The Poldip2 binding inhibits the Clp function by sequestration of its necessary component. Lipoic acid-activating medium chain acyl-CoA synthetase ACSM1 is one of the Clp substrates, so Poldip2 deficiency leads to ACSM1 degradation and impaired PDC and KGDC lipoylation, which negatively affects their activity, as well as the respiration [46]. Moreover, the Poldip2 knockdown induces hypoxia-like metabolic reprogramming, regardless of the oxygen concentration. This reprogramming is manifested by enhanced glycolysis, upregulation of HIF-1α, and activation of PDKs, which phosphorylate PDH E1 and exert even deeper PDC inhibition. The HIF-1α stabilization in Poldip2-deficient cells, which leads to a pseudohypoxia state, was the most likely consequence of the impaired Krebs cycle flux due to inactive PDC and KGDC and low aKG levels. This mechanism may significantly contribute to glycolytic phenotype and OXPHOS restriction in cancer cells. Indeed, certain breast cancer cell lines (MB231, BT549) that exhibit highly glycolytic metabolism also have a very low Poldip2 protein level [46].

7.5 Regulation of pyruvate dehydrogenase complex by mitochondrial sirtuins The activity of the pyruvate dehydrogenase complex determines the fate of major energy substrates: the rate of glucose utilization, the Krebs cycle flux, and the production of reducing equivalents used in the electron transfer chain (ETC) and oxidative phosphorylation (OXPHOS), as well as provision of intermediates for lipid synthesis. Glucose utilization has far-reaching consequences for the systemic energy balance in the multicellular organism, so the PDC activity is tightly regulated by various mechanisms: by covalent modifications such as phosphorylation and acetylation; by allosteric regulation; and by lipoamide cofactor removal. The PDC phosphorylation and dephosphorylation is carried out by pyruvate dehydrogenase kinases (PDK1
4) and phosphatases (PDP1, and 2), respectively. The enzymatic removal of lipoamide is a recently discovered regulatory mechanism carried out by Sirt4, present both in Pro- and Eukaryota [27,28]. The other regulatory mechanism, well-studied in the case of PDC, involves serine phosphorylation of the PDH E1 subunit (S232, S293, and S300) by PDKs, which leads to inhibition of catalytic function. PDKs’ activity is also regulated either by allosteric activators (NADH, acetyl-CoA, and ATP) or by phosphorylation by signal transducing kinases. Phosphorylation of PDKs is associated with their increased activity. Various PDK isoforms (PDK1
4) are frequently overactive in cancer: their expression is upregulated by oncogenes (c-Myc) and by HIF-1α during hypoxia, and their activity is increased by phosphorylation, for example, phospho-Tyr 243 in PDK1, by oncogenic kinases that transduce signal from growth factor receptors [47
49]. The PDK-mediated inhibition of PDC significantly contributes to the, so-called, Warburg effect and metabolic reprogramming of cancer cells toward glycolysis dependence and limited ATP generation through OXPHOS. Recent studies have pointed to the role of PDP1, which removes serine phosphorylation induced by PDKs. PDP1 is loosely associated with PDC through the interaction with the lipoyl-binding domain of E2 component in the presence of calcium ions [50]. PDP1 itself is regulated by posttranslational modifications: phosphorylation and acetylation, posing a higher-level control of PDC activity. PDP1 is phosphorylated on two tyrosine residues (Y94 and Y381) by kinases that transduce signal from growth factor receptors (e.g., epidermal and fibroblast growth factor receptors). Y94 phosphorylation inhibits PDP1 ability to bind lipoic acid and destabilizes the association with the E2 PDC component, whereas Y381 phosphorylation recruits acetyl-CoA acetyltransferase (ACAT1) to the PDP1-PDC complex with the simultaneous dissociation of Sirt3 [16]. Actually, PDP1 being a part of the PDC complex, provides a physical connection between PDC and ACAT1 and Sirt3, the two proteins that exert opposite effects on the PDC activity [16]. ACAT1 catalyzes acetylation of both PDP1 and PDH E1 components on K202 and K321, respectively. The binding of ACAT1 facilitates the recruitment of PDK1 and subsequent PDH E1 phosphorylation and inhibition. On the other hand, when PDP1 is not phosphorylated, Sirt3 is associated with the complex, keeping PDH E1 deacetylated and active. In this fashion, the PDP1 phosphorylation and acetylation status decides the overall PDC activity [16]. Multiple phosphorylation events (activating phosphorylation of PDKs, inhibiting phosphorylations of PDP1 and PDH E1) result from growth factor receptor kinases, which are frequently hyperactive in cancer cells. This growth factor-stimulated signaling serves as a trigger for the metabolic switch from oxidative pyruvate metabolism and OXPHOS (active PDC) toward intensive glycolysis and reduction of pyruvate to lactate (inactive PDC). Indeed, epidermal growth factor (EGF) signaling, for instance, stimulates glycolysis and promotes proliferation

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7.6 Alpha ketoglutarate dehydrogenase complex regulates gene expression

111

FIGURE 7.2 Mitochondrial sirtuins, Sirt3, Sirt4 and Sirt5 differentially affect the enzymatic activity of several key mitochondrial dehydrogenases: pyruvate dehydrogenase complex (PDC), alpha-ketoglutarate dehydrogenase complex (KGDC), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH2). The arrows indicate positive modulation and blunt endings indicate negative impact on the enzymatic activity. Sirt4 lipoamidase activity and KGDC inhibition has been demonstrated in gram-positive and gram-negative bacteria [28], it is possible but not yet demonstrated in mammalian cells, therefore is indicated by question mark. Altered activities of these dehydrogenases lead to the imbalance of the intracellular concentrations of organic acids: succinate, fumarate, alpha-ketoglutarate (aKG). IDH1/2 mutations frequently present in particular types of cancer cells gain a neomorphic activity to form an oncometabolite 2-hydroxyglutarate. Succinate and 2-HG are inhibitors of aKG-dependent dioxygenases (TET and JmjC-domain proteins) and protein hydroxylases (PHD). Further consequences involve hypoxia-inducible factor HIF-1α stabilization, metabolic reprogramming (aerobic glycolysis, so called Warburg effect in cancer cells) and modified methylation patterns (see text).

in cancer cells [51]. In this context, Sirt3, which contributes to sustaining PDC activity, counteracts metabolic reprogramming during oncogenic transformation. Apart from deacetylation by Sirt3, PDC is also a target for Sirt5-mediated desuccinylation. A proteomic analysis revealed multiple succinylation sites on all the PDC subunits, with the highest abundance on PDH E1 [31]. PDC in embryonic fibroblasts isolated from Sirt52/2 mice was hypersuccinylated, and the treatment of purified PDC with Sirt5 resulted in desuccinylation and inhibition of the holoenzyme activity. Similarly, Sirt5 knockdown increased PDC activity as compared to control [31]. In summary, it seems that PDC is a subject of an intense activity from all three mitochondrial sirtuins, with Sirt3 exerting upregulation in contrast to Sirt4 and Sirt5 that inhibit or decrease PDC activity (Fig. 7.2). Correspondingly to the case of PDC, Sirt3 and Sirt5 exert the opposite actions on another protein crucial both for the Krebs cycle function and respiration, namely, succinate dehydrogenase (SDH). SDH is a heterotetramer consisting of two membrane-bound heme-possessing SDHC and SDHD subunits: a SDHB subunit carrying three Fe
S clusters and a SDHA subunit with a covalently bound FAD cofactor. SDHC and D along with the B subunit form a ubiquinone-binding site. Thirteen lysine residues of SDHA located on the protein surface can be modified by acetylation (Table 7.2). Sirt3 directly interacts with SDHA and removes this modification, which leads to increased activity of the respiratory complex II [52,53]. SDHA is also a target of succinylaton with a considerable degree of overlap with acetylation, that is, K179, 250, 335, 485, 498, 538, and 547 can be either acetylated or succinylated [31,53]. Interestingly, SDHB K276 has been identified as a succinylation, but not acetylation site. Sirt5driven desuccinylation was shown to suppress SDH activity, and concordantly, knockdown of Sirt5 induces SDH activity and overall respiration rate, as compared to the control [31].

7.6 Alpha ketoglutarate dehydrogenase complex regulates gene expression Recently, an unexpected activity of KGDC in the histone modification has been discovered in human glioma cell lines [54]. The KGDC complex mainly resides in mitochondria, but one of its components, alpha ketoglutarate dehydrogenase (KGDH), possesses a putative nuclear localization signal and a small fraction of KGDC acts in the nucleus as a binding partner for a histone acetyltransferase KAT2A. The KAT2A enzyme utilizes succinylCoA, generated by KGDC, as a substrate for histone H3 succinylation on K9. The highest enrichment in H3 succinylation was detected in the promoter regions of genes involved in cell cycle regulation or activated growth

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factor signal transduction pathways. The KAT2A knockdown significantly reduced the histone succinylation in these regions and inhibited the expression of the target genes [54]. These results point to the important epigenetic regulatory function of KGDC, apart from its metabolic roles. This context encourages us to imagine Sirt4 as a KGDC lipoamidase that could modulate gene expression patterns through this mechanism, too (Fig. 7.2).

7.7 Fluctuations of the intracellular concentration of organic acids has far-reaching implications SDH is believed to act as a tumor suppressor, and mutations in the genes coding its subunits are observed in certain hereditary malignancies, particularly of neuroectodermal origin, such as paraganglioma and pheochromocytoma [55,56]. Inactivating mutations of SDH leads to the impaired Krebs cycle flux and accumulation of succinate. Depletion of SDH in yeast cells leads to eightfold increase in succinate with a concomitant decrease in intracellular alpha-keto glutarate (aKG) to a barely detectable level [57]. Succinate is a competitive inhibitor of aKG-dependent protein hydroxylases, such as ten-eleven translocation (TET) dioxygenases, Jumonji-C domain (JmjC)-containing histone demethylases, and prolyl hydroxylases (PHDs). TET proteins and JmjC-domain histone demethylases play an important role in the epigenetic control over the expression of protooncogenes and tumor suppressor genes. TET dioxygenases (TET1, 2, 3), including aKG and ferrous ion (Fe(II))-dependent dioxygenases, are involved in removing a methylation pattern in DNA through iterative oxidations of 5-methylcytosines (mainly present in CpG dinucleotides) to 5-hydroxymethyl-, 5-formyl- and 5-carboxylcytosine [58,59]. In order to achieve a complete demethylation, the latter two cytosine derivatives are further subjected to base-excision repair mechanism by DNA glycosylases and substituted by cytosines [60,61]. DNA methylation in the gene promoter regions is usually associated with transcriptional repression and gene silencing. 5-Methyl-C (5-mC), 5-hydroxymethyl-C (5-hmC), 5-formyl-C (5-fC), and 5-carboxyl-C (5-caC) are recognized by the specialized distinct set of “reader” proteins. Methyl-CpG binding protein 2, methyl-binding domain proteins (MBPs) and Uhrf1 among others, recognize methylated cytosines, whereas another set of “chromatin readers” prefer binding to nonmethylated cytosines or 5-hmC with a very little overlap [62]. The pattern and abundance of cytosine methyl-modification is not only tissue specific (with a particularly high hmC presence in brain), but also depends on the developmental stage and is associated with the transition from pluripotency to a differentiated state [62,63]. It is not surprising then, that TET proteins dysregulation (e.g., loss of function mutations, missense mutations) and subsequent alterations in cytosine methyl- and oxidized methyl derivatives are frequently observed in cancers, such as acute myeloid leukemia (AML) [64]. JmjC-domain-containing histone demethylases (KDMs) are an important group of enzymes involved in epigenetic regulation on the level of modulation of histone methylation pattern. Recent studies revealed that a removal of some or all methyl residues from lysines (they can be mono-, di-, or tri-methylated) in histone tail regions is quite a dynamic process, important for chromatin remodeling, and activation or repression of gene transcription. The characteristic JmjC domain is a double-stranded β helix fold that coordinates Fe(II) and aKG during the catalysis. The demethylation reaction is carried out by hydroxylation of a methyl group with oxygen, aKG, Fe(II) as substrates and succinate, CO2, and Fe(III) are released as products. Ascorbate is necessary for the regenerative reduction of Fe(III) to Fe(II), during which dehydroascorbate is formed [65]. The newly formed hydroxymethyl group (i.e., hydroxymethyl hemiaminal intermediate) spontaneously decomposes to formaldehyde, leaving a demethylated lysine [66]. KDMs are subdivided into several families (KDM2
7) that demethylate mainly histone H3 at the K4, K9, K27, and K36 sites [67]. KDMs have been implicated in cancer both as tumor suppressors or oncogenes, depending on the cancer cell type [68]. For instance, in the case of KDM5 (JARID1) the tumor suppressive activities included improving genomic stability, cooperation with retinoblastoma protein (pRB) in pancreatic cancer, and induction of cellular senescence [69,70], whereas in breast cancer JARID1B has been revealed as an oncogene [71]. JARID1B has been also implicated in the maintenance of cancer stem cell potential in melanoma cells and support of continuous tumor growth [72]. Scarce levels of aKG lead to inhibition of prolyl hydroxylases such as EglN1. Normally, these enzymes catalyze protein hydroxylation on proline residues using aKG and oxygen as substrates and producing carbon dioxide and succinate. HIF-1α is one of the most important substrates of PHD, implicated in oncogenesis, malignant transformation, and tumor metabolic adaptation to hypoxia [47]. Hydroxylated HIF-1α is ubiquitinated by von Hippel Lindau (VHL tumor suppressor) ubiquitin ligase and subjected to proteolytic degradation [73]. HIF-1α has a very short halflife (roughly several minutes), but in the low oxygen conditions PHD are not active, HIF-1α is not hydroxylated,

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which remarkably improves its stability [74]. In case of aKG depletion this situation also takes place regardless of oxygen concentration, which leads to a pseudohypoxia state. HIF-1α is responsible for enforcing glycolysis, block of respiration, and development of an invasive, metastatic phenotype. As described above, the decreased intracellular concentration of aKG and high level of succinate may have profound consequences in the alterations of DNA and histone methylation pattern, as well as facilitate adaptation of cancer cells to hypoxia through the stabilization of HIF-1α. With an even more disturbing effect on the activity of aKG-dependent enzymes are two glutaric acid derivatives, namely D-2-hydroxyglutarate and L-2-hydroxyglutarate (2-HG), which structurally resemble closely aKG. D-2-HG is a product of particular mutants of isocitrate dehydrogenase IDH1 or IDH2 that gain neomorphic activity of aKG reduction to D-2-HG. The mutations responsible for that change affect one of the three arginine residues crucial for isocitrate binding: R132 in IDH1 or R140 and R172 in IDH2 protein and R residue is substituted with another polar amino acid, usually H, C or S [75]. These mutations are remarkably frequent (60%
80%) in grade II and III astrocytomas, secondary gliomas, and AML (20%) [76
78]. IDH1 (present mostly in cytoplasm and peroxisomes) and IDH2 (mitochondrial) are structurally similar and in usual conditions perform NADP-dependent oxidative decarboxylation of isocitrate to aKG. The third IDH enzyme, mitochondrial IDH3 is not related to IDH1 and 2, and is a bona fide Krebs cycle enzyme using NAD. In hypoxic conditions, the cancer cells, particularly those addicted to avid glutamine consumption, perform glutaminolysis, which is glutamine deamination to glutamate and subsequent oxidation of glutamate to aKG (anaplerosis). In these cells, IDH1 and 2 perform reductive carboxylation of aKG to isocitrate, which is further converted to citrate and exported from mitochondria to cytoplasm, where it is a substrate for fatty acid synthesis. Hypoxic conditions and low pH promote accumulation of both D- and L-2-HG, as result of mutant IDH1/2 or moonlighting activities of lactate and malate dehydrogenases (LDH, MDH) [79
81]. Both D- and L-2-HG inhibit aKG-dependent enzymes in a similar fashion to succinate (see above), and indeed the hypoxia-induced L-2-HG accumulation was shown to alter the histone methylation pattern through inhibition of KDM4C [79]. Both 2-HG enantiomers can be oxidized to aKG by FAD-dependent D-2-HG and L-2-HG dehydrogenases (D2HGDH and L2HGDH, respectively). These flavoproteins are located at the inner mitochondrial membrane and transfer the electrons derived from 2-HG to FAD and later to ubiquinone in the ETC, supporting the mitochondrial membrane potential maintenance during hypoxia [80]. 2-HG aciduria and the D2HGDH and L2HGDH deficiencies have been linked to development of certain malignancies, such as diffuse large B-cell lymphoma and invasive brain tumors [82,83]. Loss of TET2 and IDH1/2 neomorphic mutations result in an overlapping genomic cytosine hypermethylation pattern in AML, which results in increased expression of stem cell markers (c-kit) and impaired myeloid differentiation [84]. These changes significantly contribute to a malignant phenotype of this type of cancer. Concluding, the perturbations in the Krebs cycle flux lead to imbalance in the concentrations of the intermediate metabolites, namely aKG, succinate, and fumarate, which exert a significant impact on the epigenetic regulation of gene transcription. Mitochondrial sirtuins, by affecting PDC, KGDC, and SDH activity, have a crucial role in the control of the Krebs cycle flux and indirectly influence the transcriptional profile (Fig. 7.2).

7.8 Ketogenic enzymes ACAT1 and HMGCS2 as substrates for Sirt3 and Sirt5 Protein acetylation by ACAT1 is quite unexpected, because this enzyme is involved in ketogenesis and produces acetoacetyl-CoA from two acetyl-CoA molecules. This novel role of ACAT1 directly links this protein with tumor progression. Fan and colleagues demonstrated that knockdown of its expression severely impairs cancer cell proliferation under hypoxia (naturally occurring in solid tumors), because it slows down glycolysis. Human lung cancer cells lacking ACAT1 grew much slower and formed significantly smaller tumors in athymic mice, showing reduced proliferative capacity [16]. Most likely, these cells had their metabolism fixed on pyruvate oxidation, which was difficult in the hypoxic tumor microenvironment, and were unable to adapt to anaerobic glucose utilization [16]. In light of these results, ACAT1 seems to contribute to the Warburg effect (intense glycolysis despite the presence of oxygen), frequently observed in transformed cells of various origins. As mentioned in Section 7.4, ACAT1 and Sirt3 affect PDC in the opposite directions through modifications of PDH E1 acetylation status. Another intriguing interaction between Sirt3 and ACAT1 in the context of regulation of the cellular response to nutritional stimuli, was reported in the liver mitochondria from fasted and refed mice [20]. A proteomic analysis showed that several proteins involved in major mitochondrial metabolic processes (Table 7.2) possess multiple sites with dynamically changing acetylation status. Among these proteins ACAT1

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was revealed to be an enzyme involved in the majority of these pathways, namely ketogenesis, fatty acid oxidation, and branched chain amino acid metabolism. Three acetylated lysin residues were identified as Sirt3 targets: K187, K260, and K265 in murine protein (K190, K263, and K268 in human protein). Acetylation at these sites (especially K260 and K265, adjacent to CoA binding site) changed the charge in the catalytic pocket and inhibited ACAT1 enzymatic activity, by affecting the basic kinetic parameters: decreasing maximal velocity (Vmax) and increasing the Michaelis constant (Km) for CoA [20]. During fasting, Sirt3 deacetylated ACAT1 enhancing its activity, which has an important role in adaptation to decreased nutrient availability. Interestingly, K265 is also succinylated and desuccinylated by Sirt5. Embryonic fibroblasts-derived Sirt52/2 mice show 120-fold increased abundance in K265 succinylation compared to the wild phenotype [31]. Sirt3 and Sirt5 have also been shown to deacylate and activate 3-hydroxy-3-methylglutaryl synthase 2 (HMGCS2), the enzyme catalyzing the rate-limiting step of ketogenesis. Both sirtuins target multiple lysine residues on HMGCS2, but K301 seems to be particularly important for the enzymatic activity [21,32,39]. In the three-dimensional structure of the protein, this residue lies in close proximity to a 30 -phosphate group of acetyl-CoA, and the acetylation or succinylation of this residue eliminates a positive charge necessary for the proper interaction with the substrate. The deacylation of HMGCS2 by Sirt3 and Sirt5 greatly induces its activity and promotes ketogenesis [21,32]. At this time Sirt3 and Sirt5 act concordantly, not oppositely, as was the case for PDC. The relation between ketone body metabolic pathway and oncogenic phenotype is puzzling, because numerous previous reports indicated that the malignant cells have difficulties in performing ketone body oxidation, which is the process that engages ACAT1 activity [85,86]. ACAT1 is involved both in ketogenesis, which is the process of ketone body (acetoacetate and 3-hydroxybutyrate) synthesis, and in ketolysis, that is, the ketone body catabolic pathway. Nevertheless, some particular cancer cells, such as melanoma cells harboring the activating mutation (V600E) in the protooncogenic kinase BRAF, have been shown to benefit from the synthesis of acetoacetate, one of the ketone bodies [87,88]. In these cells, acetoacetate-generating HMG-CoA lyase (HMGCL) expression is crucial for sustaining proliferative potential [87]. The activating point mutation of BRAF (V600E) is very frequent in melanoma, present roughly in 50% cases, and confers a highly proliferative, particularly invasive, and glycolytic phenotype [89,90].

7.9 Antagonistic roles of mitochondrial sirtuins in fed and fasted state The cooperation between Sirt3 and Sirt5 in the promotion of ketogenesis is a part of the bigger picture of these two sirtuins as the chief coordinators of the cellular response to fasting. Their expression is higher in the liver mitochondria of fasted mice, compared to the fed animals [91]. Both Sirt3 and Sirt5 are also involved in the positive regulation of fatty acid beta oxidation, by deacylation and activation of the enzymes involved in this process, such as long-chain and very-long-chain acyl-CoA dehydrogenases (LCAD, VLCAD). Other enzymes, for example, trifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketothiolase), have also numerous Sirt3- and Sirt5-targeted lysine residues [22,39]. Particularly, Sirt3 and Sirt5 target K299 of VLCAD, which is important for FAD cofactor binding and the catalytic activity. Sirt5 desuccinylates also K482, K492, and K507, which are responsible for the interaction with cardiolipin and the inner mitochondrial membrane binding [23]. In contrast to Sirt3 and Sirt5, Sirt4 is active in the nutrient abundant conditions and inhibits fatty acid oxidation in muscle and promotes lipid anabolism in adipose tissue [30]. Malonyl-CoA (a substrate for cytosolic de novo fatty acid synthesis) inhibits carnitine palmitoyltransferase 1 (CPT1), the enzyme that controls long fatty acyl chains’ entrance to mitochondria for beta oxidation. Malonyl-CoA decarboxylase (MCD) cleaves malonyl-CoA into CO2 and acetyl-CoA. MCD is acetylated on six lysine residues: K58, K167, K316, K388, and K444 and on evolutionary conservative K210 and K471 [30,92]. Sirt4 possesses a weak deacetylase activity specifically toward K471. MCD with K471Ac exhibits elevated enzymatic activity that promotes malonyl-CoA decomposition and intensifies fatty acid oxidation. In the fed state, Sirt4 deacetylates MCD K471, which reduces its enzymatic activity and the malonyl-CoA can enter fatty acid synthesis as a substrate. Depletion of Sirt4 in hepatocytes increases fatty acid oxidation and the Sirt4 2/2 mice present better exercise capacity and lower weight gain than wild-type littermates, when fed with a high-fat diet [30,93]. In this way, Sirt4 stimulates lipogenesis in nutrient-rich conditions and plays an opposite physiological role to Sirt3 and Sirt5.

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7.10 The interplay between Sirt3 and isocitrate dehydrogenase in cancer cells The studies performed in MEFs derived from Sirt32/2 mice directly demonstrated the role of Sirt3 as a mitochondrial tumor suppressor: maintaining the proper cellular defense against reactive oxygen species (ROS), genotoxic and metabolic stress factors, and protection of genome stability [94]. Sirt3-deficient cells exhibited a tumor permissive phenotype that facilitated immortalization and subsequent oncogenic transformation with tumorforming ability in vivo [94]. The metabolic consequences of Sirt3 loss were typical for neoplastic reprogramming, with increased glycolytic rate and decreased activity of respiratory complexes I and III, leading to overall inhibition of OXPHOS. This action of Sirt3 as a tumor suppressor is linked to maintaining redox homeostasis through maintaining the proper reduced-to-oxidized glutathione ratio (GSH/GSSG). This is carried out by deacetylation and activation of IDH2 that generates NADPH, a reducing equivalent important for glutathione reductase/thioredoxin cycling [95]. The acetylation of IDH2 K413 prevents the homodimerization of the enzyme and severely impedes its activity. Sirt3-mediated deacetylation of IDH2 restores the dimerization and increases the activity (Fig. 7.2). The cells expressing acetylation-mimetic IDH2 mutant (K413Q) show elevated intracellular ROS levels, metabolic shift toward glycolysis, and tumor-permissive phenotype [96]. The dependence of Sirt3 tumor suppressing activity on IDH2 seems unexpected, bearing in mind the high frequency of IDH1/2 mutations in glioma and AML. However, the analysis of randomized controlled trials and prospective and retrospective studies on malignant brain tumor patients indicate that these IDH1/2 R132H/R172 mutations are associated with better prognosis and higher overall survival rate [97]. Sirt3 or IDH1/2 loss create a transformation-permissive environment, but of course are only small elements of the whole picture.

7.11 Tumor-suppressing and tumor-promoting activities of sirtuins in the context of glutamine and glucose metabolism In healthy nontransformed cells, the exposure to DNA damaging agents induces DNA repair response and concomitantly, the cell cycle is arrested. Cell cycle block provides a safety mechanism meant to limit genomic instability and to prevent the increased mutation incidence in the cell progeny. The failure to block cell proliferation in the conditions of genotoxic stress leads to genomic instability, which is an intrinsic feature of oncogenic transformation and frequently observed in various cancer types. Highly proliferative cells, including cancer cells, have obviously an increased demand for deoxyribonucleotide supply to satisfy replication and DNA repair. These cells avidly consume glutamine, which fuels the Krebs cycle flux through anaplerosis. In this process, glutamine is first deaminated to glutamate by glutaminase (GLS1) and next glutamate is converted to aKG by glutamate dehydrogenase (GDH). aKG acts as a carbon supply for the Krebs cycle, and can be either oxidized to succinyl-CoA by KGDH, as happens in the nontransformed cells, or reductively carboxylated to isocitrate by the reverse reaction catalyzed by IDH2 in cancer cells, mainly during hypoxia. The glutaminolytic flux is an important metabolic signal that supports proliferation. Sirt4 has been shown to act as a metabolic tumor suppressor through the repression of glutamine-driven anaplerosis in response to genotoxic stress and DNA damage [98]. The mechanism of this action involves Sirt4-driven inhibition of both GLS1 and GDH. The latter enzyme is ADP-ribosylated by Sirt4, which negatively impacts its catalytic activity [26]. It is not entirely clear if there is a direct interaction between GLS1 and Sirt4, nevertheless GLS1 has two putative succinylation sites: K130 and K164, that might be sirtuin deacylation targets. The inhibition of glutaminolysis by Sirt4 results in decreased glutamine-derived carbon flux to the Krebs cycle, which is a blocking signal for cell cycle progression during the genotoxic stress of either irradiation or exposure to chemical DNA-damaging agents [98]. Sirt4-deficient cells exhibited much faster clonogenic growth and tumor formation in the mouse xenograft model than wild-type counterparts, which the illustrates tumor suppressive activity of Sirt4. Interestingly, recent studies by Greene and collaborators have shown that Sirt5 has a totally opposite effect on the glutamine metabolism than Sirt4. Sirt5-mediated desuccinylation of GLS1 protects it from ubiquitination and proteolytic degradation and therefore contributes to maintenance of stable high protein levels of GLS1 [34]. Sirt5 activity supports glutaminolysis, which translates into enhanced proliferation and breast cancer tumorigenesis. The study revealed also that the high Sirt5 expression level correlated with poor prognosis for the breast cancer patients [34]. Sirt5 is also overexpressed in nonsmall cell lung cancer (NSCLC) and has been linked to drug resistance and higher probability of recurrence, appearing therefore as a poor survival prognostic factor [99].

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The cancer-promoting activities of Sirt5 are not limited only to glutaminolysis. This sirtuin demalonylates and activates eight out of ten glycolytic enzymes (including GAPDH, aldolase, phosphoglucose isomerase, and pyruvate kinase) [38], therefore facilitating acquisition of glucose-addicted phenotype and the Warburg effect. A study by Xiangyun and coauthors [35] presents pyruvate kinase M2 isoenzyme (PKM2) as a direct target of Sirt5 in lung cancer cell line. PKM2 is often highly expressed in embryonic and cancer cells, because it provides a growth advantage in highly proliferative, glycolytic cells. Pyruvate kinases catalyze an irreversible step of glycolysis, which is dephosphorylation of phosphoenolpyruvate (PEP) to pyruvate with simultaneous ATP formation, therefore their activity is a major contribution to the overall glycolysis rate. PKM2 is one of the PK isoenzymes (along with hepatic PKL, PKR expressed in erythrocytes, and a constitutively active PKM1), but not a particularly active one, and frequently inhibited by posttranslational modifications, such as phosphorylation and acetylation [100]. Its expression enables the redirection of glycolytic intermediates into biosynthetic processes: the pentose phosphate pathway (PPP) and amino acid synthesis, important for the proliferating cells [100]. The PKM2 succinylation on K498 is necessary for the catalytic activity. Sirt5 physically binds and desuccinylates PKM2 leading to its inactivation. PKM2 inactivation shunts glucose catabolism almost entirely toward the PPP, which is one of the major sources of intracellular NADPH (apart from IDH1/2 activity). Sirt5 activity helps to sustain high NADPH levels and therefore provides the better protection against oxidative stress, which translates to higher proliferation rate and accelerated tumor formation in the mouse xenograft model, as compared to the cells treated with Sirt5 inhibitor, suramin [35]. In support of the evidence of a positive role of Sirt5 in tumor growth promotion, Sirt5 overexpression has been demonstrated in numerous hepatocellular carcinoma cell (HCC) lines, in comparison to normal liver cell line (LO2) [101]. Sirt5 knockdown significantly reduced proliferation rate in HCC lines and induced apoptosis, suggesting a prosurvival role of Sirt5. The authors infer that the possible mechanism of antiapoptotic action of Sirt5 involves cytochrome c deacetylation [101]. Summing up, Sirt3 and Sirt4, despite their contrasting roles in the several aspects of the cellular metabolism, for example, fatty acid oxidation, act as tumor suppressors via repression of glutamine catabolism and promotion of respiration. Sirt5 clearly stands on the opposite side, by activating the metabolic routes important for sustaining proliferation and promotion of tumor growth in vivo.

7.12 Sirtuins regulate iron
sulfur cluster assemblage Sirt3 regulates iron absorption and intracellular nonheme iron content. The Sirt3-deficient cells have an inappropriate regulation of iron metabolism [102]. Normally, in sufficient intracellular iron concentration the iron regulatory proteins 1 and 2 (IRP1, IRP2) induce ferritin expression for safe iron storage and inhibit transferrin receptor 1 (TfR1) expression by a respective stabilization or degradation of their transcripts [103,104]. Sirt2/2 MEFs show upregulated expression of transferrin receptor 1 (TfR1) and iron uptake, while downregulating ferritin and ferroportin 1 expression [102]. These cells show also the symptoms of oxidative stress and higher ROS levels in comparison to wild-type controls. The exposure to ROS leads to destruction of Fe
S clusters and induces conversion of cytosolic aconitase to IRP1 that controls stability of transcripts of genes involved in iron metabolism [103,104]. Apart from the role in iron storage regulation, Sirt3 is responsible for cellular protection against ROS [94]. Proliferating cancer cells have an increased demand for iron as a necessary cofactor for numerous metabolic enzymes, as well as the proteins engaged in nucleic acid synthesis. Indeed, TfR1 expression is activated in c-Myc transformed cells and contributes to sustaining proliferation [105]. Sirt3 inhibits IRP1 in pancreatic cancer cells decreasing their clonogenic growth [102]. In conclusion, Sirt3 acts as a tumor suppressor by keeping a tight regulation of iron uptake and storage and Sirt3 loss provides a growth advantage for the transformed cells. Apart from the indirect impact of Sirt3 on the aconitase activity through oxidative stress management, there is also a direct interaction between these two proteins. The mass spectrometric analyses revealed that mitochondrial aconitase possesses multiple acetylation sites and their acetylation is associated with a high enzymatic activity. Sirt3 inhibits it through deacetylation and this process is implicated in the modulation of response to high fat diet [17].

7.13 Mitochondrial fatty acid synthesis is linked to Fe
S cluster assembly and protein lipoylation—implications for cancer cell metabolism As discussed above, Sirt3 emerged as a regulator of iron metabolism and storage, which affects Fe
S cluster formation. The most common Fe
S cluster cofactors present rhombic [2Fe
2S] or cubic [4Fe
4S] geometry

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(Fig. 7.3), whereas [3Fe
4S] and higher order complexes, such as [8Fe
7S] are less common [106]. The Fe
S clusters are inserted into target apoproteins by mitochondrial ISC and cytoplasmic CIA systems. The ISC machinery consists of a scaffold protein ISCU, cysteine desulfurase NFS1, desulfurase-interacting protein ISD11 (LYRM4), frataxin (FXN), and mitochondrial acyl-carrier protein (mtACP) [107
109]. Mammalian CIA system includes scaffold protein NUBP1/2, IOP1 involved in the cluster transfer, CIAO1, and CIAPIN1 (Anamorsin) [110]. The ISC components FXN and mtACP are also the key players in the regulation of mitochondrial metabolism. FXN is an evolutionary conservative nuclear-encoded mitochondrial protein acting as an iron chaperone, and involved in the defense against ROS [111]. Frataxin deficiency is associated with a neurodegenerative disorder named Friedereich’s Ataxia (FRDA), multiple metabolic abnormalities, and organ dysfunction, as well as liver cancer development in the mouse model of this disease [112]. In ISC complex, FXN allosterically activates persulfide formation by NFS1, which is an essential step in Fe
S biogenesis [113]. Frataxin protects the aconitase Fe
S cluster from disassembly and stabilizes its enzymatic activity [114], therefore contributing to sustaining the Krebs cycle flux [115]. The loss of mitochondrial aconitase activity is an indicator of high superoxide production and oxidative stress [114]. Through supporting the function of respiratory complexes, FXN promotes OXPHOS and also acts as a tumor suppressor: targeted disruption of frataxin in hepatocytes induces liver tumor formation in mice [116,117]. This latter function might also be linked with FXN’s role in improving the efficiency of DNA repair mechanisms after the exposure to genotoxic agents and reduces mutation frequency [112]. In cardiomyocytes isolated from FRDA model mice, FXN dysfunction leads to an 85-fold increase in NAD/NADH ratio, as a consequence of the mitochondrial metabolic imbalance [118]. Such a large increase in NAD concentration inhibits Sirt3 and leads to mitochondrial protein hyperacetylation and respiratory defects on the cellular level, which translate into FRDA-associated cardiomyopathy on the systemic level [118,119]. mtACP is a crucial member of the ISC complex that physically interacts with Isd11 and facilitates the Fe
S assembly. The Isd11 (LYRM4)-NFS1-FXN complex provides Fe and S atoms to form a complete cluster at the scaffold protein ISCU. The cluster is next transferred onto a target protein by the ISCU-HSPA9-HSC20 chaperone complex [120,121]. Apart from this role, mtACP is involved in a highly conserved mitochondrial mtFAS, which is inherited from an alpha-proteobacterial mitochondrial predecessor (see Chapter 3, Sirtuins and Metabolic Regulation: Food and Supplementation). mtFAS is carried out by separate proteins: acyl-carrier protein (mtACP), malonyl-CoA:ACP transferase (MCAT), ketoacyl-ACP synthase (OXSM), 3-ketoacyl-ACP reductase (KAR1), 3-hydroxyacyl-ACP dehydratase (HTD2), and 2-enoly-ACP reductase (MECR) [10]. In contrast to the mitochondrial pathway, the cytoplasmic fatty acid synthesis (FASI) is carried out in mammalian cells by fatty acid synthase, a dimer

FIGURE 7.3 Schematic illustration of the most common types of iron
sulfur (Fe
S) clusters with examples of proteins harboring them. ETC, Electron transfer chain; LIAS, lipoic acid synthase; RNR III, ribonucleotide reductase class III; SDHB, succinate dehydrogenase subunit B.

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formed by two large multidomain polypeptides that possesses seven enzymatic activities (starting from N-terminus, they are as follows: 3-ketoacyl synthase, acyl-S-transferase, 3-ketoacyldehydratase, enoyl reductase, 3-ketoacyl reductase, acyl carrier protein, and thioesterase). Cytoplasmic FASI produces fatty acids for triglyceride and phospholipid formation, whereas the main function of mtFAS is the synthesis of lipoic acid and several sphingolipid species. mtFAS utilizes acetyl-CoA and malonyl-CoA affecting their mitochondrial concentrations. The fluctuations of these active acyl intermediates are reflected in the protein acylation and the pathways regulated by mitochondrial sirtuins. The mtACP activity depends on the attachment of a 4-phophopantetheine (4-PP) prosthetic group to the conserved S82 residue [122]. Therefore mitochondrial phosphopantetheinyltransferase 2 (Ppt2) which moves 4-PP moiety from CoA to ACP, is an important auxiliary protein for mtFAS [123]. In mtFAS, mtACP serves as a soluble scaffold that facilitates transfer of acyl intermediates among the other enzymes involved in the pathway [122]. mtFAS is able to synthesize acyl chains of up to 14 carbon atoms in length, but octanoic acid, a lipoic acid precursor, is its most important product. The experiments on yeast cells expressing mtACP mutant S82A, which cannot be 4-phosphopantetheinylated, demonstrated that the defects in Fe
S biogenesis in these cells result from an inability to form acyl-ACP, and not from the lack of lipoic acid synthesis [122]. Therefore the treatment with exogenous lipoic acid does not rescue the cells with mtFAS defects [122]. mtACP is also a structural component and the accessory subunit of the respiratory complex I (NADH:ubiquinone oxidoreductase) [124,125]. To play this role, mtACP must possess the 3-hydroxymyristoyl chain bound to the 4-PP group. During the ETC complex assembly, mtACP binds to LYR (Leu-Tyr-Arg) motif proteins (LYRMs). LYRMs are small hydrophilic proteins that act as assembly factors for respiratory complexes I, II, III, and V, and take part in the Fe
S cluster formation [120]. mtACP deficiency destabilizes also respiratory complex II (succinate:CoQ dehydrogenase), probably due to the defects in Fe
S assembly and leads to inactivation of lipoamide-dependent enzymatic complexes, such as PDC and KGDC [123]. As was shown on the example of Isd11, the formation of LYRM-ACP complex requires mtACP acylation (usually 3-hydroxymyristoylation). The acyl chain is inserted into the LYRM hydrophobic pocket [10,126]. This interaction leads to the allosteric activation of LYRM proteins and supports of ETC complex biogenesis. During lipoic acid synthesis, LIAS is an important recipient of Fe
S clusters and mtACP provides octanoyl chain as a substrate. To insert sulfur atoms into octanoyl chain, LIAS probably uses Fe
S cluster from the Isd11/NFS1/mtACP complex [120]. The mtACP activity affects also mitochondrial tRNA processing [127,128]. One of mtFAS’ peculiarities is the fact that in vertebrates HTD2 and RPP14 subunits of RNAse P are encoded by the same bi-cistronic transcript, which is extremely unusual for Eukaryota. The experiments on yeast cells also revealed connections between mtFAS and RNA processing: the defects in mtFAS are associated with problems in RNAse P activity in mitochondria [128]. RNAse P performs endonucleolytic cleavage of the 50 end of tRNAs during their maturation. Additionally, mtACP is a structural component of human mitochondrial ribosomes and cooperates with LYRMs in the assembly of large ribosomal subunits [129] and therefore is indispensable for the mitochondrial translation. In summary, mtFAS controls mitochondrial metabolism by several mechanisms: (1) lipoic acid supply for PDC, KGDC etc.; (2) support of Fe
S cluster assembly for the Krebs cycle enzymes; (3) ETC complex assembly; and (4) RNAse P-mediated tRNA processing and mitochondrial translation (including the mitochondrially encoded ETC components) [123]. mtFAS is also a part of the general mechanism that controls the mitochondrial homeostasis by responding to the nutritional status. The control is exerted through acetyl-CoA fluctuations: acetyl-CoA is a carbon source for the Krebs cycle, but also for mtFAS, which generates both the lipoic acid and acyl chain for ACP, which is crucial for ETC complex assembly and respiration. In general, the mitochondrial acetyl-CoA level is the resultant of anabolic (citrate export for fatty acid synthesis, ketogenesis) and catabolic (input from glucose, amino acid, and fatty acid catabolism) processes. As described in previous sections, mitochondrial sirtuins control all these pathways and therefore have a crucial impact on the acetyl-CoA level. On the other hand, mitochondrial acetyl-CoA determines the rate of protein acetylation which impacts their activity. So, the balance between acetyl-CoA level and sirtuin activity serves as a nutritional sensor for maintenance of mitochondrial homeostasis.

7.14 Consequences of Fe
S cluster defects in cancer cells Fe
S cluster-containing proteins play several crucially important cellular functions (as already mentioned previously): (1) they are the components of the ETC complexes (e.g., 8 Fe
S centers in the complex I, 3 centers in the complex II, Rieske proteins in the complex III); (2) they belong to the Krebs cycle (aconitase, fumarase,

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119

FIGURE 7.4 Mitochondrial sirtuins, particularly Sirt3, affect iron
sulfur (Fe
S) cluster assembly and maintenance by two mechanisms: (1) through supporting effective cellular mechanisms of antioxidative protection, (2) by the control of the labile iron storage and iron metabolism. Fe
S containing proteins (indicated by gray box arrows) play multiple and diverse roles, crucial for cellular homeostasis (indicated by smaller blue arrows). The numerous metabolic pathways that employ Fe
S proteins, are the source of reactive acyl thioesters (acetyl-CoA, succinyl-CoA, malonyl-CoA, etc.) that can nonenzymatically acylate cellular proteins that are sirtuin targets. ETC, Electron transfer chain.

succinate dehydrogenase); (3) they take part in lipoic acid synthesis (LIAS); (4) they control tRNA modifications (TYW1, CDKAL1, CDK5RAP1, ELP3 catalyzing tRNA nucleotide thiolation and isoprenylation); (5) they regulate iron homeostasis (IRP1/cytosolic aconitase); (6) they are required for DNA repair (MUTYH, XPD and RTEL1 helicases, FancJ, Endonuclease III, DNA glycosylases) and replication (δ-, ε-, ζ- DNA polymerases, primase); and finally (7) they take part in reduction of ribonucleotides to deoxyribonucleotides (RNRs). Mitochondrial sirtuins, particularly Sirt3 contribute to the control of labile iron pool through various mechanisms including antioxidative protection which has a direct impact on the Fe
S cluster assembly. Defective Fe
S cluster assembly, which happens in cancer cells, therefore, translates into metabolic perturbations: impaired OXPHOS and deregulation of the Krebs cycle, which may lead to succinate accumulation and has consequences in altered epigenetic regulation (Figs. 7.2 and 7.4). The consequences of Fe
S absence were studied in the cells expressing the conditional dominant negative ISCU C69S mutant, which are unable to ligate the nascent Fe
S cluster [130]. These cells exhibited dramatic alterations in the metabolism: the loss of PDC and KGDC activity (due to lipoic acid deficiency), OXPHOS blockade, and inactivation of aconitase (due to lack of Fe
S centers) resulted in acute citrate accumulation. The restriction in the Krebs cycle flux caused the export of citrate pool to cytoplasm and its subsequent conversion to lipids via the cytoplasmic FASI pathway and abundant lipid droplet formation [130]. A heavy lipid droplet load is very common in cancer cells, being one of the adaptations to hypoxia and oxidative stress and provides a significant growth advantage [131]. The therapeutic implications of lipid droplet accumulation are severe, because lipophilic cytotoxic anticancer drugs can be absorbed and stored in lipid droplets, and sequestered from their molecular targets, leading to drug resistance [132]. The impairment of Fe
S cluster machinery significantly contributes to oncogenic transformation and tumor progression (Fig. 7.4). One example of such a process involves MMS19, an adaptor protein from the cytosolic CIA system. MMS19 interacts with numerous DNA repair proteins (MUTYH, XPD, DNA glucosylases, FancJ) and helps them acquire Fe
S clusters necessary for the proper function [133]. MAGE-F1 modulatory protein, overexpressed in several types of cancer (including lung cancer, esophageal cancer, head and neck carcinoma), binds to MMS19 and induces its ubiquitination and degradation [133]. It is possible that induction of MAGE-F1 is a response to insufficient intracellular iron content, which also happens in cancer cells [133]. When cytosolic Fe
S cluster transfer is impaired the DNA repair machinery does not function properly, which in turn leads to progressive genomic instability. High mutation rate and genomic instability are cancer driving force, frequently observed for instance in breast cancer with loss of function mutations in BRCA1/2 genes [134].

7.15 Deoxyribonucleotide synthesis—toward the as yet unexplored areas of sirtuin research The actual engagement of sirtuins in the evolution of DNA via deoxyribonucleotide synthesis is still more than enigmatic. Discovery of RNR shed new light on the classical “chicken and egg” problem of the origin of DNA and the encoded proteins [135]. DNA is synthesized from deoxyribonucleotides, which are produced from

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ribonucleotides (not just by assemblage of deoxyribose and nucleobases) via reduction of carbon 20 of the ribose ring with the use of RNR [6]. The mechanism of this reaction is strictly free radical-dependent and demands sophisticated processes of initiation and then transfer of the free valency electron through the chain of aminoacyl radicals placed, for example, in the class Ia RNR in the interphase between two subunits coded in human in different chromosomes to the enzyme active site [7,8]. To achieve such a long-distance transfer of electrons highly specialized complexes of proteins revealing surface-active structural properties must be involved [8]. Consequently, DNA must have evolved in the world of highly specialized proteins encoded in RNA genomes. The problem is that nowadays RNR are divided into several surprisingly different groups varying primarily in the mechanism of initiation, and appearing in various groups of organisms: i) the already mentioned “aerobic” class I RNR (divided into Ia and Ib group), ii) the “in-between” class II RNRs present with no dependence on the oxygen demand in microorganisms, and iii) the “anaerobic” class III RNRs present in strictly anaerobic and facultatively aerobic organisms under strictly anaerobic conditions, besides other “atypical” variants of RNRs. Altogether, these enzymes occur in all the main domains of organisms [7]. The common feature of all RNRs is the ultimate appearance of the thiyl radical (RS•) being the direct donor of the electron to reduce the ribose moiety [8,136]. The actual role of mitochondrial sirtuins in this process is not known. But remembering their engagement in the intracellular iron sensing, it becomes extremely intriguing also from this point of view. Firstly, because of the cosmopolitan presence and universal engagement of both groups of enzymes in the metabolism of all major groups of organisms, and secondly, because of the role of nonheme iron in the reduction of ribonucleotides. Either in the form of diiron-oxo complexes in the RNR class I, and as [4Fe
4S] clusters in RNR III [137]. There is almost no doubt, at present, that the most ancient is the RNR III class of the enzymes [7]. It appears almost exclusively under strictly anaerobic conditions even in facultative anaerobes. It is dependent on iron
sulfur clusters (the [4Fe
4S] type), which generate the oxygen-sensitive glycyl radical depending on formate as the electron donor, and S-adenosylmethionine and flavodoxin [7,136]. There is a convincing hypothesis that RNR I may have originated only in the world containing atmospheric dioxygen [7]. Namely, the initiating agent (thioredoxin or glutaredoxin, or nucleotide reductase “supporting” enzyme NrdH) serving to reduce the tyrosyl residue to the tyrosyl radical cooperates with the diiron-oxo class of iron complexes [138]. Here a question arises concerning the assemblage of this type of iron complex versus their evolutionary origin in the context of Fe
S clusters. Perhaps there was an interphase in the evolution, when the dioxygen-dependent diiron-oxo complexes were generated in trace, catalytic amounts, as the trace-like presence of oxygen in the primordial neutral or reducing atmosphere cannot be excluded [139]. There is an interplay between this form of iron and iron
sulfur clusters, because diiron-oxo complexes have been detected in the system repairing Fe
S cluster in E. coli (RIC) [140].

7.16 Perspectives—evolutionary implications and new directions in cancer treatment Numerous metabolic disorders that result from improper functioning of mitochondria, are related to sirtuins (Sirt4) [141]. On the other hand, mitochondria are critically dependent in their functions on the support of deoxyribonucleotides, which in mammals are produced in the cytoplasm or even depend on the cytoplasmic transit of extracellular deoxyribonucleotides to mitochondria [7]. Not accidentally, the first steps of elementary, “prebiotic” evolution, as strongly depended on the supply of monomers for template-dependent replication, were heading for the process of autonomic nucleotide synthesis [142]. One must be reminded that the support of deoxyribonucleotides is important not only for DNA synthesis in the S-phase (thus proliferation of cancer cells), but also for DNA repair (in the nucleus but also in the mitochondrion [143]), so that the expression of RNR is even regulated by the absorbed doses of ionizing irradiation [144]. Consequently, it seems a good idea to develop new strategies for cancer treatment via targeting ribonucleotide reduction. And indeed, based on targeting RNR activity, however, via sirtuin-independent glutaredoxin and thioredoxin inhibition, Haffo et al. have reactivated the p53 pathway in human osteosarcoma, and caused an inhibition of cell growth [145]. There are several other concepts of RNR-targeted cancer therapies [146]. But more recently, Chen et al. have shown that Sirt2 (admittedly, a nonmitochondrial sirtuin) is really engaged in the control of RNR activity and responsible for deoxyribonucleotide support in human lung cancer cells, constituting such a potential target. Interestingly, the anticancer activity of Sirt2 is based on its deacetylase activity [147]. The further dissection of the sirtuin-RNR interdependence is therefore of crucial importance. The evolutionary aspects of these problems, leading to the better understanding of the coevolution of irontriggered and iron-sensing processes, sirtuins, and DNA synthesis/repair may, consequently, identify new

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targets for therapies, and new concepts for cancer treatment. A good example may be the actual mitochondrial localization of the interested processes. Mitochondria are believed to evolutionary originate from alphaproteobacteria, which still exist as intracellular “superparasites” depending on the cellular metabolism to the degree only slightly weaker than some viruses [148]. Rickettsias, for instance, are considered to be organisms closely related to eukaryotic protomitochondria, but they do not produce ATP, instead exchanging it for ADP with the host’s cellular sources [149]. Meanwhile, closely related intracellular pathogens, for example, Chlamydia, produce their own deoxyribonucleotides, with a modified class Ia RNR, and this modification has been proven to reveal adaptive value in the intracellular parasitism [150,151]. Moreover, the most enigmatic anaerobic RNRs were “suddenly” discovered in the genome of bacteriophage T4 [152], where the presence of a type I RNR gene was itself found as an astonishing phenomenon in the late 1960s [153]. Clearly, a focus on sirtuin functioning in Rickettsias, Chlamydias, or DNA-phages, still poorly understood, should contribute to the better exploration of mitochondrial sirtuin-related targets for therapeutic purposes. Little is known about the interactions between sirtuins and RNRs, and every newly discovered fact brings new applicative ideas. There is, for example, the problem of the actual origin of sirtuins, which, as transferases, may be rooted in the elementary processes of biogenesis as deeply as RNRs. As there is no “evolutionary vacuum,” many executors of biological functions in the present biological systems possessed other roles in the past. For protoribosomes, for instance, it may have been just template-dependent synthesis of RNA [154,155]. The idea of evolutionary horror vacui demands to name the primary function of sirtuins, as well as of RNRs, as in the preDNA world it must have NOT been reduction of ribonucleotides, unless to satisfy the demand for DNA synthesis. This is, obviously, a clearly unexplored field in the development of new anticancer strategies, and the same for understanding the neoplastic transformation itself. In the light of the above-described phenomena the cancer cell is a cell revealing genetic disorders which, speaking in a common mode, not only “move it backward in the ontogenesis (to gain a more embryonic phenotype),” but also in phylogeny (to take advantage of more primitive metabolic pathways and regulations). The mitochondrial sirtuins (Sirt3) strongly affect the iron metabolism, in particular, control of nonheme iron turnover, serving as a support for “the iron sensor,” the already mentioned, but inactive cytosol aconitase serving as IRP-1 [114,156]. The action of this system is dependent on Sirt3 (in Sirt32/2 mice the assemblage of Fe
S clusters is impaired, see Section 7.12). The main active form of sulfur in the process of Fe
S cluster formation is the persulfide anion RSS2, itself quite easily oxidizable to perthiyl radical (RSS•) which, contrary to the thiyl radical, is stabilized by resonance, thus responding for the electron transfer properties of persulfide [157,158]. This is a very ancient trace of biogenesis. Generation of [4Fe
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C H A P T E R

8 Sirtuins and the hallmarks of cancer Talita H.B. Gomig, Tayana S. Jucoski, Erika P. Zambalde, Alexandre L.K. Azevedo, Daniela F. Gradia and Enilze M.S.F. Ribeiro Genetics Postgraduation Program, Genetics Department, Federal University of Parana´, Curitiba, Brazil O U T L I N E 8.1 Introduction

8.7 Sirtuins in genome instability and replicative immortality 138

129

8.2 Sirtuins in sustaining proliferative signaling and evading growth suppressors 132 8.3 Sirtuins and resisting cell death

135

8.8 Sirtuins in reprogramming energy metabolism

139

8.9 Sirtuins and cancer therapy

141

8.4 Sirtuins in tumor-promoting inflammation and immune system function 136

8.10 Concluding remarks

142

8.5 Sirtuins in angiogenesis

137

References

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8.6 Sirtuins in invasion and metastasis

137

8.1 Introduction Cancer is a major cause of morbidity and mortality worldwide. GLOBOCAN, based on 2018 data, reports 18.1 million new cases and 9 million deaths by cancer, projecting 29.5 million new cases in 2040, making it clear that cancer is a public health problem for all countries [1]. This increase in incidence results not only by the global increase of life expectancy, but also from the current lifestyle that leads to cancer development [2]. The high numbers justify the continuing basic research on cancer to reveal the mechanisms of initiation and progression as well as new approaches of prevention and treatment. Cancer is a generic name given to a group of over 100 diseases, since each organ in the body can develop different types of cancer. In common, these diseases have an uncontrolled growth of cells and the ability to invade neighboring and distant tissues and organs, characterizing the metastasis. The process is multistep and complex, and the basis is genome instability which generates genetic changes [3]. The role of sirtuins is expanding each year in several age-related diseases, including cancer [4,5]. Sirtuins have been described in several sites of cancer. Considering their roles as a master regulator of many cellular and physiologic functions, they have effects pro- and antitumor and previous studies have shown that the sirtuins family (SIRT1
7) plays a role in tumorigenesis, promoting or inhibiting tumor formation [6
9]. The dual roles of sirtuins in malignant phenotype development are exemplified in Table 8.1. In an attempt to organize all capabilities acquired during the multistep process of a tumor, Hanahan and Weinberg [139] proposed six hallmarks of cancer as a “distinctive and complementary capabilities that enable tumor growth and metastatic dissemination.” These hallmarks are (1) sustaining proliferative signaling; (2) evading growth suppressors; (3) activating invasion and metastasis; (4) enabling replicative immortality; (5) inducing

Sirtuin Biology in Cancer and Metabolic Disease. DOI: https://doi.org/10.1016/B978-0-12-822467-0.00013-9

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8. Sirtuins and the hallmarks of cancer

TABLE 8.1 Sirtuins dual role in cancer process. Biological Sirtuin process SIRT1

SIRT2

SIRT3

SIRT4

Regulation by sirtuins As cancer promoter

As cancer suppressor

Cancer type

Tumor growth

SFRP-1 [10], SFRP-2 [10], E-cadherin [10], GATA4-GATA5 [10], MLH1 [10], P53 [11], MMP2 [12], FOXO3A [12], E2F1 [13], FXR [14], H3K4 [14], H3K9 [14], HIF1ɑ [15]a, HIF2ɑ [15]a, eNOS [16], NOTCH1 [16]

ERɑ [17,18], PTEN1 [19], NF-κB [20], CYCLIN D1 [20], β-Catenin [21], STAT3 [22], BCA3-NEDD8 [23], P65 [24], FOXM1 [25], PCAF [26], BIRC5 [27], MYC [28]

Adenocarcinoma [25,28], breast [10
12,17,18,23,27], Colon [10,15], Colorectal [21], Gastric [20,22], Liver [14,15], Lung [13,16,24], Osteocarcinoma [13,26], Osteosarcoma [25], Prostate [19,21,23]

Invasion and metastasis

MMP-2 [12], FOXO3A [12], BIRC5 STAT3 [22], SMAD4 [33,34] [29], AKT [30], HIFɑ [31], PGC-1ɑ [32]

Adenocarcinoma [31], breast [12,29,30,33], fibrocarcinoma [31], gastric [22], hepatocellular [31,32], kidney fibrosis [33], ovarian [29], oral [34], squamous cell [31]

Genome alterations

P53 [11], E2F1 [13], RB [35], H3K4AC [36], H4K16 [36], H3K9 [36]

KU70 [37], XPA [38], NBS1 [39], WRN [40]

Adenocarcinoma [38,39], breast [11,36], bone osteossarcoma [37], lung [13,38], osteocarcinoma [13], osteosarcoma [40], retinoblastoma [35]

Metabolism

FXR [14], H3K4 [14], H3K9 [14], ETHE1 [41], LKB1 [42], LXR [43], SREBP-1C [44]




Colorectal [41], hepatocellular [42], liver [14,43,44]

Drug resistance

XRCC1 [45], FOXO1 [46], CREB [47]




Breast [46], gastric [47], lung [45]

Angiogenesis stimulation

MMP2 [12], FOXO3A [12], eNOS [16],
NOTCH 1 [16]

Breast [12], lung [16]

Tumor growth

P73 [48], AKT1 [49], G6PD [50], PGAM [51], LDH-A [52], P53 [53], ɑtubulin [54]

ACLY [55], HIF1ɑ [56], FOXO1 [57]

Adenocarcinoma [56], colon [57], glioblastoma [48], leukemia [50], liver [54], lung [51,53,55], myeloid leukemia [49], pancreatic [52]

Invasion and metastasis

LDH-A [52], PEPCK1 [58], AKT [59]




Gastric [58], hepatocellular [59], pancreatic [52]

Genome alterations




E-Cadherin [60], CDC20 [60], H4K16 [61], PR [61], Set7 [61], CDK9 [62]

Adenocarcinoma [62], breast [60], osteossarcoma [62], papiloma [61]

Metabolism




ACLY [55], PKM2 [63]

Breast [63], lung [55]

Angiogenesis stimulation

STAT3 [64]




Colorectal [64]

Tumor growth

GDH [65], PYCR1 [66], LADH [67], NMNAT2 [68], KU70 [69], ACC1 [70]

AceCS2 [71], JNK2 [71], NOTCH1 [72], MDM2 [73], OGG1 [74], PDHA1 [75,76], PDP1 [75], HIF1ɑ [76], PDK1 [76], TFAM [77]

Breast [66], cholangiocarcinoma [76], clear cell renal [65], cervical [70], colorectal [71], gastric [67,72], glioblastoma [74], glioma [69], hepatocellular [73], lung [66,75], nonsmall cell lung cancer [68], osteosarcoma [66,71], renal cell adenocarcinoma [77]

Invasion and metastasis

SKP2 [78], ACC1 [70]

SRC [79], FAK [79]

Breast [78], cervical [70]

Genome alterations




OGG1 [74], FOXO3A [80]

Colon [80], glioblastoma [74]

Metabolism

LADH [67], ACC1 [70], SHMT2 [81], SOD2 [81]

PDHA1 [75], PDP1 [75], TFAM [77], HIF1ɑ [82], CYPD [83]

Breast [82,83], colorectal [81], cervical [70], gastric [67], lung [75], ovarian [81], renal cell adenocarcinoma [77]

Tumor growth

IDE [84]

GDH [85
88], Fis-1 [89], ECADHERIN [90], ERK [91],

Breast [84], burkitt lymphoma [87], colorectal [90], esophageal squamous (Continued)

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131

8.1 Introduction

TABLE 8.1 (Continued) Biological Sirtuin process

SIRT5

SIRT6

SIRT7

Regulation by sirtuins As cancer promoter

As cancer suppressor

Cancer type

CYCLIN-D [91], CYCLIN-E [91], AMPKɑ [92], SIRT1 [93]

cell [86], gastric [91], glioma [85], hepatocellular [92], lung [84], neuroblastoma [93], nonsmall cell lung [89], thyroid [88]

Invasion and metastasis




GDH [86,88], FIS1 [89], E-Cadherin [90], SIRT1 [93]

Colorectal [90], esophageal squamous cell [86], neuroblastoma [93], nonsmall cell lung [89], thyroid [88], neuroblastoma [93]

Genome alterations




GDH [94], SIRT1 [93]

Adenocarcinoma [94], lung [94], neuroblastoma [93]

Metabolism




E-Cadherin [90], GDH [87,94], SIRT1 [93], ANT2 [95]a

Adenocarcinoma [94], Burkitt lymphoma [87], colorectal [90], kidney [95], lung [94], neuroblastoma [93]

Drug resistance




IL-6 [96]

Breast cancer [96]

Tumor growth

Cyt-C [97], SOD1 [98], BIRC2 [99], GLUD1 [100], E2F1 [101], LDBH [102], PKM2 [103], GLS [104]

SHMT2 [105], SDHA [106]

Breast [104], colon [105], colorectal [100], E2F1 [101], LDBH [102], PKM2 [102,103], clear cell renal cell [106], hepatocellular [97,101], lung [98,103], nonsmall cell lung [99], osteosarcoma [105]

Invasion and metastasis

E2F1 [101]




Hepatocellular [101]

Genome alterations




ACOX1 [107]

Liver [107]

Metabolism

GLS [104]




Breast cancer [104]

Drug resistance

BIRC2 [99]




Nonsmall cell lung [99]

Tumor growth

H3K9 [108], H3K9Ac [109], H3K56Ac [109], BAX [109], AMPK [110]

MYC [111], HIF1ɑ [112], VEGF [112], H3K9Ac [113], H3K56Ac [109,113], LIN28B [113], NF-κB [114], H3K9ac [115], TWIST1 [116], ERK1/2 [117], NOTCH3 [118], PKM2 [119]

Adenocarcinoma [108], erythroleukemia [111], fibrossarcoma [108], glioma [115], hepatocellular [109,117,119], lung [112], nasopharyngeal [114], nonsmall cell lung [116], ovarian [118], pancreatic [113], skin [110]

Invasion and metastasis

Ca 1 Levels [120], H3K9 [120], NUDT9 [120]

PKM2 [119]

hepatocellular [119], pancreatic [120]

Metabolism

TGFB [121], H2O2 [121], HOCL [121]

MYC [111]

erythroleukemia [111], hepatocellular [121]

Drug resistance

FOXO3 [122]




liver [122]

Angiogenesis stimulation

Ca21 levels [120], H3K9 [120], NUDT9 [120]

HIF1ɑ [112], VEGF [112]

pancreatic [120]

Tumor growth

MAPK [123], H3K18Ac [124], P53 [125], miR-34a [126], H3K18ac [126], % [127], MTOR [128], NF-κB [127], P65 GTF3C3 [128], CDC4 [129], SMAD4 [130], P38 [131], MAPK [131], DBC1 [132], ERK [133], STAT3 [133]

DDB1 [134], ELK4 [135], H3K18Ac [135]

adenocarcinoma [128,134], breast [131], colon [123], gastric [126], glioma [133], glioblastoma [124], hepatocellular [125,135], ovarian [127], osteosarcoma [129], thyroid [132] (Continued)

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TABLE 8.1 (Continued) Biological Sirtuin process

Regulation by sirtuins As cancer promoter

As cancer suppressor

Cancer type

Invasion and metastasis

MAPK [123], NF-κB [127], P65 [127], CDC4 [129], SMAD4 [130], DBC1 [132], ERK [133], STAT3 [133], Ecadherin [136]

TGFB [137], BTRCP1 [137], SMAD4 [138]

breast [137], colon [123], glioma [133], ovarian [127], osteosarcoma [129], oral squamous cell [138], prostate [130,136], thyroid [132]

Genome alterations

H3K18Ac [124]

DDX21 [138]

breast [138], glioblastoma [124]

a

Role is controversial.

angiogenesis, and (6) resisting cell death. Ten years later, the same authors [140] revisited the hallmarks and added new ones: (7) avoiding immune destruction; (8) tumor-promoting inflammation; (9) genome instability and mutation; and (10) deregulating cellular energetics. Considering the role of sirtuins in the majority of the mechanisms involved in cancer, in this chapter we will discuss some of their specific roles as hallmarks of cancer, in an attempt to demonstrate their potential as markers in diagnosis, prognosis, and therapeutics in cancer (Fig. 8.1).

8.2 Sirtuins in sustaining proliferative signaling and evading growth suppressors Cancer development and progression involves complex and interconnected signaling that sustains chronic proliferation and evades antigrowth signaling and cell death programs. Cancer cells can support proliferative signaling independently of growth factors through the constitutive activation of pathways that impact cell proliferation and fate [140,141]. Moreover, excessive proliferative signaling can also be sustained by disruption of negative-feedback mechanisms that generally operate to attenuate proliferative signaling [140]. Sirtuin functions impact some of the major aberrant signaling pathways in cancer, such as Wnt/β-catenin [142,143], PI3K/AKT/mTOR [30,143
145], NF-κB [6,146], and MAPK [147]. Sirtuins are functionally related to β-catenin, the main effector in the canonical Wnt signaling pathway [143]. Interestingly, both Wnt signaling and SIRT1 orchestrate many of the same biological processes [148], and other downstream effectors of the Wnt pathway are impacted by sirtuin expression and/or activity. For example, the disheveled proteins that act as crucial messengers for several Wnt ligands are regulated by SIRT1, which may explain the diverse responses of Wnt signaling in different cellular contexts [142]. Sirtuins play dual roles in the PI3K/AKT/mTOR signaling pathway. For example, SIRT1 directly interacts with AKT promoting SIRT2 activity [30]. SIRT2 is required for optimal AKT activation and SIRT2 overexpression enhances AKT signaling and its downstream targets [149]. SIRT6 and SIRT7 negatively regulate AKT signaling at the chromatin level and through a protein complex that impairs AKT function, respectively [145,150]. Furthermore, SIRT3 overexpression can suppress the phosphorylation of AKT which impacts on the PI3K/AKT/mTOR signaling pathway [151]. SIRT-mediated regulation of mTOR has been described in response to nutrients and cellular stresses. The SIRT1 expression is induced by calorie restriction and affected by IGF signaling [144]. SIRT1 levels can negatively regulate the mTOR pathway [152]. In addition, the activation of mTORC1 (a mTOR complex) stimulates glutamine metabolism through the dehydrogenase GDH and promotes cell proliferation by repressing SIRT4, which inhibits GDH and suppresses bioenergetics and tumor proliferation [153]. Sirtuin activity impacts some of the most critical regulatory pathways involving cancer drivers, such as Ras oncoprotein, PTEN phosphatase, and mTOR kinase. SIRT1 binds to the promoters of Ras [154] and also binds, deacetylates, and activates K-Ras, a member of the Ras family most frequently mutated in cancer [155], which activates a wide range of signaling pathways, such as PI3K and RAF/MEK/ERK (members of the MAPK pathway) [147]. The regulation of Ras-ERK1/2 and the AKT pathway may be modulated by SIRT7 impacting H3K18 expression, which is essential to cell viability, migration, and cell cycle progression [156]. In contrast, another member of the Ras family, R-Ras2, can be defatty-acylated by SIRT6, which attenuates its functions in the PI3K pathway and inhibits growth of cancer cells [157].

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FIGURE 8.1 Sirtuins as hallmarks of cancer. Pathways and molecules regulated by sirtuins in cancer development.

PTEN is a critical regulator of proliferation, survival, and cell growth that functions by antagonizing signaling through the PI3K/AKT/mTOR pathway [84]. Unregulated cell proliferation occurs in the absence of functional PTEN due to activation of a signaling cascade downstream of the AKT pathway and inhibition of the FOXO transcription factors family of tumor suppressors, which are direct substrates of AKT [158]. Several cellular stresses, such as nutritional starvation, can induce SIRT4 expression, which interacts with and reduces the stability of PTEN. Low PTEN levels inhibit the mTOR pathway and promote cell survival by accelerating autophagy to increase cell survival in a hostile environment [84]. SIRT6 also has been shown to modulate the PTEN/AKT pathway by regulating AKT and its downstream genes, MTOR, CCND1, and MYC via PTEN, which impacts on tumor progression [159]. In addition, SIRT1 promotes tumorigenesis via the PTEN/PI3K/ AKT signaling pathway [160].

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Multiple sirtuins modulate MYC activity, one of the most highly amplified oncogenes in various tumor types [161], and whose activation antagonizes Ras-induced senescence and is critical for cellular immortalization [162]. SIRT1 plays an essential role in MYC-driven tumorigenesis through feedback loops. The overexpression of MYC leads to increased SIRT1 expression, which in turn deacetylates and downregulates MYC and its target genes (i.e., telomerase reverse transcriptase, TERT) and suppresses cell growth and transformation [28]. Moreover, the MYC-mediated increase in SIRT1 protein levels also involves sequestering the SIRT1 inhibitor DBC1 which controls SIRT1 proteasomal degradation and increasing amounts of NAD1 via MYC-induced NAMPT (a NAD1 biosynthetic enzyme) expression [163]. These data suggest a MYC-NAMPT-DBC1-SIRT1 positive feedback loop that allows enhanced proliferation and survival of cancer cells. This feedback loop exemplifies SIRT1’s dual role in tumorigenesis as dependent on the stage of cell transformation [28]. In contrast, SIRT2 negatively regulates MYC and can effectively suppress cancer cell proliferation, since SIRT2 inhibition leads to MYC ubiquitination and degradation [161]. Upregulation of SIRT3 can also lead to MYC degradation [151]. Importantly, MYC target gene promoters are enriched for SIRT6, which deacetylates its H3K56 residues, inhibiting MYC transcriptional output and suppressing genes involved in ribosomal biogenesis (members of RPL protein family) and glutamine metabolism [111,164]. In fact, one of the most important functions of MYC in cancer is regulating the metabolism of the nonessential amino acid glutamine, which is primarily utilized by cancer cells and for macromolecular synthesis [164]. Low levels of SIRT6 result in an increased transcriptional output of MYC, and consequent accelerated cell proliferation, increased expression of the apoptosis inhibitor surviving, and impaired p53 and p73 apoptotic functions [111]. Furthermore, to sustain growth-stimulatory signals, cancer cells must circumvent antigrowth signaling programs, which greatly depend on tumor suppressor activities. Evasion of growth control mechanisms allows unchecked cell proliferation and prevents apoptosis, cell cycle arrest, and senescence by tumor suppressors [158]. Tumor cells may evade tumor suppressors by epigenetic mechanisms, which implicates sirtuin deacetylation activity in disrupting upstream and downstream targets of antigrowth signaling pathways. Tumor suppressor genes can stop or decelerate tumorigenesis, and Rb, p53, PTEN, Hippo, and Notch pathways are among the most important tumor suppressor genes in cancer [158]. Sirtuins play complex and important roles in regulating tumorigenesis and cancer progression by disrupting these antigrowth signals. Rb and p53 are typical tumor suppressors and gatekeepers of the cell cycle and play a role in determining cell fate: cellular proliferation, senescence, or apoptotic programs [158]. Multiple signals from intra- and extracellular sources are integrated by Rb protein and drive cell cycle progression, controlling S-phase entry when Rb is hypophosphorylated [35,158]. Rb acetylation operates as an additional regulatory mechanism, and SIRT1 has been described as a potent deacetylase for Rb. Thus active Rb is hypophosphorylated and acetylated, and SIRT1-mediated deacetylation deactivates Rb [35]. Interestingly, SIRT1 may deacetylate Rb and p53 at a common lysine motif [35]. Antigrowth functions of the p53 family involve SIRTs activities. p53 is the most widely known substrate of SIRT1, and its deacetylation by SIRT1 negatively regulates p53-mediated transcriptional activity, promoting cellular senescence in response to DNA damage [165]. p53 can also be deacetylated in the cytoplasm by SIRT2 [166], and SIRT2 inhibition leads to p53 accumulation inducing p53 canonical target genes (i.e., CDKN1A, PUMA, and NOXA) [53]. Furthermore, SIRT3 can partially abrogate p53 function, and SIRT3 overexpression upregulates p53 and its downstream factor p21 [167]. SIRT6 expression is directly activated by p53, leading to FOXO1 deacetylation and nuclear exclusion, and loss of FOXO1-induced gluconeogenesis [168]. Hippo and Notch signaling are also critically important in tumor development [140,158]. Interestingly, the Hippo pathway influences the output of multiple signaling pathways that regulate the hallmarks of cancer [140]. Activated Hippo protein has an antigrowth function by inhibiting the complex formed by YAP protein and its transcriptional coactivator TAZ [158]. YAP functions are also regulated by SIRT1-mediated deacetylation, which leads to increased cell proliferation [169]. Further, MST1, a key component of the Hippo pathway, can upregulate SIRT1 protein levels and, in contrast, also promote p53 function via SIRT1 phosphorylation, inhibiting the SIRT1-p53 interaction and SIRT1 deacetylation activity [170]. An important role of histone deacetylation by SIRT1 occurs in the Notch pathway. Transcription of the NOTCH target genes can be controlled by sirtuins by modulating histone modifications associated with gene repression, which involves SIRT1 activity [171]. SIRT3 and SIRT6 have also been shown to inhibit the expression of NOTCH1 and NOTCH3, respectively, and in both cases, this NOTCH inhibition impairs cancer cell proliferation [72,118]. In sum, sirtuins impact the main proliferative and antigrowth signaling pathways, and their dual roles in tumorigenesis can impair several growth control mechanisms, supporting excessive proliferation through many cancer drivers.

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8.3 Sirtuins and resisting cell death During tumor development, it is essential that cancer cells evolve strategies to limit or circumvent programmed cell death by apoptosis, a natural barrier to cancer progression. Autophagy is another mechanism utilized in tumor development to sustain cell survival in a stressed tumor environment. Apoptosis occurs by two major mechanisms: extrinsic and intrinsic pathways, and involves an activation cascade of cysteine proteases called caspases and regulatory proteins of the Bcl-2 family (proapoptotic: Bax, Bak, Bid, Bim/Bad, Noxa, and Puma; and antiapoptotic: Bcl-2, Bcl-W, Bcl-XL, Mcl-1, and A1) [140,172]. The sirtuin proteins can interact with additional signaling pathways and cancer drivers that regulate apoptosis, including JNK kinase, hypoxia-related HIF-1α, p53, the FOXO transcription factors, the signal transducer STAT3, and reactive oxygen species (ROS) [173,174]. In this context, SIRTs may promote prosurvival functions or, in contrast, may facilitate cell death. SIRT1 negatively regulates p53, inhibiting p53-dependent apoptosis under DNA-damage stresses and promoting DNA repair and cell survival [6]. The p53 activity can also be suppressed through MYC-induced SIRT1 expression, which decreases the transcription of proapoptotic genes and MYC-related apoptosis [162]. SIRT1 plays a role in TNF-α-induced apoptosis [24], by deacetylating NF-κB and attenuating its transcriptional activity, thus increasing apoptosis in response to TNFɑ [6]. SIRT2 and SIRT6 have also been described in deacetylation and consequently suppression of p53 and NF-κB signaling [6,175,176]. Moreover, SIRT1 and SIRT6 regulate survivin, an essential inhibitor of the apoptosis machinery [6,177]. The expression of FOXO target genes, such as Bim, is increased by deacetylation of FOXOs by sirtuins [178]. For example, SIRT1 deacetylates FOXO1, repressing its proapoptotic activity [6]. The proapoptotic FOXO3a, a downstream target of AKT, is a substrate of multiple sirtuins, including SIRT1 [179], SIRT2 [178], and SIRT5 [180]. Another member of the FOXO superfamily, the transcription factor FOXOM1, is involved in apoptosis via the SIRT7/mTOR/IGF2 pathway [181]. SIRT3 extensively participates in the regulation of apoptosis. SIRT3 regulates the levels of proapoptotic and antiapoptotic proteins (Bax/Bcl-2 ratio), mediates apoptosis involving Bcl-2 and the JNK pathway (a MAPK subfamily that promotes the expression of downstream pathways of c-Jun, p53 and p21 [182]), exerts a prosurvival role via p53 deacetylation [183], and, like SIRT1, SIRT3 can inhibit mitochondrial apoptosis by deacetylating Ku70, which prevents Bax translocation to the mitochondria [144,184,185] and induces Ku70-dependent DNA repair [184]. SIRT3 also promotes tumorigenesis through resistance to anoikis, an apoptotic cell death mechanism triggered by the loss of extracellular matrix (ECM) contacts [186]. SIRT3 overexpression sustains cell survival by reducing the ROS levels, which are thought to activate antiapoptotic proteins [187]. Under nutrient restriction, SIRT3 and SIRT4 exert antiapoptotic activity with NAMPT, protecting cells from cell death programs [188]. Furthermore, SIRT4 plays a role in apoptosis through downregulation of the apoptosis-related proteins Bax, NOX1, and p38 [189], and plays a protective role in hypoxia-induced apoptosis [190]. SIRT5 expression levels can impact proapoptotic and antiapoptotic pathways [97]. SIRT5 has been shown to deacetylate cytochrome c (Cyt c), inhibiting mitochondrial apoptosis [97,191]. Interestingly, SIRT5’s impact on apoptosis is localization-dependent: mitochondrial SIRT5 stimulates cell death, while nuclear or cytosolic SIRT5 supports cell survival [180]. SIRT6-related apoptosis can be induced through the inhibition of JAK2/STAT3 signaling [192], and modulation of the PTEN/AKT and ERK1/2 pathways [159,193]. SIRT6 overexpression drives apoptosis in cancer cells through p53 or p73 [176]. In contrast, high levels of SIRT6 upregulate Bcl-2 expression and phosphorylated-ERK, and downregulate cleaved-CASP3 and Bax, consequently increasing proliferation via the ERK1/2 pathway and suppressing apoptosis [193]. SIRT7 promotes proliferation of tumor cells through ERK and STAT3 signaling, and SIRT7 depletion suppresses ERK and STAT3 activities. which triggers apoptosis [133]. In fact, SIRT7 knockdown results in upregulation of proapoptotic proteins (cleaved-CASP3, cleaved PARP, Bax, and Bim) and downregulation of antiapoptotic proteins (Bcl-2 and Mcl-1) [126]. SIRT7 also regulates apoptosis through deacetylation and deactivation of ATM, a central kinase in the response to DNA damage whose persistent activation induces cell death [194]. In addition, SIRT7 can affect apoptosis induced by endoplasmic reticulum (ER) stress by repressing certain MYC functions [195] and can also play a role in NF-κB-mediated apoptosis [196]. Like apoptosis, autophagy is an important physiological cell response that mediates both tumor cell survival and death, and is strongly induced in cellular stress conditions and produces nutrients for cell survival during starvation. Autophagy is frequently linked to cancer initiation and progression through many autophagy-related (ATG) proteins [197] and apoptosis-interconnected programs based on regulatory circuits involving p53, PI3K, AKT, and mTOR kinases, as well as MAPK, ERK1/2, and FOXOs, which interact with multiple sirtuins.

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SIRT1 levels increase under calorie restriction in a FOXO3a-p53-nutrient sensitive manner [144,198]. p53 deacetylation by SIRT1 impacts on p53-induced apoptosis, induces autophagy, and moreover, SIRT1 deacetylates essential components of autophagy machinery, such as ATG5, ATG7, and LC3 (ATG8), stimulating autophagy [199]. In fact, SIRT1 is known to control the expression of ATG-related genes [197,200]. SIRT1 can also stimulate autophagy via FOXO1 deacetylation and Rab7 upregulation [200]. In contrast, SIRT1 inhibition can also lead to autophagy [197]. SIRT2 can inhibit autophagy under basal conditions by forming a complex with and deacetylating FOXO1, which is highly acetylated in response to oxidative stress or starvation and interacts with ATG7 to enhance autophagic cell death [199]. In addition, SIRT2 regulates mitophagy, a selective type of autophagy that removes damaged mitochondria, as well as mitochondrial function, and its protein deacetylation [201]. SIRT3 can also act as a positive regulator of mitophagy via the hypoxia pathway [199,202]. In contrast, SIRT5 depletion has been reported to promote mitochondrial fragmentation and degradation during autophagy [203]. SIRT4 can accelerate autophagy promoting cancer cell survival via downregulation of PTEN and inhibition of the mTOR pathway [84], which is also inhibited by SIRT1 and SIRT6 activities, which positively regulates cell autophagy [204]. SIRT5 has been associated with glutamine metabolism and ammonia-induced autophagy and mitophagy [205]. Interestingly, a potential crosstalk between SIRT5 in regulating SIRT3 and SIRT1 activities in autophagy has been described in cancer [206]. SIRT3 and SIRT5 positively modulate autophagy and exert a proproliferative function under certain stress conditions, while SIRT1 can affect autophagy both positively and negatively [206]. SIRT6 is a known autophagy regulator involved in cancer development in several ways, and its expression is correlated with autophagy biomarkers, such as LC3 and p62. The effects of SIRT6 on autophagy can also involve the IGF/AKT pathway, highlighting a crucial role of the SIRT6/AKT/autophagy axis in tumorigenesis [207]. SIRT7 in turn modulates autophagy to regulate TGF-β signaling via maintenance of TGF-β receptor levels [208]. Furthermore, SIRT7 may regulate tumor cell proliferation and autophagy via androgen receptor (AR) signaling and interactions with coregulators, such as SMAD4 [130]. In sum, sirtuins play essential roles in resisting cell death through their activities in cancer-related apoptosis and autophagy.

8.4 Sirtuins in tumor-promoting inflammation and immune system function Tumor progression is supported by different cell types that constitute the microenvironment. Inflammation modulates this microenvironment through bioactive molecules such as growth factors, ECM-modifying proteins, and proangiogenic factors, which function as hallmark pathways of cancer progression. Several SIRTs, such as SIRT1, SIRT3, and SIRT6, are involved in inflammation [209,210]. SIRT1 and SIRT6 interact with NF-κB, a transcription factor involved in inflammation and immune cell proliferation [24,211]. SIRT1 deacetylates K310 of the RelA/p65 subunit of NF-κB operating as a negative regulator. SIRT6 interacts with p65 and deacetylates H3K9 in target gene promoters, reducing the accessibility of NF-κB and attenuating downstream signaling pathways. SIRT6 reduction increases NF-κB transcriptional activity, which stimulates the production of inflammatory cytokines such as IL-1B, matrix metalloproteinases (MMPs), and adhesion molecules [212]. High expression of both SIRT1 and SIRT6 observed in cancer cells reduces inflammation and immune response [108]. Furthermore, SIRT1 controls proinflammatory cytokine production in immune cells through deacetylation of key transcriptional factors or, indirectly, via metabolic pathways playing an important role in immune response regulation [213,214]. SIRT1-NF-κB interactions suppress the macrophage proinflammatory phenotype that plays a role in the tumor microenvironment. In contrast, SIRT1 silencing increases activation of the c-Jun pathway by proinflammatory stimuli [215]. c-Jun interacts with c-Fos to form the AP-1 transcriptional factor, an essential molecule of tumor cell proliferation, that positively regulates c-Jun expression and prolongs AP-1 activity independent of extracellular signals. In this immune scenario, SIRT1 interacts with transcription factors and reduces T cell activation, thus SIRT1 is responsible for the immune response against the tumor. In CD41 T cells, SIRT1 reduces FOXP3 activity contributing to the more inflammatory immunological phenotype [216]. SIRT1 overexpression in macrophages reduces prostaglandin E2 production and leads to increased phagocytic percentage and tumoricidal activities of peritoneal macrophages. Low levels of SIRT1 are prejudicial to the antitumor activities of macrophages [217,218].

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SIRT3 and SIRT4 have tumor-suppressive activity in B cell malignancies. High expression of SIRT4 can inhibit the growth of Burkitt lymphoma cells, and SIRT1 and SIRT2 inhibition can increase apoptotic activity and ROS production in B-cell chronic lymphocytic leukemia (CLL) [87,219]. SIRT3 also positively regulates gene expression of antioxidant molecules that reduce ROS, and consequently inflammation [220]. The reduction of SIRT3 contributes to the increase of ROS in the tumor microenvironment and angiogenesis [82,221]. Tumor initiation involves angiogenic insufficiencies and creates hypoxic areas with high ROS concentrations favoring tumor expansion. ROS produced by hypoxia promotes the activation of HIF1α, a transcriptional factor that controls invasion and metastasis, through inactivation of its inhibitor and increasing the VEGF growth factors and their receptors (VEGFRs) that are crucial in angiogenesis [222]. In this context, sirtuins can regulate ROS production and detoxification [223] through HIF1α deacetylation. There is a lack of consensus as to whether hypoxia increases or decreases SIRT1 levels in tumor cells [224,225]. SIRT1 action on HIF1α can vary depending upon the tissue or organ. SIRT3 control of HIF stability regulates ROS levels as well as other metabolic pathways. At reduced ROS levels, SIRT3 stabilizes the HIF-degrading enzyme PHD, thus lowering HIF1α levels [82,226]. Low SIRT3 expression is observed in several tumors and cancer cell lines [82]. In hypoxia adaptation, VEGFs, VEGFRs, MMPs, adhesion molecules, PDGF, and chemokines are essential to angiogenesis, invasion, and migration, and all of these can be epigenetically regulated by sirtuins. In the tumor microenvironment sirtuins seem to interact and modulate immune system compounds to favor tumorigenesis rather than prevent it.

8.5 Sirtuins in angiogenesis Angiogenesis is the formation of new blood vessels from preexisting ones and is essential to cancer progression because it supplies oxygen and nutrients to the tumor. The ability to induce and sustain angiogenesis during tumor development occurs via an “angiogenic switch.” Different tumor cells use distinct molecular strategies to activate angiogenesis. SIRT1 deacetylates FOXO1, which is a negative regulator of angiogenesis. Effects of SIRT1 in FOXO1 activity remain controversial, with some data suggesting that deacetylation increases FOXO activity, and other data showing the opposite effect, consequently reducing or promoting angiogenesis, respectively [227,228]. SIRT1 action on FOXO family function may vary depending on the cell type and specific FOXO target genes. SIRT1 reduction in endothelial cells reduces blood vessel formation due to lowered MMP14 expression [229]. SIRT1 can also deacetylate the NOTCH intracellular domain, modulate endothelial nitric oxide synthase (eNOS) synthesis via enhanced nitric oxide (NO) production, and deacetylate AKT K19 and K20 which permit interaction with PIP3 and AKT pathway activation in endothelial cells during tumor angiogenesis [230
233]. SIRT1, SIRT3, and SIRT7 are all capable of deacetylating and inactivating p53, which acts as antiangiogenic factor [82,183,234]. SIRT2 interacts with β-catenin and alters MMP expression during angiogenesis. Depletion of SIRT2 prevents STAT3 phosphorylation, reducing the VEGF transcription [64,235]. SIRT3 in turn increases endothelial cell survival in hypoxic conditions via increased deacetylation of FOXO3 [236]. In breast cancer, loss of SIRT3 increases transcription of HIF1α-induced target genes, such as VEGF, that can be produced and secreted by cancer cells to sustain endothelial cell proliferation. However, low expression of SIRT2 reduces angiogenesis, thus acting as negative regulation of cancer progression [221]. VEGF produced and secreted by cancer cells drives endothelial cell proliferation contributing to new blood vessel development. SIRT4, SIRT5, and SIRT6 make ADP-ribosylation of PARP, which regulates VEGF and VEGFR2 [237
239]. Further, SIRT6 can modulate intracellular Ca21 levels and promote expression of IL-8, TNFα, and proangiogenic factors [120]. SIRT7 inhibits HIF1α and regulates downstream gene expression, such as VEGFA [240]. In some cancer metastases, SIRT7 activates the TGF-β pathway [137]. In the angiogenesis process, sirtuins play an essential role in regulating genes that control blood vessel development, providing nutrients, and oxygen to the tumor.

8.6 Sirtuins in invasion and metastasis During tumorigenesis, cells can escape the primary tumor, acquire an invasive phenotype, and travel to distant sites to metastasize. In most cancer patients, mortality is correlated with the metastatic events [241].

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Successful tumor cell invasion and migration depends on epithelial
mesenchymal transition (EMT). EMT alters the cell state, through loss of cell-to-cell and cell
to
extracellular matrix adhesion, loss of basement membrane adhesion, and the capacity of tumor cells to evade the immune system [140]. Sirtuins play different roles in EMT depending on the cellular context, tissue of origin, and microenvironment architecture. Positive regulation of EMT can occur by TGF-β pathway activation which increases SIRT1 expression. Interaction between SIRT7, SIRT1, Zeb1, and the Twi deacetylation of the st-MBD1 complex represses the transcription of epithelial genes (i.e., E-cadherin) by H3K18 which causes promoter silencing and increases mesenchymal gene expression (i.e., N-cadherin and vimentin) [136]. Additionally, fibronectin and γ-catenin are substrates of SIRT1 during the EMT process in cancer cell lines [242]. High and low SIRT1 levels correlate with increased angiogenesis in several tumor types [243
248]. Negative regulation of EMT by sirtuins involves deacetylation of SMAD3, SMAD4, and SMAD7 by SIRT1, which reduces MMP7, MMP9, and E-cadherin, inhibiting the TGF-β pathway. In metastasis, SIRT1 regulates MMP7 by deacetylating the K37 of SMAD4, which results in repression of TGF-β signaling [34]. SIRT3 negatively regulates cancer-associated fibroblasts (CAFs), known EMT inductors, where suppressed Wnt/β-catenin signaling causes EMT-associated processes. SIRT3 can also interact with FOXO3A and Twist, controlling EMT, invasion, and migration [249
251]. SIRT2 induces EMT via Akt/GSK/β-catenin activation and the RAS/ERK/JNK/MMP9 pathway [58,59]. SIRT2 overexpression leads to an increase in Slug protein, which will result in the repression of transcriptional targets, like cellular adhesion molecules, and E-cadherin, associated with invasiveness and an aggressive phenotype [252]. In contrast, SIRT4 suppresses EMT by restricting mitochondrial glutamine metabolism [87,90,253]. Additionally, SIRT4 inhibits invasion and migration by blocking MAPK/ERK/MEK activity through upregulation of E-cadherin expression [89,90]. SIRT5 contributes to invasion by interacting with the transcription factor E2F1, inducing vimentin acetylation, and positively regulating SNAIL, an essential transcription factor to repress E-cadherin expression [101,254]. SIRT6 overexpression enhances ERK1/2 phosphorylation and activates MMP9, which promotes invasion and migration [255]. The deletion of SIRT6 by modulating Lin28b also contributes to metastasis [113]. Through multiple mechanisms, sirtuins can regulate expression of target genes that promote invasion and metastasis, an essential hallmark of cancer progression.

8.7 Sirtuins in genome instability and replicative immortality Tumor growth and cancer progression are often triggered by DNA mutations, including those in genes that regulate DNA repair pathways and, subsequently, affect genomic stability [256]. DNA damage response (DDR) mechanisms, including base-excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR), and nonhomologous end-joining (NHEJ), are activated both from external agents, like UV radiation, and by molecules of cellular metabolism, like ROS [257]. During genotoxic stress, which sets foci for DDR-related proteins by recruiting DNA damage factors [258
261] SIRT1 promotes HR by deacetylating WRN (a RecQ DNA helicase) [262,263]. In the NHEJ pathway, SIRT1 enhances DNA repair through Ku70 and KAP1 deacetylation [264,265]. Lastly, SIRT1 regulates NER by deacetylating xeroderma pigmentosum proteins XPA and XPC, which, in turn, recruit other NER factors at the breaks for DNA repair [261]. SIRT2 is also implicated in mitotic progression and cell cycle regulation [266]. Regarding DNA damage pathways, SIRT2 modulates the mitotic methylation deposition of H4K20, which is essential for genome stability and DNA repair signaling [61] and directs the replication stress response through the deacetylation of kinase CDK9 [62]. SIRT3, SIRT4, and SIRT5 are mainly associated with mitochondrial metabolism [267], and by distinct pathways, they indirectly contribute to DNA stability through their participation in DDRs [107,268,269]. SIRT6 mediates H3K9 and H3K56 deacetylation, and SIRT6 deficiency leads to genomic instability and hypersensitivity to certain forms of genotoxic damage, thus interconnecting chromatin dynamics with metabolism and DNA repair [270,271]. In the BER pathway, SIRT6 acts as a regulator in a PARP1-dependent manner [272]. SIRT6 interacts with the enzymes MYH and APE1, and with the Rad9
Rad1
Hus1 checkpoint clamp, which stimulates BER [273]. Recently, SIRT6 has been identified as a double-strand break (DSB) sensor, since it recognizes DSBs,

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relocates to sites of damage, and activates downstream signaling for repair by triggering ATM recruitment, H2AX phosphorylation, and recruiting proteins of the HR and NHEJ pathways [274]. SIRT7 deacetylates H3K18 and affects the focal accumulation of the DDR factor 53BP1, thus reducing DSBs repair and leading to genome instability [275]. The activation of DDR signaling, can be achieved through a mechanism by which cells control their number of divisions through telomere shortening, leading to genomic destabilization [276]. This process results in replicative cell senescence. However, cells that deviate from this regulatory pathway can reach replicative immortality. Replicative immortality is a key feature in cancer and is defined as a hallmark of tumor development [139,140]. Several works have shown that sirtuins are essential in controlling senescence and extending cell life span. In humans SIRT1 and SIRT6 are the most studied chromatin remodeling factors that interact with telomere repeats, which are related to telomere integrity [277
280]. The role of SIRT1 and SIRT6 in cell senescence has been elucidated by analysis of their expression in various cells and conditions [281
284], by chemical inhibitors [36], and also by modulating their expression through overexpression [281,285
287] or silencing [288,289]. Those distinct approaches have shown that SIRT1 and SIRT6 expression exhibit an inverse relationship with senescence, suggesting their participation in promoting this process. Localized at the telomeres, SIRT1 recruits WNR and promotes its deacetylation, which contributes to its function in maintaining genome stability [40]. Telomeric chromatin is maintained in a hypoacetylated, heterochromatic form via H3K9 deacetylation by the deacetylase SIRT6 [290]. Depletion of SIRT6 results in end-to-end chromosomal fusions and premature cellular senescence [277]. In these ways sirtuins participate in various DNA repair pathways, affecting genomic integrity and contributing to the maintenance of cell viability. Therefore they are key molecules in cancer development.

8.8 Sirtuins in reprogramming energy metabolism Otto Warburg was the first to observe metabolic reprogramming in tumor cells. He termed this phenomenon as “the Warburg effect,” a process where tumor cells use glycolysis in the cytosol to produce energy rather than the oxidative phosphorylation (OXPHOS)/tricarboxylic acid (TCA) cycle in the mitochondria as most normal cells do [291]. In addition, to acquire necessary nutrients for tumor development, tumors increase utilization of glutamine, an amino acid that is ultimately converted to ɑ-ketogluterate in the mitochondria as a substrate in the TCA cycle [292]. Furthermore, tumors increase utilization of the intermediates in glycolysis/OXPHOS/lipometabolism for biosynthesis and nicotinamide adenine dinucleotide phosphate (NADPH) production. This promotes alterations in metabolite-driven gene regulation and metabolic interactions with the tumor microenvironment [293]. Sirtuins can directly interact with metabolism-related genes and enzymes, and/or indirectly regulate upstream factors or pathways of glycolysis or OXPHOS, such as HIF-1/2, MYC, LKB1-5-AMPK, and p53 [294]. SIRT1 can positively regulate SIRT6 through a complex formed with FOXO3a and, also with NRF, both SIET1 and SIRT6 negatively regulate glycolysis, triglyceride synthesis, and lipid metabolism [295,296]. SIRT1 can also upregulate ATGL through FOXO1 activation, and therefore increase the rate of lipolysis [297]. SIRT1 indirectly interacts with major regulators, such as HIF-1/2, MYC, and LKB1-AMPK. SIRT1 deacetylates HIF-1α and HIF-2α, and thus triggers metabolic reprogramming. The deacetylation of HIF-2α and its increase in activity drive the expression of various transporters and enzymes that support the reductive and oxidative glutamine metabolism [298]. Alternatively, HIF-1α deacetylation inhibits its transcriptional activity decreasing the rate of glycolysis and promoting oxidative metabolism [223]. Another way that SIRT1 can inhibit the glycolysis is through the PGAM-1 deacetylation, reducing its catalytic activity [299]. The regulation of HIF-1α by SIRT2 occurs in a different manner than by SIRT1. HIF-1α induction by SIRT2 increases the expression of the glycolytic genes GLUT1 and LDHA, supporting tumor growth [56]. Moreover, the increased degradation of HIF-1α by SIRT2-mediated deacetylation causes an increase in the intracellular NAD1/NADH ratio [56]. These findings suggest that SIRT2-mediated HIF-1α deacetylation is critical for HIF-1α destabilization and the hypoxic response of cancer cells, and thus may affect their metabolism [300]. SIRT2 also regulates the activity of PGAM and G6PD, two key metabolic enzymes involved in glycolysis and the pentose phosphate pathway (PPP) [294]. SIRT2-mediated deacetylation of G6PD activates its enzymatic activity and stimulates PPP to supply cytosolic NADPH, thus promoting cell survival during oxidative stress [301]. SIRT2 also significantly increases OXPHOS and inhibits glycolysis via deacetylation of glycolytic enzymes, such as ALDOA, GAPDH, ENO1, and PGK1, which modulate cancer metabolism [302,303].

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SIRT2 is involved in fatty acid (FA) metabolism. SIRT2 deacetylates ACLY protein, causing its destabilization. ACLY destabilization leads to fatty synthesis inhibition, another alternative mechanism used by tumor cells to generate energy [55]. The absence of SIRT3 leads to overproduction of ROS, which causes stabilization of HIF-1ɑ in the nucleus and upregulation of its glycolytic targets, implicating SIRT3 as a potential regulator of the Warburg effect and tumor growth [82,174,221]. Another way that SIRT3 promotes upregulation of glycolytic genes is by interacting with LDHA [67]. Moreover, SIRT3 may protect mitochondrial DNA from oxidative damage via OGG1 deacetylation, which acts in DNA repair [300]. SIRT3 can modulate the OXPHOS/TCA cycle. SIRT3 increases mitochondrial oxygen consumption, by promoting GLUD1 expression to regulate glutamine-dependent oxidation [65,304]. GOT2 deacetylation by SIRT3 alters the malate
aspartate NADH shuttle, which sustains high rates of glycolysis in tumor cells [304]. Moreover, when localized into the mitochondrial matrix, SIRT3 deacetylates and increases the activity of PDHA1, which increases the conversion of pyruvate into acetyl-CoA. This promotes the utilization of glucose and represses lactate production by tumor cells [305]. Furthermore, SIRT3 may lead to increased ATP production by deacetylating protein components of the electron transport chains [300,306
308]. In addition, SIRT3 can regulate ROS production through modulation of SOD2, altering tumor cell metabolism [309,310]. Concerning FA oxidation, SIRT3 deacetylates long-chain acyl CoA and upregulates its enzymatic activity, creating an alternative route for energy production [299,311]. The mitochondrial sirtuin, SIRT4, also plays an important role in glutamine metabolism regulation via GLUD1, GDH1, and GLDH1 [312]. Reduction in utilization of glutamine by SIRT4 sensitizes tumors to glucose depletion [87]. In addition, SIRT4 can also function as a lipoamidase to diminish PDH activity, a hallmark of the Warburg effect [313]. SIRT4 catalyzes an alternative reaction involving the transfer of ADP-ribose from NAD1 to target substrates, causing inhibition of GDH, an enzyme that converts glutamate into ɑ-ketogluterate, and which, along with GLS1, drives glutamine catabolism to replenish the TCA cycle [87,314]. The ADP-ribosylation mediated by SIRT4 leads to GDH inactivation and inhibits mitochondrial glutamine metabolism [314]. Moreover, it was found that DNA damage induced a massive increased in SIRT4 expression, which triggers a shutdown of glutamine metabolism that is sufficient to cause cell cycle arrest [94]. In addition, SIRT4 can affect the transportation of ATP into the cytosol and ADP into the mitochondrial matrix, thereby providing substrates for ATP synthase [315]. SIRT4 also participates in FA oxidation, where it deacetylates malonyl-CoA decarboxylase, thus repressing its enzymatic activity and inhibiting the OXPHOS pathway [316]. The suppression of FA oxidation by SIRT4 can also occur through modulation of PPARα and AMPK activity [95,317]. In addition, SIRT4 is also responsible for leucine metabolism [294]. SIRT5 is also located in the mitochondria matrix and regulates the function of mitochondria. SIRT5 regulates cellular NADPH homeostasis and redox potential by deglutarylation of G6PDH [318]. SIRT5 desuccinylates PKM2, which contributes to the antioxidant response and suppresses tumor cell proliferation [103]. Moreover, the suppression of SIRT5 by CDK2 positively regulates aerobic glycolysis [319]. In addition, SIRT5 controls glucose oxidation by directly desuccinylating and repressing the activity of the enzymes PDH-E1 and DH [318,320]. SIRT5 also promotes IDH2 desuccinylation, thus regulating cellular NADPH homeostasis and redox potential [318]. SIRT5 can be translocated into the mitochondrial intermembrane space (MIS), where it deacetylates Cyt c, a protein of the MIS with a central function in the electron transport chain (ETC), as well as apoptosis initiation [191]. SIRT5 plays an important role in ROS detoxification, through desuccinylation and activation of Cu/Zn SOD1, a key antioxidant enzyme [98]. In addition, SIRT5 regulates lysine succinylation in the mitochondria and presents a mechanism for the inhibition of ketogenesis through HMGCS2 [300]. Regarding glutamine metabolism, SIRT5 uses its desuccinylate function to inhibit GLS, the enzyme that catalyzes the conversion of glutamine into glutamate in a reaction producing ammonia, thereby fostering glutamine utilization [205]. The participation of SIRT5 in FA oxidation involves the desuccinylation and activation of ECHA, an enzyme that catalyzes the formation of hydroxyacyl-CoA from enoyl-CoA, another alternative pathway to generate energy for tumor cells [321]. Interestingly, SIRT5 is the only sirtuin known to be involved in the urea cycle, where SIRT5 deacetylates, desuccinylates, and deglutarylates CPS1, increasing its activity. CPS1 is the enzyme that catalyzes the first step of the urea cycle for ammonia detoxification [237,322
324].

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SIRT6 can repress multiple enzymes involved in the homeostatic control of glucose metabolism in cancer cells [111,325,326]. Lack of SIRT6 leads to an increase in glutamine metabolism and ribosomal gene expression [313]. Like SIRT1, SIRT2, and SIRT3, SIRT6 acts as a corepressor of HIF1ɑ transcriptional activity, deacetylating promoters of HIF1ɑ target genes and suppressing the expression of several key glycolytic genes, such as GLUT1, PFK1, PDK1A, and LDH [327]. In this way, SIRT6 maintains an efficient glucose flux into the TCA cycle under normal nutrient and oxygen conditions [328]. SIRT6 is also a MYC corepressor and the loss of SIRT6 results in derepressed expression of GLS1. This allows OXPHOS, but not glycolysis, to generate efficient ATP levels [300]. As mentioned before, SIRT6 is positively regulated by a complex between SIRT1 and FOXO3a, or with NRF1, all of which are involved in metabolic control [295,296]. This regulation results in the inhibition of two ratelimiting enzymes for gluconeogenesis, G6P and PEPCK-C. Increased expression of SIRT6 by p53 can contribute to inhibition of gluconeogenesis and repression of tumor growth [168]. In addition, the expression of SIRT6 can also negatively regulate triglyceride synthesis and fat metabolism [294]. SIRT7 also regulates HIF proteins by binding and decreasing the stability of both HIF-1α and HIF-2α, although the molecular mechanism involved in this regulation is not known [240]. Moreover, SIRT7 suppresses ribosomal gene expression acting as a corepressor of MYC in response to ER stress [329]. Nevertheless, the complex role of SIRT7 in cancer-associated metabolic reprogramming remains to be elucidated [313]. The ability of tumor cells to alter their metabolism and promote cell survival still has many unanswered questions. Sirtuins are involved in several steps of metabolic modulation, acting either directly or indirectly with genes or as cofactor for enzymes involved in metabolism. Sirtuins have been demonstrated to be key factors in this process and therefore deserving of further investigation in this field.

8.9 Sirtuins and cancer therapy Considering that one main role of sirtuins is to deacetylate histones, and that caloric restriction does not extend life span when sirtuins are deleted [144,330], the potential of sirtuins as a therapeutic target has been widely explored [167,331
333]. Pharmacological manipulation of sirtuins began with the use of resveratrol (trans-3,40 ,5-trihydroxystilbene; Rsv), a compound found in grapes that confers protection against several age-related diseases, including cancer. Based on the hypothesis that sirtuins are critical proteins mediating caloric restriction and life span extension, Howitz et al. [334] identified multiple polyphenols as potential SIRT1 activators, and among them Rsv was the most potent. Currently, several SIRT1 activators and inhibitors have been described, with activators presenting more encouraging therapeutic potential than inhibitors [333]. Despite being the most promising as therapeutic targets, fewer activators were identified compared to inhibitors. Some activators have been described for SIRT1 [167,331], while many inhibitors have been reported for SIRT1 and SIRT2 [331
333,335], and a small number for SIRT3 and SIRT5 [167]. In spite of the considerable efforts to develop these molecules, there has been limited success, mainly due to the lack of specificity, poor availability [331], and incomplete knowledge of downstream mechanisms [336]. This field of research is still in its infancy, and greater understanding of sirtuin function will have the potential to identify more promising therapeutic targets. Fig. 8.2 summarizes some of the SIRTs activators and inhibitors with reported effects on cancer. We have listed only the main activators/inhibitors and not their analogs. Also, we cited only the activators and inhibitors with scientific evidence of their roles in cancer, as follows: Resveratrol (Rsv)—Reduced tumorigenesis in SIRT11/2; p531/2 mice [247]. Unrelated to Rsv (SRT 1720)—Apoptosis in myeloma multiple cells via ATM/CHEk2 pathway [337]. Isoflavone/genistein—Binds to hormonal receptors in breast cancer cell lines upregulating SIRT1 to get lower oxidative stress and greater mitochondrial functionability [338]. Curcumin—Deacetylation of p53 and reduction of apoptosis [339]. Combrestatin analog YK-3
237—Deacetylated both mutated and wild-type p53; inhibited the proliferation of TNBC cell lines carrying WTp53; arrested cell cycle in the G2/M phase in mtp53 TNBC cells by inducing apoptotic cell death [340]. Nicotinamide—Blocked proliferation and promoted apoptosis in leukemic cells and OSCC; inhibited the growth and viability in human prostate cells [341
343]. Tenovin-1—Cytotoxic to Burkitt lymphoma and melanoma cells with wild-type p53 [344]. Tenovin-6—Reduced disease progression of CML [344]; eliminated cancer stem cell in uveal melanoma [345]. AGK2—Inhibited growth of glioblastoma multiforme [346]. Salermide—Inhibition of leukemic, colon, and lymphoma cell lines . Sirtinol—Inhibited viability of breast, lung, prostate, and oral cancer cells [342,343,348,349]. Cambinol—Induced apoptosis in BCL6-expressing Burkitt Lymphoma and reduced tumor growth in xenograft mouse; reduced neuroblastoma formation in N-Myc transgenic mice [350,351].

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FIGURE 8.2 Activators and inhibitors of sirtuins in cancer.

8.10 Concluding remarks It is clear in the current literature that sirtuins play a key role in multiple mechanisms of cancer progression. This chapter has summarized numerous research studies describing the role of specific sirtuins in each of the “hallmarks” of cancer. Many challenges remain to be addressed, for example, understanding the dual role of sirtuins as oncogenes or tumor suppressors, and identifying how modulation of sirtuin expression can be employed therapeutically. Certainly, the coming years should bring us abundant growth in sirtuin research, providing us with a better knowledge of their potential for understanding and treating several human diseases, including cancer.

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9 The bifunctional roles of sirtuins and their therapeutic potential in cancer Yeuan Ting Lee*, Yi Jer Tan*, Pei Yi Mok, Ayappa V. Subramaniam and Chern Ein Oon Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Penang, Malaysia O U T L I N E 9.1 The mammalian sirtuins 9.1.1 SIRT1 9.1.2 SIRT2 9.1.3 SIRT3 9.1.4 SIRT4 9.1.5 SIRT5 9.1.6 SIRT6 9.1.7 SIRT7

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9.1 The mammalian sirtuins Sirtuins (SIRTs) are highly conserved class III histone deacetylases that utilize NAD1 as a cofactor to sense energy fluctuations resulting from oxidative, metabolic, or genotoxic stress followed by the coordination of appropriate responses [1,2]. The enzymatic activities of SIRTs are lysine deacetylase and/or mono-ADP-ribosyltransferase that targets both histone and nonhistone proteins [3,4]. The seven members of the mammalian SIRT family (SIRT1
7) have varied specificity, catalytic activity, localization, and substrates; and share a catalytic domain of B275 amino acids with variable lengths of unique additional N-terminal and/or C-terminal sequences [3,5]. Furthermore, mammalian SIRTs differ in subcellular localizations: SIRT1 is prominently present in the nucleus but also found in the cytosol; SIRT2 in the cytoplasm; SIRT3
5 mainly mitochondrial; SIRT6 in the nucleus; and SIRT7 in the nucleolus [1,5]. Generally, SIRT functions can be classified into four main processes: chromatin regulation, cell survival under stress, metabolic homeostasis regulation, and developmental and cell differentiation [6]. SIRTs are highly conserved enzymes implicated in many biological processes linked to longevity, aging, DNA repair, epigenetic regulation, and metabolism homeostasis. Dysregulation of either one of these processes could result in tumorigenesis [2,7
9]. Increasing evidence has demonstrated the crucial role of SIRTs in cancer initiation and progression, and thus SIRTs have become the focus of increasing attention as potential targets in anticancer therapy [3,6,9
11]. However, various reports have highlighted SIRTs to possess bifunctional and contradicting roles in cancer. In the event of cellular stress, the opposed roles of SIRTs can be exhibited, for instance, via the maintenance of DNA integrity (antitumor) versus promotion of cell survival under stress (protumor) (Fig. 9.1). This chapter will discuss the roles of mammalian SIRTs in tumorigenesis and the development of SIRT modulators as anticancer agents. * These authors contributed equally to this work and are joint first authors.

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FIGURE 9.1 Dual role and function of sirtuins in cancer.

9.1.1 SIRT1 SIRT1 is a deacetylase targeting a variety of substrates involved in the modulation of cancer cell proliferation, survival, and apoptosis. SIRT1 levels were found to be dysregulated and overexpressed in many types of cancer, including breast, colon, lung, liver, pancreatic, prostate, leukemia and lymphoma, ovarian, and cervical cancers [12]. SIRT1 has been linked to tumor-suppressive or tumor-promoting roles in cancer, through the targeting of diverse signaling pathways, depending on the cancer type and cellular context. 9.1.1.1 SIRT1 as a tumor suppressor SIRT1 is an important chromatin regulator that is necessary for the maintenance of genome stability and repair of damaged DNA [13,14]. Events of DNA damage such as double-stranded breakage (DSBs) are repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ) modulated by SIRT1 [15,16]. Recent studies have revealed that SIRT1-deficient cells are more susceptible to chromosomal aberrations and fusions upon DNA damage, which is indicative of an impaired DNA repair mechanism [13]. Interestingly, the degree of SIRT1 expression was found to negatively correlate with skin cancer progression, mainly through the deacetylation of skin pigment xeroderma pigmentosum C (XPC) protein which is essential for the initiation of global genome nucleotide excision repair (NER). Furthermore, high SIRT1 activity was reported to reduce the tumorigenesis of liver cancer in vivo by protecting liver tissues from metabolic and DNA damage [14,17]. Low SIRT1 levels were also found to correlate with BRCA1 mutations in breast cancer [18]. In addition, SIRT1 possesses inhibitory roles in cancer cell growth through the modulation of signaling pathways and deacetylation of several targets [19
21]. The effect of varied SIRT1 levels on similar targets may yield contradictory responses and result in different outcomes due to the contrasting roles of numerous SIRT1 targets. In the context of cancer suppression, the moderate increase in SIRT1 levels has been shown to reduce spontaneous carcinomas, sarcomas, and the incidence of tumorigenesis in APC1/min mice [17,22]. SIRT1 also deacetylates HIF-1α and blocks the transcription of downstream genes to subsequently impede tumor growth and angiogenesis in vivo [19]. Moreover, activated SIRT1 is capable of deacetylating the c-MYC protein for degradation in a negative feedback loop and results in the blockade of cervical cancer cell proliferation [21,23]. SIRT1 is the regulator of the forkhead O family (FOXO) transcription factors that are crucial for cell proliferation, differentiation, and survival, which are all implicated in tumorigenesis [24
27]. SIRT1-mediated activation of FOXO3a was found to induce cell cycle arrest and oxidative stress resistance but inhibits FOXO3a-induced cell death, highlighting the partial antitumor role of SIRT1 [28]. Next, SIRT1 can promote autophagosome maturation for the removal of cellular waste and substances harboring malfunctions and mutations through autophagy [29]. The depletion of SIRT1 was found to associate with and may promote the development of prostatic intraepithelial neoplasia (PIN) [30]. The Wnt/β-catenin signaling pathway which is constitutively expressed in many cancer types, is critical for cell differentiation, embryo developments, as well as tumorigenesis [31
34]. SIRT1 was found to deacetylate and block cytoplasmic-to-nuclear translocation of β-catenin which impeded cell proliferation and tumorigenesis in pancreatic cancer cells and colon cells, respectively

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[35,36]. NF-κB plays a central role in the regulation of immune responses and inflammation, such that its dysregulation can lead to tumorigenesis [37,38]. NF-κB is a target of SIRT1 deacetylase activity, and the removal of acetylated NF-κB subunits can result in the suppression of downstream transcriptional activity leading to an inhibition of its prosurvival function [39,40]. A decrease in SIRT1 activity as a result of cigarette smoke exposure, can induce NF-κB acetylation at the RelA/p65 proinflammatory mediator, which is a precursor of non-small cell lung carcinoma [41,42]. As higher SIRT1 levels were linked to the recurrence-free survival rate of prostate cancer patients, in vivo studies revealed that the increased SIRT1 level may be due to the reaction to cancer rather than the cause of tumorigenesis [43]. 9.1.1.2 SIRT1 as an oncoprotein Conversely, SIRT1 has been identified to promote cancer cell survival and tumor progression, given that SIRT1deleted APC1/min mice showed an increase in tumor apoptosis [44]. The knockdown of SIRT1 was also observed to inhibit the expression of HIF-1α downstream target genes in mice harboring hepatocellular carcinoma (HCC) [45]. In addition, one study reported that deacetylation of c-MYC by SIRT1 could reinforce its stability for NAD1 synthesis which could, in turn, enhance tumorigenesis [46]. In prostate cancer, inhibition of SIRT1 resulted in lower cell viability, cell growth, and chemoresistance which provided insights into the oncogenic roles of SIRT1 [47]. However, the oncogenic function of SIRT1 may require additional factors given that the overexpression of SIRT1 alone did not increase tumor formation in vivo [43]. SIRT1 can display oncogenic properties through crippling the functions of several tumor suppressors such as p53, p73, and HIC1 proteins [48]. One possible oncogenic role of SIRT1 is through the suppression of p53 and p300 (histone acetyltransferase of p53) activities via deacetylation of these substrates [49,50]. As p53 modulates cell fate such as DNA repair, cell cycle arrest, and apoptosis, p53 deacetylation by SIRT1 could reduce p53-mediated apoptosis and hamper p53-induced cellular senescence [51,52]. The TGF-β signaling pathway is involved in cell growth, differentiation, migration, apoptosis, and other key physiological and pathological processes [53,54]. SIRT1 has been demonstrated to regulate TGF-β through deacetylating Smad7 leading to its ubiquitination, which subsequently inhibits TGF-β-dependent apoptosis [55]. One factor that could have contributed to the contradictory roles of SIRT1 is the subcellular localization in different cell lines. Both SIRT1 and p53 contain nuclear localization signals and nuclear export signals which facilitate the shuttling of both types of proteins through the nuclear envelope under different circumstances [56,57]. For instance, some cancers such as prostate, lung, breast, and skin cancer showed mainly cytoplasmic expressions of SIRT1 and nuclear p53 compared to normal tissues which are both found localized primarily in the nucleus [58,59]. The difference in localization allows for a reduced interaction between SIRT1 and p53, thus minimizing the regulation on p53 transcription. Therefore the tumorpromoting or suppressing properties of SIRT1, in this case, depended on other SIRT1 targets present in the cytoplasm. The difference in the cellular localization of SIRT1 in different cancer types could explain why SIRT1 can act as a tumor suppressor in some, but as an oncoprotein in others. Another factor that could contribute to the contrasting effect of SIRT1 in cancer is the mutation profile of SIRT1-targeted proteins. Several studies have reported the antitumor role of SIRT1 in cells with nonfunctional p53 in vivo and displayed oncogenic behavior in PTENdeficient prostate cancer mice models [60,61]. Furthermore, the role of SIRT1 in tumorigenesis may be agedependent, probably due to an accumulation of mutations of SIRT1 regulators and targets [62].

9.1.2 SIRT2 SIRT2 is a deacetylase that is involved in the regulation of chromatin assembly, promotion of chromosomal stability, and control of the cell cycle during early metaphase checkpoint regulation; involving the deacetylation of α-tubulin and interaction with histone methyltransferase PR-Set7 [63
66]. SIRT2 is also involved in the regulation of mitotic processes, cell motility, differentiation, oxidative metabolism, and cell death [63]. The role of SIRT2 is tumor-specific and dependent on cellular context. Generally, SIRT2 deacetylates various substrates involved in the modulation of the cell cycle, which include H4K16, p53, p65, FOXO1, FOXO3, and CDK4 [67]. SIRT2 has bifunctional roles in cancer. SIRT2 has been shown to inhibit cancer cell growth through deacetylating β-catenin and KDM4A [68,69], but increase cell proliferation and decrease the overall survival of patients with pancreatic cancer, HCC, or neuroblastoma [70
72]. In addition, SIRT2 can also play contrasting roles in tumors of different grades and classifications. Low SIRT2 expression in moderately differentiated grade 2 breast tumors contributed to impaired cell cycle and poor prognosis. Conversely, the high SIRT2 levels in poorly differentiated grade 3 breast tumors are responsible for more aggressive tumors, a lower relapse-free period, and mortality contributed by cell cycle dysregulation and crippled DNA repair mechanism [73].

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9.1.2.1 SIRT2 as a tumor suppressor SIRT2 is downregulated and depleted in several types of cancers including breast cancer, glioma, HCC, neck squamous cell carcinoma, non-small cell lung cancer (NSCLC), and prostate cancer [63,73
77]. The low expression of SIRT2 was linked to cancer progression and promotion of cancer cell migration, invasion, and metastasis [78]. SIRT2 exhibits antitumor properties mainly through deacetylating key proteins involved in cell proliferation, cell integrity, and DNA damage pathways [72]. SIRT2 also acts to maintain proper cell cycle checkpoint and chromatin regulation to prevent the formation of hyperploid cells. A study by Kim and colleagues found that SIRT2-deficient mice developed liver, intestinal, and mammary tumors as a result of cell cycle disruption [76]. Moreover, SIRT2 was reported to suppress skin cancer in vivo, and reduced SIRT2 expression was observed in both breast and liver cancer tissues from clinical samples [66,79]. Furthermore, SIRT2 can induce apoptosis in the event of DNA damage either via deacetylating p53 and repressing p53-mediated transcriptional activation or through deacetylating FOXO3a, and triggers caspase-dependent apoptosis in breast cancer and kidney cells [80,81]. 9.1.2.2 SIRT2 as an oncoprotein SIRT2 is overexpressed in some cancers such as neuroblastomas and pancreatic cancer cells; while knockdown of SIRT2 resulted in apoptosis of glioma and cervical cancer cell lines [12]. SIRT2 is also highly expressed and responsible for cell proliferation, invasion, and tumor growth in leukemia, neuroblastoma, HCC, and pancreatic cancer. SIRT2 is known to inhibit p53 such that the diminishment of SIRT2 activity was shown to reduce p53 acetylation and activate apoptosis in cancer cell lines. In addition, SIRT2 overexpression has been shown to cause MYC activation, which subsequently increased antioxidant production and inhibited apoptosis in cholangiocarcinoma [82]. SIRT2 activity was shown to promote cell proliferation via increasing N-MYC and c-MYC protein stability in neuroblastomas and pancreatic cells respectively [71]. Furthermore, SIRT2 was reported to positively regulate epithelial
mesenchymal transition (EMT) by reducing E-cadherin expression, leading to increased cancer cell migration and invasion in vivo [68]. The deacetylation of protein kinase B (AKT) by SIRT2 will also result in the activation and enhancement of EMT through the RAS/ERK/JNK/MMP-9 pathway and the AKT/GSK3β/β-catenin signaling pathway. Subsequently, this has been shown to promote the migration and invasion of gastric cancer and HCC, respectively [77,83]. A study by Jing and colleagues reported that SIRT2 inhibition resulted in MYC reduction through ubiquitination and degradation in vitro and in vivo, which is required for the growth and survival of transformed breast cancer cells [84]. Interestingly, tumors expressing high levels of SIRT2 were found to possess enhanced tumorigenesis and exhibit enhanced resistance to chemotherapy due to the deacetylation of KRAS (a SIRT2 substrate) leading to its activation [85].

9.1.3 SIRT3 SIRT3 is a NAD1-dependent deacetylase that is mainly localized in the mitochondria [86]. The full-length SIRT3 protein (44 kDa) is inactive, and proteolytic cleavage is required for the release of the active enzyme (28 kDa) [87]. SIRT3 regulates intracellular processes which include metabolism, longevity, aging, cancer, and stress responses [88]. The function of SIRT3 may be different depending on the cell-type and tumor-type; thereby, SIRT3 may act as an oncogene or a tumor suppressor. High SIRT3 expression was observed in various cancer types, such as NSCLC, breast cancer, oral squamous cell carcinoma (OSCC), renal cancer, colon cancer, gastric cancer, melanoma, and esophageal cancer [69,89
95]. On the other hand, low level of SIRT3 was also reported in different cancer types including hepatocellular cancer, pancreatic cancer, osteosarcoma, prostate, ovarian cancer, B-cells malignancies, and breast cancer [96
98]. 9.1.3.1 SIRT3 as a tumor suppressor Among all types of cancers, a total of 20% of cancer types consisted of deleted SIRT3 gene [99]. Moreover, in testicular, prostate, and hepatocellular tumors, the gene expression level of SIRT3 was found to be lower in cancer cells relative to normal cells [98,99]. SIRT3 protein levels were also found to be lower in human breast tumors compared to normal tissues, and mice lacking SIRT3 were more susceptible to mammary cancer [98
100]. The lack of SIRT3 can lead to poor prognosis and result in low patient survival rate [100]. Compared to SIRT1 and SIRT2, SIRT3 is a highly context-specific tumor suppressor. The major tumor suppression function of SIRT3 is repressing ROS-mediated damage which may otherwise cause DNA mutations, genomic instability, and activation of metabolism, cell survival, and proliferation pathways, leading to tumorigenesis [101]. One of the functions of SIRT3 is to promote the regeneration of antioxidant glutathione via deacetylating and activating both mitochondrial manganese superoxide dismutase (MnSOD2) and isocitrate dehydrogenase 2 (IDH2) [102
104].

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In addition, SIRT3 activates mitochondrial respiration via the pyruvate dehydrogenase complex (PDC) which is usually inactivated in cancer cells [105
107]. The mitochondria play central roles in the production of ROS with a tightly controlled threshold [108]. High ROS levels may cause damage in DNA, proteins, and lipids, which can result in mitochondrial dysfunction and carcinogenesis [109]. One major function of SIRT3 is to prevent oxidative damage by limiting ROS production through the regulation of MnSOD and IDH activities [98,109]. MnSOD is involved in superoxide (O2 2 ) detoxification via the conversion of superoxide to hydrogen peroxide, and then to water [110,111]. On the other hand, IDH is responsible for converting isocitrate to α-ketoglutarate for the production of nicotinamide adenine dinucleotide phosphate (NADPH) [103]. In the event of oxidative stress, SIRT3 deacetylates and activates FoxO3a to promote the transcription of oxidative stress response genes (such as SOD2, IDH, and catalase) to reduce the level of ROS [112]. SIRT3-knockout have resulted in increased ROS in vitro and in vivo. The unregulated high levels of ROS may cause genomic, metabolic, and microenvironmental-associated instabilities, leading to enhanced cancer survival and induced therapeutic resistance [98,108,111]. The loss of SIRT3 appears to drive HIF activation and enhance the hypoxia-inducible factor-1 (HIF) response to hypoxia through a ROS-dependent mechanism, such that the upregulation of HIF activity is strongly associated with cancer cell survival and proliferation [98,99]. A study by Kim and colleagues demonstrated that SIRT3-deficient cells can cause invasive and tumorigenic phenotypes in vitro and in vivo; mainly through the expression of MYC and RAS oncogenes which resulted in increased chromosomal aberrations and tumor growth in athymic nude mice [98]. The downregulation of SIRT3 in breast cancer was reported to promote cancer glycolysis and cell proliferation [97]. Moreover, SIRT3 was reported to play proapoptotic roles in colorectal cancer (CRC) and HCC cells through the JNK and GSK-3β/Bax signaling pathways, respectively [113,114]. SIRT3 was also shown to downregulate glutathione S-transferase P1 (GSTP1), activate the JNK and c-Jun pathway, and upregulate the proapoptotic Bim protein to sensitize HCC cells to chemotherapeutic agents [115]. 9.1.3.2 SIRT3 as an oncoprotein The overexpression of SIRT3 in cancer, such as glioma, was shown to inhibit apoptosis, disrupt cell cycle, and promote CpG island hypermethylation [116]. SIRT3 is also elevated in breast and OSCC cancer cells, and reportedly inhibited p53-induced growth arrest in human bladder cancer cells [95,117,118]. In addition, SIRT3knockdown was reported to inhibit the proliferation and sensitize OSCC cells to radiation and chemotherapy treatments in vivo [95]. High SIRT3 expression is positively correlated with tumor progression, poor prognosis, and low patient survival [94,96]. Overexpression of SIRT3 was found to promote cell proliferation and enhance ATP generation, glucose uptake, glycogen formation, increased MnSOD activity, and lactate production, as well as inducing an aggressive phenotype in gastric cancer cells [69]. Silencing of SIRT3 led to the inhibition of cancer cell growth, proliferation, invasion, migration, and sensitized cancer cells to chemotherapy and radiation [90,92]. SIRT3 knockdown also reduced cell proliferation, colony formation, and migration, as well as induced senescence and G1-phase arrest in human melanoma cells [93]. Furthermore, SIRT3 was described to deacetylate histone proteins H4K16Ac and H3K9Ac which could lead to genomic instability. H4K16Ac is linked to the regulation of cell cycle progression, transcription, DNA repair, and DNA replication; deacetylation of H3K9Ac is required for H3K9me2/3 methylation [4]. SIRT3 also deacetylates human 8-oxoguanine-DNA glycosylase 1 (OGG1) which is a base excision repair (BER) enzyme to prevent degradation and limit apoptotic death in cells subjected to oxidative stress. Cheng and colleagues reported that the loss of SIRT3 led to increasing acetylation and degradation of OGG1 which can promote stress-induced apoptosis in glioblastoma cancer cells [119]. SIRT3 was reported to deacetylate tumor suppressor p53 and partially abrogate p53 activity, to promote cancer cell proliferation, and enact cellular growth arrest and senescence in human bladder cancer cells [117]. SIRT3 also inhibits mitochondrial translocation of pro-apoptotic protein Bax for the maintenance of mitochondrial membrane integrity.

9.1.4 SIRT4 SIRT4 is present primarily in the mitochondrial matrix and is involved in various mitochondrial metabolic processes. Unlike the other SIRTs, SIRT4 was initially known to be ADP-ribosyltransferase but lacked NAD1-dependent deacetylase activity [120,121]. However, in a recent study, SIRT4 was reported to exhibit lysine deacetylase activity. SIRT4 is involved mainly in regulation of cell metabolism. SIRT4 was found to regulate the leucine metabolism and insulin secretion to maintain the glucose homeostasis in the cells [122]. In lipid metabolism, SIRT4 deacetylates and inhibits malonyl CoA decarboxylase (MCD), which is involved in fatty acid oxidation, as well as functions as lipoamidase [123,124].

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9.1.4.1 SIRT4 as a tumor suppressor Reduced SIRT4 mRNA levels have been correlated with poor prognosis in colorectal, bladder, breast, lung, esophagus, and gastric cancers [125
129]. SIRT4 was reported to regulate glutamine metabolism which is a key player in cancer cell growth and proliferation [130]. Under normal conditions, SIRT4 suppresses tumor growth via ADP-ribosylation and inhibition of glutamate dehydrogenase (GDH) [121,131]. GDH is a mitochondrial enzyme that converts glutamate into α-ketoglutarate in the tricarboxylic acid (TCA) cycle, which is a series of ATP-synthesis metabolic reactions. SIRT4 downregulates GDH activity via ADP-ribosylation, resulting in reduced insulin secretion which negatively affects energy supply. A study conducted by Jeong and colleagues demonstrated that overexpression of SIRT4 significantly inhibited the proliferation of Burkitt lymphoma cells via repression of the mitochondrial glutamine metabolism [132,133]. Moreover, SIRT4 has been linked to the maintenance of genomic stability and cancer initiation. The loss of SIRT4 could lead to both increased glutaminedependent proliferation and stress-induced genomic instability which can promote tumorigenesis in lung cancer, as evident in SIRT4 knockout mice [132]. This study also reported that SIRT4 may inhibit glutamine utilization upon DNA damage and results in cell cycle arrest. The mitochondrial regulator proteins including mitofusins, optic atrophy protein 1 (OPA1), and dynamin-related proteins (DRPs) are responsible for mitochondrial fusion and fission that promote cancer cell invasion and migration. Overexpression of SIRT4 has been found to deregulate DRP1 via an extracellular signal-regulated protein kinase (ERK) activity, resulting in the reduction of mitochondrial fission in NSCLC [134,135]. The overexpression of SIRT4 was also reported to negatively regulate CRC invasion and metastasis through increase of E-cadherin protein expressions resulting in blockade of EMT [129]. 9.1.4.2 SIRT4 as an oncoprotein Mounting evidence has revealed SIRT4 to exhibit oncogenic properties upon prolonged endoplasmic reticulum (ER) stress and DNA damage, specifically after exposure to chemotherapeutic drugs and ultraviolet light [136,137]. Jeong et al. reported that SIRT4 is capable of protecting cancerous cells against these stresses by inhibiting apoptosis and promoting liver cancer cell survival [137]. The upregulation of SIRT4 expression in liver cancer cells was shown to reduce caspase-3 protein expression and resulted in increased cancer cell survival and growth [137]. SIRT4 overexpression was also found to positively affect breast cancer cell proliferation, migration, and invasion [136]. As such, tumor cells may have utilized SIRT4 as a tool to hijack cellular stress response pathways upon oncogenic stress to increase their growth and survival. The limitations of currently available data on the roles of SIRT4 as an oncogene demonstrate the need for future research.

9.1.5 SIRT5 SIRT5 is a strong lysine desuccinylase, demalonylase, and deglutarylase but a weak deacetylase. SIRT5 is localized in the mitochondria. It targets the urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS-1) involved primarily in maintenance of the cellular ammonia homeostasis [138]. The activation of CPS-1 via the removal of glutaryl and succinyl moieties by SIRT5 catalyzes the detoxification of ammonia, which otherwise could induce oxidative stress and subsequently damage the host tissues and drive tumorigenesis [139,140]. In addition, SIRT5 is also involved in the regulation of glycolysis by activating the glyceraldehyde 3-phosphate dehydrogenase (GADPH) activity via demalonylation of a key residue, K184 located on the enzyme’s homodimerization interface, thereby enhancing glycolytic flux in primary hepatocytes [141]. 9.1.5.1 SIRT5 as a tumor suppressor SIRT5 has been reported to regulate cellular ROS levels by desuccinylating Cu/Zn superoxide dismutases (SODs). Recent studies have revealed that binding of SIRT5 to SOD1 could initiate the reduction of ROS level to hamper H1299 lung cancer cell proliferation [142]. In addition, upregulation of SIRT5 was found to correlate with decreased ACOX1, an enzyme involved in acyl-coA catalysis which produces hydrogen peroxide (H2O2) as a byproduct. The desuccinylation activity of SIRT5 on ACOX1 was shown to reduce H2O2 and oxidative stress levels in HCC cells, thus preventing DNA damage and suppressing tumor development [143]. The suppression of HCC development and progression may also be attributed to the negative modulation of EMT [144]. For example, SIRT5 was found to negatively interact with SNAIL and deacetylates vimentin to reduce EMT and consequently inhibit HCC cell migration and metastasis [144]. In tumorigenesis, SNAIL plays vital roles in maintaining cancer stem-like properties to increase replicative ability and drive drug resistance [145,146]. The increase in SIRT5 levels may inhibit SNAIL gene expression which subsequently suppresses metastasis. II. Sirtuins and cancer

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9.1.5.2 SIRT5 as an oncoprotein Elevated SIRT5 mRNA expression in colon, lung, breast, ovarian, melanoma, and pancreas relative to the corresponding normal tissue type suggested its role as a tumor promoter [147]. Overexpression of SIRT5 protein has been reported to be positively linked to larger tumor size, increased lymph node metastasis, and advanced colorectal tumor stage through glutamine metabolic rewiring [148]. SIRT5 can activate glutamate dehydrogenase 1 (GLUD1) to provide an adequate supply of energy to the CRC cells, leading to enhanced cell proliferation and survival. In contrast, when SIRT5 gene expression is silenced, the formation of α-ketoglutarate is inhibited and thereby prevents the entry of glutamine-derived metabolites into the TCA cycle. This will, in turn, suppress the proliferation of CRC cells via increased apoptosis and cell cycle arrest [148]. Overexpression of SIRT5 has been reported to facilitate drug resistance in NSCLC and ovarian cancer [149,150]. Both in vitro and in vivo studies have shown the overexpression of SIRT5 to promote cell proliferation and colony formation, whereas the opposite effect was observed in SIRT5 knockdown cells. The NSCLC cancer cells with depleted SIRT5 are more susceptible to the anticancer drugs, including cisplatin (CDDP), 5-fluorouracil (5-FU), and bleomycin [149]. In addition, SIRT5 has been shown to inhibit ROS-mediated DNA damage and apoptosis upon cisplatin treatment by activating the Nrf2/HO-1 pathway which controls the expression of antioxidant-responses and drug resistance genes in NSCLC cancer cells [149,150]. The deacetylation of FOXO3a and deactivation of BCL-XL by SIRT5 prevent cells from suicide via apoptosis [147,150]. The percentage of apoptosis cells was increased in HCC cells upon SIRT5 knockdown, in correspondence to the upregulation of proapoptotic proteins (i.e., cl-cas3, cl-PARP, and Bax) and downregulation of apoptosis suppressor Bcl-2 [151].

9.1.6 SIRT6 SIRT6, a Sir2 homolog, is a chromatin-bound sirtuin and located primarily in the nucleus. SIRT6 plays a vital role in cancer pathways as SIRT6 mostly functions in the regulation of metabolic homeostasis and DNA repairs [152]. SIRT6 was found to act as mono-ADP-ribosyltransferase [153] and therefore was involved in mono-ADP-ribosylation (MARylation) of PARP1 in DNA repair [154]. During DNA repair, SIRT6 is relocalized to the site of DNA damage, in order to conduct base excision and double-stranded repair by activating the ADP-ribosyltransferase diphtheria toxin-like (ARTD1) enzyme through MARylation [155]. Recently, SIRT6 was found to exhibit histone deacetylase activity on two substrates, H3K9Ac and H3K56Ac, which is essential to promote chromatin modification and support genomic stability. 9.1.6.1 SIRT6 as tumor suppressor A role of SIRT6 has been found in pancreatic ductal adenocarcinoma (PDAC) [156]. The loss of SIRT6 in PDAC can activate the Lin28 oncogene promoter to increase the rate of PDAC progression and metastasis. In contrast, the activation of SIRT6 reduces the hyperacetylation of H3K9Ac and H3K56Ac at the Lin28b promoter region, thereby inhibiting the cellular proliferation and tumor progression [157]. Colon cancer patients harbouring lower levels of SIRT6 protein have a significantly shorter overall survival or disease-free survival time [158]. In CRC, P13K/Akt signaling is an important intracellular pathway in regulating nutrient uptake, cell proliferation, migration, and apoptosis [159]. The inhibition Akt protein upon BKM120 P13K-inhibitor treatment resulted in the dephosphorylation of FOXO3a, to promote apoptosis in colon cancer cells [158]. The loss of SIRT6 expression in CRC cells suppressed the BKM120-induced cell apoptosis by inhibiting the cyto-c release and caspase-9/3 activity. This correlates with the increased cell viability and survival rate among SIRT6-knockdown cells, highlighting the significance of SIRT6 in governing cell death. Besides, the overexpression of SIRT6 protein could inhibit H3K9 acetylation and hamper the protein expression of survivin, thereby causing the cells to undergo apoptosis. In another study, Lin and coworkers have discovered the positive relationship between ubiquitinspecific peptidase (USP10) and SIRT6 in colon cancer. Reduced expression of both USP10 and SIRT6 have been observed in colon cancer cells [160,161]. USP10 works as a deubiquitinase for SIRT6 to protect it from degradation due to ubiquitination in human colon cancer cells. USP10 interacts closely with SIRT6 to inhibit the activation of c-Myc, an oncogene involved in colon cancer cell cycle progression and tumor development. Knockdown of both USP10 and SIRT6 resulted in cell cycle arrest at the S and G2/M phases. 9.1.6.2 SIRT6 as an oncoprotein SIRT6 has been shown to govern cell proliferation and apoptosis in HCC by activating the ERK1/2 signaling pathway [162]. The upregulation of SIRT6 protein expression is positively correlated with the expression of transforming growth factor (TGF)-β1 and H2O2/HOCl ROS to promote anoikis resistance and enhanced tumorigenesis in HCC [163]. In contrast, inhibition of SIRT6 expression will lead to cellular senescence, resulting in the suppression in HCC cell tumorigenicity. The overexpression of SIRT6 also enhanced the invasiveness and migratory ability II. Sirtuins and cancer

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of NSCLC cells via the ERK1/2/MMP9 signaling pathway [164]. In a human skin squamous cell carcinoma (SCC) mice model, a reduction of tumor growth was reported in the SIRT6 skin-specific conditional knockout (cKO) group compared to the wild-type group post treatment with UVB, DMBA, and TPA. In another study, SIRT6 was found to increase cell proliferation and survival through positive regulation of cyclooxygenase-2 (COX-2) expression in skin cancer cells [165].

9.1.7 SIRT7 SIRT7 is the least studied among the SIRT family members. SIRT7 is associated with RNA polymerase I (Pol I) transcriptional machinery, which plays a role in regulating rRNA expression. Although it presents predominantly in the nucleolus, SIRT7 exhibits a weak deacetylase activity compared to SIRT1 and SIRT6. Its role in modulating cellular stressors (ER, mitochondrial, and oxidative stress) to aid cell survival is well documented as a tumor suppressor [166
170]. SIRT7 is also involved in DNA damage repair and maintaining genomic stability via the regulation of the p53 tumor suppressor [171]. 9.1.7.1 SIRT7 as a tumor suppressor In pancreatic, breast, and oral cancer patients, the low expression of SIRT7 is positively correlated with increased tumor aggressiveness and reduced survival [172
174]. In a well-designed study by McGlynn and colleagues, the survival analysis showed that pancreatic cancer patients with low SIRT7 protein expression have a shorter diseasefree and disease-specific time, suggesting high SIRT7 protein expression could prolong patient survival and tumor recurrence [174]. The downregulation of SIRT7 protein resulted in breast cancer metastasis through the activation of the TGF-β1 signaling pathway, leading to enhanced EMT [172]. In contrast, the overexpression of SIRT7 blocked TGF-β1 signaling and therefore inhibited the EMT process and metastasis in breast cancer. In OSCC, the overexpression of SIRT7 promoted Smad4 deacetylation, which significantly reduced the expression of N-cadherin, vimentin, and MMP7, but increased expression of E-cadherin, indicating the suppression of EMT [173]. 9.1.7.2 SIRT7 as an oncoprotein The H3K18Ac histone is a novel substrate for SIRT7 [175]. The hypoacetylation of H3K18Ac is related to poor prognosis and an aggressive form of pancreatic adenocarcinoma [176,177]. SIRT7 is important to maintain cancer cell phenotypes. The downregulation of SIRT7 in fibrosarcoma cell lines led to impairment of anchorageindependent cellular growth. The SIRT7 knockdown of U251 xenografts study of human glioblastoma revealed disruption to the tumor development, suggesting the protumorigenesis roles of SIRT7 in maintaining cancer development. In another study, SIRT7 protein expression is associated with the metastatic phenotypes of epithelial and mesenchymal tumors [178]. The loss of SIRT7 reduced the migratory ability and invasiveness of the PC3 prostate cancer cell line. The metastatic properties of PC3 cells were linked to the declined expression level of E-cadherin (CDH-1) protein [179]. This indicates that SIRT1 could act as a scaffold protein to recruit SIRT7 in which the latter may suppress the E-cadherin protein expression via H3K18Ac deacetylation. In contrast, there is little effect of SIRT1 activity on E-cadherin repression [178]. SIRT7 is reported to be overexpressed in several cancers, including colon, ovarian, and liver cancers [171,180,181]. Yu and colleagues reported that the overexpression of SIRT7 protein was found to positively linked to the aggressive form of CRC, resulting in a reduced survival rate among patients [182]. SIRT7 overexpression has been demonstrated to promote CRC cell proliferation by increasing ERK1/2 phosphorylation and cyclin D1 protein expression via the activation of the RAS-RAF-MEK-ERK pathway. The study demonstrated the role of SIRT7 in the promotion of cell migration and tumor metastasis via EMT induction in vitro and in vivo. On the other hand, downregulation of SIRT7 significantly inhibited cell growth, proliferation, invasiveness, and migratory ability of CRC cells [182].

9.2 Sirtuin modulators In the previous part, we have discussed the dual roles of each SIRTs in tumor progression. Different tumor type and tumor microenvironments can induce different responses to SIRT regulation. A wide range of sirtuin inhibitors (SIRTi) and sirtuin-activating compounds (STACs) have been discovered and developed (Figs. 9.2 and 9.3). In the following section, different classes of SIRTi and STACs and their effects on the progression of various cancers will be discussed.

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161 FIGURE 9.2 Different classes of sirtuin inhibitors with their relative chemical structures.

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FIGURE 9.3 Structures of cancer-related sirtuin-activating compounds.

9.2.1 Sirtuin inhibitors Increasing evidence indicates that inhibiting SIRTs may interrupt cancer cell biology and tumor progression. Compared to SIRT activators, SIRTi have been widely studied in anticancer research. Many SIRTi have been developed and are available in the market (Table 9.1). SIRTi can be classified into two categories: general SIRTis targeting the NAD1 pocket (e.g., Nicotinamide) and SIRT isotype-specific inhibitors (e.g., EX527, AGK2, sirtinol, cambinol, and tenovins) [11]. These SIRTis have been reported to exert anticancer properties in various cancer types. Below is the summary of different classes of SIRTi with their roles in inhibiting tumor progression. 9.2.1.1 Nicotinamide and its analogues Nicotinamide (NAM) is a form of vitamin B3 (nicotinic acid) that provides NAD1 as a substrate to non-redox enzymes including the SIRTs family proteins [198]. NAM maintains the NAD1 redox homeostasis and could modulate SIRT activity through noncompetitive product inhibition mechanisms and regenerating NAD in salvage pathways [199]. In brief, NAM is the by-product generated by SIRTs and is an endogenous inhibitor of the SIRTs deacetylation activity [200]. NAM is cleaved by nicotinamidase (Pnc1), to maintain a low level of NAM. NAM is a well-known water-soluble SIRTi shown to exhibit an inhibitory effect toward SIRT1 in vitro [198,200].

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TABLE 9.1 Commercial SIRTi used in anticancer research. Sirtuin inhibitor

Target

IC50 (cell free assay)

References

Nicotinamide

SIRTs

SIRT1: 120 μM; SIRT2: 100 μM; SIRT3: 50 μM; SIRT5: 150 μM; SIRT6: 184 μM

[183
185]

3-Triazolylpyridine (3-TYP)

SIRT1; SIRT2; SIRT3

SIRT1: 0.088 μM; SIRT2: 0.092 μM; SIRT3: 0.016 μM

[186,187]

Sirtinol

SIRT1; SIRT2

SIRT1:131 μM; SIRT2: 38 μM

[188]

Salermide

SIRT1; SIRT2

SIRT1: 100 μM; SIRT2: 25 μM

[189]

Cambinol

SIRT1; SIRT2

SIRT1: 56 μM; SIRT2: 59 μM

[190]

Selisistat (EX 527)

SIRT1

38 nM

[191]

Inauhzin

SIRT1

0.7
2 μM

[192]

Thiomyristoyl

SIRT2

0.028 μM

[193]

Suramin

SIRT5

22 μM

[194]

Tenovin-1

SIRT1; SIRT2; SIRT3

SIRT1: 21 μM; SIRT2: 10 μM; SIRT3: 67 μM

[195]

Tenovin 6

SIRT1; SIRT2

SIRT1: 21 μM; SIRT2: 10 μM

[195]

AGK2

SIRT2

3.5 μM

[196]

SirReal2

SIRT2

0.14 μM

[197]

NAM treatment was reported to abolish Sir2p activities in yeast and could induce inhibition at a sufficiently high concentration, if it is assumed that it can be readily transported to cells [198]. NAM inhibits SIRT1 in chronic lymphocytic leukemia (CLL), leading to the blockade of CLL cells’ proliferation and induction of p53-mediated apoptosis [201]. NAM has also been reported to inhibit cell growth, proliferation, and promote apoptosis by targeting SIRT3 in OSCC cells [95], as well as targeting SIRT1 in prostate cancer cells [202]. A recent study conducted by Yousafzai and colleagues demonstrated that NAM potentiated the inhibition of cisplatin-induced viability and the activation of apoptosis in lung cancer cells [203]. NAM analogs, such as acridinedione derivatives (compound 4d) and 30 -phenethyloxy-2-anilino benzamide analogues (compound 33a and 33i), demonstrated anticancer effects by inhibiting SIRT1 and SIRT2 in breast and colon cancer cells, respectively [204,205]. 3-Triazolylpyridine (3-TYP) is another NAM analog which exhibits specific SIRT3 inhibitory activity, whilst also inhibiting both SIRT1 and SIRT2 protein activity. 3-TYP has exhibited a cytotoxic effect in the HeLa cervical cancer cell line [186,187]. 9.2.1.2 β-Naphthol-containing inhibitors β-Naphthol-containing inhibitors are a well-known category of SIRTi comprising the first generation of SIRTi. Some SIRTis such as Splitomicin and Sirtinol are commercial, while others like salermide, cambinol, and JGB1741 are still under research. Both Splitomicin and Sirtinol were identified through cell-based screens in Saccharomyces cerevisiae yeast by accessing Sir2p inhibition [206]. Splitomicin is a Sir2p inhibitor with a weak activity on human SIRTs. Splitomicin has a short half-life in vitro due to its labile δ-lactone toward aqueous hydrolysis in neutral conditions [207]. Splitomicin analogs, β-(4-methyl) phenyl-8-bromo-splitomicin and R-enantiomer of β-(4-methyl) phenyl-8-methyl-splitomicin, inhibit SIRT2 activity [208]. The Splitomicin analogs demonstrated an antiproliferative effect and induced α-tubulin hyperacetylation in breast cancer cells [208]. Another Splitomicin analogue, Benzodeazaoxaflavins, is a SIRT1/2 inhibitor that promotes apoptosis in leukemia cells and exhibits an antiproliferative effect in colon carcinoma and glioblastoma multiforme cancer stem cells [209]. Sirtinol 2-((2-Hydroxynaphthalen-1-yl methylene) amino)-N-(1-phenethyl) benzamide is a SIRT1 and SIRT2 inhibitor. Sirtinol is more potent against SIRT2 compared to SIRT1 [188]. Sirtinol inhibits cell proliferation, induces apoptosis and cell cycle arrest in breast, lung (NSCLC), adult T-cell leukemia-lymphoma (ATL), and colon cancer cells [210
213]. Sirtinol has also been reported to sensitize prostate cancer and cervical cancer cells to cisplatin and camptothecin treatments [214,215]. Combination treatments of Sirtinol with sodium dichloroacetic acid (DCA) in NSCLC cells have been shown to synergistically induce G0/G1 phase arrest, enhance cancer cell death in vitro, and inhibit tumor growth in vivo [216].

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Salermide N-(3-[2-hydroxy-naphthalen-1-ylmethylene]-amino)-phenyl-2-phenyl-propionamide is a drug modified from Sirtinol which exhibits a greater inhibition of SIRT1 and SIRT2 compared to Sirtinol [189]. Salermide inhibits 80% of the enzymatic activity of SIRT1 and SIRT2 at a concentration of 100 and 25 μM, respectively [189]. Salermide treatment demonstrated a reduction of cell viability in a panel of cancer cells including leukemia, lymphoma, colon, and breast cancer cell lines. Salermide demonstrated a strong cytotoxic effect toward blood malignancy (MOLT4) cells but does not kill nontumorigenic cells (MRC5 cells). The main mechanism of Salermide is apoptosis induction via SIRT1 inhibition [189]. Cambinol inhibits SIRT1 and SIRT2 proteins whilst exhibiting a weak inhibitory activity against SIRT5 and no inhibition against SIRT3 [190]. Cambinol-mediated inhibition of SIRT1 sensitizes a NCI H460 lung cancer cell line to DNA-damaging agents (i.e., etoposide, paclitaxel) in a p53-independent manner by increasing the acetylation of p53, Ku70, and FOXO3a. Cambinol also induced apoptosis in Burkitt lymphoma cells and human HCC, as well as inhibiting tumor growth in tumor xenografts [190,217]. JGB1741, (4,5,6,7-tetrahydro-2-[(E)-[(2-hydroxy-1-naphthalenyl)methylene]amino]-N-(phenylmethyl)-benzo[b] thiophene-3-carboxamide) inhibits SIRT1 in K562, HepG2 and MDA-MB 231 cancer cell lines [218]. 9.2.1.3 Indole derivatives Indole is a heterocyclic bioactive moiety that is widely found in nature, amino acids (e.g., tryptophan), animal hormones (e.g., melatonin), and plant hormones (e.g., indol-3-ylacetic acid/auxin). The indole group consists of a number of SIRTis containing an indole moiety [219]. EX-527 (6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) was the first potent, selective, and cellpermeable SIRT1i, screened using a bacterial-expressed recombinant human SIRT1 [191,220]. EX-527 strongly inhibits SIRT1, but displays a weak potency against SIRT2 and SIRT3 [191]. Upon the release of nicotinamide from the SIRT1 enzyme postcatalysis, EX-527 prevents the release of both products, deacetylated peptide and O-acetyl-ADP-ribose, from the enzyme [9]. However, the use of EX-527 as a single agent with concentrations up to 100 μM did not increase the level of p53 upon SIRT1 inhibition [195]. By exposing the SIRT1 abundant, p53 wild-type human NSCLC cell lines to DNA-damage agents, the inhibition of SIRT1 by EX-527 conversely led to an increase in p53 acetylation. At the same time, no p53-mediated cell survival or cell proliferation was detected [221]. EX-527 exhibited a strong antiproliferative effect in pancreatic cancer cells [222,223]. EX-527 was reported to enhance the sensitivity of pancreatic cancer cells to gemcitabine treatment by inducing DNA damage with micronuclei formation and elevation of apoptotic cell death [223]. Zhang and coworkers also reported that EX-527 sensitizes pancreatic cancer cells to gemcitabine treatment by a significantly inducing G0/G1 cycle arrest, senescence, and apoptosis [222]. In addition, EX-527 also increased the chemosensitivity of drug-resistant lung cancer cells (H460-R) to cisplatin treatment [203], as well as induced G1 phase cell cycle arrest in cervical cancer cells [224,225]. EX-527 inhibited SIRT1 in H460-R cells leading to an increase of X-ray cross-complementing-1 (XRCC1) ubiquitination, which promotes chemoresistance in lung cancer [203]. Inauzhin, 10-[2-(5H-[1,2,4] triazino[5,6-b] indol-3-ylthio)butanoyl]-10H-phenothiazine inhibits SIRT1 and leads to p53 acetylation [192]. It was demonstrated that this results in a reduction in cell viability across a panel of cancer cell lines including human lung, ovarian, colon, osteosarcoma, glioma, and breast cancer. Inauzhin inhibits cell proliferation and induces senescence and apoptosis through a p53-dependent mechanism [192]. Inauzhin was also shown to suppress tumor growth in xenograft models derived from human lung cancer H460 and colon cancer HCT 116 cells. BZD9L1 is a benzimidazole derivative SIRTi that inhibits SIRT1 and is more potent against SIRT2 [226]. According to Yoon et al., inhibition of SIRT1 and SIRT2 resulted in hyperacetylation of α-tubulin with no obvious changes in p53. One unique feature of this SIRTi is its autofluorescent properties which can be used for pharmacokinetic studies. BZD9L1 exerts cytotoxic activity against colorectal, leukemia, and breast cancer cells. The mechanism of BZD9L1 in monitoring CRC cells growth has been thoroughly studied in vitro [148]. BZD9L1 inhibits cell proliferation and migration, and promotes apoptosis without inducing cell cycle arrest in CRC cell lines. Combination treatment of BZD9L1 with 5-FU synergistically reduced CRC cell viability and induced cell cycle arrest, senescence, micronucleation, and apoptosis in HCT 116 CRC cells [227]. Combination treatment of BZD9L1 and 5-FU was also demonstrated to inhibit tumor growth in a HCT 116 CRC tumor xenograft [227]. Other indole derivative of SIRTis include AC-93253 [228] and GW5074 [229,230]. AC-93253 is a highly potent SIRT2i which also possesses inhibitory activity against SIRT1 and SIRT3 [228]. AC-93253 exhibits potent cytotoxic effects towards prostate, lung, and pancreatic cancer cells in nanomolar concentration ranging from 10 to 100 nM. AC-93252 treatment leads to an upregulation of acetylated α-tubulin and hyperacetylation of p53 in NCI-H460 cells.

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9.2.1.4 Thioacyllysine-containing compounds Thioacyllysine-containing compounds inhibit SIRT activities by forming a covalent ADP-ribose-adduct (10 -S-alkylimidate intermediate) at the active site to block the SIRT-catalyzed deacetylation reaction [231]. BH-TM-4 is a thioacyllysine-containing SIRTi targeting SIRT1
3 and SIRT6 [193]. Among them, BHJH-TM4 is the potent cell-permeable SIRT6 inhibitor which also inhibits SIRT1, SIRT2, and SIRT3 [193]. Nε-thioacetyl-lysinecontaining tri-, tetra-, and pentapeptides are α-tubulin- and p53-based SIRTis with potent SIRT1 and SIRT2 inhibitory activities [232]. The most potent SIRT2i discovered by Kiviranta et al. is H-K-K(ε-thioAc)-L-A. A few potent SIRT1is have also been developed, exhibiting an equipotency to EX-527. The histone H3 lysine 9 (H3K9) thiosuccinyl peptide (H3K9TSu) inhibits SIRT5 with no inhibitory activities against SIRT1
3 [233]. Thiomyristoyl (TM) strongly inhibits SIRT2 but weakly inhibits SIRT1 and exhibits no activity against SIRT3 [84]. TM inhibition of SIRT2 leads to an increase of acetylated α-tubulin in breast cancer cells as well as downregulated c-MYC in various cancer cell lines. TM also inhibits tumor growth in vivo [84]. Compound 27 and 30 from Mellini and coworkers were SIRT is shown to be cell-permeable and nontoxic against SIRT1
3 [234]. Both compound 27 and 30 inhibit cell proliferation and induce cell cycle arrest at the G1 phase of breast cancer (MCF-7) and ARPE-19 cells [234]. Although thioacyllysine-containing compounds have effective inhibition properties, their poor selectivity, weak cell permeability, and instability have limited further development into potential drug candidates [235].

9.2.1.5 Tenovin Tenovin-1 and its water-soluble analog, Tenovin-6 are small molecules that activate p53 via inhibition of SIRT1 and SIRT2 [195]. Tenovin-1 increases p53 protein expression and the p53 downstream target p21CIP1/WAF1 protein through SIRT1 inhibition. Tenovin-1 is more cytotoxic against p53 wild-type cells including the BL2 Burkitt’s lymphoma cells, NTera2D cells, HCT 116 CRC cells, and ARN8 melanoma cells [195]. Compared to Tenovin-1, Tenovin-6 exhibits a greater inhibiting capacity against SIRT1 and SIRT2 [195]. Tenovin-6 has been demonstrated to be more cytotoxic against ARN8 melanoma cells than Tenovin-1, inhibiting the growth of ARN8-derived xenograft tumors [195] and neuroblastoma xenografts [236]. Many studies have also demonstrated Tenovin-6 to effectively induce apoptosis in various cancer cells such as colon cancer, uveal melanoma, B-cell lymphoma, acute lymphoblastic leukemia, and gastric cancer cells [237
241]. Besides enhancing the apoptotic effect in cancer cells, Tenovin-6 has also been shown to suppress cancer cell proliferation, inhibit migration, and block autophagy in vitro [238,239,242]. Tenovin-6 is also an effective adjuvant in combination therapies. Synergistic anticancer effects have been observed in a HCT 116 tumor xenograft treated with Tenovin-6 and oxaliplatin or 5-FU combination treatment [237]. In addition, Tenovin-6 combined with metformin synergistically induced caspase-3dependent apoptosis in NSCLC cells [243]. Combined treatment of Tenovin-6 and BCR-ABL tyrosine kinase inhibitors (TKI), imatinib, significantly improved survival in mice xenografts and increased apoptosis in imatinib-resistant chronic myelogenous leukemia (CML) stem and progenitor cells in vivo [244]. The development of Tenovin-6 as single-agent treatment option is limited by its low specificity; however, Tenovin-6 is a good agent to be used in combination treatments [243,244]. Tenovin-D3 is an analog of Tenovin-6 [245]. Unlike Tenovin-6, which functions as a SIRT1i, Tenovin-D3 does not increase p53 levels or transcription factor activity but inhibits SIRT2 which contributes to the elevation of acetylated α-tubulin and p53-independent induction of p21CIP1/WAF1 (CDKN1A) [245].

9.2.1.6 Suramin Suramin (8,80 -[carbonylbis [imino-3,1-phenylenecarbonylimino (4-methyl-3,1-phenylene) carbonylimino]] bis-1,3,5-naphtalenetrisulfonic acid) is a symmetric polyanionic naphthylurea originally developed for trypanosomiasis and onchocerciasis treatments [221]. Suramin is a SIRT5i [194]. Suramin showed anticancer activities by inhibiting cell proliferation and inducing angiogenesis in adrenocortical cancer, human brain-metastatic melanoma, cervical, and ovarian cancer [246
248]. However, Suramin is not a specific inhibitor targeting only SIRTs, but also targets other proteins such as the heparinase (HPA) enzyme [248] and cell surface receptors (e.g., EGFR and PDGFR) [249]. Suramin is currently under phase 1 clinical trial for bladder cancer, but not as a SIRTi [250,251]. Although Suramin analogs, such as aminoanthranilic acid derivative (NF675), exhibit preferential inhibitory activity against SIRT1 and SIRT2, suramin and its analogs have a highly polar sulfonic acid structure which causes cell impermeability and limits their use as a SIRTi [194]. In addition, Suramin induces serious systemic side effects including neurologic toxicity, which has restrained its clinical use [251].

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9.2.1.7 Other SIRTi 9.2.1.7.1 AGK2

AGK2 (2E)-2-cyano-3-[5-(2,5-dichlorophenyl)furan-2-yl]-N-(quinolin-5-yl)prop-2-enamide is a SIRT2i with reported slight inhibition of SIRT1 and SIRT3 [196]. AGK2 displayed anticancer activities in cervical cancer [224] and glioma cells [252]. AGK2 downregulates both SIRT1 and SIRT2 protein expression and triggers sub-G0 phase arrest, leading to an increase in cervical cancer cells death [224]. The AGK2 and DCA combination treatment has been demonstrated to synergistically inhibit 80%
90% of NSCLC cancer cell survival by inhibiting SIRT2 [216]. 9.2.1.7.2 MHY2256

MHY2256 showed potent inhibition against SIRT1. MHY2256 decreased the viability of breast (MCF-7) and ovarian (SKOV-3) cells after a 48-h treatment period [253]. MHY2256 significantly induced cell cycle arrest in the G1 phase, leading to an effective increase in apoptotic cell death in MCF-7 and SKOV-3 cells. MHY2256 treatment also resulted in a significant increase in acetylated p53, upregulated LC3-II, and induced autophagic cell death in MCF7 tumor xenograft model. 9.2.1.7.3 SirReal2

Sirtuin-rearranging ligands 2 (SirReal2) is a family of aminothiazoles that selectively inhibits SIRT2 [197]. Binding of SirReal2 to the SIRT2 protein leads to the rearrangement of its active site and locking it in an open conformation. Incubation of HeLa cells with SirReal2 showed an increase expression of acetylated α-tubulin and a downregulation of BubR1. 9.2.1.7.4 MC2494

MC2494, 3-(4-(2-chlorobenzoyl)-1H-pyrrol-2-yl)-2-cyano-N-(quinolin-5-yl) acrylamide, inhibits both SIRT1 and SIRT2. MC2494 was recently found to be a pan-SIRT1 inhibitor which displays promising anticancer activity in vitro, in vivo (xenograft and allograft cancer models), and in leukemic blasts ex vivo [254]. MC2494 inhibits cancer cell proliferation and migration, induces apoptosis, and suppresses tumor growth [254,255]. 9.2.1.7.5 Toxoflavin

Toxoflavin, also known as xanthothricin, is a SIRT1/2 inhibitor with higher inhibition against SIRT1 than SIRT2. Toxoflavin was originally identified as a toxin produced by bacteria with antibiotic function. Toxoflavin has been demonstrated to exhibit cytotoxic effects in various cancer cells including NSCLC, breast, prostate, ovarian, gastric, central nervous system, leukemia, and pancreatic cancer cells [256]. Toxoflavin is an electron-carrier which facilitates H2O2 production during glycolysis and the citric acid cycle [257]. The H2O2 produced accounts for its poison and nonspecific toxicity effects, limiting its clinical development.

9.2.2 Sirtuin activators The mechanism by which STACs activate SIRTs remains controversial. Two opposing models have been proposed to account for STAC activity: (1) direct allosteric activation of SIRTs through the lowering of peptide substrate Km, and/or (2) indirect activation resulting from off-target effects [258
261]. There are two different types of STACs: natural and synthetic. Chalcones, flavones, and stilbenes are plant polyphenols which were the first natural SIRT1 STACs discovered in 2003 [262
266]. As research on SIRTs progressed over the years, synthetic STACs were also developed. The different classes of synthetic STACs consist of imidazothiazoles, oxazolopyridine, thiazolopyridines, benzimidazoles, and bridged ureas [235,263]. These STACs activate SIRTs allosterically [235]. The more commonly known synthetic STACs which are commercially available and reported to target cancer include SRT1720, SRT3025, SRT1460, SRT2183, and UBCS039 [235,267
270]. The drugs which have been developed to target and activate SIRTs are mainly focused on antiaging, antiinflammatory, antidiabetic, and antiobesity activity. Resveratrol (RSV) is a natural polyphenol compound containing two phenyl rings separated by a methylene bridge. RSV has been found in many plants, such as cranberries, grapes, peanuts, and Japanese giant knotweed [271]. The anticancer roles of RSV have been widely studied and indicate that RSV can inhibit tumorigenesis by inducing S-phase cell cycle arrest and apoptosis, as well as inhibiting metastasis, aromatase, and angiogenesis in liver, breast, prostate, leukemia, skin, and myeloma cancer cells [271,272].

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Despite SIRT1 being the most studied SIRT and possessing a wide range of activators and inhibitors, SIRT1 still yields controversial results regarding whether or not its activation or inhibition can produce an anticancer effect [273
275]. SIRT1 deficiency induced an abnormal accumulation of cells in the early phases of mitosis-impaired DNA damage repair, and chromosome instability which could subsequently cause cancer [60]. RSV is a potent SIRT1 agonist with implicated antitumor capability. RSV regulates SIRT1 through the AMP-dependent kinase (AMPK) pathway which activates SIRT1 by increasing the intracellular concentration of NAD1 [273,276]. Wang and colleagues have previously shown that in vivo SIRT11/2; p531/2 mice xenograft models with different malignancy types (i.e. sarcomas, lymphomas, teratomas, and carcinomas) exhibited SIRT1 activation after treatment with RSV, which in turns effectively inhibited the tumorigenesis of SIRT11/2; p531/2 mice [60]. RSV treatment in highproliferative SIRT1 knockdown prostate cancer cells have a reverse effect by inhibiting cancer cells’ growth and proliferation [277]. This study also reported that RSV treatment induces autophagy through SIRT1/S6K pathways and inhibits the incidence of precursor lesions of prostate cancer through the Akt/mTOR signaling pathway [277]. RSV was also reported to reduce cell viability and induce apoptosis in siRNA-SIRT1 transfected human chondrosarcoma (a malignant primary bone tumor) cells, via activation of caspase-3 and inhibition of NF-kB pathways [278]. Besides, RSV has been shown to alleviate EMT processes in lung and ovarian cancer in vitro and in vivo [279,280]. Expression of SIRT1 was suppressed in these two cancer cell lines due to hypoxic stress, thus promoting EMT. RSV activates SIRT1 expression which consequently overcomes the SUMOylation-mediated EMT process [279,280]. Although increased evidence indicates RSV to be an effective SIRT1 activator with anticancer properties, its poor solubility, low bioavailability, rapid elimination, and unwanted toxicity effects are the major factors that limit its development as a cancer drug candidate [281]. Alternatively, synthetic STACs such as SRT2170 and SIRT1460 have been synthesized to overcome the RSV limitation. These synthetic STACs are potent SIRT1 activators with 800- to 1000-fold efficacy compared to RSV [259,267]. SRT1720 has been shown to reduce cancer cell viability and sensitize cancer cells to chemotherapy drugs. A study conducted by Sonnemann and coworkers demonstrated that combination treatments of SRT1720 with chemotherapy drugs largely enhanced etoposide- and vincristine-induced cell death, whereas RSV inhibited cell death in Ewing’s sarcoma cells [282]. Furthermore, another study also reported an enhanced effect when SRT1720 was used in combination with dexamethasone or bortezomib [269,283]. SRT1720 induced apoptosis in multiple cancer cells including breast and myeloma [283,284] through the increase in SIRT1 deacetylase activity. SRT1720 induced apoptosis through caspase 8/9/3 activation and ATM-CHK2 signaling pathways which will subsequently inhibit the NF-κB signaling pathway, which otherwise maintains cell growth and survival [283]. Similarly, SRT1720 was shown to inhibit the growth of pancreatic cancer in vitro and in vivo through a SIRT1
lysosomal-dependent apoptotic pathway in another study [285]. This study also reported that SRT1460 and SRT3025 were effective in inhibiting the growth and survival of pancreatic cancer. SRT1720 and SRT3025 were shown to potently hinder cell survival when used in adjunction with paclitaxel and gemcitabine, whereas SRT1460 only showed equal potency when used alone [285]. However, SRT1720 has also been reported to promote breast cancer metastasis [274], suggesting that the effects of STACs may function in the opposite manner. Thus further evaluation of STACs activity in a cancer-specific manner is required. SIRT1 STACs, SRT2183, and SRT501 have demonstrated significant inhibition of growth and induction of apoptosis in malignant lymphoid cell lines. Furthermore, a significant inhibition of growth and induction of apoptosis were demonstrated in malignant lymphoid cell lines when these compounds were used in adjunct with panobinostat [275]. It is noteworthy that SRT501 is an improved formulation of RSV possessing improved bioavailability [270,273]. UBCS039 is a newly synthesized pyrrolo[1,2-a]quinoxaline derivative, the first synthetic SIRT6 activator [286]. UBCS039 was reported to activate SIRT6 activities without affecting its expression level regardless of cancer histotype [287]. UBCS039 induced a time-dependent activation of autophagy in various cancer cells, such as human NSCLC, fibrosarcoma, colon, HeLa, and epithelial cervix carcinoma [287]. Iachettini and colleagues reported that UBCS039-mediated activation of autophagy was strictly dependent on SIRT6 deacetylating activity. In this study, antioxidants such as NAC or Trolox were reported to completely counteract UBCS039-induced autophagy, indicating that increased ROS had a key role in upstream events that commit the cells to autophagy. A sustained activation of SIRT6 as a result of UBCS039 treatment also resulted in autophagy-related cell death [287]. Another recent SIRT6 activator that has been discovered is MDL-800 [288]. Huang et al. reported that MDL-800 increases the deacetylase activity of SIRT6 by up to 22-fold via binding to an allosteric site. The activation of SIRT6 by MDL-800 inhibits cell proliferation and induces cell cycle arrest in HCC. The in vivo study of MDL-800 in a HCC tumor xenograft model and in SIRT6 KO mice also demonstrated a significant impediment of tumor growth by activating the deacetylase activity of SIRT6 [288].

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9.3 Conclusion and future perspectives SIRTs are important biological regulators that are implicated in many cellular processes. Their physiological functions and critical roles in maintaining genome stability and stress responses are fundamental in the onset of most cancers. SIRTs are also the command centre regulating the signaling pathways which are highly associated with tumor development. The molecular mechanisms of SIRTs are yet to be fully elucidated. For instance, some SIRTs share common targets and regulate the same cellular process in either the same or different contexts, suggesting that they could demonstrate synergistic or antagonistic effects among each other when responding to stress. SIRTs also have a dual role in regulating tumor initiation and progression. These sometimes-contradictory roles of SIRTs in cancer are highly dependent on the tumor type and tumor microenvironment. However, knowledge of what specific factors in the cancer type and stroma that underpin the oncogenic or suppressor roles of SIRTs is still far from complete. Over the last decade, the dual roles of SIRT1 have been extensively studied in many cancers, which in turn has provided us with some ideas on the mechanisms of SIRTs in cancer modulation. However, the dual roles of other SIRTs in cancer are not well established and remain largely unresolved. Efforts to identify the unique functionality of each SIRT in the bid to unravel how different SIRTs regulate the cancer biological processes and signaling pathways are critical steps toward the development of effective SIRT-targeting cancer therapies. Depending on their specific roles as oncogenics or tumor suppressors, targeting SIRTs is a potential therapeutic option in sensitizing tumor cells to anticancer treatments. The discovery and development of novel and highly specific SIRT modulators will facilitate the development of an effective cancer therapy. The commercially available SIRTi are well-known for their inhibitory activities against SIRT1/2, which affect the acetylation of p53 and α-tubulin, to produce anticancer effects. These SIRTi inhibitors target the NAD1 binding site in the active site of SIRT protein. Nevertheless, most of the SIRT modulators have not been tested against other SIRT family members. These SIRT inhibitors may lack selectivity, the requisite pharmacological properties, or fail to display a significant potency with high micromolar IC50, ultimately limiting their potential for the development as treatment modalities. Growing evidence has indicated that the anticancer effects of available SIRTis are most likely be attributed to the nonspecific targeting of multiple SIRTs at once. This action may improve treatment outcomes but may also induce antagonist effects and unwanted toxicity consequences. The other aspect that deserves consideration includes its effects against the other NAD1-consuming proteins, such as poly(ADP-ribosyl) polymerases (PARPs) and cyclic ADP-ribose synthases (CD38 and CD157). In addition, little is known about how these commercially-available SIRTis affect the NAD1-consuming proteins. Thus the polypharmacology properties of SIRTi are an important factor to understand in order to establish the feasible use of SIRTi in anticancer therapies. Moreover, only tenovin, cambinol, and inauhzin have demonstrated an anticancer effect in in vivo models. At the same time, none of these SIRTis has entered clinical trials as possible anticancer agents. Therefore, research emphasised on developing a highly potent, specific, and low-toxic SIRTi is crucial. SIRTs are highly dependent on NAD1 for their activities, therefore the activation of SIRTs could lead to an overconsumption or overproduction of NAD1, resulting in many downstream effects, given that SIRTs are implicated in several pathways. Furthermore, SIRT activation is not necessarily therapeutic in all types of cancer as it has shown contrasting reports in different cancer types, though there is still plenty of work to be done to fully understand the nature of SIRTs activation. The potential for SIRT activation as a possible anticancer treatment is a huge therapeutic step in cancer therapy. Many studies have also emphasised on SIRT inhibition as an anticancer therapy, underscoring the need for research on SIRT activation as a potential therapeutic strategy. All together, these warrant further investigations to elucidate the mechanisms of SIRT activity in diverse cancer types and in cancers harbouring different mutations before directing attention towards the development of an activator or inhibitor. The lack of efficient enzymatic activity assays has also limited the development of SIRT modulators, especially for SIRT4
7 family members. Structural proteomics is one of the important aspects that can be employed to address the unresolved issues of SIRT members. In silico studies involving the use of bioinformatics tools, docking, and molecular modeling can enhance the understanding on the intricate relationships among the SIRT family members. The information derived from analyses of the SIRT/effector complexes is also essential to uncover the mechanism underlying SIRT inhibition and/or activation, which in turn facilitates the structure-based design of specific SIRT modulators with therapeutic potential. In vitro and in vivo studies involving the overexpression, knockdown, or knockout of SIRTs may also provide a deeper insight into the molecular functions of this protein family. Collectively, better characterization and understanding of each SIRT family member is crucial to fill the knowledge gaps between SIRTs and malignancy.

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Acknowledgment The authors would like to acknowledge Chris Malajczuk from the Biomolecular Modeling Group in the School of Pharmacy and Biomedical Sciences at Curtin University for proofreading the manuscript.

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10 Sirtuins and next generation hallmarks of cancer: cellular energetics and tumor promoting inflammation Robert Kleszcz and Wanda Baer-Dubowska ´ Poland Department of Pharmaceutical Biochemistry, Poznan University of Medical Sciences, Poznan, O U T L I N E 10.3.1 SIRT3 10.3.2 SIRT4 10.3.3 SIRT5

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10.1 Introduction: an overview of sirtuins involvement in inflammation and cancer metabolism The activities of mammalian sirtuins (SIRTs) depend on NAD1, as a cosubstrate for their histone deacetylase activity. In addition to its role as a cofactor in many enzymes, NAD1, regulates key metabolic processes involved in energy production and usage. Therefore SIRTs along with NAD1, play a critical role in guarding cellular homeostasis by sensing bioenergy needs and restoring it during stress response. Inflammation is the immune system’s response to different harmful stimuli and defends against cellular stress. However, uncontrolled acute inflammation may become chronic, contributing to a variety of diseases including cancer. Receptor activation by detrimental stimuli triggers intracellular signaling pathways such as the mitogen-activated protein-kinase (MAPK), nuclear factor kappa-B (NF-κB), activator protein 1 (AP-1), Janus kinase (JAK)-signal transducer, and activation of transcription (STAT) pathways [1 4]. Sirtuins, such as SIRT1, known to be a major metabolic regulator, epigenetically reprogram inflammation by altering histones and transcription factors, including NF-κB and AP-1 [5,6]. Inflammation links the immune, metabolic, and mitochondrial bioenergy pathways and SIRTs seem to be important modulators of these networks. Moreover, the inflammation connection with metabolism might be an important contributor to the development of cancer. One of the most intriguing features of cancer cells is the appearance of aerobic glycolysis, known as the Warburg effect. This phenomenon, which was first observed by Otto Warburg in the 1920s, was initially thought

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to be an adaptation to hypoxic conditions, but later studies showed that the mutations that lead to tumorigenesis also cause aerobic glycolysis by upregulating the expression of glycolytic genes at the transcriptional level [7]. It is now widely accepted that the Warburg effect is one of the key features of tumorigenesis [8]. It not only promotes rapid uncontrolled proliferation, but also confers invasive properties. The proposed mechanisms that underlie the Warburg effect include: (1) mitochondrial uncoupling, which can promote aerobic glycolysis in the absence of permanent and transmissible alterations to the oxidative capacity of cells; and (2) aerobic glycolysis, representing a shift to the oxidative metabolism of nonglucose carbon sources, particularly glutamine, an amino acid that is ultimately converted to α-ketoglutarate in the mitochondria in order to enter the citric acid cycle (CAC). Moreover, alterations in the glycolytic pathway itself may be equally or even more important. For example it was suggested that pyruvate kinase M2 (PKM2) or hexokinase 2 (HK2) might be the key mediators of aerobic glycolysis and promote tumor growth at least in certain types of tumors [9,10]. Epigenetic alterations, both on DNA and chromatin level in which SIRTs might play a particularly important role, seem to be equally important in glucose metabolism in cancer cells [11]. It has been shown that the loss of SIRT6 promotes cell glycolytic production toward lactate production, even in aerobic conditions [12]. This observation suggested that the lack of SIRT6 might provide a growth advantage for tumor cells. Additionally, it seems that promoter methylation of genes encoding enzymes affecting glycolysis may interfere with the same signaling molecules, namely NF-κB and hypoxia-inducible factor HIF1α [13]. Thus aerobic glycolysis in cancer cells and inflammation might be linked through epigenetic modulators of those transcription factors. In this regard multiple studies have showed that sirtuins, such as SIRT1 and SIRT2, limit inflammation. SIRT1 directly impacts inflammation by deacetylating and inactivating the p65 subunit of NF-κB, reducing the expression of NF-κB-dependent proinflammatory genes [14], as well as indirectly among the others via inhibition of the NLRP3 inflammasome activated by mitochondrial damage and reactive oxygen species (ROS) production [15]. Moreover, in macrophages activated by Lipopolysaccharide (LPS), the switch from oxidative phosphorylation to glycolysis occurs. As a result increased levels of intermediates of glycolysis and the pentose phosphate pathway (PPP) can be observed. This metabolic shift directly influences the inflammatory state of the cell [16]. It is likely that the increase in glycolysis allows for a rapid increase in adenosine triphosphate (ATP) production since, although less productive in terms of ATP synthesis than the CAC, glycolysis can be strongly induced [17]. This allows the macrophages to persist during the host defense response. The increase in the PPP allows for the production of intermediates for biosynthesis, such as purines and pyrimidines. The increased aerobic glycolysis and downregulation of the CAC refer to the Warburg effect observed in cancer cells. Interestingly, antiinflammatory M2 macrophages have decreased glycolysis and utilize oxidative metabolism. Therefore a movement toward high glycolysis might be indicative of inflammatory cells, whereas a shift toward oxidative phosphorylation is a hallmark of antiinflammatory cells. In turn, inflammatory cells promote cancer development sustained by aerobic glycolysis [15]. The above facts clearly point out the involvement of SIRTs in cancer metabolism and inflammation. As ROS formation is a key element of inflammatory defense, the SIRTs requirement for NAD1 further supports their function also in redox sensing as one of the mechanisms linking cancer and inflammation. In the next sections the role of specific sirtuins in the processes linking cancer metabolism, oxidative stress, and inflammation will be reviewed, taking into consideration their varied subcellular location.

10.2 Nuclear and cytosolic sirtuins involvement in metabolism of cancer and inflammatory cells 10.2.1 SIRT1 Overwhelming evidence suggests that SIRT1 senses nutritional availability and passes this information to proteins that control fuel utilization and energy adaptation. SIRT1 binds to and deacetylates a number of important transcription factors, such as peroxisome proliferator activated receptor gamma (PPARγ), PPARα, PPAR gamma coactivator 1 alpha (PGC-1α), and forkhead box, subgroup O (FOXO) family of transcription factors to drive metabolic responses such as gluconeogenesis and fatty acids oxidation [18]. In this regard deacetylation of FOXO1 increases the expression of the gene encoding adipose triacylglycerol lipase (ATGL), which is involved in adipose lipids mobilization [19,20]. In turn, deacetylation of PPARγ suppresses adipogenesis in concert with decreased cholesterol and lipids production resulting from SIRT1 modification of sterol regulatory element-binding protein 1 (SREBP1) [21]. Deacetylating of PGC-1α causes a shift in mitochondrial substrate usage promoting fatty acids oxidation and preserving glucose. This switch is supported by increased transcription of mitochondrial fatty acid

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oxidation and oxidative phosphorylation genes suggesting that SIRT1 may couple fatty acid oxidation with ATP production [18,22]. Downregulation of glycolysis by SIRT1 may result on one hand from deacetylation of HIF1α, and phosphoglycerate mutase 1 (PGAM1) on the other. The gene encoding the latter is the only glycolytic enzyme which is not controlled by HIF1α [23,24]. Aerobic glycolysis and limited mitochondrial activity are the hallmarks of cancer cells as well as inflammatory cells, which in addition promote cancer development that is sustained by aerobic glycolysis. The SIRT1-induced metabolic switch from glucose to fatty acid oxidation may be considered as one of the mechanisms of the calorie restriction (CR) protective effect against cancer [25]. In turn CR induces SIRT1 upregulation [26]. Besides metabolic effects, SIRT1 protects against oxidative stress and inflammation via the upregulation of FOXO3-dependent antioxidant enzymes, for example, catalase and interaction with RelA/p65 subunit of NF-κB leading to its downregulation [14,27 29]. Overall, SIRT1’s influence on bioenergetic metabolism can counteract the aerobic glycolysis both in cancer cells and inflammatory cells and thus limit their survival.

10.2.2 SIRT2 SIRT2 is basically a cytoplasmic enzyme, but also can be found in the nucleus and inner mitochondrial membrane [30,31]. It was suggested that the major function of SIRT2 in cooperation with SIRT1 is induction of mitotic arrest in critically damaged cells, allowing them to proceed to apoptosis. Therefore the inhibition of SIRT2 may predispose cells to uncontrolled growth [18]. SIRT2, similarly to SIRT1, is involved in the regulation of glucose metabolism affecting the expression and/or activity of the key enzymes of its catabolic pathways. In this regard PGAM, a glycolytic enzyme, commonly upregulated in several cancers, is deacetylated by SIRT2 at lysines 100/106/113/138 in its central region. Overexpression of SIRT2 or mutations at gene sites encoding the acetylatable lysines of PGAM attenuates cancer cell proliferation with a concomitant decrease in PGAM activity [32]. The isoform of pyruvate kinase PKM2 was also shown to be a critical target of SIRT2 that is implicated in cancer. When PKM2 is in its active tetramer state pyruvate is directed into mitochondria, ultimately providing ATP via oxidative phosphorylation [33]. In contrast, when PKM2 dissociates a decrease in enzymatic activity, it is observed that it blocks pyruvate production. The study of Park et al. showed that acetylation reduced PKM2 activity by preventing the formation of the active tetrameric form of enzyme. Overexpression of deacetylated PKM2 mutant in SIRT2-deficient mammary tumor cells altered glucose metabolism and inhibited malignant growth. Therefore the loss of SIRT2 in cancer reprograms glycolytic metabolism via PKM2 toward enhanced aerobic glycolysis [34]. A more recent study using primed pluripotent stem cells showed that SIRT2 critically regulates metabolic reprogramming during induced pluripotency by targeting glycolytic enzymes including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, and enolase. Moreover, knockdown of SIRT2 in human fibroblasts resulted in significantly decreased oxidative phosphorylation and increased glycolysis [35]. On the other hand, the combination of SIRT2 inhibitor with dehydrogenase kinase inhibitor efficiently activated pyruvate dehydrogenase E1 component (PDHA1), and shifted the metabolism of nonsmall cell lung cancer (NSCLC) to oxidative phosphorylation, enhancement of ROS generation, and activation of AMP-activated protein kinase (AMPK) signaling [36]. The PPP plays a pivotal role in meeting the anabolic demands for cancer cells. SIRT2 deacetylates the first and rate-limiting enzyme glucose-6-phosphate dehydrogenase (G6PD) and promotes NADPH production. It was shown that activation of G6PD by SIRT2 supports the proliferation and clonogenic activity of leukemia cells. Moreover, SIRT2 is overexpressed in clinical acute myeloid leukemia (AML), while G6PD acetylation is downregulated and its activity is increased compared to that of normal cells [37]. Similarly to SIRT1, SIRT2 affects the lipid metabolism, blocking adipogenesis through the repression of transcriptional activity of PPARγ [38]. Recently, the involvement of SIRT2 in mitochondrial metabolism was described [31]. In this regard, acetylation of several metabolic mitochondrial proteins was found to be altered in SIRT2-deficient mice resulting in increased oxidative stress and decreased ATP levels in some tissues. In summary, SIRT2 involvement in energy metabolism in cancer cells rather facilitates their survival. However, its effects on cell cycle and subsequent induction of apoptosis indicates that increased activity of SIRT2 in certain cancers could be beneficial.

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10.2.3 SIRT6 SIRT6 is a nuclear sirtuin which has both ADP-ribosyltransferase and deacetylase activity preferentially acting on histone H3K9 and H3K56 [39]. SIRT6 plays various roles in metabolism, stress resistance, and life span. SIRT6 appears to function as a corepressor of the transcription factor HIF1α by deacetylating H3K9 at HIF1α target gene promoters. SIRT6-deficient cells exhibit increased HIF1α activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration [12,29]. This sirtuin is described as a master regulator of glucose metabolism and tumor suppressor that regulates aerobic glycolysis in cancer cells. Lack of this chromatin factor leads to tumor formation even in nontransformed cells. Notably, inhibition of glycolysis in SIRT6-deficient cells completely rescues their tumorigenic potential, suggesting that enhanced glycolysis is the driving force for tumorigenesis in these cells [40]. Early studies of Zhong et al. demonstrated that SIRT6 regulates glucose homeostasis via inhibiting multiple glycolytic genes, including the glucose transporter GLUT1, by promoter binding and histone deacetylation. This allows mitochondrial oxidative phosphorylation, but not glycolysis, to generate efficient ATP production. Consequently, the loss of SIRT6 increases glycolysis and diminishes mitochondrial respiration [12]. Evidence exists that SIRT6 may exert this activity by interacting not only with HIF1α, but also MYC transcription factor, acting as its corepressor. SIRT6 deacetylates H3K56 of MYC target genes. Although knocking out SIRT6 may lead to neoplastic transformation in cell lines, the interaction with MYC is rather necessary to exert this effect in vivo indicating a key role of this sirtuin in controlling cancer cell proliferation by corepressing MYC transcriptional activity [40]. However, the loss of SIRT6 corepressor activity engages also glutamine metabolism and ribosomal biogenesis independent of mutations in growth factor signaling pathways. Activation of each of these distinct metabolic components is necessary for tumorigenesis, and together, this metabolic reprograming is sufficient to transform cells [41]. The observation that tumors showing lack of SIRT6 activity grow to be bigger and more aggressive than SIRT6-expressing tumors further adds to the evidence for the crucial importance of SIRT6-mediated regulation of glycolysis and cell proliferation in tumorigenesis. Therefore the tumor suppressor role for SIRT6 is strictly dependent on its ability to repress aerobic glycolysis and maintaining glucose homeostasis is well documented. It was suggested that the latter is exerted by at least three different mechanisms: (1) inhibiting the activity of HIF1α, a transcription factor that drives glycolysis and simultaneously inhibits oxidative phosphorylation [12]; (2) attenuating insulin IGF-1-like signaling pathway and glucose uptake by reducing JUN transcriptional activity [42,43]; and (3) inhibiting hepatic gluconeogenesis by promoting acetylation of the transcription factor PGC-1α [44]. SIRT6 deacetylates the histone acetyltransferase GCN5, which in turn acetylates and activates the transcriptional regulator PGC-1α, reducing de novo production of glucose, that is, gluconeogenesis in the liver [45]. SIRT6 also affects cholesterol synthesis repressing the transcription of SREBP1/SREBP2 and that of their target genes as well as their activation by inhibition of SREBP1/SREBP2 cleavage to their active forms. On the mechanistic level, SIRT6 is recruited by FOXO3 to the Srebp2 gene promoter where SIRT6 deacetylates histone H3 at lysines 9 and 56, thereby promoting a repressive chromatin state. Remarkably, SIRT6 or FOXO3 overexpression improves hypercholesterolemia in diet-induced or genetically obese mice [46,47]. Cholesterol plays an important role in cancer development. Both clinical and experimental studies have found that hypercholesterolemia and a high-fat high-cholesterol diet can affect cancer development. External cholesterol can directly activate the oncogenic Hedgehog pathway, and internal cholesterol can induce mTORC1 signaling. Cholesterol is a key component of lipid rafts, which are subdomains of the plasma membrane that contain high concentrations of cholesterol and glycosphingolipids [48]. Lipid rafts are the major platforms for signaling regulation in cancer, and chelating membrane cholesterol is an effective anticancer strategy that disrupts the functions of lipid rafts. Cholesterol metabolism is often reprogrammed in cancer cells [49]. Thus reducing cholesterol synthesis as a result of SIRT6 interference with its regulating elements/proteins indicates its additional role in cancer suppression. Besides the mediation of HIF1α signaling, SIRT6 also interacts with RelA subunit of NF-κB and deacetylates histone H3K9 at NF-κB target gene promoters, which leads to their repression. The disruption of the SIRT6 gene results in the hyperactivation of NF-κB signaling [50]. Min et al. have shown using genetic mouse models specific for liver cancer initiation, that survival of initiated cancer cells is controlled by c-Jun, independently of p53, through suppressing c-Fos-mediated apoptosis. Mechanistically, c-Fos induces SIRT6 transcription, which represses survivin by reducing histone H3K9 acetylation and NF-κB activation [51]. Moreover, in human dysplastic liver nodules, but not in malignant tumors, a specific expression pattern with increased c-Jun-survivin and attenuated c-Fos and SIRT6 levels was identified. These observations indicate the existence of a regulatory

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FIGURE 10.1 SIRT6’s interaction with transcription factors HIF1α, MYC, and NF-κB. SIRT6 suppresses glutaminolysis through interaction with MYC. Glycolysis is downregulated as a result of its interaction with HIF1α and MYC. Decreased oxidative stress results from reduced NF-κB activity and normalized energy metabolism (HIF1α and MYC). In this way SIRT6 may lower the risk of chronic inflammation and transformation of malignant cells.

network connecting stress response, inflammation, and histone modification in liver tumor initiation, which could be targeted to prevent liver tumorigenesis [29]. SIRT6’s interaction with HIF1α, MYC, and NF-κB target genes importantly affects cellular energy metabolism, oxidative stress, and subsequent also (cancer-related) inflammation (Fig. 10.1). Although SIRT6 is considered to be a tumor suppressor it may also act as a tumor promoter. High SIRT6 levels have been reported in breast cancer, pancreatic cancer, and prostate cancer, and are associated with drug resistance and poor prognosis [29]. High SIRT6 levels promote cellular proliferation through deacetylation of the cell cycle control proteins FOXO3a and p53, and increase IL-8- and TNF-mediated inflammatory responses, angiogenesis, and tumor metastasis in part through activation of the Ca21 channel TRPM2 [45]. Moreover, in BCPAP papillary thyroid cancer cells upregulated SIRT6, thus promoting the aerobic glycolysis through upregulation of GLUT1, HK2, and GAPDH genes, and increased ROS production. Treatment with ROS scavengers normalized the expression of these genes as well as glucose uptake and lactate production, suggesting that SIRT6 promoted the Warburg effect of papillary thyroid cancer cells via the upregulation of ROS. Thus the inhibition of ROS in SIRT6-upregulated cells could rescue activation of the Warburg effect [52]. As ROS production is essential for killing pathogens, it is also central to the progression of many inflammatory diseases, including cancer. Thus increased ROS production in cancer cells with upregulated SIRT6, along with the other events, for example, development of angiogenesis, contributes to SIRT6’s tumor-promoting activity.

10.2.4 SIRT7 SIRT7 is arguably the least studied of the mammalian sirtuins and the only known lysine deacetylase selectively removing acetylation from Lys 18 at histone 3. This specific histone mark is particularly associated with highly malignant cancer and poor prognosis [53]. SIRT7 was reported to activate transcription of rRNA by RNA polymerase I through specific interaction of its subunit PAF53 with the transcription factor UBF, which allows the pre-rRNA processing [54,55]. Since rRNAs are essential elements of ribosomes, SIRT7 is involved in their biogenesis. This process, along with enhanced glycolysis and excessive PPP activity, fits the highly proliferative potential of cancer cells [56]. On the other hand, SIRT7 was described as a negative regulator of the hypoxia responsive pathway. SIRT7 overexpression decreased HIF1α and HIF2β protein levels, transcriptional activity, and target gene expression [57]. In this way SIRT7 indirectly can downregulate the aerobic glycolysis in cancer and/or inflammatory cells. Moreover, SIRT7 has been shown to suppress endoplasmic reticulum stress and prevent the development of fatty liver disease. Mechanistically, this effect was explained by a recent finding that SIRT7 maintains energy homeostasis by deacetylating GABPβ1, a central regulator of mitochondrial function. As deacetylation is required for GABPβ1 activity, SIRT72/2 mice displayed pathologies that resemble phenotypes of mitochondrial diseases, which include cancer. Energy starvation induces ubiquitin-independent degradation of SIRT7, which leads to

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attenuation of ribosome biogenesis and maintenance of cellular energy homeostasis [55]. This supports the widespread functions of SIRT7 in response to metabolic demands. Overall, this sirtuin may contribute to cancer metabolic phenotypes in different ways. That is why some authors have described SIRT7 as protein having seven faces [55].

10.3 Mitochondrial sirtuins Mitochondria generate high-energy phosphate bonds of ATP by an electrochemical proton gradient created by the transfer of electrons through a series of electron carriers embedded in the mitochondrial membrane. Transfer of electrons to molecular O2 is tightly controlled, and only 1% 2% of electrons that leaked out in this process react with O2 resulting in ROS, namely superoxide radical formation [58]. The main sites of superoxide radical production in the electron transport chain (ETC) are complex I and III [59,60]. Superoxide radicals generated by mitochondria react with manganese superoxide dismutase (MnSOD, SOD2) in the mitochondrial matrix to generate H2O2, which can cross the mitochondrial outer membrane to access cytosolic targets. This can lead to multiple functional outcomes such as activation of redox-sensitive transcription factors, including HIF1α, and NF-κB activation of proinflammatory cytokines and activation of inflammasomes. Therefore mitochondrial sirtuins activity may be directly or indirectly involved in mitochondrial-derived oxidative stress and subsequently chronic inflammation and cancer [61]. Mitochondrial sirtuins obviously do not target histone proteins but deacetylate or modify lysine residues in proteins important for processes occurring in this compartment, particularly those linked with the ETC.

10.3.1 SIRT3 SIRT3 is the best characterized mitochondrial sirtuin. Acting as a protein deacetylase, it regulates metabolism, respiration, and stress control in that cellular compartment [62]. SIRT3 may enhance the ATP production via oxidative phosphorylation by catalyzing the deacetylation of protein components of the ETC, including NDUFA9 (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9), which is a member of complex I of the ETC [11,63], SDHA (succinate dehydrogenase subunit A) in complex II, and ATP synthase subunit β [64 66]. Moreover, it was shown that SIRT3 is involved in fatty acid oxidation, where it deacetylates long-chain acyl CoA dehydrogenase and upregulates its enzymatic activity along with activation of 3-hydroxy-3-methylglutaryl CoA synthase 2 involved in ketogenesis during fasting [67,68]. The quantitative acetyl-proteomic analysis applied to examine the function of resident deacetylase SIRT3 in mediating the CR response revealed that SIRT3 is a major regulator of the mitochondrial acetylome in response to CR [69]. Since CR is linked with beneficial effects on cancer and the way of its prevention, these findings provide an additional argument for the role of SIRT3 in the suppression of tumor growth [25]. SIRT3 also interacts with acetyl-CoA synthetase 2 and by deacetylation of lysine 642 increases the enzyme activity in both in vitro and in vivo systems [70,71]. SIRT3 may be involved in the redirection of pyruvate, a glycolysis end product, into the CAC by activation of PDH1 as a result of deacetylation of its lysine residues [72], which ultimately reduces cancer cells proliferation [73]. The most important function of SIRT3 in the context of ROS and inflammation is involvement in the regulation of ROS production, most probably through modulating SOD2 [74,75]. SIRT3-mediated deacetylation of Lys122 of SOD2 increases the enzyme activity and allows ROS scavenging [74]. SIRT3 also deacetylates Lys144 of mitochondrial isocitrate dehydrogenase IDH2, which uses NADP1 as a cofactor, producing the NADPH required for maintaining a reduced form of glutathione and self-maintaining active IDH2, which might be inactivated by oxidized glutathione [76]. SIRT3 deficiency leads to the overproduction of ROS and HIF1α stabilization by protection against proteasomal degradation and subsequent increased expression of its target genes, including those encoding glycolytic enzymes [77,78]. Recent studies indicate that SIRT3 may increase the expression of genes encoding antioxidant enzymes through the deacetylation of FOXO3 transcription factor [79,80]. Overall, SIRT3 is a key NAD1-dependent protein deacetylase in the mitochondria of mammalian cells, functioning to prevent cell transformation via regulation of mitochondrial metabolic homeostasis. However, SIRT3 is also found to express in some human tumors; its role in these SIRT3-expressing tumor cells needs to be elucidated [81].

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10.3.2 SIRT4 SIRT4 is a mitochondrial enzyme that was initially thought to be deprived deacetylating activity and to catalyze only an alternative reaction involving the transfer of ADP-ribose from NAD1 to the substrates [82]. Now it is known that SIRT4 deacetylates and inhibits malonyl-CoA decarboxylase (MCD), an enzyme that produces acetyl-CoA from malonyl-CoA. Malonyl-CoA provides the carbon skeleton for lipogenesis and also inhibits lipids oxidation [83,84]. More recently SIRT4 deacetylating activity has been shown to influence the stability of mitochondrial trifunctional protein α-subunit (MTPα). This protein subunit contains the long-chain hydratase 3-hydroxyacyl-CoA dehydrogenase and catalyzes the second and third steps of fatty acid oxidation [85,86]. SIRT4-mediated deacetylation of MTPα induces destabilization of the enzyme by promoting its ubiquitination and degradation. In this way, SIRT4 also functions to inhibit fatty acid oxidation in the liver [87]. Overall, SIRT4 is considered to be a regulator of lipid homeostasis and is active in nutrient-replete conditions to repress fatty acid oxidation while promoting lipid anabolism. Recently SIRT4 has emerged as an enzyme capable of removing diverse postranslational modifications (PTM) from its substrates, thereby modulating their functions. One such PTM is lipoylation, which is thought to be an exceedingly rare PTM. There are only four known lipoylated multicomponent enzymatic complexes in mammals: pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, branched-chain α-ketoacid dehydrogenase complex, and the glycine cleavage system [87,88]. Lipoyl modifications are essential for the enzymatic activities of these complexes, but also for capturing and presenting substrate intermediates between the active sites of the multicomponent. It was found that SIRT4 interacts with subunits from all four lipoylated multicomponent enzymatic complexes, as well as biotin-dependent carboxylases in humans. SIRT4 was shown to remove lipoyl groups from the pyruvate dehydrogenase (PDH) subunit dihydrolipoyl transacetylase (DLAT). The lipoamidase activity of SIRT4 was confirmed in cell culture and in vivo in mice and indicated that removal of lipoylation inhibits the activity of PDH [87,89]. Therefore SIRT4 might be an important regulatory point between glycolysis and the CAC. SIRT4-catalyzed transfer of ADP-ribose from NAD1 to the substrates might be involved in glutamine metabolism leading to the inhibition of glutamate dehydrogenase (GDH). This enzyme converts glutamate into α-ketoglutarate [82] and along with glutaminase 1 (GLS1), which transforms glutamine to glutamate, it serves to replenish the CAC [90]. The flow of amino acids metabolites into the CAC is limited by the inactivation of GDH. This effect can be reversed, because GDH is activated by the mammalian target of rapamycin complex 1 (mTORC1) via repression of SIRT4 and thereby increases glutamine metabolism [91]. Glutamine metabolism plays an important role in the proliferation of cancer cells by replenishing the CAC intermediates to support increased growth and by producing ammonia, which neutralizes the acidic metabolites frequently produced as a result of increased glycolysis in cancer cells. Therefore SIRT4 inhibition of glutamine metabolism via repression of GDH activity is thought to contribute to its function as a tumor suppressor [87]. It was found that DNA damage induced a massive increase in SIRT4 expression, which triggered a shutdown of glutamine metabolism that was sufficient to arrest progression of the cell cycle [92]. Thus this mechanism may prevent tumor development through a checkpoint which combines a significant decrease in glutamine use with the induction of cell cycle arrest, thus allowing cells to repair the damage [93,94]. SIRT4 displays features of a tumor suppressor in experimental systems, and its expression is reduced in a substantial number of human cancers [91,93]. Thus SIRT4 may exert its tumor suppressor function by both repressing glutamine metabolism and promoting genome stability. However, SIRT4 is upregulated in several tumors, can induce cellular stresses that are associated with tumorigenesis, and can prepare tumor cells to gain selective advantages. Therefore in several tumors and clinical cases, SIRT4 may play an oncogenic role, which requires further studies for confirmation [95].

10.3.3 SIRT5 SIRT5 in contrast to the other sirtuins shows low, although detectable, deacetylase activity. The only known physiological substrate with regard to its deacetylating activity is carbamoylphosphate synthetase 1 (CPS1). This enzyme catalyzes the first and rate-limiting step of the urea cycle. SIRT5 deacetylates and activates CPS1, and thus plays an important role in the regulation of the urea cycle [96]. However, SIRT5 enzymatic activity is unique compared with that of other sirtuin proteins. The analysis of the crystal structure of SIRT5 bound to a substrate peptide revealed that SIRT5 possesses a larger binding cavity compared with other sirtuins, with three unique active site amino acid residues, Ala86, Tyr102, and Arg105. It was established that SIRT5 efficiently removes acidic acyl groups—succinyl, malonyl, and glutaryl moieties—from lysine residues [97]. Therefore SIRT5 targets hundreds of

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protein substrates, particularly those localized in mitochondrial matrix, with many involved in cellular metabolism including the CAC, urea cycle, and fatty acids oxidation, but also was shown to act in cytosol affecting multiple glycolytic enzymes [98 102]. In general, SIRT5’s involvement in cell metabolism, particularly concerning glucose, favors metabolic reprogramming and a tumor-promoting cellular metabolism [97]. In this regard the lack of SIRT5 causes downregulation of glucose transporter GLUT1, for example, in NSCLC H1299 cells [103]. SIRT5 was shown to demalonylate GAPDH, which catalyzes the conversion of glyceraldehyde 3-phosphate to glycerate 1,3-bisphosphate in glycolysis, resulting in increased GAPDH and elevated glycolytic flux [97,98]. SIRT5-dependent inhibition of PKM2 in A549 and 293T NSCLC cells as result of Lys498 desuccinylation led to reduced glycolytic flux, reduced mitochondrial respiration, and enhancement of cell proliferation [104]. On the other hand it was also shown that Lys311 desuccinylation enhanced PKM2 activity [105]. In fact, both actions may positively influence the PPP, because disrupted flux of metabolites via PKM2 lower activity, as well as PKM2 in a low-active dimer state, enables the increase of the PPP glucose-6-phosphate entry. A conversion into 6-phosphogluconolactone is conducted by G6PD, which is activated by deglutarylation via SIRT5 [106]. Thus accelerated PPP produces elements for macromolecules synthesis and improves antioxidant defense. SIRT5 has been shown to repress the activity of PDH complex E1α subunit by desuccinylation [99]. Moreover, SIRT5 catalyzed deacetylation of STAT3. This cytosolic transcription factor undergoes mitochondrial translocation and subsequently regulates mitochondrial pyruvate metabolism. Deacetylation inhibits its function [107]. On the other side, SIRT5 deficiency correlates with lower lactate dehydrogenase A (LDHA) expression [103], so in SIRT5 active state pyruvate can be redirected to LDHA to produce lactate, a metabolic marker of the Warburg effect. Recently in colorectal cancer cells an interaction between lactate dehydrogenase B (LDHB) isoenzyme and SIRT5 was demonstrated. SIRT5 is a binding partner of LDHB and deacetylates Lys329 to promote LDHB enzymatic activity, followed by increased autophagy and accelerated growth of colorectal cancer cells, but also cancer progression in patients [108]. The SIRT5-mediatied regulation of the CAC and subsequently the ETC and oxidative phosphorylation is not unidirectional. In 293T cells SIRT5 the desuccinylation of SDHA, which is also the complex II of the ETC was described. Lack of succinylation disrupts conversion of succinate to fumarate, coupled with reduction of NAD1 to NADH and the generation of ubiquinol from ubiquinone [99]. However, it was also shown that SIRT5 electrostatically binds to cardiolipin and desuccinylates inner mitochondrial membrane proteins, including all four ETC complexes and ATP synthase, and finally promotes cellular respiration [109]. SIRT5 also interacts with complex I subunit NDUFA4 and cytochrome C [105]. SIRT5 involvement in the CAC is related mainly to its effect on IDH2 activation [106]. In mitochondria exist two isoforms of IDH, both transforming isocitrate to α-ketoglutarate. One, IDH3 uses NAD1 as an electron acceptor and acts in the CAC by irreversible decarboxylation of isocitrate to yield α-ketoglutarate, while IDH2 catalyzes reversible decarboxylation of isocitrate to α-ketoglutarate while reducing NADP1 to NADPH or acting in the reductive carboxylation reaction to convert α-ketoglutarate to isocitrate while oxidizing NADPH to NADP1 [76]. The importance of IDH2 activation is bifunctional. Mutations in IDH1 (cytosolic form of IDH) and IDH2 have been identified as driving events, for example, in gliomas and AML [110]. These heterozygous mutations cause the production of the oncometabolite 2-hydroxyglutarate (2-HG), which inhibits α-ketoglutarate-dependent dioxygenases related to DNA and histone demethylation, in turn leading to protumorigenic epigenetic dysregulation [97]. In addition, the increase of NADPH production by IDH2 enhances oxidative stress defense, because NADPH is necessary in the regeneration of glutathione (GSH) from its oxidized (GSSG) form by glutathione reductase [111]. SIRT5 tends to protect cancer cells from ROS also via the desuccinylation and activation of Cu/Zn superoxide dismutase (SOD1) [112] and G6PD, a key enzyme of the PPP [106]. In NSCLC cells induced overexpression of SIRT5 was positively correlated with expression of the Nrf2 gene, thus the expression of Nrf2-target genes involved in cellular redox homeostasis was also improved [113]. Antioxidant effects of SIRT5 and its further influence on cytochrome C related with intrinsic apoptotic cascade or antiapoptotic B cell lymphoma-XL (BCL-XL) protein suggests SIRT5 engagement in cancer cells’ evasion of apoptotic death [30,97,105]. Fatty acids β-oxidation is a four-step process of fatty acid breakdown into acetyl-CoA. For very long fatty acylCoA (VLCFA) substrates, very-long-chain acyl-CoA dehydrogenase (VLCAD) converts these intermediates to enoyl-CoA, and further enoyl-CoA hydratase (ECHA, a subunit of the mitochondrial trifunctional complex) catalyzes conversion of enoyl-CoA to ketoacyl- and acetyl-CoA to break down fatty acids for energy [97]. Two mitochondrial sirtuins, SIRT3 and SIRT5, together promote fatty acids β-oxidation via the deacetylation of VLCAD, thereby promoting its mitochondrial membrane localization, increasing its activity and enhancing conversion of acyl-CoA to enoyl-CoA [97,105,114]. However, SIRT5, similarly to SIRT3, may stimulate the ketone body production by the desuccinylation of 3-hydroxy-3-methylglutaryl CoA synthase 2 [101].

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Influence on nitrogen metabolism is also related to the wide metabolic function of SIRT5. As previously mentioned, SIRT5 supports ammonia detoxification via the stimulation of CPS1 activity and conversion of ammonia to carbamoyl phosphate to initiate the formation of urea for excretion [97]. But SIRT5 may also reduce the production of ammonia in the glutaminolysis pathway. The influence on CPS1 is limited to liver and partly kidney. However, in human breast cancer cell lines MDA-MB-231 and mouse myoblast C2C12 overexpressing SIRT5 ammonia production by repressed glutaminase was reduced [115]. To sum up, SIRT5 regulates several aspects of glucose metabolism across multiple subcellular compartments. It reprograms cells to use glucose rather than producing lactate and stimulates the PPP to produce biomolecules and NADPH. Furthermore, it promotes fatty acids β-oxidation together with the production of ketone bodies. SIRT5 protects cells from oxidative stress and ammonia toxicity by interaction with, for example, glutaminolysis or activity of CPS1 and SOD1. In some cancer tissues with an IDH2 mutation SIRT5 can contribute to the production of the novel oncometabolite 2-HG, which modulates epigenetic changes for tumor survival. Negative regulation of the ETC complex II might potentially affect ATP production, but SIRT5 deficiency in HEK293T leads to suppression of mitochondrial NADH oxidation and inhibition of ATP synthase activity, and subsequently activates AMPK [116]. Thus SIRT5 promotes metabolic reorganization that favors tumor promotion. Nowadays, little is known on its impact on innate immune defenses and inflammation. A recent study using SIRT5 knockout mice showed that SIRT5 deficiency does not affect immune cell development, cytokine production, and proliferation by macrophages and splenocytes exposed to microbial and immunological stimuli [117]. However, SIRT5-dependent inhibition of PKM2 may potentially affect inflammatory cells. To summarize the general roles of sirtuins in cellular metabolism, Fig. 10.2 presents the tendency of action of particular sirtuins according to glycolysis, fatty acids metabolism, and glutaminolysis.

FIGURE 10.2 The effect of sirtuins in cellular energy metabolism. Due to varied tissue specificity, the dominant effect is presented. CAC, Citric acid cycle; ETC, electron transport chain.

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10.4 Sirtuins indeed link metabolism, inflammation, and cancer? The causal relationship between inflammation, innate immunity, and cancer is now widely accepted. However, many of the molecular and cellular mechanisms mediating this relationship are not clear yet. Furthermore, tumor cells may take over key mechanisms by which inflammation interfaces with cancers to further host invasion [118]. In this regard angiogenesis, which is one of the hallmarks of cancer, is closely linked with inflammation [119]. Energy metabolic pathways have a direct influence on pro- and antiinflammatory responses in macrophages, which, in response to danger signals, reprogram the nutrient metabolic pathway toward aerobic glycolysis. Thus in both cancer cells and activated macrophages similar metabolic changes occur. SIRTs seem to be important regulators of these processes. However, as it was described in previous sections, SIRTs can function both as suppressors or tumor promoters depending on the specific form, tissue, and microenvironment. Moreover, an important part of SIRTs’ metabolic effects is related to cellular redox balance [28]. An imbalance in redox processes favors the creation of a proinflammatory microenvironment, suitable for cancer development [8]. The family of FOXO transcription factors controls expression of genes encoding proteins diminishing oxidative stress. Catalase and MnSOD are the two most important FOXO3a target genes related to the ROS defense and their expression is partly controlled by SIRT1, 2, and 3 [28,79,80,120]. SIRT5 can also protect cancer cells from oxidative stress by desuccinylation and activation of SOD1 [112]. Inversely, SIRT6-dependent deacetylation can suppress FOXO3a, thus impairing the ROS balance [121]. In view of mitochondrial localization, SIRT3, 4, and 5 mostly influence ROS production. Beyond promoting SOD2, SIRT3 and 5 augment IDH2 activity, resulting in enhancement of NADPH synthesis [76]. SIRT3 activity is particularly involved in the decrease of oxidative stress as the result of the normalization of cellular energetics. As previously mentioned, SIRT3 interacts with the ETC proteins, for example, NDUFA9 and SDHA, thus it may improve the electron flow through the ETC and in this way may protect against mitochondria-derived ROS. Excessive production of ammonia might be another stress-inducing event for cells. SIRT3 targets ornithine transcarbamoylase to increase ammonia detoxification via the urea cycle [122]. Similarly, SIRT5 by deacetylation, desuccinylation, and deglutarylation stimulates the activity of CPS1 [96,102]. Significant amounts of ammonia are created also as a by-product of glutaminolysis. According to previously cited data, SIRT3 enhances the activity of GDH and glutamine influx to the CAC, but SIRT4 and 5 repress this metabolic pathway. Nuclear functions of HIF1α and MYC transcription factors not only affect energetic metabolism, but indirectly also regulate redox status. In short, HIF1α controls glycolysis, while MYC also affects the efficiency of glutaminolysis. An overview of SIRTs metabolic roles suggests antagonistic activity toward HIF1α in the case of SIRT1, 2, 3, 6, and 7, although various mechanisms are related to this interaction. Moreover, MYC functions are affected by SIRT1, 6, and 7, but also SIRT4 via the inhibition of GDH reducing MYC effects on glutaminolysis. Only SIRT2 rather stabilizes MYC transcription factor [36,123]. Irrespective of some exceptions, the active state of sirtuins favors the maintenance of redox balance. In inflammation ROS excessive production is the response to some harmful stimuli, but paradoxically aims to restore previous homeostasis [124]. Proinflammatory signals induce initially acute systemic inflammation. This process starts with the proinflammatory phase, which switches then to the adaptation phase that should finally restore homeostasis [125] (Fig. 10.3A). The proinflammatory phase of inflammation is typically promoted by NF-κB transcription factor activation. In the canonical NF-κB signaling pathway harmful stimuli activate toll-like receptors (TLRs), tumor necrosis factor α (TNFα) induces tumor necrosis factor receptor (TNFR), and interleukin-1 (IL-1) interacts with interleukin-1 receptor (IL-1R). Binding to the receptor leads to an activation of IKKβ (IκB kinase β) in the IKK complex, which can then phosphorylate IKKα (IκB kinase α) on Ser32 and Ser36 residues. Phosphorylation mark is followed by polyubiquitination and proteasomal degradation of IKKα and the release of NF-κB p50 and RelA/p65 subunits, which can translocate into the nucleus and stimulate target genes expression [126,127]. Transformation from the proinflammatory to adaptation phase requires deactivation of NF-κB signaling. De novo synthesis of RelB enables its binding to p50 and replacement of p65/RelA. However, previously p50 p65 heterodimer transcription activity is in addition disrupted by chromatin deacetylation [125 127]. That suggests sirtuins involvement in the switch from acute to adaptation phase. Thus SIRT1 and 6 were shown to interact with NF-κB target genes expression at H3K9 lysine position [27,28,50]. Moreover, SIRT1 directly interacts with and represses the p65/RelA subunit of NF-κB by its deacetylation at Lys310 [14]. The proinflammatory and adaptation phases of inflammation require, as was mentioned earlier, quite different metabolic support. In fact, glycolysis and fatty acids provide the nutritional needs of immune cells [6,128].

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FIGURE 10.3 Overview of metabolic changes in inflammatory cells in relation to cancer. Cellular energy metabolism switches from metabolic balance to the dominance of glycolysis in the proinflammatory phase of inflammation, and further to the dominance of fatty acid β-oxidation in the adaptation phase of inflammation. In addition, the metabolic state is related to NAD1 level and subsequent sirtuins activation/inhibition. (A) Metabolic state of cells and related level of SIRTs activity during classical acute inflammation. (B) Metabolic state of cells and related level of SIRTs activity during chronic inflammation and possible malignant transformation of cell.

Macrophages derived from hematopoietic stem cells are functionally divided into classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages). M1 macrophages are characteristic for the proinflammatory phase in a host defense against bacterial and viral infections, while M2 macrophages are associated, for example, with responses to antiinflammatory reactions [129]. The proinflammatory phase relies on high glycolytic activity, which enables rapid energy production, and additionally activation of the PPP to improve eradication of bacterial infections via the promotion of NADPH oxidase [6,125]. The induction of glucose flux through the glycolytic pathway results from HIF1α transcriptional activity. A high rate of glycolysis decreases AMP/ATP and NAD1/NADH ratios [125]. Sirtuins as the NAD1 level sensors [130] in proinflammatory phase are repressed. The switch to the adaptation phase coexists with energetic reprograming. Cells sense the resulting increase of adenosine monophosphate (AMP) levels, which activates Nicotinamide phosphoribosyltransferase (Nampt) and further Nicotinamide mononucleotide adenylyltransferase (Nmnat) enzymes to produce NAD1, which eventually elevates SIRTs activity [125]. M2 macrophages related to the adaptation phase of inflammation require oxidative metabolism based on fatty acids β-oxidation [128,129]. Three sirtuins were pointed to be crucial in describing the transition of inflammatory phases. Both AMP (particularly AMPK) and NAD1 are the activators of SIRT1—an effective inducer of PGC1 transcription factor that promotes fatty acids β-oxidation and mitochondrial biogenesis [22,131]. There is an advanced antagonistic crosstalk between NF-κB and SIRT1 signaling pathways, with domination of NFκB in the proinflammatory phase and SIRT1 prevalence in the adaptation phase [128]. SIRT6 was described as a master regulator of glucose metabolism, and its high activity suppresses glycolysis mechanistically via contracting the HIF1α metabolic rearrangement [12]. Moreover, SIRT3 is importantly involved in mitochondrial oxidative respiration, being in line with metabolic changes in the second phase of inflammation. Thus after the postinflammation restoration of homeostasis also SIRTs activity tends to be normalized [6,125] (Fig. 10.3A).

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The chronic inflammation together with oxidative stress creates an environment for malignant cells transformation. This facilitates the fact that in chronic inflammation there is a high activity of NF-κB, and also ROS overproduction becomes permanent and no adaptation phase is observed. At low AMP and NAD1 cellular levels sirtuins activity also remains low [6,125]. Finally, a high glycolytic rate can be maintained in an inflammatory environment favoring tumorigenesis (Fig. 10.3B). One of the possibilities to deal with an unstable microenvironment might be promotion of autophagy. In general, autophagy is a process by which damaged proteins and organelles, including mitochondria (mitophagy), are targeted for lysosomal degradation. Such a reutilization process is useful for providing metabolic intermediates during metabolic disabilities [132]. Interplay between sirtuins, cellular energetics, oxidative stress, and inflammation highlights the possible role of SIRTs also in regulation of autophagy. An increase in LC3 (autophagy-related protein 8, Atg8) represents a well-known marker of autophagy taking place in cells. Starvation, as a stress factor, induces translocation of LC3 from the nucleus to the cytoplasm. It was demonstrated that SIRT1 forms complexes with components of autophagy drivers, including LC3, Atg5, and Atg7, to improve its activity via deacetylation [133]. SIRT1 nuclear localization is also related to the preparation of LC3 for translocation to cytoplasm. Only LC3 deacetylated at Lys49 and Lys51 by SIRT1 is proper for interaction with DOR (diabetes- and obesity-regulated nuclear factor), which serves as a carrier for LC3 to translocate it to the cytoplasm and create there autophagosomes [134]. In contrast, SIRT2 was shown to inhibit autophagy, but also improve mitophagy [132]. In glioma cells subjected to hypoxia the lack of SIRT3 expression decreased mitochondrial localization of LC3. Additionally, depletion of mitophagy led to a further decrease in the mitochondrial membrane potential, and increased accumulation of ROS was demonstrated, eventually triggering the degradation of antiapoptotic proteins Mcl-1 and survivin through the proteasomal pathway [135]. Thus the proper activity of SIRT3 and the related induction of mitophagy allow cancer cells to survive. Another example is the role of SIRT5 in forcing LDHB enzymatic activity (deacetylation of Lys329) to convert lactate to pyruvate, with concomitant production of NADPH and H1. Protons generated by LDHB promote lysosomal acidification and autophagy in colorectal cancer cells, finally enhancing tumor growth [108]. SIRT6 is also an autophagy regulator and its expression differs between primary and metastatic melanomas. Particularly, in primary cancer SIRT6 is downregulated, which enables intensive glycolytic metabolism, but it is also correlated with a decrease of autophagy biomarker LC3. However, in advanced, metastatic stages SIRT6 undergoes upregulation to support autophagy as a following mechanism of tumorigenesis maintenance [136].

10.5 Conclusions and perspectives The engagement of sirtuins in the control of cellular energetics and tumor-promoting inflammation represents complex interactions at the level of all cellular compartments. It is impossible to point to an unidirectional role of a particular sirtuin in the tumorigenesis process. Sirtuins, as cellular regulators of homeostasis, normalize bioenergetics metabolism both in inflammatory and cancer cells. Reduced ROS production by inflammatory cells prevents epithelial tissue injury and the creation of microenvironment favoring tumor progression. However, sirtuins are often overexpressed in cancer tissue and act rather as tumor promoters. What decides the net balance between the suppressing and promoting activities of sirtuins requires more comprehensive studies. Similarly a more profound molecular understanding of how specific sirtuins control glycolytic enzymes in resting and activated inflammatory cells, such as macrophages, is likely to deliver new opportunities for manipulating metabolism in these cells as an antiinflammatory strategy for cancer prophylaxis and treatment. Finally, sirtuins can also support cellular functions in stress conditions via the induction of autophagy, although this ability in cancer cells is predominantly negative.

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C H A P T E R

11 Sirtuins and cellular metabolism in cancers Zhen Dong1,2 and Hongjuan Cui1,2 1

State Key Laboratory of Silkworm Genome Biology, Institute of Sericulture and Systems Biology, College of Sericulture and Textile and Biomass Science, Southwest University, Chongqing, P. R. China 2Cancer Center, Medical Research Institute, Southwest University, Chongqing, P. R. China O U T L I N E 11.1 The metabolic characteristics of cancers 11.1.1 Glucose metabolism in cancers 11.1.2 Lipometabolism in cancers 11.1.3 Other kinds of metabolism in cancers

11.4.2 Direct posttranslational control of OXPHOS by sirtuins 11.4.3 Direct posttranslational control of lipometabolism by sirtuins 11.4.4 Direct posttranslational control of amino acid metabolism by sirtuins

195 196 197 197

11.2 The regulatory modes of sirtuins in controlling cellular metabolism 197 11.3 Direct epigenetic control of cellular metabolism by sirtuins 198 11.3.1 Direct epigenetic control of glucometabolism by sirtuins 198 11.3.2 Direct epigenetic control of lipometabolism by sirtuins 200 11.3.3 Direct epigenetic control of amino acid metabolism by sirtuins 200 11.4 Direct posttranslational control of cellular metabolism by sirtuins 200 11.4.1 Direct posttranslational control of glycolytic enzymes and transporters by sirtuins 200

11.5 Indirect control of cellular metabolism by sirtuins 11.5.1 Indirect control of glycolysis by sirtuins 11.5.2 Indirect control of OXPHOS by sirtuins 11.5.3 Indirect control of lipometabolism by sirtuins 11.5.4 Indirect control of amino acid metabolism by sirtuins

203 204 205 206 208 209 210 211

11.6 Conclusions

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References

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11.1 The metabolic characteristics of cancers Changes in cellular metabolism are considered as one of the important signs of cancer [1]. Cancer cells change their metabolism, getting the necessary nutrients from an undernourished environment to gain energy and build new biomass [2]. Abnormal cancer metabolism is mainly characterized by upregulation of glucose and amino acid uptake; opportunistic modes of accessing nutrients; increased demand for intermediates in glycolysis/oxidative phosphorylation (OXPHOS), lipometabolism for biosynthesis, and nicotinamide adenine dinucleotide phosphoric acid (NADPH) production; urgent demand for nitrogen; changes in metabolite-driven gene expression and regulation; and metabolic interactions with the tumor microenvironment [3]. From current studies, metabolic reprogramming is considered one of the most important contributors for tumor malignancy and interfering with the metabolism may be a promising strategy for cancer treatment.

Sirtuin Biology in Cancer and Metabolic Disease. DOI: https://doi.org/10.1016/B978-0-12-822467-0.00008-5

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11. Sirtuins and cellular metabolism in cancers

11.1.1 Glucose metabolism in cancers In 1924, German physiologist Otto Heinrich Warburg (1983
1970) observed that cancer cells prefer to use glycolysis to generate energy in the cytosol, which differs from most normal cells that produce adenosine triphosphate (ATP) by using the OXPHOS/tricarboxylic acid (TCA) cycle in mitochondria (Fig. 11.1) [4]. This phenomenon was later referred to as the “Warburg effect.” This process catalyzes a cytoplasmic pathway that breaks down glucose into two three-carbon compounds, companied by the generation of a small quantity of energy. However, dysfunctional energy metabolism was not recognized as a new hallmark of cancer until 2011 [1]. Recently, more and more evidence indicates that changes in energy metabolism, especially glucose metabolism, such as glycolysis, OXPHOS/TCA cycle, pentose phosphate pathway (PPP), gluconeogenesis, etc., play important roles in tumorigenesis and cancer development [5]. Glucose metabolism is a biochemical process mediated by a series of enzymes and some major regulators that target these enzymes through transcriptional or posttranslational control. Aerobic glycolysis is controlled by a series of enzymes and glucose transporters, including hexokinase II (HK II), aldolase (ALDOA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase 1 (PGK1), enolase (ENO1), lactate dehydrogenase A (LDHA), pyruvate kinase M2 (PKM2), glucose 6-phosphate dehydrogenase (G6PDH, G6PD), and glucose transporter 1 (GLUT1), which are direct effectors during glycolytic alterations in tumors. These enzymes can also be regulated by some major regulatory factors, such as hypoxia-inducible factors (HIF-1/2), c-Myc and liver kinase B1 (LKB1), and AMP-activated protein kinase (AMPK), or other new regulatory factors can affect aerobic glycolysis through targeting glycolytic enzymes and glucose transporters, thus affecting tumor growth [2]. Actually, the relationship between oncogenes and glucose metabolism shows high crosstalk and has established vicious circles for tumorigenesis [6]. The OXPHOS/TCA cycle is a process in mitochondria in the downstream of glycolysis and a crossroad of the three major metabolic pathways, including glucometabolism, lipometabolism, and protein metabolism. The OXPHOS/TCA cycle—also known as the citric acid cycle (CAC) or the Krebs cycle—is a series of enzymatic chemical reactions that oxidize acetyl-CoA derived from carbohydrates, fats, and proteins, into ATP and carbon dioxide (CO2). The OXPHOS/TCA cycle is controlled by a series of enzymes, such as isocitrate dehydrogenase 2 (IDH2), pyruvate dehydrogenase E1 α1 subunit (PDH-E1, PDHA1), phosphoinositide-dependent kinase-1 (PDK1), and succinate dehydrogenase (SDH). In addition, OXPHOS also has some important regulators, such as the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), manganese-containing superoxide dismutase (MnSOD), dynamin-related protein 1

FIGURE 11.1 Metabolic changes in glucose metabolism in tumors. Normal cells prefer to use OXPHOS in the mitochondria for the production of energy, whereas tumor cells prefer to use aerobic glycolysis for the production of energy and metabolic intermediates.

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(Drp1), GA-binding protein transcription factor (GABPα/β), and nuclear respiration factor 1/2 (NRF-1/2), thereby regulating mitochondrial biogenesis, fission/fusion, or function, which is closely related to the energy generated by the OXPHOS/TCA cycle [7]. Although aerobic glycolysis is important, recent studies also reveal that OXPHOS can be also upregulated in certain cancers, even in the face of active glycolysis [8,9]. Increased OXPHOS protects cancer mitochondria from apoptosis-associated permeabilization, reduces aspartate production that leads to impaired nucleotide biosynthesis, or restricts energy support in tumor cells [10
12].

11.1.2 Lipometabolism in cancers The reprogramming of lipometabolism, also known as lipid metabolism, has recently been recognized as a new hallmark of cancer progression [13
17]. Due to uncontrolled cell growth and rapid metastasis, tumors need to develop efficient production pathways for energy and biomass components. Tumors can strongly activate the endogenous biosynthetic pathway of lipids or increase the uptake of exogenous lipids from the diet. Lipometabolism mainly includes fatty acid (FA) metabolism and cholesterol metabolism. Many reports indicate that fatty acid synthesis (FAS) and oxidation (FAO) are important aspects of lipometabolism reprogramming in tumor cells [18]. In addition, cholesterol metabolism plays an equally important role in tumors as FA metabolism [19]. FA metabolism consists of mitochondrial or peroxisomal FAO and cytoplasmic FAS, both of which are controlled by a series of enzymes. Recently, fat metabolism enzymes such as medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD), very-long-chain acyl-CoA dehydrogenase (VLCAD), carnitine palmitoyl transferase 1a (CPT1 a), and enoyl-CoA hydratase (ECHA) in FAO, as well as ATP citrate lyase (ACLY) and malonyl-CoA decarboxylase (MLYCD) in FAS have been shown to play important roles in tumorigenesis.

11.1.3 Other kinds of metabolism in cancers In addition to glucose metabolism and lipid metabolism, many other metabolic pathways, including amino acid metabolism [20], the urea cycle [21], and nucleotide metabolism [22], are also important for tumor growth, metastasis, and immune response. Among them, glutamine metabolism, also known as glutaminolysis, is the most important metabolic pathway for cancers. Since glutamine is an important carbon source that supplements the TCA cycle, thereby promoting OXPHOS [23], glutamine metabolism has been shown to be related to the cellular biology, physiology, and clinical opportunities of cancers [24,25].

11.2 The regulatory modes of sirtuins in controlling cellular metabolism Recently, increasing evidence has shown that epigenetics plays a pivotal role in cellular metabolism during the tumorigenesis of many kinds of tumors [26
30], including glioblastoma [31], melanoma [32], hepatocellular cancer [33], pancreatic cancer [34], and leukemia [35,36]. Even in mitochondria, epigenetics also plays emerging roles during carcinogenesis and tumor progression [37]. There are seven sirtuin members (SIRT1
SIRT7) that are expressed in different subcellular locations [7,30]: SIRT1, SIRT6, and SIRT7 are usually located in the nucleus; SIRT2 is usually located in the cytoplasm; SIRT3, SIRT4, and SIRT5 are usually located in the mitochondria. However, sometimes, SIRT1 is also located in the cytoplasm, and SIRT3 is also located in the nucleus [38,39]. This makes sirtuins widely participate in cellular functions, especially cell metabolism regulation from the nucleus, plasma, and mitochondria. The most important regulatory way is epigenetic modulation in the nucleus. Importantly, sirtuin-knockout mice exhibit impressive syndromes related to metabolic alterations [40] (Table 11.1). Unlike other known histone deacetylases that catalyze only acetyllysine residues, sirtuin-mediated deacetylation in acetyllysines depends on nicotinamide adenine dinucleotides 1 (NAD1) hydrolysis [55]. This catalyzing process produces 20 -O-acetyl-ADP-ribose (a substrate for deacetylation) and nicotinamide (NAM), which are feedback inhibitors of the activity of sirtuins [56]. Due to the dependence of sirtuins on NAD1, fluctuations in NAD1 levels will regulate the catalytic activity of sirtuins, which is directly related to the energy state through the ratio of NAD1:NADH in the cell [57]. Sirtuin family proteins share a highly conserved NAD1 binding domain, but have diverse functional domains. Therefore, sirtuins are found to have other catalytic functions [7]. For example, SIRT4 has lower deacetylase activity, but has adenosine diphosphate (ADP)-ribosyltransferase activity;

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TABLE 11.1

Syndrome of sirtuin knockout mice models.

Knockout sirtuin

Syndrome and molecular alterations

References

SIRT1

Majority of mice die prenatally with retinal; cardiac defects; cartilage degradation; increased PPARγ activity, adipogenesis, and insulin sensitivity in adipocytes; enhanced inflammation in kidney and myeloid; hepatic steatosis and inflammation; attenuation in spermatogenesis and germ cell function; repression of forkhead transcription factors

[41
46]

SIRT2

Tumor development in mammary (Q) or liver and intestine (R)

[47]

SIRT3

Age-related cardiac hypertrophy; hyperacetylation of mitochondrial proteins without affecting global metabolic homeostasis; change in ACCS2 activity, ATP levels, and mitochondrial protein acetylation

[48,49]

SIRT4

Spontaneously develop several types of tumors; increased mitochondrial GDH activity and upregulation of mitochondrial glutamine metabolism

[50]

SIRT5

No any overt metabolic abnormalities; defect in the urea cycle

[51,52]

SIRT6

Death within 1 month; progeroid syndrome; low levels of serum IGF-1; complete loss of subcutaneous fat; lymphopenia; osteopenia; and acute onset of hypoglycemia, leading to death.

[53]

SIRT7

Reduced life span; ageing-related phenotypes; progressive heart hypertrophy

[54]

SIRT5 also has lysine demalonase, desuccinase, and deglutamylase activities; SIRT6 also has ADP-ribosyltransferase, palmitoylase, and myristoylase activities; SIRT7 also has ADP-ribosyltransferase activity. These enzymes are important erasers of protein modifications both in histones and nonhistone proteins. Therefore sirtuins play essential roles in both epigenetic and posttranslational regulation of cancer metabolism. As important erasers of histones, sirtuins are important epigenetic regulators of key factors regarding cellular metabolism [7,58]. As protein modifying enzymes, sirtuins can regulate metabolic factors in posttranslational levels. Firstly, sirtuins rely on their deacetylase activity to epigenetically regulate the expression of these metabolic genes at the translational level. For instance, SIRT1 deacetylates histone 1 lysine 9 (H3K9) and H4K16 [59]; SIRT2 deacetylates H3K18 and H4K16 [60,61]; SIRT3 deacetylates H4K16, H3K9, and H3K56 [62]; SIRT6 deacetylates H3K9 and H3K56 [63,64]; SIRT7 deacetylates H3K18 [65,66]. SIRT7 also can desuccinylate succinyl-H3K122 in the promoter of the target gene [67]. Besides, they also recruit some transcription factors to initiate transcription. Secondly, sirtuins rely on their catalytic activities such as deacetylation, desuccinylase, demalonylase, demyristoylase, depalmitase, and/or mono-ADP-ribosyltransferase activities to regulate these key enzymes via modulating enzymatic activity, sublocation, and/or stability [7]. Finally, sirtuins regulate major regulators, such as transcription factors, signaling molecules, ubiquitin-ligases, or other factors regarding cellular metabolism by direct or indirect transcription/posttranslational regulation [7].

11.3 Direct epigenetic control of cellular metabolism by sirtuins From current studies (Table 11.2), SIRT1 and SIRT6 are key epigenetic regulators that can modify the status of chromosome remolding by deacetylating the lysines in histone 3 (H3), thereby recruiting transcriptional factors to the promoter of their target metabolic genes, some of which are tightly related to cancer initiation and progression (Fig. 11.2).

11.3.1 Direct epigenetic control of glucometabolism by sirtuins Sirtuins, especially SIRT6, can affect the expression of glycolytic genes encoding enzymes and glucose transporters via an epigenetic route [78]. For example, in the liver, SIRT1 forms a complex with the forkhead box O3 (FOXO3a) and nuclear respiration factor 1 (NRF1, GABPα) on the SIRT6 promoter, and positively regulates the expression of SIRT6, which in turn leads to the H3K9 deacetylation in the promoter of glycolytic genes including glucokinase (GK) and pyruvate kinase liver type (PKL), thereby suppressing their expressions [76]. In tumors, SIRT6 functions as a corepressor of MYC transcription activity by deacetylating histone H3 lysine 9 (H3K9) and histone H3 lysine (H3K56) at the promoter of their target glycolytic genes, including glucose transporter 1 (GLUT1), pyruvate dehydrogenase kinase 1 (PDK1), pyruvate dehydrogenase kinase 4 (PDK4), aldolase/fructose-bisphosphate C (ALDOC), phosphofructokinase1 (PFK1), LDHA, LDHB, triosephosphate isomerase (TPI5), and glyceraldehyde-3-phosphate dehydrogenase

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TABLE 11.2

Epigenetic regulation of cell metabolism by sirtuins.

Sirtuin Transcriptional member Modification factor

Targeted genes

Cell metabolism

Tumors/cells

References

SIRT1

H3K4 Unknown deacetylation

ACC2

Inhibition of the production of malonyl-CoA in triglyceride synthesis

Cervical cancer, pharyngeal squamous, and colorectal cancer

[68]

SIRT6

H3K9 and c-Myc H3K56 deacetylation

GLUT1, PDK4, PDK1, ALDOC, PFK1, LDHB, LDHA, TPI5, and GAPDH

Inhibition of glycolysis

Glioblastoma, melanoma, colorectal cancer, SIRT6 KO H-RasV12/shp53transformed mouse embryonic fibroblasts

[69
73]

SIRT6

H3K9 Hif1α deacetylation

GLUT1, PFK1, PDK1, and LDHA

Inhibition of glycolysis

Breast cancer and 293FT

[74,75]

SIRT6

H3K9 Unknown deacetylation

FATP, ACC1, ACC2, Inhibition of FASN, SCD1, ELOVL6, triglyceride synthesis GPAT, DGAT and fat metabolism

Cervical cancer, pharyngeal squamous, and colorectal cancer

[68,76]

SIRT6

H3K56 c-Myc deacetylation

GLS

Inhibition of glutamine uptake

Sirt6 KO H-RasV12/shp53transformed mouse embryonic fibroblasts

[69]

SIRT6

Histone ATF4 lysine deacetylation

SLC6A9, SLC7A1, SLC7A5, SLC36A1, SLC36A4, SLC38A1, and SLC38A3

Inhibition of amino acid uptake

Colon cancer

[77]

FIGURE 11.2 Models of SIRT6-controlled glycolysis in the transcriptional regulation levels. In normal cells, SIRT6 binds to the promoter of a series of glycolytic genes and catalyzes H3K9 and/or H3K56 in the histones, thereby inhibiting the transcriptional activity of transcriptional factors c-Myc or HIF1α and blocking the transcription of these glycolytic genes. In tumor cells, SIRT6 is downregulated and the functions of c-Myc and HIF1α are activated, which further promote the translation of glycolytic genes, thus enhancing the glycolysis and promoting tumorigenesis.

(GAPDH), resulting in the inhibition of aerobic glycolysis and impeding cell proliferation [69,70]. In addition, SIRT6 binds and cosuppresses HIF-1α translational activity and deacetylates H3K9 in the promoter region of several key glycolytic genes, such as GLUT1, PFK1, pyruvate dehydrogenase kinase 1 (PDHK1, also known as PDK1), and LDHA, thus inhibiting its expression [74,79]. In bladder cancers, SIRT1 also promotes the transcriptional activity and expression of GLUT1, resulting in an increase of glucose uptake and tumor progression [80].

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The sirtuins-controlled metabolic processes can also be regulated by many upstream factors. For example, forkhead box O3 (FOXO3a) can also transcriptionally regulate SIRT6, which contributes to tumor growth in both glioblastoma [71], melanoma [72], and colorectal cancers [73] via regulating aerobic glycolysis. In breast cancer, Runt-related transcription factor 2 (RUNX2) negatively regulates SIRT6, inhibiting PDK1 and activating pyruvate dehydrogenase (PDH), which controls the pyruvate metabolism into the TCA cycle [75].

11.3.2 Direct epigenetic control of lipometabolism by sirtuins Sirtuins directly control the transcription of genes encoding lipometabolic enzymes or fatty acid transporters through remodeling chromatin. For example, SIRT1 forms a complex with FOXO3a and NRF1 (GABPα) on the SIRT6 promoter, and positively regulates the expression of SIRT6, and then deacetylates H3K9 in the gene promoter of a series of lipometabolic genes including fatty acid transporter (FATP), acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), stearoyl-CoA desaturase 1 (SCD1), elongation of very-long-chain fatty acid protein 6 (ELOVL6), CPT1a, 3-phosphoglycerol acyltransferase (GPAT), and diacylglycerol O-acyltransferase (DGAT), thereby negatively regulating triglyceride synthesis and fat metabolism [76]. Another example in cancer cells is the expression of acetyl-CoA carboxylase 2 (ACC2) controlled by SIRT1/6. FAS and FAO are two mutually exclusive pathways that depend on the regulation of malonyl-CoA. This process not only participates in the elongation of fatty acid, but also negatively regulates FAO by inhibiting CPT1a [81]. SIRT1 and SIRT6 can deacetylate H3K9 and histone 4 lysine 8 (H4K8) in the promoter of ACC2, an enzyme that mediates the produce of malonyl-CoA in acidic pH-adapted tumor cells. Therefore the activation of SIRT1/6 results in the promotion of FAO and the reduction of glucose metabolism, while the inhibition of SITR1/6 results in an increase in FAS and a decrease in FAO [68].

11.3.3 Direct epigenetic control of amino acid metabolism by sirtuins Sirtuins also directly control the glutamine metabolism via an epigenetic route. For example, SIRT6 can also deacetylate H3K56 in the promoter region of the glutamine metabolism-related gene glutaminase (GLS) and inhibits its expression by cosuppressing the transcription factor c-Myc and inhibiting c-Myc-regulated glutamine uptake in SIRT6 KO H-RasV12/shp53-transformed mouse embryonic fibroblasts (MEFs) [69]. SIRT6 also functions as a corepressor of the transcriptional activity of activating transcription factor 4 (ATF4), thereby restricting AAT (amino acid transporter) gene expression, including SLC6A9, SLC7A1, SLC7A5, SLC36A1, SLC36A4, SLC38A1, and SLC38A3. However, this axis can be disrupted by NRF2 under glutamine deprivation, leading to the upregulation of amino acid uptake [77]. Recently, SIRT1 was also shown to affect glutamine metabolism. Haploinsufficiency of SIRT1 induces c-Myc expression and several transcriptional targets of c-Myc, including GLUT1, PDK1, SLC1A5, and SLC7A5 that promote glutamine metabolism and sustain colon cancer development [82]. However, whether SIRT1 also epigenetically controls these genes remains unknown.

11.4 Direct posttranslational control of cellular metabolism by sirtuins As important posttranslational enzymes, sirtuins can also directly modify the metabolic enzymes in the posttranslational levels, thereby affecting cancer metabolism and progression (Table 11.3). These modifications usually lead to three different kinds of effects on these enzymes or transporters, that is, enzymatic activity, protein stability, or translocations (Fig. 11.3).

11.4.1 Direct posttranslational control of glycolytic enzymes and transporters by sirtuins Sirtuins affect the enzymatic activity of glycolytic genes through deacetylation or other posttranslational changes. For example, in human fibroblasts, SIRT2 significantly increases OXPHOS and inhibits glycolysis through the deacetylation of glycolytic enzymes such as ALDOA, GAPDH, enolase 1 (ENO1), and PGK1 [83,84]. In HEK293T cells, SIRT2 also deacetylates glucose-6-phosphate dehydrogenase (G6PD) and activates its enzymatic activity, thereby stimulating PPP to provide cytoplasmic NADPH and thus supporting cell survival under oxidative stress [85]. SIRT2 deacetylates PKM2 at K305, thereby reducing PKM2 activity by preventing its tetramerization to develop the active form of enzymatic activity which further inhibits glycolysis and inhibits the

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TABLE 11.3

Direct posttranslational control of ketone body metabolism by sirtuins in cancers.

Sirtuin member Modification

Targeted proteins

SIRT2

Deacetylation

SIRT2

The molecular effect of modifications

Cell metabolism

Tissues/cells

References

ALDOA, Decrease of GAPDH, enzymatic activity ENO1, and PGK1

Promotion of OXPHOS and inhibition of glycolysis

Fibroblasts

[83,84]

Deacetylation

G6PD

Increase of enzymatic activity

Stimulation of PPP pathway

HEK293T cells

[85]

SIRT2

Deacetylation

PKM2

Decrease of enzymatic activity

Inhibition of glycolysis

Tumors

[86]

SIRT2

Deacetylation

PGAM

Increase of enzymatic activity

Increase of glycolysis

Cancer cells and HEK293T cells

[87,88]

SIRT3

Deacetylation

LDHA

Increase of enzymatic activity

Promotion of glycolysis

Gastric cancer

[89]

SIRT5

Deglutarylation

G6PD

Increase of enzymatic activity

Maintenance of cellular NADPH homeostasis and redox potential

murine embryonic fibroblasts (MEFs)

[90]

SIRT5

Desuccinylation

PKM2

Increase of enzymatic activity

Divertion of glucose flux into the pentose phosphate pathway

293T and A549 cells

[91]

SIRT1

Deacetylation

GAPDH

Cytoplasm location Promotion of glycolysis

HeLa and 293FT cells

[92]

SIRT3

Deacetylation

HK2

Mitochondria location

Inhibition of glycolysis

Ovarian cancer

[93]

SIRT3

Deacetylation

CS

Increase of enzymatic activity

Promotion of OXPHOS

Neuroblastoma

[94]

SIRT3

Deacetylation

IDH2

Increase of enzymatic activity

Promotion of OXPHOS and glycolysis

HEK-293T, MCF7 and NIH3T3 cells

[95,96]

SIRT3

Deacetylation

PDHA1, PDP1

Activation of their Promotion of OXPHOS and activity and decrease of glycolysis dissociation of the mitochondrial PDC

H1299 293T, HCT116, HeLa, T47D, MMT, and MCF7 cells

[97,98]

SIRT5

Desuccinylation

PDH-E1, SDH, IDH2

Decrease of enzymatic activity

Maintenance of cellular NADPH homeostasis and redox potential

MEFs and HEK293

[90,99]

SIRT5

Desuccinylation

OGDH

Decrease of enzymatic activity

Reduction of OXPHOS, and induction of mitochondrial membrane potential (ΔΨm), ATP products and increases the ROS levels

Gastric cancer

[100]

SIRT2

Deacetylation

PEPCK

Stabilization of protein

Promotion of gluconeogenesis and supporting TCA cycle and glycolysis

HEK293, 293T, HepG2, and Chang’s liver cells

[101]

SIRT3

Deacetylation

NDUFA9, SDHA, MTATP6

Increase of enzymatic activity

Promotion of ETC function

Drosophila, MEFs, HIB1B and K562 cells

[102
104]

SIRT5

Deacetylation

Cyto C

Unknown

Unknown

N.A.

[105] (Continued)

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TABLE 11.3

(Continued)

Sirtuin member Modification

Targeted proteins

SIRT4

ADP-ribosylation

ANT2/3

SIRT3

Deacetylation

SIRT5

The molecular effect of modifications

Cell metabolism

Tissues/cells

References

Increase of enzymatic activity

Inhibition of insulin secretion

Pancreatic β cells

[106]

LCAD

Increase of enzymatic activity

Promotion of FAO

HEK293 cells and mice liver

[107,108]

Desuccinylation

VLCAD

Increase of enzymatic activity

Promotion of FAO

Mice liver

[109]

SIRT3

Deacetylation

VLCAD

Increase of enzymatic activity

Promotion of FAO

Mice liver

[109]

SIRT5

Desuccinylation

ECHA

Increase of enzymatic activity

Promotion of FAO

Mice heart

[110]

SIRT2

Deacetylation

ACLY

Stabilization of protein

Promotion of FAS

HEK293 cells

[111]

SIRT4

Deacetylation

MLYCD

Decrease of enzymatic activity

Promotion of FAS

MEFs and C2C12 cells

[112]

SIRT3

Deacetylation

LCAD

Increase of enzymatic activity

Promotion of FAO

Tibialis anterior [113] and spinal cord of mice

SIRT3

Deacetylation

HMGCS2

Increase of enzymatic activity

Promotion of ketone body formation

HEK293 cells

[114]

SIRT5

Desuccinylation

HMGCS2

Decrease of enzymatic activity

Inhibition of ketone body formation

Mice liver

[115]

SIRT1

Deacetylation

ACSS1

Increase of enzymatic activity

Promotion of FAS

Cos-7 cells

[116]

SIRT3

Deacetylation

ACSS2

Increase of enzymatic activity

Promotion of fatty acid synthesis

HEK293, COS1, and HeLa cells

[117]

SIRT4

ADP-ribosylation

GDH1

Inhibition of enzymatic activity

Inhibition of glutamine metabolism Hepatoma cells

[118]

SIRT4

Delipoylation

DLAT

Diminishment of PDH activity

Glutamine

MEFs

[119]

SIRT3

Deacetylation

GDH1

Increase of enzymatic activity

Support of glutamine metabolism

Renal cell carcinoma

[82]

SIRT3

Deacetylation

GOT2

Enhancement of Regulation of malate-aspartate the protein NADH shuttle interaction between GOT2 and MDH2

Pancreatic tumor

[120]

SIRT5

Desuccinylation

GLS

Inhibition of enzymatic activity

MDA-MB-231 [121] and C2C12 cells

SIRT4

Demethylglutarylation, dehydroxymethylglutarylation and de-3-methylglutarylation

MCCC

Increase of stability Promotion of leucine oxidation

Liver, heart, and/or skeletal muscle of mice

[122]

SIRT5

Deacetylation, desuccinylation, CPS1 and deglutamylation

Increase of enzymatic activity

Mice liver and 293T

[52]

Conversion of glutamine to glutamic acid in the production of ammonia

Promotion of urea cycle

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FIGURE 11.3 Models of sirtuins’ demodifications and their effects on proteins (some examples). Typically, release of these covalent modifications (most of them are deacetylation) leads to three different alterations in protein: alterations of enzymatic activity, affects on protein stability, and translocations.

growth of malignant tumors [86]. SIRT2 can also deacetylate phosphoglycerate mutase (PGAM) on lysine 100/106/113/138 to regulate its enzymatic activity, which coordinates the energy produced by glycolysis and decreases reducing power and the biosynthesis of uridine precursors and amino acids, thereby playing an important role in regulating cell proliferation and tumor growth [87,88]. However, the exact promotion or inhibition of PGAM by SIRT2 deacetylation should be further clarified. SIRT3 also directly participates in the glycolytic process by regulating the activity of glycolytic enzymes. SIRT3 interacts with LDHA and enhances its activity by deacetylation to promote the expression of glycolysis-related genes in gastric cancer cells overexpressing SIRT3 [89] and may also contribute to tumor progression of cervical cancer [123] and ovarian cancer [124]. SIRT4 also shows a potential to regulate glycolysis; however, the detailed molecular mechanism remains unknown [125]. SIRT5 is also located in mitochondria and regulates mitochondrial function. Unlike other sirtuins, SIRT5 shows a unique affinity for deacetylation and catalyzes desuccinylation, demalonylation and deglutarization of proteins [126]. For example, SIRT5 promotes G6PD deglutarylation to regulate cell NADPH homeostasis and redox potential [90]. SIRT5 desuccinylates PKM2 on K49, which contributes to the antioxidant response and inhibits tumor cell proliferation [91]. SIRT5 also can be suppressed by CDK2, resulting in the downregulating of aerobic glycolysis in gastric cancer [127]. In addition, sirtuins also affect the sublocations of these enzymes, thereby controlling glucose metabolism. For example, SIRT1 interacts with GAPDH and retains it in the cytoplasm, thereby protecting the enzyme from nuclear translocations, promoting glycolysis, and inducing apoptosis through radiation and other means to regulate cell survival [92]. SIRT3 also increases the level of HK2 localization at the mitochondria, thereby inhibiting the glycolysis of ovarian cancer cells [93].

11.4.2 Direct posttranslational control of OXPHOS by sirtuins As a mitochondrial factor, SIRT3 is not only involved in the regulation of glycolysis, but also in the regulation of mitochondrial function. Its biological process is directly related to the OXPHOS/TCA cycle. SIRT3 directly

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regulates the OXPHOS/TCA cycle by regulating enzyme activity in the TCA cycle. For instance, SIRT3 deacetylates acetyl groups of citrate synthase (CS) and activates its activity in neuroblastoma SH-SY5Y cells [94]. In addition, SIRT3 deacetylates IDH2 and promotes OXPHOS, thereby increasing NADPH levels and the abundance of active glutathione [95]. SIRT3 deacetylates IDH2 at K413, thereby increasing its enzymatic activity by reducing the formation of IDH2 dimers, which further increases the cell’s reactive oxygen species (ROS) and glycolysis [96]. In addition, in epidermal growth factor (EGF) stimulated cells and cancer cells, SIRT3 deacetylates PDHA1 at K321 and pyruvate dehydrogenase phosphatase catalytic subunit 1 (PDP1) at K202, thereby activating their activity and dissociating the mitochondrial pyruvate dehydrogenase complex (PDC), which further promotes OXPHOS and reduces glycolysis in cancer cells, leading to subsequent tumor growth [97,98]. Another mitochondrial sirtuin, SIRT5, also regulates enzymes in the OXPHOS/TCA cycle. For example, SIRT5 controls glucose oxidation by directly desuccinating these enzymes to inhibit PDH-E1 and SDH activity [99]. SIRT5 also promotes desuccinylation of IDH2, which regulates the homeostasis and redox potential of cell NADPH [90]. Recently, SIRT5 was also shown to desuccinate oxoglutarate dehydrogenase (OGDH) and inhibit its enzymatic activity, thereby inhibiting cell proliferation and migration in gastric cancer [100]. In addition to mitochondrial sirtuins, other members of the sirtuin family are also directly involved in the regulation of the OXPHOS/TCA cycle. For example, SIRT2 deacetylates and stabilizes phosphoenolpyruvate carboxykinase (PEPCK), thereby catalyzing the rate-limiting, irreversible step of gluconeogenesis, thereby generating pyruvate to provide energy for the TCA cycle and glycolysis [101]. Importantly, sirtuins also affect the function of the electronic transmission chain (ETC), thereby regulating the OXPHOS/TCA cycle. SIRT3 deacetylates several components of the ETC including complex I (such as NADH dehydrogenase (ubiquinone) 1α subcomplex 9, NDUFA9), several complexes II (succinate dehydrogenase complex flavin protein subunit A, SDHA), and complex V (ATP synthase subunit β, MTATP6) [102
104]. Importantly, SIRT3 has been shown to aggravate mitochondrial complex I inhibitors, metformin-induced energy stress, and induce cell apoptosis in ovarian cancer. In addition, other sirtuins also play an important role in ETC. SIRT5 can be transferred to the mitochondrial intermembrane space (MIS) and deacetylated cytochrome c (Cyto C), a protein of MIS that has important functions in ETC and can initiate apoptosis [105]. It has been shown that SIRT4 binds to the ADP/ATP carrier protein adenine nucleotide transporter 2/3 (ANT2/3), which transports ATP into the cytoplasm and ADP into the mitochondrial matrix, thereby providing a base for ATP synthase [106]. In addition, it appears that SIRT1 can promote mRNA levels of NDUFA10, one of the subunits of complex I [128]. However, the regulatory mode of SIRT1 and SIRT4 in the regulation of ETC should be further confirmed.

11.4.3 Direct posttranslational control of lipometabolism by sirtuins Generally, sirtuins bind to lipometabolism-related enzymes and modify them under translational levels to activate their activity. For example, in FAO, SIRT3 stimulates FAO through the deacetylation and activation of longchain specific acyl-CoA dehydrogenase (LCAD) [107,108], a key enzyme that catalyzes a long-chain acyl-CoA to a long-chain trans-2,3-dehydroacyl-CoA. SIRT5 desuccinylation and SIRT3 deacetylation stimulate VLCAD, a key enzyme that can catalyze a very-long-chain acyl-CoA to a long-chain trans-2,3-dehydroacyl-CoA, thereby promoting FAO [109]. SIRT5 can also desuccinylate and activate ECHA, an enzyme that catalyzes the formation of hydroxyacyl-CoA from enoyl-CoA, thereby inducing FAO [110]. There is also a similar effect of SIRT5 in the peroxisome lipid metabolism pathway [129]. Deletion of SIRT5 results in the accumulation of medium- and longchain acylcarnitines and inhibits FAO [115]. At the same time, sirtuins also play important roles in deacetylating enzymes involved in FAS. For example, SIRT2 deacetylates and destabilizes ACLY, which regulates the production of acetyl-CoA and causes FAS inhibition [111]. SIRT4 deacetylates the enzyme MLYCD, which catalyzes the formation of malonyl-CoA, and inhibits its activity. Malonyl-CoA provides a carbon backbone for lipid anabolic metabolism and reduces FAO [112]. Acetate is transported into cells by members of the monocarboxylate transporter family, and is catalyzed into acetyl by acyl-CoA synthetase short-chain family member 1 (ACSS1) and acyl-CoA synthetase short-chain family member 2 (ACSS2). As a substrate for fatty acid synthesis, acetyl-CoA is present in most cancer cells to establish sufficient lipid biomass as a fuel to support cell proliferation [130,131]. Recently, sirtuins including SIRT1 and SIRT3 have been shown to play an important role in directly regulating the enzyme activity of ACSS1 and ACSS2, respectively. SIRT1 deacetylates and activates ACSS1, thereby enhancing FAS [116]. SIRT3 deacetylates and stimulates ACSS2, causing a 10-fold increase in carbon dioxide [117].

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Ketone bodies, including β-hydroxybutyrate (PHB), acetoacetate, and acetone, are usually produced in the liver during starvation and function as mitochondrial fuels [132]. Recently, ketone bodies function as cancer metabolites that drive tumor growth and metastasis, such as hydroxymethylglutaryl coenzyme A synthase 2 (HMGCS2), acetyl coenzyme A acetyltransferase 1/2 (ACAT1/2), and 3-oxo acid coenzyme A transferase 1/2 (OXCT1/2) [133]. Importantly, sirtuins have also been shown to have direct regulatory capabilities in regulating the activity of these enzymes. SIRT3 promotes ketone body production in FAO by activating HMGCS2, the rate-limiting enzyme for ketone body formation, and indirectly regulates FAO [113,114]. SIRT5 introduces the desuccinylation of HMGCS2, reduces HMGCS2 activity, and leads to reduced PHB production in the body, which means that SIRT5 is involved in ketone body metabolism [115].

11.4.4 Direct posttranslational control of amino acid metabolism by sirtuins SIRT4 catalytic efficiency for lipoyl-, biotinyl-lysine, and ADP-ribosyl modifications is superior to its deacetylation activity. Recent reports indicate that mitochondrial SIRT4 plays an important role in regulating glutamine metabolism through ADP-ribosylated glutamate dehydrogenase 1 (GLUD1, also known as GDH1 or GLDH1) [118]. Alternatively, SIRT4 hydrolyzes the lipoamide cofactors from the E2 component dihydrolipoyllysine acetyltransferase (DLAT), diminishing PDH activity, leading to a metabolic flux switching via glutamine stimulation [119]. SIRT4 inhibits tumorigenesis by inhibiting mitochondrial glutamine metabolism, which responds to DNA damage and inhibits tumor activity, including c-Myc-driven human Burkitt lymphoma and colorectal cancer cells [50,134
136]. Importantly, the reduced glutamine utilization induced by SIRT4 makes tumors sensitive to glucose consumption [135]. In thyroid cancer cells, SIRT4-suppressed glutamine metabolism also impedes the proliferation, migration, and invasion abilities [137]. In addition, glutamine metabolism regulated by SIRT4 is also affected by other regulators. For example, C-terminal binding protein 1 (CtBP) inhibits SIRT4 expression, thereby promoting glutamine breakdown in cancer cells [138]. The CtBP-SIRT4 axis also coordinates the glycolysis and glutamine metabolism in cancer cells. CtBP represses SIRT4 expression only under a high level of glucose but not low, because in the latter condition, CtBP binding to SIRT4 promoter is abolished [139]. The mechanistic target of rapamycin kinase complex 1 (mTORC1) promotes proteasome-mediated instability of cAMP response element binding protein 2 (CREB2), a factor that promotes SIRT4 transcription [136]. SIRT4 can also be transcriptionally inhibited by HUTF1, thereby mediating UHRF1-promoted cell metabolism switching in pancreatic cancers possibly through SIRT4-suppressed PDH activity [123]. In addition to SIRT4, other sirtuins also have the ability to regulate glutamine metabolism through a variety of regulation methods. For example, SIRT3 enhances GLUD1 ability to regulate glutamine-dependent oxidation, increases mitochondrial oxygen consumption, and supports tumor growth in renal cell carcinoma [82]. In diffuse large B cell lymphomas (DLBCLs), SIRT3 depletion impairs glutamine flux to the TCA cycle, leading to a reduction in acetylCoA pools that in turn induce autophagy and cell death [140]. In clear cell renal carcinoma, SIRT3-regulated glutaminosis also promotes cell proliferation [141]. In addition, SIRT3 deacetylates mitochondrial glutamate oxaloacetate aminotransferase (GOT2), a key enzyme that controls the malate
aspartate NADH shuttle and is located at K159, K185, and K404. This shuttle is important for transferring NADH from the cytosol to the mitochondria to maintain a high ratio of glycolysis in tumor cells. Increased GOT2 acetylation in the absence of SIRT3 promotes a net transfer of cytosolic NADH to the mitochondria, thereby supporting the production of ATP and the proliferation of cancer cells. Importantly, increased acetylation of GOT2 K159 in human pancreatic tumors is associated with decreased SIRT3 expression [120]. At the same time, SIRT5 has also been reported to desuccinylate and inhibit GLS, which catalyzes the conversion of glutamine to glutamic acid in the production of ammonia and ammonia-induced autophagy in nonammonia cells [121]. Importantly, SIRT5 has also been reported to regulate ammonia production and ammonia-induced autophagy in nonhepatocytes by regulating glutamine metabolism. Mechanistically, SIRT5 can desuccinylate at K158 and K164 sites of GLS and inhibit GLS, an enzyme that catalyzes the conversion of glutamine to glutamic acid in a reaction that produces ammonia, thereby promoting autophagy [121,139]. In transformed cells and human breast tumors, the SIRT5-GLS axis also plays an important role in glutamine metabolism [142]. This evidence suggests that sirtuins regulate cellular metabolism in a highly intertwined manner. SIRT5 is also responsible for the urea cycle. SIRT5 regulates the urea cycle by deacetylating, desuccinylating, and deglutamylating carbamoyl phosphate synthase 1 (CPS1), thereby increasing its activity [143,144]. CPS1 catalyzes the first step of the urea cycle for ammonia detoxification in the liver and, to a lesser extent, in the kidney [52].

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Aside from glutamine metabolism, SIRT4 is also responsible for leucine metabolism. In SIRT4 KO mice, leucine metabolism disorders lead to increased basal metabolism and insulin secretion, which gradually develop into glucose intolerance and insulin resistance [122]. In addition, SIRT4 removes three acyl moieties from lysine residues, including methylglutaryl (MG)-, hydroxymethylglutaryl (HMG)-, and 3-methylglutaryl (MGc)-groups, thus providing an intermediate for leucine oxidation [122].

11.5 Indirect control of cellular metabolism by sirtuins Cellular metabolism is also controlled by some major regulators, including metabolism-specific transcriptional factors, signaling transduction pathways, as well as some growth factors, oncogenes, and tumor suppressors. Recently, sirtuins also regulate these factors in both transcriptional, posttranslational, or upstream factors that modulate these major regulators (Table 11.4). For instance, the PGC1α is key regulator of glucogenesis which can be regulated by SIRT1 and SIRT6 (Fig. 11.4). TABLE 11.4

Modulation of major regulators of cellular metabolism by sirtuins.

Targeted Sirtuin proteins/ member Modification genes

The molecular effect of modifications

Cell metabolism

Tumors/cells

References

SIRT1

Deacetylation HIF1/2

Inhibition of translational activity

Inhibition of glycolysis

Hep3B, HT1080, and HEK293 cells

[145,146]

SIRT2

Deacetylation HIF-1

Increase of stability

Promotion of glycolysis

HeLa cells

[147]

SIRT3

Deacetylation HIF-1α

Decrease of stability

Decrease of glycolysis

Breast cancer, colorectal cancer, and cholangiocarcinoma

[148
152]

SIRT7

Deacetylation HIF-1/2

Decrease of stability

Decrease of glycolysis (possible)

Hep3B and HeLa cells

[153]

SIRT1

Deacetylation c-Myc

Promotion of c-Myc/Max association

Promotion of glycolysis

HeLa, HO15, K562, HEK293, 293A cells, and Burkitt lymphoma cells

[154
157]

SIRT2

H4K16 NEDD4 Deacetylation

Repression of NEDD4 gene Promotion of glycolysis expression and protection of N- (possible) Myc and c-Myc from ubiquitination degradation

Neuroblastoma and pancreatic cancer

[158]

SIRT7

H3K18 c-Myc Deacetylation

Inhibition of Myc transcriptional activity

Inhibition of lipogenesis.

Mice liver

[159]

SIRT1

Deacetylation LKB

Activation of LKB activity

Inhibition of FAS

HEK293T, HepG2 cells, and osteosarcoma

[160
162]

SIRT3

Deacetylation PTEN

Decrease of stability

Inhibition of glycolysis

p53 wild-type cancer

[163]

SIRT2

Deacetylation APC(CDH1) and CDC20

Decrease of the anaphasepromoting complex/cyclosome activity

Inhibition of the SH-SY5Y and HEK293 cells Warburg effect (possible)

[47,164]

SIRT3

Deacetylation Cyclophilin D

Diminishment of the peptidylprolyl cis-trans isomerase activity

Inhibition of glycolysis

Breast cancer

[165]

SIRT1/6 Deacetylation hnRNPA1

Reduction of the alternative splicing capacity of the pyruvate kinase (PK) mRNA

Inhibition of glycolysis

Hepatocellular carcinoma

[166]

SIRT1

Deacetylation PGC-1α

Increase of transcriptional activity

Increase of OXPHOS

Colon cancer

[167,168]

SIRT3

Deacetylation MnSOD

Increase of enzymatic activity

Maintenance of mitochondrial metabolism

Mice liver, MEFs, and breast [169
171] cancer (Continued)

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11.5 Indirect control of cellular metabolism by sirtuins

TABLE 11.4

(Continued)

Targeted Sirtuin proteins/ member Modification genes

The molecular effect of modifications

Cell metabolism

Tumors/cells

References

SIRT7

Deacetylation GABPβ1

Transcriptional activation of the GABPα/GABPβ heterotetramer

Maintenance of mitochondrial homeostasis

heart, liver, and inner ear of mice

[172]

SIRT1

Deacetylation PGC-1α

Increase of transcriptional activity

Promotion of fatty acid esterification

Mice heart

[173]

SIRT1

Unknown

Activation of PPARα coactivator PGC-1α

Inhibition fatty acid metabolism

Mice liver and HEK293 cells

[44]

SIRT2

Deacetylation FOXO1

Promotion of FOXO1 binding to PPARγ and subsequent repression on PPARγ transcriptional activity

Inhibition of adipogenesis

Mice white adipose tissue

[174]

SIRT1

Deacetylation SREBP1

Inhibition of transcriptional activity

Inhibition fatty acid metabolism and cholesterol synthesis

Caenorhabditis elegans

[175]

SIRT2

Deacetylation SREBP2

Control of the nuclear trafficking and transcriptional activity

Inhibition of sterol biosynthesis

Drosophila eye

[176]

SIRT6

Deacetylation CLOCK: BMAL1

Control of circadian chromatin recruitment of SREBP-1 to circadian gene promoters

Cyclic regulation of fatty Mice liver and HEK293 cells acid and cholesterol metabolism

[177]

SIRT6

Deacetylation H3K9 and Inhibition of SREBP H3K56 on transcription the promoter of SREBP

Lower liver cholesterol and increased serum cholesterol

[178]

SIRT7

Unknown

SIRT6 SIRT1

PPARα

DCAF1/ DDB1/ CUL4B complex

Mice liver

Inhibition of the degradation of Increased uptake of fatty Mice liver TR4 acid and synthesis and storage of triglycerides

[179,180]

H3K9 PI3K Deacetylation

Transcriptional inhibition of PI3K signaling

Inhibition of lipid metabolism

Cancer stem cells

[181]

Deacetylation HIF-2α

Increase of transcriptional activity

Maintenance of the reductive and oxidative glutamine metabolism under chronic acidosis

SiHa cervix cancer cells, FaDu pharynx squamous cell carcinoma cells, and HCT-116 colon cancer cells

[182]

FIGURE 11.4 Models of sirtuins’ function in major regulators of cell metabolism (using PGC1α in the regulation of glucogenesis as an example). SIRT6 catalyzes the deacetylation of the transacetylase GCN5, which further catalyzes the acetylation of PGC1α. The deacetylation of PGC1α is catalyzed by SIRT1. Deacetylated PGC1α recruits transcriptional factors FOXO1 and HNF4α, leading to the transcription of PEPCK and G6Pase, which are enzymes that catalyze the glucogenesis.

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11.5.1 Indirect control of glycolysis by sirtuins In addition to direct regulation, sirtuins also indirectly regulate some major regulatory factors, such as HIF-1/2, c-Myc, and LKB1-AMPK, or other new regulatory factors in aerobic glycolysis, thus affecting tumor growth. 11.5.1.1 HIF-1/2 HIF-1 and HIF-2 are major regulators that balance oxygen supply and demand, and mediate metabolic changes, especially glycolysis, which will drive cancer progression [183
185]. Recently, many sirtuins have been shown to have the ability to affect their activities. SIRT1 can deacetylate HIF-1 and HIF-2 [145,146]. First, SIRT1 deacetylates HIF-1 and inhibits its translational activity by inhibiting acetyltransferase recruitment to the promoter of HIF-1 targeting genes through p300, which may be involved in cell metabolism [146]. However, SIRT1 has also shown a positive effect on HIF-1-dependent glycolytic switches [186,187], which may depend on the cell type [188]. In addition, in solid tumors, SIRT1 also regulates glucose metabolism in myeloid suppressor cells (MDSCs), which exhibit an immature phenotype and can exhibit a classically activated (M1) or alternative activated phenotype (M2). When the cells enter the periphery from the bone marrow, SIRT1 inhibits the transition of M2 to the M1 lineage through glycolytic activation mediated by the mTOR-HIF-1α pathway, thereby enhancing the inhibitory function and facilitating the proinflammatory M1 phenotype that attacks tumor cells [187]. Besides which, SIRT2 deacetylates HIF-1 at K709, thereby increasing its stability and activity, which promotes the expression of GLUT1 and LDHA, thereby promoting tumor growth [147]. SIRT3 also disrupts the stability of HIF-1α, thereby further suppressing tumorigenesis and opposing metabolic reprogramming of a variety of cancers, including human breast cancer, colon cancer, and cholangiocarcinoma [148
152]. SIRT4 is also negatively correlated with HIF-1α in pancreatic cancer, but it may not be through direct interaction between them [123]. In addition, SIRT7 also binds HIF-1 and HIF-2 and reduces its stability dependent on its deacetylase activity, thereby downregulating its downstream genes in response to hypoxia-induced stress [153]. 11.5.1.2 c-Myc c-Myc is a translation factor that controls the expression of GLUT1 and many glycolytic genes, such as phosphoglucose isomerase (PGI), phosphofructokinase (PFK), GAPDH, PGK1, and ENO1 [189,190]. Recently, aside from functioning as a transcriptional factor that cooperates with SIRT1/SIRT6-mediated histone lysine deacetylation, many sirtuins also affect glycolysis by regulating c-Myc at posttranslational level in various tumors. SIRT1 physically interacts with c/N-Myc. c-Myc can directly bind to the SIRT1 promoter to increase its expression, and in turn SIRT1 deacetylates c-Myc in its C-terminal domain, promoting c-Myc/Max association [154
156]. Besides, SIRT1 stimulates c-Myc-induced LDHA expression, which may affect the Warburg effect in cancer [157]. In addition, SIRT1 promotes and stabilizes mitogen-dependent protein kinase phosphatase 3 (MKP3)-dependent N-Myc phosphorylation and induces SIRT1 expression in neuroblastomas [191]. Similarly, SIRT2 deacetylates and stabilizes c-Myc and N-Myc, thereby inducing the expression of SIRT2. This feedback loop promotes cell proliferation of neuroblastoma and pancreatic cancer cells [158]. In addition, SIRT7 synergizes with c-Myc and inhibits its transcriptional activity [159], suggesting that SIRT7 may also play a potential role in the Warburg effect. 11.5.1.3 LKB1-AMPK LKB1-AMPK is also the main regulatory axis of glycolysis [192]. Recently, SIRT1 has also been shown to deacetylate LKB1 and activate LKB1 while increasing its activity, cytoplasmic/nuclear ratio, and binding to the LKB1 activating factor STE20-related kinase adaptor alpha (STRAD) [160], which may subsequently activate AMPK and affect glycolysis and probably one of the causes of osteosarcoma progression [161]. SIRT4 deletion also inactivates the LKB1/AMPK pathway, leading to the increase of mTOR signaling through upregulation of glutamine metabolism [193]. 11.5.1.4 p53 p53 also functions as a major regulator of glycolysis. It promotes its targets (such as HKs and TP53-induced glycolysis-regulated phosphatase (TIGAR)) and inhibits its targets (such as PGAM, GLUT1/4, IKK/NF-κB, etc.) [194,195]. SIRT3 has been reported to deacetylate and stabilize PTEN, which further inhibits MEM2 transcription and induces p53 degradation, thereby inhibiting glycolytic genes, including GLUT4, TIGAR, and PGAM, in p53 wild-type cancer cells [163]. In turn, p53 directly activates expression of SIRT6, which can interact with the forkhead box O1 (FOXO1) and lead to FOXO1 deacetylation and export to the cytoplasm, thereby downregulating PCK1 and G6PC expression and affecting glucose metabolism via the suppression of gluconeogenesis [196].

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11.5.1.5 Other In addition to the aforementioned regulators, sirtuins also affect other regulators of glycolysis. For example, SIRT2 deacetylates E3 ubiquitin ligase APC/C-Cdh1 which leads to inhibition of the Warburg effect and tumor progression via ubiquitinating and degrading 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3, PFK2) [164]. SIRT3 is indirectly involved in glycolysis through regulators that control the glycolysis process. SIRT3 deacetylates and inactivates cyclophilin D (peptidylprolyl isomerase D, PPID), resulting in the detachment of mitochondrial HK II and inhibition of glycolysis [165]. SIRT1 and SIRT6 also deacetylate hnRNPA1, leading to deacetylated hnRNPA1, which further reduces PKM2 and increases PKM1 alternative splicing. Afterwards, the metabolic activity of PK and the nonmetabolic PKM2-β-catenin signaling pathway are reduced, resulting in inhibition of tumorigenesis in hepatocellular carcinoma (HCC) [166].

11.5.2 Indirect control of OXPHOS by sirtuins In addition to direct epigenetic and posttranslational regulation, sirtuins also indirectly regulate OXPHOS via some regulators, such as peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), MnSOD, and dynein-related proteins 1 (Drp1), a GA-binding protein transcription factor (GABPα/β, nuclear respiration factor 1/2, NRF-1/2), thereby regulating mitochondrial biogenesis, fission/fusion, or function, which are closely related to the OXPHOS/TCA cycle and energy production. 11.5.2.1 PGC-1α Translational coactivator PGC-1α is a major regulator of mitochondrial biogenesis and the OXPHOS/TCA cycle, thus promoting tumor malignancy [197,198]. SIRT1 activates PGC-1α and increases OXPHOS by increasing mitochondrial biomass, respiratory chain enzyme expression, and oxygen consumption rate, thereby making patient-derived spheres and tumor xenografts sensitive to chemotherapy for colon cancer [167,168]. In addition, the deletion of mouse SIRT1 can impair the transcription factor peroxisome proliferator-activated receptor alpha (PPARα) coactivated by PGC-1α and reduce the expression of PPARα targets, including CytoC, CytoC oxidase subunits IV (COX4) and CytoC oxidase subunit 5B (COX5B) involved in OXPHOS [44]. 11.5.2.2 MnSOD MnSOD is the main mitochondrial ROS scavenging enzyme inside the mitochondrial matrix, which is targeted by stress response proteins to maintain the fidelity of mitochondrial function in cells producing effective ATP [199]. SIRT3 deacetylates K122 and K68 of MnSOD through an electrostatic repulsion mechanism, thereby increasing its activity and reducing intracellular superoxide levels, which maintains mitochondrial integrity and metabolism during stress [169
171]. Therefore SIRT3 acts as a mitochondrial-localized tumor suppressor. 11.5.2.3 Drp1 Drp1-mediated mitochondrial fission is a complex process that affects the dynamics of cells and organs, including development, apoptosis, cell metabolism, acute organ damage, and various diseases such as tumors [200]. SIRT4 inhibits Drp1 phosphorylation by blocking MEK/ERK activity and attenuating Drp1 recruitment to the mitochondrial membrane through interacting with Fis-1 (Fis-1), thereby reducing mitochondrial fission and inhibiting the malignant progression of nonsmall cell lung cancer (NSCLCs) [201]. 11.5.2.4 GABPα/GABPβ complex Mitochondrial biogenesis requires GABPα/GABPβ transcription factors [202]. Sirt72/2 mice exhibit pleiotropic characteristics of mitochondrial dysfunction, which can be reflected by increased blood lactate levels, decreased athletic performance, cardiac dysfunction, liver microcapsule steatosis, and age-related hearing loss. SIRT7 stabilizes and activates the GABPα/GABPβ complex (the main regulator of nuclear-encoded mitochondrial genes) through the deacetylation of GABPβ1 and the translational activation of GABPα/GABPβ heterotetramers, which may involve the activation of cytochrome oxidase expression and nuclear control of mitochondrial function [172].

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11.5.3 Indirect control of lipometabolism by sirtuins In addition to direct regulation, sirtuins also indirectly affect fatty acid metabolism through activation or inactivation. Some of the major factors that regulate fatty acid metabolism are PPARα/γ, PGC-1α, SREBP1/2, TR4/ TAK1, and PI3K-Akt. 11.5.3.1 PPARα/γ and PGC-1α PPARα/γ can regulate lipid synthesis and mediate adaptive responses to fasting and starvation [203]. It is indicated that specific deletion of SIRT1 in mouse liver cells can impair PPARα signaling and reduce PPARα target expression, including fatty acid transporters (CD36), CPT1a, CPT2, acyl-CoA dehydrogenase (ACAD), MCAD, liver bifunctional enzyme (alkenyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase, EHHADH, ECHD), palmitoyl CoA carboxylase (AOX), and microsomal cytochrome P450 enzymes (CYP4A10 and CYP4A14), which are involved in beta- and omega-oxidation of fatty acids in tissues. GPAT, DGAT1, and DGAT2 are involved in esterification in the FAS pathway. In addition, SIRT1 interacts with PPARα and is necessary to activate the PPARα coactivator PGC-1α [44]. SIRT1 also directly interacts and deacetylates PGC-1α, thereby activating the expression of MCAD and CPT1a involved in FAO. DGAT2 is involved in fatty acid esterification into glycerol. Fasting FASN, ABCA1, scavenger receptor class B Member 1 (SR-B1), low-density lipoprotein receptor (LDLr), and cytochrome P450 family 7 subfamily A member 1 (CYP7A1) are involved in cholesterol degradation and bile acid synthesis in the liver [44]. SIRT2 and SIRT4 appear to have the ability to inhibit PPARγ involved in lipid metabolism. SIRT2 deacetylates FOXO1 and promotes its binding to PPARγ, thereby inhibiting PPARγ translational activity and causing adipogenesis inhibition [174]. SIRT4 KO mice show increased expression of liver PPARα target genes, including lipase G (LIPG), acyl-CoA thioesterase 1 (ACOT3), PDK4, acyl-CoA oxidase 1 (ACOX1), CPT1a, and MCAD associated with FAO by inhibiting SIRT1 activity [204]. Consistently, SIRT1 reduction and PPARγ induction have a common gene expression profile involved in lipid metabolism, which means that SIRT1 and PPARγ jointly control lipid metabolism [205]. These results suggest that SIRT1, SIRT2, and SIRT4 collectively regulate lipid metabolism by inactivating PPARα/γ. However, a better understanding of the underlying mechanisms by which sirtuins regulate lipid metabolism is needed. 11.5.3.2 SREBP family Cholesterol metabolism has also been shown to be closely related to tumorigenesis and cancer development [206]. The sterol regulatory element-binding protein (SREBP) family is the major transcription factor for genes that control adipogenesis and cholesterol production [207]. In the fed state, SREBP promotes the expression of these genes and promotes fat storage. During fasting, SIRT1 deacetylates and inhibits SREBP1, and results in inhibition of the expression of SREBP target gene clusters related to fatty acid synthesis, such as FASN, ELOVL6, SREBP1c, ACC2, and ACLY, and cholesterol synthesis, such as the squalene ring oxygenase (SQE), mevalonate diphosphate decarboxylase (MVD), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), and mevalonate phosphate (MVK) [175]. The inhibitory effect of miR-132 on SIRT1-SREBP downregulates the levels of cholesterol and fatty acids and SREBP1c targets in glioma cells, including HMGCR and FASN [175]. In addition, SIRT2 inhibits sterol biosynthesis by suppressing SREBP2 [176]. SIRT6 controls the circadian chromatin recruitment of SREBP1, which leads to cyclic regulation of fatty acid and cholesterol metabolism [177]. SIRT6 overexpression inhibits SREBP1c/SREBP2 mRNA levels and its target genes, such as FASN, HMGCR, and HMGCS, and reduces SREBP1/SREBP2 activity formation in HepG2 cells, leading to inhibition of cholesterol synthesis [208]. In a liver-specific SIRT6 knockout mouse model, SIRT6 recruits FOXO3 to the SREBP2 gene promoter and deacetylates H3K9 and H3K56, leading to lower liver cholesterol and increased serum cholesterol [178]. There are also some reports of sirtuins regulating cholesterol metabolism, probably also through SREBPs. In a fasting state, SIRT1 promotes systemic cholesterol while reducing liver cholesterol. Knockdown of SIRT1 results in total cholesterol accumulation in the liver. Overexpression of SIRT1 reduces liver cholesterol without altering the ratelimiting enzyme HMGCR in the cholesterol synthesis pathway. While altering the ability of ATP-binding cassette subfamily A member 1 (ABCA1) and ATP-binding cassette subfamily G member 1 (ABCG1), cholesterol efflux plays a role in the cellular lipid removal pathway [209]. This evidence suggests that SREBPs play important roles in sirtuin-regulated lipid metabolism, especially cholesterol metabolism.

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11.5.3.3 TR4/TAK1 TR4/TAK1 are nuclear receptors involved in lipid metabolism, thus activating TR4 target genes, including CD36, DFFA-like effector A (CIDEA) that induces cell death inducing DFFA-like effector C (CIDEC), monoacylglycerol O-acyltransferase 1 (MOGAT1), and PPARγ. Liver SIRT7 binds to DDB1-CUL4-related factor 1 (DCAF1)/damagespecific DNA-binding protein 1 (DDB1)/cullin 4B (CUL4B) E3 ubiquitin ligase complex, thereby inhibiting degradation of TR4/TAK1 and increasing the uptake of fatty acid and synthesis and storage of triglycerides [179,180]. However, the role of SIRT7 in lipid metabolism is somewhat controversial. Another study showed that SIRT7 acts as a c-Myc translational repressor and delineates a druggable regulatory branch of the ER stress response to inhibit fatty liver development [159]. Therefore the role of SIRT7 in lipid metabolism needs to be further explored. 11.5.3.4 PI3K-Akt By directly activating ACLY or indirectly activating SREBP, the PI3K-AKT signaling pathway is also important for homeostasis of lipid metabolism [210,211]. SIRT6 inhibits the PI3K pathway at the transcriptional level independent of its histone deacetylase activity, thereby controlling the expression of lipid metabolism-related genes in cancer stem cells [181]. 11.5.3.5 LKB1 In addition to glycolysis, LKB1-AMPK is an important regulatory axis involved in fat metabolism [192]. SIRT1 deacetylates and activates LKB1 at lysine 48 while increasing its activity and cytoplasmic/nuclear ratio, and association with LKB1 activator STRAD, thereby promoting its target ACC phosphorylation and inactivation, which may inhibit FAS [160,162].

11.5.4 Indirect control of amino acid metabolism by sirtuins In addition to regulating glucose metabolism, HIF-2α is also an important transcriptional factor that controls glutamine metabolism. SIRT1 deacetylates HIF-2α and increases its activity to drive expression of various transporters and enzymes, including GLS, SLC1A5, MCT1, and IDH1, which maintain the reductive and oxidative glutamine metabolism under chronic acidosis [182].

11.6 Conclusions As a family of NAD1-dependent deacetylases, sirtuins have been shown to play a key role in regulating cellular metabolism, including glucose, lipid, nucleotide, and amino acid metabolism, which are important for both tumorigenesis and cancer development. Sirtuins regulate cellular metabolism by regulating key enzymes or transporters involved in these metabolic pathways at various regulatory levels in various ways, such as transcription, posttranslational levels, and levels of enzyme activity, sublocations, and signaling pathways. These modulations of sirtuins show a complex network in cellular metabolism and will provide some clues for the diagnosis, treatment, and prevention of cancer.

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12 Dual role of sirtuins in cancer Margalida Torrens-Mas1 and Pilar Roca1,2 1

Translational Oncology Multidisciplinary Group, Research Institute of Health Sciences (IUNICS), University of Balearic Islands, Health Research Institute of the Balearic Islands (IdISBa), E-07122 Balearic Islands, Palma, Spain 2 Ciber Obesity and Nutrition Physiopathology (CIBEROBN, CB06/03), Health Institute Carlos III, Madrid, Spain O U T L I N E 12.1 Introduction

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12.1 Introduction The seven mammalian sirtuins, SIRT1 7, are classified as NAD1-dependent deacetylases and differ in their subcellular localization and function. Sirtuins 1, 6, and 7 are found in the nucleus, sirtuin 2 in the cytoplasm, and sirtuins 3, 4, and 5 are present in mitochondria [1]. However, some sirtuins have also been found in other compartments of the cell exerting their functions. For instance, SIRT1 is also present in cytoplasm in cancer cells [2], while SIRT3 has been reported to be present also in the nucleus [3]. Furthermore, apart from their lysine deacetylase activity, SIRT4 can also act as a deacylase [4] and ADP-ribosyl transferase [5], SIRT5 possesses deglutarylation [6], desuccinylation, and demalonylation activities [7], and SIRT6 also functions as a deacylase [8] and ADP-ribosyl transferase [9]. Sirtuins have been widely studied in the cellular response to several stresses and as key players in aging. However, sirtuins have gained a lot of attention in the field of cancer research, as several reports show that their expression is altered in several types of tumors and could be involved in cancer development, progression, and resistance to treatment. However, the role of sirtuins in cancer remains controversial, with evidence existing for both oncogenic and tumor suppressor activities of these proteins. Here, we review the roles described for the seven sirtuins in different aspects of cancer, including tumor metabolism, redox homeostasis, DNA damage response, metastasis, cancer cell stemness, and response to treatment.

12.2 Sirtuins and cancer metabolism SIRT1 is considered a major regulator of metabolism by deacetylation of several important targets. First, SIRT1 activates serine-threonine liver kinase 1 (LKB1), which in turn activates AMP-activated protein kinase (AMPK) signaling and other kinases that regulate metabolism and activate peroxisome proliferator-activated receptor

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gamma coactivator 1-alpha (PGC-1α) [10]. PGC-1α is also activated by SIRT1, and it is a major regulator of mitochondrial biogenesis, promoting mitochondrial metabolism. Finally, SIRT1 also enhances FOXO3 function, which in turn activates PGC-1α and LKB1 expression [11]. The main metabolic targets of SIRT2 are phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate kinase muscle isozyme M2 (PKM2). PEPCK deacetylation promotes its stabilization [12], while PKM2 deacetylation enhances its activity [13], leading to enhanced mitochondrial function and respiration. SIRT3 targets several enzymes involved in mitochondrial metabolism, including the Krebs cycle, oxidative phosphorylation, fatty acid oxidation, and glutamine metabolism. For instance, SIRT3 deacetylates and activates acetyl-CoA synthase 2 (AceCS2), increasing the flux of acetyl-CoA into the Krebs cycle [14]. SIRT3 also regulates the pyruvate dehydrogenase complex (PDC) direct and indirectly through deacetylation of the complex itself or some of its regulators, such as pyruvate dehydrogenase phosphatase 1 [15]. SIRT3 also regulates glycolysis by deacetylating and inhibiting cyclophilin D, which produces the detachment of hexokinase II from mitochondria and inhibits this metabolic pathway [16]. On the contrary, in gastric cancer, SIRT3 was reported to enhance glycolysis through the deacetylation and activation of lactate dehydrogenase (LDH) [17]. Other metabolic targets of SIRT3 include long-chain acyl-CoA dehydrogenase (LCAD) [18], which is involved in fatty acid oxidation; 2-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) [19], the rate-limiting step for β-hydroxybutyrate synthesis; glutamate dehydrogenase (GDH) [20], which promotes amino acid catabolism; pyrroline-5-carboxylase reductase 1 (PYCR1), a regulator of proline synthesis [21]; and ornithine transcarbamylase (OTC), which is involved in the urea cycle [22]. Finally, SIRT3 also enhances mitochondrial function through the deacetylation and activation of several subunits of the electron transport chain (ETC) complexes [23 25] and ATPase [26,27]. Consistent with this role, SIRT3 knockdown in colon cancer cells limits mitochondrial metabolism [28]. This way, SIRT3 can have an important impact in several metabolic pathways and affect tumor cell proliferation. SIRT4 has been identified as an important regulator of glutamine metabolism through ADP-ribosylation and inhibition of GDH. Interestingly, Jeong et al. described that an increase in DNA damage produced an increase in SIRT4 levels, which led to the repression of mitochondrial glutamine metabolism and cell cycle arrest to promote a damage repair response [29]. Deacetylation of malonyl-CoA decarboxylase (MCD) by SIRT4 has also been described, suggesting a role of this sirtuin in the regulation of fatty acid metabolism [30]. Interestingly, SIRT4 was reported to possess lipoamidase activity on PDC, hydrolyzing a lipoyl group from one of its cofactors and inhibiting the activity of this complex [31]. Finally, SIRT4 is thought to participate in the regulation of mitochondrial ATP production presumably through the interaction with adenine nucleotide translocator 2 (ANT2), indirectly modulating the activity of the ETC [32]. SIRT5 has been associated with an increase in glycolysis in cancer cells, by inducing the expression of glucose transporter 1 (Glut1) and Ldh in lung cancer [33], as well as the deacetylation and activation of the latter in colorectal cancer [34]. Furthermore, Nishida et al. showed that SIRT5 catalyzes the demalonylation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enhancing the glycolytic flux [35]. On the other hand, SIRT5 desuccinylates complex II of ETC, succinate dehydrogenase (SDH), as well as PDC and 2-oxoglutarate dehydrogenase (OGDH), inhibiting mitochondrial respiration [36 38]. These studies suggest that SIRT5 is a promoter of the Warburg effect. SIRT5 also supports cancer cell growth by regulating amino acid metabolism. SIRT5 desuccinylates glutaminase (GLS) in breast cancer cells and prevents its degradation, enhancing the conversion of glutamine to glutamate [39]. Another report showed that SIRT5-mediated deglutarylation of GDH increases its activity, resulting in an increased flux of α-ketoglutarate into the Krebs cycle [40]. Finally, SIRT5 desuccinylates and activates mitochondrial serine hydroxymethyltransferase (SHMT2), involved in serine metabolism, and promotes tumor cell growth [41]. SIRT6 is involved in the regulation of glucose metabolism by inhibiting glycolysis and increasing oxidative phosphorylation in some types of cancer [42,43]. Presumably, SIRT6 can bind to the promoters of Glut1, phosphofructokinase (Pfk1), pyruvate dehydrogenase kinase 1 (Pdk1), and Ldh and act as a corepressor of hypoxiainducible factor 1α (HIF1α) by deacetylating histone H3 [42]. In this way, SIRT6 directs glucose to the Krebs cycle and ATP production through oxidative phosphorylation, suggesting a role of SIRT6 in the inhibition of the Warburg effect. Interestingly, Choe et al. described that the runt-related transcription factor 2 (RUNX2) can interact with SIRT6 and reduce its levels, which results in increased glucose uptake and glycolysis in breast cancer [44]. Furthermore, E2F1 transcription factor also binds to the SIRT6 promoter and inhibits its transcription to promote glycolysis [45]. SIRT6 also deacetylates FOXO1, avoiding the translocation of this transcription factor into the nucleus. In this way, SIRT6 indirectly reduces the transcription of glucose-6-phosphatase and PEPCK, key enzymes of gluconeogenesis [46].

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Finally, very few metabolic targets of SIRT7 have been described. At the moment, only HIF [47] and GA-binding protein β1 (GABPβ1) [48] have been identified as deacetylation targets of this sirtuin, which results in the promotion of mitochondrial function. SIRT7 is also involved in the regulation of rRNA transcription and of RNA polymerases [49], which could impact on different cell processes, including metabolism.

12.3 Sirtuins and oxidative damage SIRT3 is the major sirtuin involved in oxidative stress regulation, probably because mitochondria are the major source of reactive oxygen species (ROS). SIRT3 deacetylates several antioxidant enzymes, contributing to the redox homeostasis. One of the most studied targets of SIRT3 is the mitochondrial superoxide dismutase (SOD2), which converts superoxide ion into H2O2, and its deacetylation promotes its antioxidant activity [50,51]. Another important SIRT3 target is isocitrate dehydrogenase 2 (IDH2), which converts isocitrate into α-ketoglutarate, generating nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is required for the cellular redox balance, as it is involved in the regeneration of reduced glutathione, one of the main antioxidants of the cell [52]. Interestingly, SIRT3 can also regulate the expression levels of these and other targets [28,53,54]. Furthermore, SIRT3 has also been reported to deacetylate FOXO3a, which induces the transcription of several antioxidant enzymes including SOD and catalase [55]. On the other hand, SOD1 is desuccinylated and activated by SIRT5, directly contributing to ROS homeostasis in lung cancer cells [56]. IDH2 is also activated by SIRT5-induced desuccinylation, as well as glucose-6-phosphate dehydrogenase (G6PD), which is also involved in NADPH production [57]. Another report showed that PKM2 is also a target of SIRT5, although its desuccinylation leads to its inhibition and repression of glycolysis [58]. These functions of SIRT5 are enhanced under oxidative stress conditions, suggesting that this sirtuin may limit ROS production by stopping the entry of pyruvate into the Krebs cycle and by stimulating NADPH regeneration. SIRT5 also demalonylates SDH producing its inactivation. This results in the accumulation of succinate, which Du et al. showed activates thioredoxin reductase 2 (TrxR2), while at the same time inhibiting dioxygenases, controlling redox homeostasis in colorectal cancer [59]. Finally, in hepatocellular carcinoma, SIRT5 also desuccinylates and prevents dimerization and activation of peroxisomal acyl-CoA oxidase 1 (ACOX1), which is the major contributor to H2O2 production in peroxisomes. In this way, SIRT5 contributes to limit ROS production and stops cancer progression [60]. Other sirtuins are also involved in redox balance. As mentioned before, SIRT1 also deacetylates and activates FOXO3 [11], which suggests that this sirtuin may be also involved in oxidative stress regulation. SIRT2 has been reported to deacetylate peroxiredoxin-1 (Prx1) and inhibit its antioxidant activity, thus making breast cancer cells more vulnerable to oxidative stress [61].

12.4 Sirtuins, genomic stability, and DNA repair SIRT1 is the main sirtuin maintaining genomic integrity and modulating the DNA damage response. Ku70, a protein involved in DNA repair, is a well-known deacetylation target of SIRT1 [62]. Furthermore, SIRT1 is recruited to sites presenting DNA damage and induces chromatin remodeling and transcription changes [63]. SIRT1 is also involved in heterochromatin modulation, recruitment of DNA repair machinery, and epigenetic modulators [64]. Although not directly involved in DNA repair, SIRT2 has been found in the nucleus and has been associated with genomic stability during mitosis and normal cell cycle progression. This sirtuin can regulate anaphasepromoting complex/cyclosome (APC/C) activity through deacetylation and activation of CDH1 and CDC20, coactivators of this complex [65]. SIRT2 is recruited to specific regions in DNA by PR-Set7, where it deacetylates both this protein and H4, regulating PR-Set7 activation and chromatin architecture [66]. Furthermore, SIRT2 deacetylates ATR-interacting protein (ATRIP) and promotes its interaction with the protein ataxia telangiectasia-mutated and Rad3-related (ATR), which is involved in the DNA damage response [67]. Finally, SIRT2 also deacetylates cyclinedependent kinase 9 (CDK9) through a mechanism dependent on ATR activation and promotes the escape from cell cycle arrest [68]. SIRT3 deacetylates 8-oxoguanine glycosylase 1 (OGG1), an enzyme involved in mitochondrial DNA repair, and prevents its degradation. Furthermore, although not in the context of cancer cells, SIRT3 was reported to deacetylate and activate Ku70 [69], suggesting also a role in DNA repair.

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SIRT6 has also been extensively studied in DNA repair responses, as this sirtuin exerts its functions in the nucleus. SIRT6 has been reported to deacetylate H3 at heterochromatin regions, which contributes to the proper function of centromeric [70] and telomeric chromatin [71,72] and prevents DNA damage during mitosis. SIRT6 is also required for the stabilization of Werner ATP-dependent helicase (WRN), which has been found mutated in Werner syndrome, which is characterized by premature aging. In fact, SIRT6 knockdown is associated with telomere dysfunction and cell senescence [71]. Furthermore, in several cancer cells, SIRT6 also deacetylates DNA-dependent protein kinase (DNA-PK) and C-terminal binding protein (CtIP), recruiting them to a DNA-damaged area to enhance DNA repair [73 75]. Finally, SIRT6 also targets poly ADP-ribose polymerase 1 (PARP1) for deacetylation and ADP-ribosylation under oxidative stress, also promoting a DNA repair response [76]. SIRT7, although less studied, is also involved in DNA repair through different mechanisms. This sirtuin detects damaged DNA, where it deacetylates and desuccynilates histone H3, which results in the recruitment of different DNA repair machinery and chromatin remodeling [77,78]. Furthermore, SIRT7 also increases the efficiency of these proteins by deacetylation [76,79]. Another proposed mechanism consists of a weakening of stress-activated kinases (SAPK) signaling and preventing p53 accumulation, inducing senescence instead of apoptosis and therefore facilitating DNA repair [80].

12.5 Sirtuins and metastasis All seven sirtuins have been found to be involved in several aspects of tumor metastasis, including cell migration, invasion, and epithelial mesenchymal transition (EMT). EMT involves the transdifferentiation of epithelial cells, which lose their normal phenotype, including their apical basal polarity and cytoskeleton organization, into mesenchymal-like cells, with increased motility, invasive capacity, and resistance to cell death. Several transcription factors regulate EMT, such as Snail, Twist, or Zeb1, which negatively control the expression of E-cadherin, which is the major change in EMT. SIRT1 has been reported to induce the activity of the regulators Snail and Twist, resulting in E-cadherin repression, thus promoting EMT in several types of cancer, such as hepatocellular carcinoma [81] or prostate cancer [82]. Moreover, SIRT1 has been also identified in the promoter of E-cadherin, directly inhibiting its transcription by association with other proteins, including SIRT7 [83]. Zinc finger E-box-binding homeobox 1 (Zeb1) is able to recruit SIRT1 to the E-cadherin promoter, where it deacetylates histone H3 and reduces gene transcription [82]. On the other hand, transforming growth factor β (TGF-β), which has been identified as a key player in acinar-toductal metaplasia, crucial for the development of pancreatic cancer, has been associated with upregulated levels of SIRT1, promoting the transdifferentiation program in these cells [84]. This role for TGF-β1 in the promotion of EMT has also been reported for breast cancer [85,86]. In these studies, knockdown of SIRT1 reversed the EMT process. On the contrary, SIRT1 has also been described as a negative regulator of EMT in some cancer models. For instance, low levels of SIRT1 in breast cancer lead to Smad4 hyperacetylation and activation of the TGF-β signaling cascade. This results in an increase of metalloproteinase expression and a higher degradation of E-cadherin in epithelial cells, ultimately leading to EMT [85]. Thus SIRT1 inhibits TGF-β activation and EMT, which has also been described in oral squamous cell carcinoma [87]. The role of other sirtuins in EMT has been less studied. SIRT2 has been reported to promote EMT through deacetylation and activation of the protein kinase B (Akt) pathway, which phosphorylates glycogen synthase kinase-3 β (GSK-3). This leads to GSK-3 inhibition, which prevents β-catenin and Snail phosphorylation and degradation, ultimately repressing E-cadherin transcription [88]. Furthermore, SIRT2 has been reported to deacetylate Slug in breast cancer [89] and Snail in colorectal cancer [90], preventing its degradation and promoting EMT. Consistent with these studies, Li et al. showed that SIRT2 may be involved in EMT promotion through the activation of the Ras signaling pathway, leading to the activation of metalloproteinase 9 (MMP-9) [12]. SIRT3 is proposed to be an inhibitor of EMT through the activation of FOXO3a which results in the repression of the Wnt/β-catenin signaling pathway in prostate cancer [91], and through the downregulation of Twist, and possibly its deacetylation, in ovarian cancer [92]. Furthermore, through the inhibition of HIF1α, SIRT3 can also modulate TGF-β signaling and suppress EMT [93]. SIRT4 has been described as an EMT suppressor since it upregulates E-cadherin expression in colon cancer cell lines, presumably through the regulation of glutamine metabolism [94]. Vimentin has been described as a deacetylation target of SIRT5, which has been associated with EMT repression. Moreover, in the same study, the authors showed that SIRT5 downregulates Snail, leading to higher expression of E-cadherin [95].

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SIRT6 has been shown to repress N-cadherin in osteosarcoma cells, in this way preventing EMT [96]. On the contrary, in nonsmall cell lung cancer and colon cancer, SIRT6 seems to be a promoter of EMT through the upregulation of the levels of Snail and Twist1 [97,98], as well as through the deacetylation and stabilization of Snail protein [98,99]. Furthermore, in breast and ovarian cancer, SIRT6 levels correlated with β-catenin levels in tissue samples and cancer cells, and SIRT6 was associated with increased expression and/or deacetylation of some proteins regulating EMT, such as Snail, vimentin, or MMP-9 [100,101]. Finally, as mentioned before, SIRT7 is an EMT promoter as it can regulate the expression of E-cadherin, β-catenin, Slug, Snail, and MMP-16 in osteosarcoma [102], prostate [83], and colorectal cancer [103]. On the contrary, Tang et al. described SIRT7 as an EMT inhibitor in breast cancer, as it suppresses TGF-β signaling and reduces the metastatic potential of cancer cells [104].

12.6 Sirtuins and cancer stem cells Cancer stem cells (CSC) consist of a population of cancer cells that display self-renewal capacity and tumorigenic ability. CSC can drive tumor growth and are also related to metastasis and resistance to treatment. Several signaling pathways are involved in the stemness properties of cancer cells, including the Wnt/β-catenin, Hedgehog, and Notch pathways. SIRT1 has been described to regulate the Wnt/β-catenin by directly binding to β-catenin and activating it by deacetylation, promoting the transcription of target genes. This way, SIRT1 expression is required for the maintenance of the CSC population in gliomas [105], leukemia [106], liver cancer [107], breast cancer [108], ovarian cancer [109], and colorectal cancer [110], and colocalizes with several CSC markers. Furthermore, in liver CSC, SIRT1 expression is higher in Nanog-positive cells and contributes to the expression of SOX2 for self-renewal, while SIRT1 levels decrease with cell differentiation. In fact, silencing or inhibition of SIRT1 usually results in the reduction of CSC population and stemness markers [111]. On the contrary, deacetylation of β-catenin by SIRT1 in colon cancer cells has been described as an inhibitor of cancer cell stemness [112]. Moreover, SIRT1 may limit CSC proliferation through the activation of the AMPK/FOXO3 pathway in gastric cancer [113], and through the activation of protein kinase A (PKA) in liver cancer, that targets β-catenin for phosphorylation and degradation [114]. In breast cancer cells, SIRT1 and SIRT2 positively regulate the Wnt/β-catenin pathway and their inhibition decreases the expression of Firzzled 7, a receptor for Wnt ligands [115]. Furthermore, in acute lymphoblastic leukemia, the inhibition of SIRT1 and SIRT2 triggered apoptosis of CSC [116]. SIRT6 is also a repressor of this pathway by binding to lymphoid enhancer-binding factor 1 (LEF1) transcription factor and deacetylating histone H3 of the Wnt promoter [117]. Some studies suggest that SIRT1 may also inhibit Notch1 by direct deacetylation [118,119] and deacetylation of histones in its promoter [120]. SIRT3 and SIRT6 have also been identified as inhibitors of the Notch signaling pathway through a downregulation of some components of this pathway, such as NOTCH1 or NOTCH3 [121,122]. Finally, SIRT1 may be involved in the negative regulation of the Hedgehog signaling pathway in medulloblastoma [123]. Other pathways have been implicated in CSC maintenance. Interestingly, Sun et al. recently showed that CSC from lung adenocarcinoma were dependent on SIRT1 activity for the maintenance of their malignant phenotype, through the enhancement of mitochondrial function. Furthermore, the activation of this metabolic pathway was associated with higher resistance to treatment in this type of cancer [124]. Another mechanism dependent on p53 inactivation by SIRT1 was reported to be important in the maintenance of leukemia CSC [106]. Liu et al. showed that SIRT6 is able to inhibit proliferation of colorectal CSC through the deacetylation of H3 in the promoter of cell division cycle 25A (CDC25A), which is involved in cell cycle progression [125]. Finally, SIRT7 has also been involved in cancer stemness in glioblastoma cells, through the maintenance of Signal transducer and activator of transcription 3 (STAT3) signaling, which is involved in the multipotency of these cells [126].

12.7 Sirtuins and chemoresistance Some studies have described the role of several sirtuins in the resistance to cancer treatment through the regulation of different pathways. As mentioned before, SIRT1 enhances mitochondrial metabolism through activation of PGC-1α, and this has been associated with resistance to therapy in lung adenocarcinoma [124] and in colon cancer [127]. Interestingly, Shuang et al. showed that tumor tissues from ovarian cancer patients that showed

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resistance to chemotherapy had higher levels of SIRT1 in the nucleus, while reduced levels of SIRT1 were associated with higher chemosensitivity [128]. Several chemotherapy drugs induce an increase in ROS production triggering apoptosis, and as redox homeostasis regulators, sirtuins have been reported to interfere with their efficacy. Our group has previously reported that SIRT3 silencing increases the efficacy of chemotherapy drugs in breast and colon cancer cells, as this modulation increases oxidative stress and induces cell death [53,54]. This effect on cancer treatment has also been reported for acute myeloid leukemia [129]. SIRT4 has also been associated with chemoresistance, as a report described that treatment with some anticancer drugs increases the expression of SIRT4, reducing DNA damage and promoting cell survival, while knockdown of SIRT4 improved the response in HepG2 cells [130]. On the contrary, SIRT4 overexpression improved 5-FU response in colorectal cancer cells by altering cell cycle progression [131]. As mentioned before, SIRT5 indirectly regulates some antioxidant enzymes such as TrxR2 or dioxygenases, which has been shown to contribute to chemoresistance to 5-FU, oxaliplatin, and cetuximab in colorectal cancer, suggesting that SIRT5 inhibition or knockdown could improve the response to therapy [59]. SIRT5 also contributes to cisplatin chemoresistance in ovarian cancer through the activation of the nuclear factor erythroid 2-related factor 2 (Nrf-2), which upregulates several antioxidant enzymes and reduces ROS production and damage [132]. Phosphorylation of SIRT6 by AKT1, activated by MDM2 signaling, results in the degradation of SIRT6, which ultimately leads to resistance to trastuzumab treatment in breast cancer. Thus SIRT6 levels could be a useful biomarker to monitor resistance to treatment in this type of cancer, and modulation of SIRT6 could improve the sensitivity of cancer cells to trastuzumab [133]. By contrast, in nonsmall cell lung cancer, high SIRT6 levels were associated to a more aggressive disease and resistance to chemotherapy, while its downregulation increased the efficacy of paclitaxel treatment [134]. In myeloid leukemia cells, SIRT6 downregulation potentiates the effect of daunorubicin by increasing the susceptibility to DNA damage of cancer cells [73]. Finally, high levels of SIRT7 have been associated with resistance to doxorubicin in osteosarcoma cells and in hepatocellular carcinoma, presumably due to the amelioration of DNA damage [80,135].

12.8 Sirtuins: tumor suppressors or promoters? SIRT1, SIRT6, and SIRT7, due to their nuclear localization, possess an important role in regulating gene expression through histone deacetylation. Of all nuclear sirtuins, SIRT1 has been, by far, the most studied in normal physiology and cancer, and several reports support its dual role as an oncogene and as a tumor suppressor. SIRT1 has been proposed to be an oncogene in ovarian cancer [136], hepatocellular carcinoma [81,137], colon cancer [110], gastric cancer [138], breast cancer [139], and lung cancer [140], while it limits proliferation in gastrointestinal cancers [112,137] and breast cancer [139]. The other two nuclear sirtuins also show this dual role in cancer, as they have been reported to have oncogenic effects in glioma [126], ovarian cancer [100,141], lung cancer [134], colorectal cancer [99,103], melanoma [142], and osteosarcoma [102], while they suppress proliferation in breast cancer [104], lung cancer [97], osteosarcoma [96], ovarian cancer [121], and colorectal cancer [125]. The direct and indirect targets of nuclear sirtuins, as well as their main effects in cancer cells, are summarized in Fig. 12.1. Although SIRT1 also targets some cytoplasmatic proteins, SIRT2 is the major cytoplasmatic sirtuin. SIRT2 has been mainly described as a tumor suppressor [65,66], although there is also evidence for an oncogenic role in breast cancer [89], gastric cancer [12], and hepatocellular carcinoma [88]. Targets of SIRT2 and their effects are described in Fig. 12.2. Finally, of the three mitochondrial sirtuins, SIRT3 has been extensively studied, as it is the major deacetylase of this organelle, while SIRT4 and SIRT5 show weak deacetylase activity. However, during the last years, these two sirtuins have gained attention as new activities besides deacetylation have been described. SIRT3 may be crucial for the development of some cancers such as breast cancer [143], gastrointestinal tract cancers [15,144,145], melanoma [146], or kidney cancer [19], while at the same time it has been described as a tumor suppressor in breast cancer [147], ovarian cancer [148], liver cancer [149], and leukemia [150]. SIRT4 seems to participate in several lysine modifications including deacylation, ADP-ribosylation, and removal of lipoyl- and biotynil groups. SIRT4 has been defined as a tumor suppressor, as mice lacking SIRT4 spontaneously develop some types of tumors and SIRT4 is downregulated in several human cancers, such as lymphoma, bladder, breast,

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FIGURE 12.1 Targets of nuclear sirtuins. The main direct and indirect targets of nuclear sirtuins SIRT1, SIRT6, and SIRT7 are represented. Their effects on several signaling pathways or cell processes in the context of cancer are also summarized.

FIGURE 12.2 Targets of SIRT2. Direct and indirect targets of SIRT2 are shown, as well as the main affected pathways involved in cancer development and progression.

colorectal, gastric, ovarian, and thyroid cancers [131,151,152]. On the contrary, SIRT4 has been found to be upregulated in breast and liver cancer [130,153]. SIRT5 is now considered a major desuccinylase in the cell and its impact in several cell functions has been described. This sirtuin promotes the growth of ovarian cancer [132], gastrointestinal tract cancers [40,154], and breast cancer [39], while it suppresses cell proliferation in kidney cancer [37], liver cancer [60], and gastric cancer [38]. The main targets of mitochondrial sirtuins and their influences on cancer progression are detailed in Fig. 12.3. While it is clear that sirtuin expression is altered in several types of cancer, their roles in tumor development and progression are still unclear. It seems that the role of sirtuins in cancer is context dependent, as they can act both as tumor suppressors and as oncogenes by regulating numerous pathways involved in proliferation, apoptosis, metastasis, and response to treatment. This way, the final impact of sirtuins in cancer progression depends on the cellular type, cancer stage, the choice of treatment, and even the tumor microenvironment. Furthermore, the potential interplay between all sirtuins may also play a role in cancer development and progression. For these reasons, more research is needed to stablish the usefulness of sirtuins as biomarkers for cancer and as possible therapeutic targets.

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FIGURE 12.3 Targets of mitochondrial sirtuins. Direct and indirect targets of mitochondrial sirtuins SIRT3, SIRT4, and SIRT5 are summarized. Their influences on different cellular pathways related to cancer progression are shown.

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13 Sirtuin signaling in hematologic malignancies Ryan A. Denu University of Wisconsin-Madison, Madison, WI, United States O U T L I N E Abbreviations

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13.2 Hematologic malignancies 13.2.1 The many facets of SIRT1 in cancer biology 13.2.2 Oncogenic roles of SIRT1 13.2.3 Tumor-suppressive roles of SIRT1 13.2.4 SIRT1 in hematologic malignancies 13.2.5 Closing thoughts on SIRT1 13.2.6 SIRT2 regulates genomic stability 13.2.7 SIRT3, the major mitochondrial deacetylase 13.2.8 The elusive SIRT4 regulates glutamine metabolism 13.2.9 SIRT5: the oncogenic desuccinylase

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13.2.10 SIRT6 and the age-old Warburg effect 13.2.11 SIRT7 is an oncogene that promotes ribosome biogenesis and DNA repair

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13.3 Sirtuins regulate pathways important for hematologic malignancies 13.3.1 MYC-driven hematologic malignancies 13.3.2 Sirtuins and the BCL-2 family of proteins 13.3.3 Sirtuins regulate NF-κB signaling 13.3.4 CD38, a major NADase, affects sirtuin activity

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13.4 Therapeutic opportunities

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13.5 Conclusions

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Abbreviations ALL AML APC/C BER BMT CLL CML DLBCL G6PD GVHD HIF-1α HR IDH MEF NAD1 NBM

acute lymphocytic leukemia acute myeloid leukemia anaphase promoting complex/cyclosome base excision repair bone marrow transplant chronic lymphocytic leukemia chronic myeloid leukemia diffuse large B cell lymphoma glucose 6 phosphate dehydrogenase graft versus host disease hypoxia-inducible factor 1α homologous recombination isocitrate dehydrogenase mouse embryonic fibroblast nicotine adenine dinucleotide NAD1 boosting molecule

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234 NF-κB NHEJ PARP PBMCs SIRT SLL STAC TNFα

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nuclear factor kappa-light-chain-enhancer of activated B cells nonhomologous end joining poly ADP-ribose polymerase peripheral blood mononuclear cells sirtuin small lymphocytic lymphoma sirtuin-activating compound tumor necrosis factor alpha

13.1 Introduction The sirtuins are a class of nicotine adenine dinucleotide (NAD1)-dependent enzymes with a conserved core domain that allows for deacylation or ADP-ribosylation [1,2]. They are the mammalian homologues of the silent information 2 (Sir2) that was first discovered in Saccharomyces cerevisiae due to a mutation that caused sterility [3]. It was later found that Sir2 is a histone deacetylase (HDAC), and that this activity is necessary for its functions in transcription silencing, recombination suppression, and extension of life span [4,5]. Because of their NAD1 dependency, sirtuins are intimately linked to metabolism and sensing redox changes in the cell. In recent years, sirtuins have emerged as major players in aging and age-related diseases in eukaryotes. This all started with the discovery that overexpression of Sir2 in yeast extends life span [6]. Subsequent studies in mice revealed that overexpression of Sirt1 in the brain, overexpression of Sirt6 systemically, and compounds that activate Sirt1 can all prolong life span [7 9]. The seven mammalian sirtuins (SIRT1 7) show distinct acylated protein substrates and are localized in distinct subcellular compartments (Fig. 13.1). SIRT1, SIRT6, and SIRT7 are in the nucleus, SIRT2 is primarily cytosolic, and SIRT3, SIRT4, and SIRT5 are in the mitochondria [10]. The length of acyl chains that can be removed by each sirtuin varies. For example, SIRT5 removes succinyl and malonyl groups, while SIRT6 can remove myristyl groups. In addition, SIRT4 and SIRT6 can catalyze ADP-ribosylation of proteins using NAD1. How exactly do sirtuins contribute to aging? Evidence from several genetic diseases of aging, such as Werner, Bloom, Rothmund-Thomson, and Hutchinson-Gilford syndromes, shows that aging phenotypes are at least partially due to DNA repair defects and NAD1 depletion [11]. The NAD1 depletion may be at least partially due to activation of the NAD1-dependent family of enzymes, poly (ADP-ribose) polymerase (PARP), to repair DNA damage; this results in NAD1 consumption, which subsequently can lead to loss of sirtuin activity. Another proposed mechanism of antiaging by sirtuins is the reduction of oxidative stress. Sirtuin activity enhances oxidative metabolism and stimulates scavengers of reactive oxygen species (ROS), such as SOD2. Sirtuins provide an interesting link between host lifestyle, including diet, exercise, and other modifiable behaviors, and the host acylome. Accumulating evidence suggests that acylation can occur by nonenzymatic processes as

FIGURE 13.1

Structure, enzymatic activities, and localization of the human sirtuins. Sirtuins are classified into four classes according to molecular phylogenetic analysis. Classes I and IV are only present in eukaryotes.

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a result of high acetyl-CoA levels in the mitochondria [12]. This suggests that sirtuins may play a role in quality control to limit cellular protein acylation. The role of sirtuins in cancer is controversial. For most of the sirtuins, there is evidence for their roles as both tumor suppressors and oncogenes. Some sirtuins help protect the cell from oxidative damage and preserve genomic stability, which would seemingly prevent cancer. On the other hand, some sirtuins promote cell survival under stressful conditions, which could promote tumorigenesis, cancer progression, and resistance to stress-inducing chemotherapies. This chapter reviews each sirtuin, its functions, its role in cancer (particularly hematologic malignancies), and potential therapeutic opportunities.

13.2 Hematologic malignancies Hematologic malignancies are a diverse group of human cancers that arise from blood cells and include leukemias, lymphomas, myeloproliferative neoplasms, mast cell neoplasms, plasma cell neoplasms, histiocytic tumors, and dendritic cell neoplasms. In general, they are classified based on lineage: myeloid, lymphoid, and histiocytic/ dendritic. In 2019 there were an estimated 74,200 new cases of non-Hodgkin lymphoma and 19,970 deaths, 61,780 new cases of leukemia and 22,840 deaths, 32,110 new cases of multiple myeloma and 12,960 deaths, and 8110 new cases of Hodgkin lymphoma and 1000 deaths. The advent of newer targeted therapies, immunotherapies, and cell therapies have improved outcomes in a number of different hematologic malignancies. However, there is still great need to better understand the biology of these devastating diseases and to pursue novel therapeutic approaches. The majority of research has assessed sirtuins in solid cancers, so this chapter will review those findings and additionally will emphasize the findings and therapeutic possibilities most relevant to hematologic malignancies.

13.2.1 The many facets of SIRT1 in cancer biology SIRT1 is the best studied of the sirtuins, and not surprisingly, was the first sirtuin to be implicated in cancer. It resides in the nucleus and deacetylates histones, among other substrates, but is unique among HDACs in that it requires NAD1. In mouse models, Sirt1 knockout or expression of a catalytically inactive mutant causes developmental defects, most notably of the retina and heart [13]. Sirt1 activation with drugs such as resveratrol [9,14] and other more potent drugs [15,16] or by genetic overexpression [17,18] result in increased glucose tolerance, promotion of oxidative metabolism, and reduced cancer incidence. SIRT1 has been shown to have both oncogenic and tumor-suppressive roles, which will be summarized in the next sections.

13.2.2 Oncogenic roles of SIRT1 With regard to the oncogenic roles of SIRT1, elevated SIRT1 levels are seen in hepatocellular carcinoma [19], colon cancer [20,21], and diffuse large B cell lymphoma (DLBCL) [22]. Mechanistically, one of the most notable oncogenic roles of SIRT1 is deacetylation of p53, which inhibits p53-mediated tumor suppression [23,24]. With loss of SIRT1, p53 is hyperacetylated and more active, resulting in increased apoptosis in response to stressors. Additionally, SIRT1 deacetylates and thereby downregulates E2F1, an E2F transcription factor that drives the expression of many cell cycle genes; with loss of SIRT1, E2F1 is hyperacetylated and drives increased apoptosis in response to DNA damage [25]. SIRT1 has also been shown to deacetylate the tumor suppressor Rb, which may inhibit Rb-mediated repression of E2F-regulated cell-cycle genes and allow for progression through the cell cycle [26]. Further favoring the oncogene theory, SIRT1 deacetylates FOXO3 [27] and Ku70 [28] to prevent apoptosis. FOXO3 is a component of the Forkhead family of transcription factors. This deacetylation event enhanced FOXO3-dependent cell cycle arrest and resistance to oxidative stress; however, it inhibits the ability of FOXO3 to induce cell death [27], which could suggest an oncogenic role for SIRT1. Next, SIRT1 deacetylates Ku70, which promotes the interaction of Ku70 with Bcl-2-associated X protein (BAX), a proapoptotic protein, thereby preventing the translocation of BAX to the mitochondria [28]. SIRT1 overexpression is often found in in drug-resistant cancer cells. One study demonstrated that this may be due to increased expression of the drug-efflux pump multidrug resistance 1 (MDR1 or ABCB1) induced by SIRT1 activity [29].

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13.2.3 Tumor-suppressive roles of SIRT1 Many studies have associated SIRT1 with tumor-suppressive functions. Notably, genetic overexpression of Sirt1 in mice decreases the incidence of carcinomas and sarcomas (mostly osteosarcomas) but has no effect on the incidence of lymphomas and histiocytic sarcomas [18]. Furthermore, tissue-specific overexpression of Sirt1 in mice decreases colon cancers [30] and radiation-induced thymic lymphomas [31]. In human cancer, SIRT1 levels are decreased in bladder cancer, breast cancer, colorectal cancer, glioblastoma, hepatocellular carcinoma, oral squamous cell carcinoma, and prostate cancer compared to nonmalignant control tissues [30,32,33]. Overexpressing SIRT1 in human cancer cell lines reduces proliferation [30]. Further supporting its tumor-suppressive role, SIRT1 is often decreased in aggressive breast cancers, and this results in decreased expression of a specific subunit of the vacuolar-type H1 ATPase (V-ATPase), which is responsible for proper lysosomal acidification and protein degradation. This has the overall effect of altering the cancer cell secretome, resulting in greater exocytosis of soluble hydrolases that degrade the extracellular matrix and promote invasion [34]. SIRT1 is also important for genomic stability, as it has been shown to regulate both single- and double-strand DNA break repair pathways. Upon DNA damage, SIRT1 relocalizes from its gene targets to DNA breaks to promote repair and genomic stability [31]. Furthermore, SIRT1 may have the ability to suppress centrosome amplification, a major cause of genomic instability in cancer, by inhibiting centriole duplication. Mechanistically, SIRT1 deacetylates PLK2, a kinase associated with centriole duplication, which promotes ubiquitination and degradation of PLK2 [35]. Additionally, Aurora A phosphorylates SIRT1 and promotes the SIRT1-PLK2 interaction in mitosis [35]. Loss of this Aurora A-mediated phosphorylation after mitosis decreases the affinity of SIRT1 for PLK2, allowing for accumulation of PLK2 at the centrosome in time for centriole duplication in late G1. Thus SIRT1 has the effect of suppressing centriole duplication, which theoretically could oppose the development of centrosome amplification and chromosomal instability. An interesting future question is whether decreased SIRT1 expression in cancer cells can cause centrosome amplification and drive chromosomal instability. SIRT1 is also involved in regulating the Wnt signaling pathway, whose overactivity is implicated in many cancers. SIRT1 deacetylates β-catenin, which prevents nuclear localization of β-catenin and therefore inhibits its ability to modulate gene expression and drive proliferation [30]. Concordantly, there is an inverse correlation between the presence of nuclear SIRT1 and nuclear β-catenin in human colon cancers. These data suggest that SIRT1 opposes colorectal tumorigenesis by suppressing β-catenin signaling. Lastly, SIRT1 deacetylates the RelA/p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and thereby decreases its transcriptional activity [36]. Sirtuin regulation of NF-κB signaling and its relation to hematologic malignancies will be discussed in a later section.

13.2.4 SIRT1 in hematologic malignancies SIRT1 has also shown to have many functions in hematologic malignancies, with most studies pointing to an oncogenic role. SIRT1 expression is increased in acute lymphocytic leukemia (ALL) [37], acute myeloid leukemia (AML) [38,39], chronic lymphocytic leukemia (CLL) [40 43], chronic myeloid leukemia (CML) [44], DLBCL [22], and cutaneous T cell lymphoma [45]. Moreover, miR-132 can downregulate SIRT1, and higher miR-132 levels are associated with better outcomes in CLL patients; this is also concordant with SIRT1 functioning as an oncogene in CLL [41]. Further, SIRT1 expression is associated with poor prognosis of DLBCL [22]. In AML, SIRT1 expression was higher in those with intermediate or high risk compared to those with low risk, and also higher in those with internal tandem duplication in FLT3 (FLT3-ITD) [38,46]. SIRT1 overexpression may be amenable to therapeutic targeting, as depletion of SIRT1 sensitizes Jurkat cells to etoposide [47]. In most cases, the mechanism of overexpression is unclear, but some have started to investigate this. For example, overexpression of SIRT1 in CLL may be due to HIC1 promoter hypermethylation, which is often seen in CML blast crisis, resulting in reduced HIC1 expression. Normally HIC1 binds to the SIRT1 promoter and represses SIRT1 transcription, so loss of HIC1 in blast crisis results in greater SIRT1 expression [48 50]. In CML, an interesting relationship has been made between SIRT1 and the BCR-ABL fusion oncogene. BCR-ABL induces SIRT1 expression in transformed hematopoietic stem cells through activated STAT5. However, BCR-ABL kinase inhibition or STAT5 knockdown can only partially reduce SIRT1 expression, which suggests that other kinaseindependent mechanisms are responsible for increased SIRT1 activity in CML [51]. In addition, SIRT1 inhibition in primary human CML specimens results in p53 hyperacetylation, leading to increased p53 target gene expression,

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cell growth inhibition, and enhanced sensitivity to imatinib [44]. Further in CML, SIRT1 inhibitors prevented accumulation of resistance mutations in BCR-ABL in the blast crisis CML cell line KCL-22 [50]. Contrary to these findings, some have reported that SIRT1 gene expression was similar in AML and CLL patient samples compared to peripheral blood mononuclear cells (PBMCs), but was actually lower in U937, Jurkat, and 697 lines compared to PBMCs [52]. As will be seen for some of the other sirtuins, there are frequently contradictory findings in terms of sirtuin expression levels in cancer. A number of reasons could explain this. First, perhaps SIRT1 can function as both an oncogene and a tumor suppressor, depending on cell of origin and other specific alterations. Second, it depends whether the investigators studied mRNA or protein expression. Given a number of discordant findings between mRNA and protein, there are likely many posttranscriptional and posttranslational mechanisms that regulate sirtuin expression. One potential way to corroborate sirtuin expression level observations is to also assess known sirtuin substrates. Third, reported discrepancies between primary human specimens and cell lines may reflect the dynamic nature of sirtuin regulation in response to cellular stresses. In general, most cell lines are grown in nutrient-rich conditions and may be expected to decrease their sirtuin expression; in contrast, many human cancer specimens are extracted from stressful in vivo conditions of hypoxia and nutrient deprivation, which may be expected to induce sirtuin expression. Further studies will be needed to address these contradictions in the literature. Interestingly, SIRT1 may play a critical tumor-suppressive role in BCL6-driven malignancies, such as DLBCL. SIRT1 may deacetylate and deactivate BCL6, an oncogene expressed in mature B cells that is necessary for germinal center formation [23,53]. This would be expected to reduce the development of lymphoma. It will be interesting to study whether SIRT1 activators reduce the incidence of DLBCL in a murine model of DLBCL. Another possible tumor-suppressive role of SIRT1 was reported in hematopoietic stem cells from patients with myelodysplastic syndrome (MDS); SIRT1 protein levels are decreased compared to normal counterparts, and this actually enhances their growth and self-renewal [54]. The mechanism is thought to be due to subsequent hyperacetylation and reduced activity of TET2, resulting in global hypermethylation and decreased expression of tumor suppressor genes [54]. This phenomenon was reversed by pharmacologic activation of SIRT1. Interestingly, there were no differences observed in SIRT1 mRNA, and it was thought that miR-9 and miR-34a mediate this phenomenon [54]. Intriguingly, SIRT1 is involved in JAK-STAT signaling, a pathway known to drive many myeloproliferative neoplasms. Mutations in JAK2 most commonly generate constitutive activation of the JAK/STAT pathway, mainly via STAT3 and STAT5. JAK1-mediated SIRT1 phosphorylation is a negative feedback suppressing the IL-6-JAK1-STAT3 pathway [55]. JAK1 phosphorylates SIRT1, allowing for greater interaction between SIRT1 and STAT3, resulting in suppression of JAK-STAT signaling [55]. SIRT1 then deacetylates STAT3 to suppress STAT3 transcriptional activity. It will be interesting to learn how sirtuin signaling affects JAK-STAT signaling in myeloproliferative neoplasms.

13.2.5 Closing thoughts on SIRT1 One potential interpretation of all of the above findings that show SIRT1 can either be an oncogene or a tumor suppressor is that SIRT1 helps prevent cancer initiation by facilitating robust DNA repair and genomic stability, but that SIRT1 may facilitate cancer progression and treatment resistance after a tumor has already initiated. Evidence for this comes from studies showing higher expression of SIRT1 correlates with metastatic potential [56,57] and decreased sensitivity to chemotherapy and targeted agents [58]. It will be critical to pursue SIRT1 modulators in hematologic malignancies.

13.2.6 SIRT2 regulates genomic stability SIRT2 homes to the cytoplasm, where it was first shown to deacetylate α-tubulin at the K40 residue [59]. Tubulin is a heavy posttranslationally modified protein, and most modifications occur in the flexible C-terminal tail, but acetylation of α-tubulin on K40 is localized to the inside of the microtubule and is associated with stable microtubules, such as those found at axonemal microtubules of cilia and flagella and in neurons [60]. Acetylated tubulin is implicated in intracellular trafficking, endoplasmic reticulum localization and its interactions with mitochondria, and the regulation of microtubule dynamics [61]. The involvement of SIRT2 in regulating all these α-tubulin-dependent processes, particularly in cancer, remains an interesting area of future investigation.

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In addition to tubulin localization, SIRT2 shows dynamic relocalization throughout the cell cycle; as the cell enters mitosis, SIRT2 localizes to critical mitotic structures, starting with the centrosome during prophase, the mitotic spindle during metaphase, and the midbody during cytokinesis [62]. It deacetylates a number of proteins during mitosis to maintain genomic stability. As the nuclear envelope breaks down, SIRT2 can migrate to chromatin and deacetylate H4K16, which contributes to chromosome condensation [63]. SIRT2 also regulates the anaphase-promoting complex/cyclosome (APC/C), a large multisubunit complex that ubiquitinates numerous proteins important for mitotic progression, including Aurora A, Aurora B, cyclins, PLK1, survivin, and securin [64]. SIRT2 deacetylates two critical coactivators of APC/C, CDC20 and CDH1, which serves to increase the activity of the APC/C and promotes progression through mitosis [65]. CDC20 activates the APC/C early in mitosis, and CHD1 activates it late in mitosis and G1 [66]. Further, Sirt2 knockout in mice causes genomic instability, as evidenced by greater aneuploidy and centrosome amplification; male mice were more likely to get hepatocellular carcinomas, while female mice were more likely to get breast cancers [65]. Further evidence for SIRT2 involvement in maintaining genomic stability comes from a study showing that SIRT2 deacetylates CDK9, which increases CDK9 activity and promotes recovery from replication arrest [67]. In glioma cell lines, overexpression of SIRT2 blocked chromosome condensation and resulted in polyploidy with a persistence of the cyclin B/cdc2 activity in response to mitotic stress. SIRT2 also deacetylates and protects the mitotic checkpoint kinase BUBR1 from ubiquitination and subsequent destruction [68]. BUBR1 is important for mitotic checkpoint functioning, ensuring that all chromosomes are properly attached to the spindle before anaphase ensues. SIRT2 is also important in the setting of mitotic stress induced by microtubule poisons, such as nocodazole; overexpression of SIRT2 prevented entry to chromosome condensation and resulted in arrest in G2 or early M phase, and this depended on the deacetylase activity of SIRT2 [69]. Sirt2 also regulates the epigenome during mitosis. At the G2/M transition, Sirt2 migrates to the nucleus and deacetylates H4K16Ac before mitosis begins. H4K16Ac is tightly regulated during the cell cycle; it peaks in S phase and drops dramatically at the G2/M transition. SIRT2 then falls off the chromosomes during metaphase [63]. Relatedly, there is an antagonistic relationship between H4K16Ac and H4K20me. In vitro studies with peptides have shown that H4K16Ac inhibits the monomethylation of H4K20 (H4K20me1) by the histone methyltransferase PRSET7 (also known as SET8, SETD8, and KMT5A) [70]. The methylation status of H4K20 is critical for mitotic control. H4K20me1 is established in late G2/early M by PRSET7 and is critical for chromosome compaction in early mitosis and mitotic exit [71], as well as for DNA repair and replication [72,73]. During late M/early G1, some H4K20me1 is further methylated into H4K20me2 (required for DNA repair) or H4K20me3 (required for heterochromatin structure formation). SIRT2 was found to mediate this interaction through its deacetylation of PRSET7 at K90, which increases PRSET7 chromatin localization, thereby increasing H4K20me1 deposition. SIRT2 deacetylation of H4K16Ac allows PRSET7 to spread along the chromatin, further increasing H4K20me1. This resulted in Sirt2-deficient mice exhibiting genomic instability (observed more polyploidy) and DNA damage (more γH2AX), chromosomal aberrations, and greater susceptibility to DMBA/TPA-induced skin tumorigenesis [74]. Glucose and oxidative metabolism are other functions regulated by SIRT2. SIRT2 deacetylates and activates glucose-6-phosphatase dehydrogenase (G6PD) [75] and the glycolytic enzyme phosphoglycerate mutase (PGAM) [76]. G6PD is critical for the pentose phosphate pathway that produces nicotinamide adenine dinucleotide phosphate (NADPH) and pentoses for nucleotide synthesis, and this deacetylation event is critical for the proliferation of leukemia cells. Chemical inhibitors of SIRT2 suppress G6PD activity, leading to reduced cell proliferation of human leukemia cell lines, but not normal hematopoietic stem and progenitor cells [75]. Further evidence for SIRT2 involvement in metabolism is demonstrated by its ability to deacetylate ATP-citrate synthase (ACLY) [77]; acetylation promotes lipid biosynthesis and tumor growth, so SIRT2-mediated deacetylation suppresses tumorigenesis. Next, SIRT2 deacetylates forkhead box protein O3 (FOXO3), which increases the expression of FOXO target genes, which in turn serves to reduce ROS (by upregulating MnSOD) and promote apoptosis (by upregulating Bim) if cells are under irreparable stress [78]. Lastly, SIRT2 deacetylates IDH1 at K224, which increases IDH1 activity and the production of α-ketoglutarate [79]. Concordantly, SIRT2 overexpression in colorectal cancer cells reduced IDH1 acetylation, increased NADPH and glutathione (GSH) to protect cells against ROS produced during rapid cell proliferation, and subsequently inhibited cell migration, proliferation, and invasion [79]. SIRT2 is also involved in cell migration. SIRT2 deacylates the GTPase RalB, which reduced GTP binding to RalB, reduced RalB localization to the plasma membrane, and reduced migration in A549 lung cancer cells [80].

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Given all the above functions of SIRT2 in genome stability, glucose homeostasis, and other pathways, there is less controversy over SIRT2’s role in cancer: it is nearly universally thought to be a tumor suppressor. Additional evidence for this notion comes from findings that Sirt2-deficient mice develop more tumors [65]. Furthermore, SIRT2 is downregulated in human breast cancer, gliomas, gastric cancer, hepatocellular carcinoma, prostate cancer, and renal cell carcinoma [65,81,82]. Lower SIRT2 expression in breast cancer correlates with worse overall survival [83]. Furthermore, increased levels of α-tubulin K40Ac are seen in cancer, consistent with reduced SIRT2 expression or activity [84]. However, in hematologic malignancies, there is also some evidence of SIRT2 being oncogenic. SIRT2 overexpression (both protein and mRNA) is seen in primary AML blasts compared to hematopoietic progenitor cells from healthy donors [85,86]. Furthermore, inhibition of SIRT2 (with AC93253) reduces proliferation and increases apoptosis in AML cell lines (NB4 and HL60) and primary blasts [86]. Likewise, the SIRT1/SIRT2 inhibitor cambinol showed activity against lymphoma cell lines in vitro and against a lymphoma cell line xenograft in mice [53]. Subsequently, more potent and more SIRT2-specific cambinol-based compounds have been developed and show toxicity in lymphoma and epithelial cancer cell lines [87]. Lastly, novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells [85]. One potential mechanisms explaining the oncogenic role of SIRT2 is its ability to deacetylate peroxiredoxin-1 (PRDX1), which decreases PRDX1 activity to clear ROS; concordantly, overexpression of SIRT2 results in greater ROS and sensitized breast cancer cells to DNA damage, arsenic trioxide, and other ROS-inducing agents [88]. It will be critical to further study the mechanisms underlying the potential discordance between the majority of reports highlighting the tumor-suppressive roles of SIRT2 and the minority of reports demonstrating the oncogenic roles of SIRT2. It will also be imperative to understand why most of the evidence supporting the oncogenic role of SIRT2 comes from studies of hematologic malignancies.

13.2.7 SIRT3, the major mitochondrial deacetylase Mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5) are involved in regulating mitochondrial metabolism and signaling. Of the three mitochondrial sirtuins, SIRT3 controls the global lysine acetylation pattern of mitochondria, as Sirt3 knockout causes global mitochondrial hyperacetylation, which is not seen with knockout of Sirt4 or Sirt5 [89]. This appears to be especially important during caloric restriction, when mitochondrial acetyl-CoA levels increase and cause an increase in the acetylation of mitochondrial proteins. SIRT3 has been shown to regulate both cell survival and death, so there is evidence for SIRT3 being an oncogene and a tumor suppressor [90]. One of the major functions of SIRT3 is the promotion of oxidative metabolism in the mitochondria. SIRT3 deacetylates and activates AceCS2, an enzyme important in converting acetate to acetyl-CoA in the presence of ATP and CoA [91 93]. Acetyl-CoA is used in the synthesis of fatty acids, some amino acids, and ketone bodies, and for entry of carbons into the citric acid cycle. SIRT3 also deacetylates glutamate oxaloacetate transaminase 2 (GOT2), which inhibits GOT2 binding to malate dehydrogenase 2 (MDH2) [94]. This prevents the malate-aspartate NADH shuttle from increasing NAD1 levels in the cytoplasm, thereby decreasing the rate of glycolysis. Additionally, SIRT3 deacetylates and activates two components of the pyruvate dehydrogenase complex: pyruvate dehydrogenase A1 (PDHA1) and PDH phosphatase 1 (PDP1); these events activate the PDH complex and promote acetyl-CoA synthesis and oxidative metabolism in the mitochondria [95]. While there are multiple reported tumor-suppressive functions of SIRT3, the most important appears to be its ability to decrease ROS. This notion is supported by two critical pieces of data. First, Sirt3 knockout mouse embryonic fibroblasts (MEFs) can be immortalized by Myc or Ras alone, whereas wild-type MEFs require both Myc or Ras and still do not immortalize as efficiently as Sirt3 knockout MEFs with either Myc or Ras; further, the addition of lentivirus expressing MnSOD prevented the immortalization of Sirt3 knockout MEFs, suggesting that tumorigenesis in the setting of Sirt3 deletion depends on elevated ROS. Second, depletion of SIRT3 from human lymphoma cell lines results in greater susceptibility to treatment with an ROS scavenger but does not result in greater sensitivity to inhibition of the hypoxia-inducible factor 1α (HIF-1α) pathway, suggesting that loss of SIRT3 increases proliferation via ROS-dependent but HIF-1α-independent mechanisms [96]. Indeed, a plethora of evidence shows that increased ROS levels promote mutagenesis and genomic instability. ROS may also be important for cell migration; SIRT3 is typically reduced during cell migration, causing elevated ROS, and overexpression of SIRT3 reduces ROS and cell migration [97]. Mechanistically, a number of critical mitochondrial substrates of SIRT3 have been identified that confer its ability to regulate ROS. SIRT3 deacetylates and thereby activates IDH2, leading to increased levels of NADPH and an increased ratio of reduced GSH to oxidized glutathione

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(GSSG), the major redox couple in the cell [98]. SIRT3 also deacetylates MnSOD (or SOD2) at the conserved K122 residue in response to oxidative stress, thus protecting cells from stress-mediated damage [99,100]. ROS promote the stabilization of HIF-1α, a transcription factor that controls a number of glycolytic enzymes, resulting in the upregulation of glycolytic enzymes, thus leading to the Warburg phenotype. In summary, the actions of SIRT3 acts to decrease ROS and promote oxidative metabolism in the mitochondria. The strongest evidence for the role of SIRT3 in cancer is that SIRT3 acts as a tumor suppressor to reduce ROS. Interestingly Sirt3 knockout mice develop hormone-receptor breast cancers [101]. SIRT3 expression is decreased in many cancers, including CLL, lymphomas, breast, testicular, glioblastoma multiforme, prostate, head and neck squamous cell, clear cell renal, hepatocellular, and pancreatic cancers [96,101 103]. In addition, SIRT3 expression is inversely correlated with metastasis in breast cancer [97] and with survival in colorectal and hepatocellular cancers [104,105]. Hormone receptor-positive breast cancers are observed in Sirt3 knockout mice after 13 months of age [101]. This decreased SIRT3 expression results in increased ROS and activation of HIF-1α and expression of its downstream target genes, notably those involved in glycolysis and angiogenesis, which promotes cancer growth [101,103,106]. Therefore SIRT3 opposes the Warburg phenotype, whereby cells undergo aerobic glycolysis [107]. This Warburg effect is seen in many tumors and provides a growth advantage. Although aerobic glycolysis is more inefficient than shuttling pyruvate into the mitochondria for energy production by the tricarboxylic acid (TCA) cycle, it yields metabolites that are important for anabolic pathways. One example is the synthesis of nucleotides via the pentose phosphate pathway. The Warburg effect has been well documented in many types of cancer, including some of the hematologic malignancies [108,109]. At the same time, there is evidence for SIRT3 being an oncogene. First, SIRT3 levels are elevated in breast cancer [110], oral squamous cell carcinoma cell lines and primary specimens [111], and esophageal squamous cell carcinoma [112]. There is also evidence that tamoxifen treatment of MCF7 breast cancer cells increases SIRT3 levels, and overexpression of SIRT3 decreases sensitivity to tamoxifen [113]. In addition, SIRT3 deacetylates pyrroline-5-carboxylate reductase 1 (PYCR1), which is involved in proline synthesis and promotes tumor cell growth [114]. Lastly, by deacetylating Ku70, SIRT3 augments Ku70-Bax interactions, prevents Bax translocation to the mitochondria, and prevents apoptosis during stress-mediated conditions [115]. Some have found that SIRT3 also resides in the nucleus, which would allow it to interact with Ku70, but this is still controversial. It is possible that expression of SIRT3 in the nucleus is cell type-specific. In hematologic malignancies, SIRT3 is downregulated in B cell malignancies, namely primary human CLL [96]. Intriguingly, there was an inverse correlation between SIRT3 protein and mRNA levels in the B cell malignancy cell lines used in these experiments, a phenomenon that has similarly been reported for SIRT1 in AML [46]. However, higher SIRT3 gene expression (mRNA) has been reported in CLL cells, though the same study reported that patients with lower SIRT3 gene expression had worse survival [116]. Another study found that higher SIRT3 gene expression correlated with higher CD44 expression, and CD44 has been associated with more aggressive CLL [40]. Interestingly, a study using the VavP-Bcl2 lymphoma mouse model found that Sirt3 knockout attenuated B cell lymphomagenesis, supporting an oncogenic role of Sirt3 in B cell lymphoma [117]. Furthermore, a mitochondrial-targeted class I sirtuin inhibitor, YC8-02, mimicked the effects of Sirt3 depletion and effectively killed lymphoma cells in this mouse model. In summary, the majority of evidence supports the tumor-suppressive role of SIRT3 to quench oxidative stress and promote mitochondrial oxidative metabolism. Interestingly, as we age, our SIRT3 levels decrease, which may create a cellular environment permissive for in vivo carcinogenesis. However, some critical evidence supporting an oncogenic function for SIRT3 in lymphoma will have to be reconciled with other data. One potential explanation for these seemingly contradictory data is that SIRT3 acts as a tumor suppressor to inhibit initiation, but after a tumor has already developed, SIRT3 promotes tumor progression by helping the tumor cells respond to stress.

13.2.8 The elusive SIRT4 regulates glutamine metabolism SIRT4 is also a mitochondrial sirtuin involved in metabolism, but it is unique from the other sirtuins in several ways. Though its activity was initially difficult to determine in vitro, it has now been shown to have deacetylase, ADP-ribosyltransferase, and lipoamidase activities [118]. In contrast to the other sirtuins, SIRT4 expression increases with nutrient-rich conditions and is downregulated by fasting and caloric restriction [119]. One of the major functions of SIRT4 is ADP-ribosylation of GDH1, a component of glutamate dehydrogenase; this ADP-ribosylation inhibits glutamate dehydrogenase activity [119]. Glutamate dehydrogenase is the rate-limiting

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step in glutaminolysis and converts glutamate to α-ketoglutarate and ammonia while reducing NADP1 to NADPH; α-ketoglutarate can then proceed through the citric acid cycle to produce ATP. In the SIRT4 literature, nearly all evidence points toward SIRT4 being a tumor suppressor. In human cancer, SIRT4 expression is decreased in breast cancer [120], colorectal cancer [121,122], endometrial cancer [123], esophageal cancer [124], gastric cancer [125], leukemia [126], hepatocellular carcinoma [127], pancreatic cancer [127], prostate cancer [127], ovarian cancer [127], renal cell carcinoma [127,128], and small cell lung cancer [129]. Furthermore, loss of SIRT4 is associated with shorter time to metastasis in breast cancer patients [130]. Overexpression of SIRT4 in DLD1 and DU145 cancer cell lines reduces cell proliferation [130]. Similarly, overexpression of SIRT4 in thyroid cancer cells reduced proliferation, migration, and invasion [131]. Further evidence for SIRT4 being a tumor suppressor comes from the finding that Sirt4 knockout mice spontaneously develop more lung cancers [132]. In cancer, loss of SIRT4 expression leads to increased glutamate dehydrogenase and therefore increased glutamine production, which promotes cancer growth. Glutamine is so important to cancer cells because it is the primary nitrogen donor for protein and nucleotide synthesis, which are critical for cell proliferation [133] It is also a critical entry point for carbon to fuel the citric acid cycle. Further, glutamine metabolism also results in ammonia production, which neutralizes the acidic metabolites produced from glycolysis. Glutamine is also an important signaling molecule and regulates the mTORC1 pathway by enhancing uptake of leucine and promoting mTORC1 assembly and lysosomal localization [130]. Relatedly, the mTORC pathway is capable of decreasing SIRT4 gene expression by promoting degradation of the transcription factor CREB2; this has the effect of increasing glutamate dehydrogenase activation and increased glutamine metabolism in cancer cells [130]. Lastly, it has been demonstrated that increased glutamine metabolism can lead to downregulation of E-cadherin expression, which promotes invasive properties and epithelial mesenchymal transition in cancer cells [122]. SIRT4 is also critical for maintaining genome stability in the setting of DNA damage. Sirt4 knockout MEFs have greater rates of aneuploidy compared to wild-type MEFs [132]. Interestingly, SIRT4 levels are induced by DNA damage to a greater extent than the other sirtuins, which allows for repression of mitochondrial glutamine metabolism in the face of DNA damage [132]. Further, SIRT4 allows for greater clearance of γH2AX foci after DNA damage. SIRT4 is also a lipoamidase [134,135]. Lipoic acid is an enzyme cofactor required for a small number of metabolic reactions, such as the oxidative decarboxylation reactions catalyzed by pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. SIRT4 catalytic efficiency for lipoyl- and biotinyl-lysine modifications is greater than its deacetylation activity [135]. SIRT4 can remove lipoic acid from the E2 component of pyruvate dehydrogenase, dihydrolipoyllysine acetyltransferase (DLAT), which serves to reduce pyruvate dehydrogenase activity, thereby reducing acetyl coA levels [135]. Oxoglutarate dehydrogenase is another lipoylated enzyme, and it catalyzes the oxidative decarboxylation of α-ketoglutarate to succinyl-CoA. It is tempting to speculate that SIRT4 may also regulate oxoglutarate dehydrogenase activity, which could serve to promote activity of the citric acid cycle, similar to the effect of SIRT4 deacetylation of GDH1. It will be interesting to learn how SIRT4 regulates the remaining lipoylated enzymes. SIRT4 also appears to function as a tumor suppressor in hematologic malignancies. Concordantly, SIRT4 expression is reduced in both acute and chronic leukemias [39,126,132] In addition, Sirt4 suppresses tumor formation in murine genetic models of Myc-induced B cell lymphoma [136]. Concordantly, SIRT4 overexpression in MYC-driven human Burkitt lymphoma cells inhibits glutamine use, inhibits cell proliferation, sensitizes the cells to glucose deprivation, and synergizes with glycolysis inhibitors to induce cell death [136]. Increasing or activating SIRT4 is of therapeutic interest in treating cancer. Interestingly, mTORC1 promotes the proteasomal degradation of CREB2, which then was shown to decrease transcription of the SIRT4 gene [130]. Rapamycin (or the drug we use clinically as sirolimus) is an mTORC inhibitor that was shown to increase SIRT4 levels [130] and therefore may be of therapeutic interest in certain cancers, including hematologic malignancies.

13.2.9 SIRT5: the oncogenic desuccinylase SIRT5 is the last of the three mitochondrial sirtuins. Its levels do not significantly change with aging or exercise, but fasting and caloric restriction induce SIRT5 expression [137]. SIRT5 is thought to primarily function as a desuccinylase in the mitochondria [134,138 140]. Subsequent studies have demonstrated that two particular pathways, fatty acid β-oxidation and ketone body production, are enriched in lysine succinylation and are therefore highly regulated by SIRT5 [139]. In contrast to SIRT3, SIRT5 has a minimal effect on the global mitochondrial

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acetylome, as Sirt5 knockout mice have minimal alterations in global mitochondrial acetylation status. In addition to its desuccinylase activity, it also is a deacetylase [141], demalonylase [142], and deglutarylase [140,143]. This preference for negatively charged substrates is thought to be conferred by the positively charged residues (Y102, R105) in the active site [144]. In general, the removal of these acyl groups increases the activity of its substrates. Similar to SIRT3, SIRT5 also regulates ROS by several mechanisms. First, it desuccinylates and thereby activates SOD1 [145] and IDH2 [146] and deglutarylates G6PD to enhance glutathione antioxidant systems. SIRT5 demalonylates and thereby inactivates succinate dehydrogenase complex subunit A, causing buildup of succinate. Succinate then binds to and activates thioredoxin reductase 2, which confers resistance to chemotherapy by scavenging ROS [147]. In addition, some evidence shows that SIRT5 may also localize to peroxisomes in addition to mitochondria. In the peroxisomes, SIRT5 is capable of desuccinylating and inactivating acyl-CoA oxidase 1, the first and rate-limiting enzyme in fatty acid β-oxidation and a major producer of H2O2 [148]. This function of SIRT5 also serves to reduce cellular oxidative stress. Reciprocally, ROS are also able to increase the activity of SIRT5 [146]. Glucose metabolism is also controlled by SIRT5. SIRT5 regulates the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase by demalonylation of the K184 residue, which increases its activity and promotes glycolysis [149]. In this way, SIRT5 promotes glycolysis. In cancer, this function could potentiate the Warburg effect, by which cancer cells perform aerobic glycolysis. SIRT5 also regulates the urea cycle. SIRT5 deacetylates and thereby activates carbamoyl phosphate synthetase 1 (CPS1) to regulate the urea cycle [150]. This serves to stimulate the urea cycle to detoxify ammonia. As was discussed with SIRT4, cancer cells are often addicted to glutamine, and glutamine metabolism increases ammonia. Therefore it is conceivably advantageous for cancer cells to upregulate SIRT5 to eliminate this excess ammonia. In human cancer, most evidence associates SIRT5 with being an oncogene. SIRT5 is overexpressed in breast cancer [151,152], ovarian cancer [153], colorectal cancer [154], hepatocellular carcinoma [155], non-small cell lung cancer [156], Waldenstrom’s macroglobulinemia bone marrow specimens, and BCWM.1 cell line [157]; many of these studies also demonstrated worse outcomes with greater SIRT5 expression. Furthermore, SIRT5 desuccinylates and stabilizes mitochondrial glutaminase, which supports breast cancer growth [151]. SIRT5 is also associated with resistance to chemotherapy; SIRT5 expression made ovarian cancer cells resistant to cisplatin by suppressing ROS [153]. Lastly, in Sirt5 knockout mice, no increase in spontaneous tumor development has been reported. However, there are also studies that point toward a tumor-suppressive role for SIRT5. First, SIRT5 expression is decreased in endometrial cancer [158] and head and neck squamous cancer [159]. Decreased SIRT5 expression is associated with worse outcomes in glioblastoma [160] and hepatocellular carcinoma [148]. Further, triple negative breast cancer patients treated with neoadjuvant chemotherapy who achieved a pathologic complete response express higher levels of SIRT5, suggesting that SIRT5 expression is protective [152]. Moreover, in IDH1-mutant gliomas, SIRT5 suppresses the oncogenic effect of the oncometabolite 2-hydroxyglutarate by reversing hypersuccinylation caused by succinyl CoA buildup [161]. Thus SIRT5 is a unique sirtuin with the ability to regulate enzymes by removing multiple different acyl groups. A preponderance of data supports its role as an oncogene. However, there is a relative dearth of data on SIRT5 in hematologic malignancies, and this represents an interesting area of future research. Such studies could reveal a role for SIRT5 inhibitors in treating hematologic malignancies.

13.2.10 SIRT6 and the age-old Warburg effect SIRT6 localizes to the nucleus where it deacetylates H3K9 [162,163] and H3K56 [164 166] to exert a number of tumor-suppressive roles. Most notably, SIRT6 is a transcriptional corepressor of HIF-1α [162], c-MYC [167], and NF-κB [168]. Sirt6 knockout mice show signs of progeria and die prematurely due to hypoglycemia [169]. Conversely, overexpression of Sirt6 in mice expands lifespan, though this was only seen in male mice [7]. Further, Sirt6 overexpression lowers low-density lipoprotein and triglyceride levels and improves glucose tolerance [170]. In the context of cancer, the majority of evidence suggests that SIRT6 functions as a tumor suppressor. Loss-offunction mutations occur in a variety of human cancers [171]. Data from The Cancer Genome Atlas indicate that SIRT6 is deleted or downregulated in 20% of all cancers [167]. Many other studies have demonstrated reduced SIRT6 gene and protein expression in many different cancer types [167,172 178]. In addition, non-small cell lung cancers with reduced nuclear expression of SIRT6 were more aggressive, and patients had worse outcomes [179].

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Furthermore, global H3K9 and H3K56 acetylation is elevated in many types of human cancer (including, breast, colorectal, hepatocellular, skin, and thyroid cancers) and correlates with higher tumor grade, consistent with reduced SIRT6 activity [178,180]. Furthermore, SIRT6 overexpression in cancer cells (breast, cervical, and fibrosarcoma), but not in normal cells (fibroblasts and mammary epithelial cell lines), can induce apoptosis [181,182] and inhibit proliferation [167,172,177,183]. Mechanistically, loss of SIRT6 results in increased expression of HIF1α, c-MYC, and NF-κB targets, most notably genes important for glycolysis, glutaminolysis, and ribosomes, all of which fuel cancer growth [162,167,168]. By repressing the expression of glycolytic genes, SIRT6 opposes the Warburg effect [167]. The relevance of SIRT6 inhibition of NF-κB to hematologic malignancies will be discussed in a later section. SIRT6 also plays an imperative role in maintaining genome stability. One of the first pieces of evidence comes from the finding that Sirt6 knockout MEFs and embryonic stem cells are more sensitive to DNA damaging agents [169]. Furthermore, these Sirt6 knockout cells demonstrate more fragmented chromosomes, detached centromeres, and translocations. First, SIRT6 maintains telomeres, which are crucial for genomic stability. Concordantly, SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence [163]. In addition, SIRT6 regulates regulate both single- and double-strand DNA break repair pathways. SIRT6 regulates DNA damage repair via its interaction with proteins in the nonhomologous end joining (NHEJ) pathway [184]. However, this is controversial, as other work shows that the formation and clearance of γH2AX foci in response to ionizing radiation was not different in Sirt6 knockout MEFs, suggesting that Sirt6 may not function in double-strand break repair; however, these knockout MEFs did show sensitivity to drugs that cause damage that is repaired by the base excision repair (BER) pathway [169]. Further, overexpression of Polβ, the major polymerase used for BER in mammalian cells, reversed the sensitivity of Sirt6 knockout MEFs to these BER-inducing agents [169]. Subsequent evidence pointed to SIRT6 involvement in double-strand break repair [185]. SIRT6 is found at centromeric DNA, where it is involved in transcriptional silencing, which is essential in mitosis to maintain genomic stability [186]. However, there are some studies that show an oncogenic role of SIRT6 in cancer. SIRT6 expression is elevated in breast cancer, CLL [40], multiple myeloma [187], pancreatic cancer [188], and prostate cancer [189]. In CLL, SIRT6 expression was even greater in ZAP70-positive disease [40]. Additionally, SIRT6 activity promotes resistance to chemotherapy in breast cancer cells, and this was thought to be due to enhancing the DNA damage response [190]. Furthermore, in pancreatic cancer, SIRT6 enhances the expression of proinflammatory cytokines and migration, favoring inflammation, angiogenesis, and metastasis [188]. Lastly, SIRT6 has been associated with the epithelial mesenchymal transition. Mechanistically, it was shown that SIRT6 deacetylates Beclin-1 in hepatocellular carcinoma cells to promote the autophagic degradation of E-cadherin; SIRT6 also deacetylates FOXO3a to promote N-cadherin and Vimentin expression [191]. A number of studies have linked SIRT6 dysregulation to hematologic malignancies. Intriguingly, the SIRT6 chromosomal locus (19p13.3) is a frequent site of breakage in human AML [192]. As mentioned above, SIRT6 expression is elevated in CLL [40] and multiple myeloma [187]. However, one might rationally conclude that loss of SIRT6 may be beneficial for multiple myeloma in particular, given that the multiple myeloma bone marrow microenvironment is highly hypoxic, and multiple myeloma cells show increased HIF-1α expression [193,194]. In addition to controlling the transcription of glycolytic enzymes, HIF-1α also controls the transcription of several potent multiple myeloma cell growth factors: IL-6, IL-8, and vascular endothelial growth factor A (VEGF A) [195]. Given all the above, the strongest data support the tumor-suppressive role of SIRT6; therefore strategies to increase or activate SIRT6 are the most promising. One potential way is to activate SIRT6 with fatty acids [134] due to the presence of a pocket that can bind long fatty acid chains. Additionally, novel activators of SIRT6 deacetylase activity have been developed [196 199], and some have shown activity in cancer cell lines [200], though these will need additional optimization before entering clinical trials.

13.2.11 SIRT7 is an oncogene that promotes ribosome biogenesis and DNA repair SIRT7 localizes to nucleoli and, most notably, regulates RNA polymerase I (PolI) transcription of rRNA to fuel ribosome synthesis [201,202]. Not surprisingly, SIRT7 is highly expressed in metabolically active and proliferative tissues, such as liver and spleen, and less in nonproliferating tissues, such as heart and brain [201]. SIRT7 deacetylates the PAF53 subunit of the PolI complex, which promotes pre-rRNA transcription [201,202]. Furthermore, SIRT7 deacetylates U3 55K, a subunit of the U3 snoRNP, to promote pre-rRNA processing and maturation [203].

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Through these deacetylation events, SIRT7 promotes ribosome biogenesis, which in turn promotes cell growth and proliferation. SIRT7 is also involved in regulating PolII, as it also deacetylates CDK9, a component of the P-TEF-b elongation factor, and this event has been shown to promote elongation of PolII-dependent transcription of mRNAs and snoRNAs [204]. During times of nucleolar or genotoxic stress, SIRT7 relocates from the nucleolus to the nucleoplasm [202]. Sirt7 knockout mice have shortened life span, genomic instability, and aging-associated pathologies such as cardiomyopathy, hepatic steatosis, hearing defects, and loss of hematopoietic stem cell regenerative potential [205 208]. SIRT7 is also involved in mitochondrial metabolism. SIRT7 regulates the expression of mitochondrial genes and has been shown to prevent mitochondrial diseases through deacetylation of the transcription factor GA-binding protein subunit β1 (GABPβ1) [209]. Since SIRT7 is involved in cell growth and is expressed more in metabolically active tissues, studying SIRT7 has implications in cancer. Most evidence supports the role of SIRT7 as an oncogene. Higher SIRT7 levels have been found in breast cancer, endometrial cancer, colon cancer, glioma, ovarian cancer, prostate cancer, renal cell carcinoma, and thyroid cancers [110,210 212]. SIRT7 expression is even higher in metastatic sites compared to primary prostate tumors [211]. Furthermore, the SIRT7 activity correlates with the expression of oncogenic genes [210]. Depletion of SIRT7 in human cancer lines reduces migration and proliferation and induces cell cycle arrest and apoptosis in vitro [205,213,214]. Consistently, SIRT7 knockdown reduces the growth of human cancer cell xenografts [210] and metastasis (using tail vein injection and observing lung metastases) in mice [211]. SIRT7 has recently been shown to deacetylate H3K18Ac and thereby stabilizes the transformed state of cancer cells by inactivating tumor suppressor gene transcription [210]. Consistent with an oncogenic role of SIRT7, H3K18 hypoacetylation is seen in cancer [215]. Moreover, SIRT7 promotes 53BP1 binding to chromatin and NHEJ by deacetylating H3K18Ac at sites of double-stranded breaks [207]. SIRT7 deacetylates ataxia telangiectasia mutated gene (ATM), which is essential for the dephosphorylation and deactivation of ATM; depletion of SIRT7 caused persistent phosphorylation and activation of ATM, which led to impaired DNA damage repair [216]. One can see how these functions in regulating DNA damage repair could have oncogenic and tumor-suppressive functions. Regarding a tumor-suppressive function, facilitating DNA repair fidelity and efficiency would be expected to prevent cancer-initiating DNA damage. On the other hand, this could also promote resistance to DNA-damaging chemotherapeutic agents, which has been seen in other contexts, such as O-6-methylguanineDNA methyltransferase (MGMT) demethylation and subsequent MGMT expression conferring resistance to the DNA alkylating agent temozolomide in gliomas. However, overexpressing SIRT7 in noncancer primary cell lines does not appear to promote oncogenic transformation [210]. As SIRT7 is important for genomic stability and DNA repair [207,217], it may be that SIRT7 is protective in nontransformed cells but promote cancer progression and chemotherapy-resistance in already transformed cells, as we have seen and discussed with several of the other sirtuins. Interestingly and relevant to hematologic malignancies, the SIRT7 locus (17q25.3) is a region which is frequently affected by chromosomal alterations in acute leukemias and lymphomas [218]. The functional significance of this is unclear, but warrants further investigation. In summary, SIRT7 is involved in promoting ribosome biogenesis and DNA repair, and most evidence supports its role as an oncogene in cancer.

13.3 Sirtuins regulate pathways important for hematologic malignancies This next section delves into pathways regulated by sirtuins that are particularly relevant to hematologic malignancies. This is summarized in Fig. 13.2.

13.3.1 MYC-driven hematologic malignancies The MYC family of transcription factors are some of the most well-studied human proteins and have long been associated with virtually every aspect of oncogenesis. They are deregulated in over half of human cancers, and this is often associated with worse outcomes. Humans have three family members: c-Myc (or MYC), N-myc (or MYCN), and L-Myc (or MYCL). These differ in their expression and potency, and together they regulate at least 15% of the human genome [219].

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FIGURE 13.2 The roles of the sirtuins in hematologic malignancies. This figure focuses on functions discussed in the text that are relevant to the biology of hematologic malignancies and is by no means exhaustive.

Many hematologic malignancies are driven by MYC. MYC was initially identified as the target oncogene dysregulated by the t(8;14)(q24;q32) translocation in Burkitt lymphoma. In DLBCL, MYC and either BCL2 or BCL6 rearrangements are termed “double-hit” lymphomas and are particularly aggressive [220]. Furthermore, amplification of MYCN is frequently found in hematologic malignancies such as lymphoma and AML. MYC is primarily regulated by phosphorylation. MYC complexes with MAX, and this complex interacts with other transcription factors to allow binding to target genes, which are involved in innumerable cellular pathways such as proliferation, glucose and glutamine metabolism, nucleotide metabolism, and ribosome biogenesis. Phosphorylation by ERK stabilizes MYC, while dephosphorylation by MAPK phosphatase 3 destabilizes MYC. However, acetylation also appears to be important for MYC regulation, and the sirtuins have shown activity in this space. SIRT1 deacetylates MYC, which stabilizes MYC and favors binding to MAX [221]. The interaction is bidirectional, as MYC activity increases NAMPT, which is the rate-limiting enzyme in NAD1 synthesis, and NAD1 is required for sirtuin activity [222]. Indeed, MYC and SIRT1 levels have been strongly correlated in hepatocellular carcinomas [19]. Similarly, there is a bidirectional relationship with SIRT1 and MYCN: SIRT1 binds to and stabilizes MYCN, and MYCN is capable of binding the SIRT1 promoter and driving SIRT1 transcription [223]. Inhibiting or depleting SIRT1 in cells with increased MYC activity results in senescence and apoptosis [19,222]. Additionally, similar bidirectional relationships have been found between SIRT2 and both MYC and MYCN; [224] MYC and MYCN drive the transcription of SIRT2, while SIRT2 represses the activity of NEDD4, an E3 ubiquitin ligase, by binding to the NEDD4 gene core promoter and deacetylating histone H4K16 at this promoter [224]; the net result is decreased NEDD4 transcription. Interestingly, SIRT2 inhibitors reactivate NEDD4 gene expression, reduce N-Myc and c-Myc protein expression, and suppressed neuroblastoma and pancreatic cancer cell proliferation [224]. SIRT4 does not directly interact with MYC, but, as discussed earlier, SIRT4 overexpression in models of Burkitt lymphoma, a MYC-driven cancer, inhibits glutamine metabolism and reduces proliferation [136]. This makes sense due to the fact that MYC-driven cancers are dependent on glutamine and utilize a lot of it.

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SIRT6 is also a major regulator of MYC signaling. SIRT6 is enriched on MYC target gene promoters and acts as a transcriptional corepressor of MYC, resulting in reduced transcription of MYC target genes [167]. Lastly, SIRT7 is also implicated in MYC-driven cancers. SIRT7 has been shown to repress MYC activity to suppress ER stress [206]. In times of ER stress, MYC recruits SIRT7 to the promoters of ribosomal genes and corepresses their expression; this effect is abrogated by loss of SIRT7 [206]. This might suggest that SIRT7 has a tumor-suppressive role in some contexts, notably MYC-driven cancers. Further work will be needed to assess this possibility. In summary, sirtuins are critical regulators of MYC signaling. Most evidence suggests that SIRT1 promotes MYC signaling, while SIRT4, SIRT6, and SIRT7 inhibit MYC signaling.

13.3.2 Sirtuins and the BCL-2 family of proteins The BCL2 family of proteins is best known for regulating the mitochondrial (or intrinsic) apoptotic pathway, and it includes at least 20 members with both pro- and antiapoptotic functions. In this pathway, BCL2 proteins promote or inhibit mitochondrial depolarization, which can lead to caspase-mediated cell death. Many cancers become resistant to chemotherapy and apoptosis by upregulating BCL2 family members. In particular, hematologic malignancies are often driven by increased expression of BCL2, an antiapoptotic protein and the first discovered of this family of proteins. For example, follicular lymphomas often harbor the t(14:18)(q32:q21) translocation, which juxtaposes BCL2 with the immunoglobulin heavy chain locus, resulting in BCL2 overexpression and preventing apoptosis [225]. As described previously, DLBCLs with BCL2 rearrangements are particularly aggressive [220]. In recent years, BCL2 inhibitors have emerged to the clinical arena. Venetoclax is a BCL2 inhibitor that is FDA-approved for the treatment of CLL/small lymphocytic lymphoma (SLL) and AML. Many of the sirtuins have been linked to this impactful BCL2 family of proteins and exploiting these interactions with sirtuin modulators and BCL2 inhibitors could be of therapeutic relevance in hematologic malignancies. SIRT1 is implicated as an antiapoptotic protein via its regulation of the BCL2 family. SIRT1 deacetylates Ku70, which is a component of a heterodimeric Ku complex that is required for NHEJ, VDJ recombination, and telomere maintenance. This deacetylation promotes the interaction of Ku70 with BAX, which blocks the translocation of BAX, a proapoptotic protein, to the mitochondria [28,226]. In addition, depletion of SIRT1 led to increased BAX and reduced BCL2, which is consistent with an antiapoptotic role for SIRT1 [227]. Lastly, SIRT1 may also contribute to BCL2 regulation via its deacetylation of p53 [23,24], which promotes p53 suppression of BCL2 through NOXA activation; [228] NOXA (also known as PMAIP1) is a proapoptotic BCL2 family member. IDH1 and IDH2 are mutated in approximately 15% of AML [229] and 20% of angioimmunoblastic T-cell lymphoma [230] and result in neomorphic enzymatic function and production of an oncometabolite, 2-hydroxyglutarate [231]. 2-Hydroxyglutarate is an oncometabolite capable of inhibiting α-ketoglutarate-dependent reactions and others, including the conversion of succinate to fumarate catalyzed by succinate dehydrogenase [161]; this results in the buildup of succinyl-CoA and subsequent hypersuccinylation in the mitochondria. Hypersuccinylation causes mitochondrial depolarization and BCL2 accumulation at the mitochondrial membrane, causing resistance to apoptosis. In IDH1-mutant gliomas, SIRT5 suppresses the oncogenic effect of the oncometabolite 2-hydroxyglutarate by reversing this hypersuccinylation [161]. Concordantly, evidence has shown that IDH1 and IDH2 mutations induce BCL2 dependence in AML [232]. This may suggest a therapeutic strategy to treat IDH1 and IDH2 mutant cancers and SIRT5-deficient cancers with anti-BCL2 drugs, such as venetoclax, or SIRT5 activators. It will be interesting to see if decreased expression of SIRT5 or other mitochondrial sirtuins, namely SIRT3, is sufficient to induce BCL2 accumulation and cause a dependence on BCL2 that might be amenable to targeting. Lastly, SIRT3 has been implicated in regulating the BCL2 pathway. SIRT3 deacetylates and activates glycogen synthase kinase-3β (GSK-3β) [233], and overexpression of SIRT3 in hepatocellular carcinoma cells activates GSK-3β, which causes GSK-3β phosphorylation of BAX and subsequent mitochondrial translocation of BAX to promote apoptosis [234]. This suggests one potential therapeutic implication worth further exploration: treatment of BCL2-dysregulated cancers with a SIRT3 activator.

13.3.3 Sirtuins regulate NF-κB signaling The NF-κB family of transcription factors forms a link between chronic inflammation and cancer transformation [235]. It is composed of five members: RelA (p65), RelB, c-Rel, NFKB1 (p105/p50), and NFKB2 (p100/p52), which form various dimeric complexes that transactivate numerous target genes via binding to κB consensus

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DNA binding sites. Normally NF-κB complexes are inactive and sequestered in the cytoplasm by inhibition by IκB proteins. NF-κB can be activated in two ways. First, the canonical or classical pathway is mediated by the RelA-p50 subunits. Second, the noncanonical or alternative pathway activates RelB-p52. Both have been implicated in hematologic malignancies, particularly lymphoid malignancies. The constitutive activation of NF-κB is capable of activating many pathways that are imperative for transformation, including inhibition of apoptosis, promotion of a dedifferentiated state, promotion of cell proliferation, and promotion of angiogenesis. It has proven difficult to drug NF-κB, perhaps because it is difficult to identify which cancers depend on NF-κB signaling and what mechanisms are in place to drive the signaling in individual cases. Interestingly, sirtuins are capable of turning off NF-κB signaling in the nucleus, and thus sirtuin activation can inhibit NF-κB signaling [236]. First, it was found that SIRT1 deacetylates RelA/p65 at K310, resulting in reduced NF-κB gene expression [36]. Further, treatment with sirtuin-activating compounds (STACs) such as resveratrol and SRTCX1003 decreases NF-κB target gene transcription and sensitizes cells to tumor necrosis factor alpha (TNFα)-induced apoptosis [36,237]. Using human plasmacytomas xenografted into mice to model human multiple myeloma, SIRT1 activation with SRT1720 decreased NF-κB signaling and showed antimyeloma activity [238]. Further evidence is provided by SIRT1 overexpression in the CML cell line K562, which inhibits the release of the inflammatory cytokines (e.g., IL-1β, IL-6, IFN-γ, TNFα, and TNFβ) in LPS-treated cells [239]. Taken together, these data suggest that SIRT1 plays a role in repressing NF-κB signaling. Interestingly, SIRT2 also deacetylates RelA at K310, but this interaction occurs in the cystoplasm [240]. Concordantly, knockout of SIRT2 results in RelA hyperacetylation and increased expression of NF-κB target genes. In vitro, SIRT2 was more efficient at this deacetylation than SIRT1, and hyperacetylation of K310 was observed after TNFα stimulation in the Sirt2 knockout MEFs but not Sirt1 knockout MEFs [240]. These data suggest that SIRT2 is the major RelA deacetylase. Sirt22/2 MEFs are more resistant to TNFα-induced apoptosis. SIRT6 also inhibits NF-κB signaling. It does not directly deacetylate RelA or other NF-κB genes, but exerts its effects on the pathway indirectly via its deacetylation of H3K9Ac on histones [168]. Concordantly, overexpressing SIRT6 in human fibroblasts and mouse chondrocytes reduces NF-κB target genes [241,242]. Lastly, SIRT6 is involved in a negative feedback pathway inhibiting NF-κB activity. In response to TNFα, SIRT6 binds to the H3K9me3-specific histone methyltransferase Suv39h1 and induces monoubiquitination of conserved cysteines in the PRE-SET domain of Suv39h1; however, this was not observed in all cell types [243]. Upon NF-κB pathway activation by TNFα exposure, SIRT6 deacetylates the nuclear localization sequence of SKP2 (an E3 ubiquitin ligase), allowing SKP2 translocation to the nucleus and ubiquitination of Suv39h1. This causes release of Suv39h1 from the IκBα promoter, allowing transcription of IκBα and subsequent repression of NF-κB signaling [243]. This appears to be a negative feedback mechanism regulating NF-κB activity. Lastly, SIRT7 may be implicated in the NF-κB pathway. Knockdown of SIRT7 in endometrial cancer cells was shown to cause apoptosis, which was associated with a decrease in the expression of NF-κB target proteins that are antiapoptotic (Bcl-xl, Bcl-2, and Mcl-1) and an increase in the NF-κB target proteins that are proapoptotic (caspase-3, Bad, and Bax) [244]. Further, SIRT7 overexpression suppresses NF-κB signaling [245]. Seemingly contradictory to this, another study found that SIRT7 deacetylates the Ran GTPase at K37, which inhibits export of RelA [246]; this would suggest that SIRT7 promotes NF-κB signaling. Interestingly, the K37 residue of Ran was previously shown to be a substrate of SIRT1, SIRT2, and SIRT3, but not SIRT7 [247]. Further work is needed to assess the role of SIRT7 in NF-κB signaling in human cancer, which could suggest ways to modulate SIRT7 activity to reduce NF-κB activity in treating hematologic malignancies. In conclusion, most evidence suggests that the sirtuins, namely SIRT1, SIRT2, and SIRT6, negatively regulate NF-κB signaling. This may suggest a therapeutic opportunity to activate these sirtuins in hematologic malignancies with constitutive NF-κB signaling.

13.3.4 CD38, a major NADase, affects sirtuin activity CD38 is a small glycoprotein and multifunctional enzyme located on the plasma membrane and inner nuclear membrane. Its expression generally correlates with poorer outcomes in a number of hematologic malignancies, namely CLL and multiple myeloma [248]. The major enzymatic activity of CD38 is the hydrolysis of NAD1. Given that sirtuins are NAD1-dependent enzymes, there is surely a link between CD38 and sirtuin activities. Indeed, CD38 has been shown to be a major regulator of cytoplasmic and nuclear NAD1 levels and sirtuin activity [249]. CD38 knockout cells have increased SIRT1 activity and decreased p53 phosphorylation [249,250]. CD38 knockout also increases Sirt3 expression and activity [251]. Concordantly, CD38 knockout in mice significantly

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increases NAD1 levels, increases intracellular GSH concentration and GSH/GSSG ratio, and decreases the contents of free fatty acids in vivo [251,252]. Further knocking out Sirt3 in these CD38 knockout mice reversed the protection against high-fat diet-induced obesity and glucose intolerance. This suggests that the effects of CD38 depletion (or inhibition) on glucose tolerance may not solely be dependent on increased NAD1 levels, but also on SIRT3 activity [252]. Taken together, these results suggest that CD38 and sirtuin activities are intimately linked. Future work will be needed to further assess the interaction between CD38 and sirtuin function in hematologic malignancies where CD38 is implicated, such as CLL and multiple myeloma. It is tempting to speculate that increased CD38, which generally correlates with more aggressive disease, reduces sirtuin activities in these malignancies. In the case of SIRT1, elevated CD38 could be expected to cause genomic instability and increased NF-κB activity. In the case of SIRT3, elevated CD38 could be expected to promote aerobic glycolysis and increased ROS. Furthermore, this might suggest adding sirtuin activators or NAD1 boosting molecule (NBMs) (discussed in the next section) to treat CD38-overactive cancers. It is unclear if the sirtuins other than SIRT1 and SIRT3 are also affected by CD38 activity, and this also represents an interesting area of future investigation.

13.4 Therapeutic opportunities Going through each of the seven sirtuins above, I have tried to highlight potential therapeutic opportunities. This section will summarize preclinical and clinical studies of sirtuin modulators and discuss some of the most impactful future avenues of investigation. STACs are allosteric modulators of SIRT1 that bind to a site on the protein other than the active site to induce a conformational change that increases the activity of the enzyme. The first and most well-known is resveratrol, a compound naturally found in grapes. A second class of sirtuin activators are the NBMs. In humans, NAD1 is salvaged from precursors (nicotinamide, nicotinamide riboside, and nicotinic acid) or synthesized de novo from dietary tryptophan. NBMs supply precursors for NAD1 synthesis. In vitro data using these compounds in models of hematologic malignancies show promise. Interestingly, both sirtuin activators and inhibitors have shown activity. Firstly regarding activators, the study of resveratrol sensitivity of cancer cell lines found a marked degree of variability based on cell type. Interestingly, U937 and MOLT-4 leukemia cell lines are the most sensitive to resveratrol, and treatment is associated with increased BAX expression [253]. Regarding sirtuin inhibitors in hematologic malignancies, sirtinol induces growth arrest, senescence, and apoptosis of human breast cancer cells, lung cancer cells, and leukemia cells, and enhances sensitivity to chemotherapy drugs of cancer cell lines [254,255]. Treatment of lymphoma cell lines and a lymphoma cell line xenograft in mice with cambinol (a SIRT1 inhibitor) causes apoptosis [53]. In addition, cambinol shows activity against Burkitt lymphoma cells [256]. The SIRT1-specific activator SRT1720 induces apoptosis of multiple myeloma cells [238]. Next, the coadministration of a HDAC inhibitor with a sirtuin inhibitor results in synergy of their cytotoxicity in primary AML and CLL samples and the following cell lines: U937 (AML), 697 (pre-B-cell leukemia), and Jurkat (acute T-cell leukemia). In comparison, these drugs were poorly active in healthy PBMCs [52]. Similarly, primary leukemia cells, leukemia cell lines, healthy leukocytes, and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol, cambinol, and EX527) in combination with HDAC inhibitors (valproic acid, sodium butyrate, and vorinostat). Sirtuin inhibitors synergized with HDAC inhibitors to kill leukemia cells, but this was not the case in healthy leukocytes and hematopoietic progenitors [52]. This suggests a potential therapeutic strategy in treating leukemia with a combination of sirtuin and HDAC inhibitors [52]. The sirtuin inhibitors tenovin-1 and tenovin-6 show activity in models of a number of hematologic malignancies, including ALL [37], DLBCL [257], and cutaneous T cell lymphoma [45]. Additionally, treatment with SRT1720 synergizes with an inhibitor of the K3K79 methyltransferase, DOT1L, in mixed lineage leukemia (MLL)-rearranged leukemia cells [258]. Intriguingly, in CML, combining imatinib with a SIRT1 inhibitor may prevent TKI-resistance and improve outcomes [51]. In preclinical animal studies, both STACs and NBMs have shown effects on cardioprotection, neuroprotection, metabolic improvements, the delay of age-related diseases, and increases in life span. Resveratrol also shows benefit in improving insulin sensitivity in nonhuman primates [259]. In cancer, resveratrol for 4 5 weeks reduces prostate cancers by about 50% in mice [260]. Specific to hematologic malignancies, mice xenografted with human plasmacytoma to model human multiple myeloma were treated with the SIRT1-specific activator SRT1720 on

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5 consecutive days per week for 4 weeks, finding that this SIRT1 agonist synergized with bortezomib or dexamethasone to reduce tumor growth [238]. However, clinical trials to date have been less promising than the preclinical studies. SIRT1 activators and inhibitors have been through the first clinical trials with evidence of safety and some efficacy. SRT2104 has shown benefit in psoriasis [261] and metabolic syndrome [262]. However, SRT2104 has been limited by poor and variable pharmacokinetics after oral intake. A number of clinical trials involving resveratrol showed benefits in glucose metabolism and cardiovascular disease risk factors [263,264], but many others have shown little to no benefits [265]. The efficacy of resveratrol is limited by its poor bioavailability and low potency. It will be interesting to see how newer, more potent, and more orally bioavailable sirtuin modulators evolve. Another strategy to modulate sirtuin activity in hematologic malignancies is by inhibiting endogenous breakdown of NAD1. CD38, as discussed above, is the major human NADase that increases with age, resulting in decreased NAD1 levels [252]. In mouse studies, flavonoids (e.g., quercetin, apigenin, and luteolindin) and thiazoloquin(az)olinones inhibit CD38 and cause an increase in NAD1 levels [266 268]. Interestingly, daratumumab, an anti-CD38 antibody, is FDA-approved for the treatment of multiple myeloma. Other treatments targeting CD38 are in development. It will be interesting to see how these affect sirtuin signaling in hematologic malignancies. Moreover, such studies might suggest ways to modulate sirtuin signaling to enhance treatment efficacy. PARPs are another major utilizer of NAD1 in humans. PARP inhibitors have been developed and show benefit in BRCA- or HR-deficient cancers. PARP inhibitors increase NAD1 levels, which in theory could activate sirtuins. Concordantly, Parp knockout mice show increased SIRT1 activity, mitochondrial metabolism, and biomass [269]. This was also seen with pharmacologic PARP inhibition in vitro and in vivo [269]. The increased availability of NAD1 and subsequent increased SIRT1 activity in the setting of PARP inhibition could result in greater repression of p53, which may partially reverse the efficacy of the PARP inhibitor. Therefore the combination of PARP inhibitors and sirtuin inhibitors represents a potentially interesting therapeutic strategy to treat cancer. It will also be critical to assess how PARP inhibitors affect the activity of the other sirtuins. In the setting of DNA damage, PARPs consume NAD1 to help repair DNA, but this probably reduces sirtuin activity. As many sirtuins have been shown to play tumor-suppressive roles, one might reason that chronic DNA damage over the life span, such as from smoking, UV light, and other environmental pollutants, may reduce sirtuin activity. An interesting question is how much of the carcinogenic effect of these exposures is conferred by the theoretical reduced sirtuin activity. As this chapter has discussed, many sirtuins have been shown to promote chemotherapy resistance. Therefore there may be interest in combining chemotherapy with sirtuin inhibitors to prevent the cancer cells from responding to DNA damage and other stresses induced by chemotherapy for treatment of cancers. However, this combination could have significant toxicity, as it would presumably prevent healthy cells from responding to damage and stresses induced by chemotherapy. These questions are worth pursuing in phase 1 clinical trials. Bone marrow transplant (BMT) is a treatment often employed in hematologic malignancies. Interestingly, there is evidence that modulating sirtuin activity could help prevent graft versus host disease (GVHD), one of the major and devastating complications associated with BMT. In a murine BMT model, genetic and pharmacologic Sirt1 inhibition attenuated GVHD while preserving the graft-versus-leukemia effect [270]. There is also evidence of SIRT3 involvement in preventing GVHD. Sirt3 knockout donor T cells cause reduced GVHD severity [271]. It is also relevant to note that SIRT3 knockout did not impact the numbers or distribution of T cell subsets. The role of sirtuins in transplant biology is a fascinating area worthy of future investigation. Modulating sirtuin activities could also be an area of prevention of hematologic malignancies. As mentioned previously, it will first be critical to reconcile the oncogenic and tumor-suppressive roles of sirtuins in hematologic malignancies, but it is tempting to speculate that certain sirtuin activators could potentially prevent the development of hematologic malignancies. The sirtuins represent an intriguing therapeutic target in hematologic malignancies. Further investigation will need to discern the nature of their seemingly contradictory oncogenic and tumor-suppressive functions. Further, searching for predictive biomarkers for sensitivity to sirtuin modulators will be a critical next step.

13.5 Conclusions One of the biggest remaining questions in the field of sirtuins in cancer biology is what role does each sirtuin play in each cancer: oncogene or tumor suppressor? As you can tell from reading this chapter, there is evidence implicating nearly all of the sirtuins as having both oncogenic and tumor-suppressive functions. There are many

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potential explanations for these findings. First, perhaps the sirtuins play different roles in each individual cancer based on tissue of origin and epigenetic and genetic alterations. It will be important to determine what factors control this. Second, perhaps this means that sirtuins are not that important in cancer biology, are merely bystanders, and are not great therapeutic targets. However, given all the many pertinent findings reviewed in this chapter, this explanation seems unlikely. Third, the contradictory findings in sirtuin expression in cancer could be explained by the fact that sirtuins play critical roles in mediating cell type-specific stress responses. Many sirtuins are induced by cellular stresses, such as hypoxia and nutrient deprivation, which are surely rampant within the tumor microenvironment. This could lend credence toward the bystander hypothesis. Lastly, the differing expression observations can be reconciled by the hypothesis that sirtuins play opposing roles in cancer initiation and progression. For example, the activities of many of the sirtuins help to prevent cancer initiation by facilitating robust DNA repair and genomic stability, among other functions, so decreased expression of many sirtuins may predispose to tumorigenesis. At the same time, the functions of many sirtuins may promote cancer progression and treatment resistance after a tumor has already initiated. A remaining open question in this field pertains to the mechanisms of underexpression or overexpression of sirtuins in cancer. It is unclear how their expression is regulated in different cancers. Perhaps they are regulated epigenetically. Alternatively, their altered expression, notably the studies that report decreased expression, could be a consequence of aging, as it is known that the expression of most sirtuins decreases with age. Also, there is some evidence for gene deletions and amplifications affecting sirtuins, as were described previously in this chapter. However, the prevalence of these copy number variants is lower than the reported rates of overexpression and underexpression, suggesting that there are other mechanisms at play. The role of alternative splicing in cancer is becoming increasingly recognized. Different splicing and transcript variants have already been reported for some of the sirtuins, such as SIRT1 [272] and SIRT3 [273]. It is unclear what mechanisms lead to these variants and what are the significance and prevalence of these variants in hematologic malignancies. This is worth further investigation. It will also be interesting to learn about how sirtuin signaling affects JAK-STAT signaling in myeloproliferative neoplasms. There are some pieces of evidence suggesting that sirtuins regulate JAK-STAT signaling, as were discussed in this chapter, specifically pertaining to SIRT1. It will be critical to continue to pursue this link, as modulation of sirtuins could be a potential therapeutic opportunity in myeloproliferative neoplasms driven by JAK-STAT signaling. The study of sirtuins in carcinogenesis and cancer therapy is an exciting and open area in cancer research, particularly involving the hematologic malignancies. The first sirtuin modulators have already entered clinical trials, and it is exciting to see how these modulators could be used to treat hematologic malignancies.

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C H A P T E R

14 Impacts of sirtuin1 and sirtuin3 on oral carcinogenesis Shajedul Islam1,2, Yoshihiro Abiko2, Osamu Uehara1, Yasuhiro Kuramitsu3 and Itsuo Chiba1 1

Division of Disease Control and Molecular Epidemiology, Department of Oral Growth and Development, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan 2Division of Oral Medicine and Pathology, Department of Human Biology and Pathophysiology, School of Dentistry, Health Sciences University of Hokkaido, Hokkaido, Japan 3Advanced Research Promotion Centre, Health Sciences University of Hokkaido, Hokkaido, Japan O U T L I N E 14.1 Introduction

259

14.2 Overview of sirtuins 14.2.1 Sirtuin1 14.2.2 Sirtuin3

260 264 264

14.3 Involvement of sirtuins in oral cancer 14.3.1 Sirtuin1 and oral cancer

264 266

14.3.2 Sirtuin3 and oral cancer

268

14.4 Potential therapeutic implications of sirtuins in oral cancer 269 14.5 Concluding remarks

271

References

271

14.1 Introduction Oral cancer is the 15th-most frequent cancer and is one of major causes of death from cancer throughout the world [1,2]. The highest prevalence rates of oral cancer have been reported in South Asian countries, including India, Pakistan, Sri Lanka, and Bangladesh [1,2]. The overall 5-year survival rates for patients with oral cancer have not significantly improved for decades in spite of advances in the field of oncology [1,2]. These findings underscore the importance of encouraging new areas of research on factors that modify oral cancer and therapeutic targets to treat it. Sirtuins (SIRTs), highly conserved nicotinamide adenine dinucleotide (NAD1)-dependent class III histone deacetylases (HDAC) enzymes, are mammalian homologs of the yeast Sir2 gene, which is known to promote the replicative life span and mediate gene silencing in yeast [3,4]. HDACs are enzymes that catalyze the removal of acetyl functional groups from lysine residues of both histone and nonhistone cellular substrates [5]. These functions of HDACs are controlled by opposing activities of histone acetyltransferases (HATs) and disturbance of this balance is reported to be involved in carcinogenesis [5 7]. Recently, SIRTs have been reported to be involved in tumor growth and differentiation [8 11]. SIRT1 and SIRT3 have been most extensively studied in oral cancer and are considered promising to treat or prevent this cancer [12 21]. Acetylation is a process of transcriptional modification that plays an important role in oral carcinogenesis by regulating protein interactions, protein catalytic activity, and stability [22]. Intriguingly, SIRT1 and SIRT3 have strong deacetylase activity and modulate different protein functions in the process of carcinogenesis [23 26]. The underlying molecular mechanisms of these SIRTs in oral carcinogenesis are still largely a matter of debate. In this chapter, we focus on the seemingly dichotomous roles of SIRT1 and SIRT3 in oral carcinogenesis and summarize the findings of recent publications, which raise the possibility of using new therapeutic approaches to treat this cancer. Sirtuin Biology in Cancer and Metabolic Disease. DOI: https://doi.org/10.1016/B978-0-12-822467-0.00002-4

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© 2021 Elsevier Inc. All rights reserved.

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14. Impacts of sirtuin1 and sirtuin3 on oral carcinogenesis

14.2 Overview of sirtuins The seven human SIRTs share an B275 amino acid catalytic core that contains two domains: a smaller zinc-biding domain and a larger Rossman-fold domain. Together, the two domains form a specific structure to unite NAD1 as cofactors [24,25] (Fig. 14.1). Each of the seven SIRTs have unique characteristics, functions, and localization [24,25]. The divergent terminal extensions account for their various subcellular localizations, enzymatic activities, and binding targets. SIRT1, SIRT6, and SIRT7 are chiefly nuclear proteins, whereas SIRT3, SIRT4, and SIRT5 predominantly reside in mitochondria and SIRT2 is primarily cytosolic [24,25] (Fig. 14.2). Since some of these proteins translocate from their typical compartments in the developmental stage, metabolic status, and certain stress conditions, localization may be FIGURE 14.1 Schematic representation of the seven mammalian SIRT proteins. The blue area represents the NAD1dependent catalytic domain. NAD1, Nicotinamide adenine dinucleotide; SIRT, sirtuin; aa, amino acids. Modified from Mei Z, Zhang X, Yi J, Huang J, He J, Tao Y. Sirtuins in metabolism, DNA repair and cancer. J Exp Clin Cancer Res. 2016;35:182. https://doi.org/10.1186/s13046-016-0461-5.

FIGURE 14.2 The subcellular localization of sirtuin (SIRT) proteins: SIRT1 is predominantly located in the nucleus, but also in the cytosol. SIRT2 is in the cytosol. SIRT3, SIRT4, and SIRT5 are mitochondrial proteins. SIRT6 and SIRT7 are localized in the nucleus.

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14.2 Overview of sirtuins

important for regulating their functions [24,25]. SIRTs are involved in several biological processes including the stress response, viability, differentiation, metabolism, apoptosis, cell survival, and carcinogenesis [12,24,25]. The biological roles of SIRTs in carcinogenesis remain a highly difficult challenge due to their dual characteristics as both tumor suppressors and tumor promoters, as revealed in different types of carcinogenesis (Tables 14.1 and 14.2). However, several activators and inhibitors of SIRTs have been applied to treat various cancers, as shown in Table 14.3. TABLE 14.1

Tumor suppressive roles of sirtuins in different cancers.

Name

Function

Cancer types

Genes or pathways involved

SIRT1

Viability

BC

BRCA1

NSCLC

K-RAS, PI3K pathway

BC

Smad4/β-catenin

NSCLC

K-RAS, and PI3K pathway

CIarcinogenesis

BC

RCA1

Viability

OC

CDK4

HCC

APC, CD20

BC

APC, CD20

NSCLC

p53

CCA

Myc

HCC

APC, CD20

BC

APC, CD20

PC

Myc, PI3K/AKT pathway

NSCLC

Bax/Bcl-2, P53

HCC

PI3K/AKT pathway

HCC

PI3K/AKT pathway

PC

Foxo3a, Wnt/β-catenin pathway

OC

Twist

OC

GDH

NSCLC

ERK/Drp1 Axis

ESCC

GDH

NSCLC

ERK/Drp1 Axis

ESCC

GDH

CRC

E-cadherin

GC

E-cadherin

HCC

LKB1/AMPKα/mTOR axis

NSCLC

ERK/Drp1 Axis

HCC

LKB1/AMPKα/mTOR axis

Metastasis

SIRT2

Apoptosis

TIumorigenesis

SIRT3

Viability

Metastasis

SIRT4

Viability

Metastasis

Carcinogenesis

SIRT5

Apoptosis

NB

Not reported

SIRT6

Viability

HCC

PKM2

CRC

PTEN/AKT signaling

ACC

NF-κB signaling

GBM

JAK2/STAT3 pathway

FSA

NF-κB signaling

CC

NF-κB signaling

HCC

PKM2

Apoptosis

Metastasis

(Continued) II. Sirtuins and cancer

262 TABLE 14.1 Name

14. Impacts of sirtuin1 and sirtuin3 on oral carcinogenesis

(Continued) Function

Carcinogenesis

SIRT7

Apoptosis

Metastasis

Cancer types

Genes or pathways involved

CRC

PTEN/AKT signaling

ACC

NF-κB signaling

HCC

PKM2

ACC

NF-κB signaling

OC

NF-κB signaling

BC

p-38 MAPK signaling

BC

TGF-β signaling

SIRT, Sirtuin; BC, breast cancer; NSCLC, non-small cell lung carcinoma; OC, ovarian carcinoma; HCC, hepatocellular cancer; CCA, cholangiocarcinoma; PC, prostate cancer; ESCC, esophageal squamous cell carcinoma; CRC, colorectal cancer; GC, gastric cancer; NB, neuroblastoma; ACC, adrenocortical carcinoma; GBM, glioblastoma multiforme; FSA, fibrosarcoma; CC, cervical cancer.

TABLE 14.2

Tumor promoting roles of sirtuins in different cancers.

Name

Function

Cancer types

Genes or pathways involved

SIRT1

Viability

TC

Myc

CRC

Oct4, Nanog, Cripto, Tert, and Lin28

Apoptosis

Metastasis

Carcinogenesis

SIRT2

Viability

Metastasis

Leukemia

STAT5 signaling, Foxo1, p53, Ku70

RB

Rb, p107, and p130

Glioma

EMT pathway

BC

AKT pathway

PC

p53

NSCLC

K-RAS, PI3K

BC

BRCA1, p53, Rb

PC

ZEB1, EGF signaling, EMT pathway

GC

DBC1

GC

DBC1

BC

AKT pathway

HCC

α-Tubulin

NB

MycN

PAC

Myc

HCC

AKT/GSK3β/β-catenin axis

GC

SIRT3

Carcinogenesis

BC

Slug

Viability

BLC

p53

CRC

AKT/PTEN signaling

NSCLC

Bax/Bcl-2, p53

Leukemia

AKT, Bax/Bcl-2 signaling

Metastasis

CRC

AKT/PTEN signaling

Viability

CRC

GLUD1, SHMT2

NSCLC

PKM2

HCC

E2F-1

RCC

SDHA

Apoptosis

SIRT5

(Continued)

II. Sirtuins and cancer

263

14.2 Overview of sirtuins

TABLE 14.2

(Continued)

Name

Function

SIRT6

Cancer types

Genes or pathways involved

Metastasis

HCC

E2F-1; Vimentin

Carcinogenesis

CRC

GLUD1

NSCLC

PKM2

RCC

SDHA

HCC

Bax

SSCC

COX-2, AKT, AMPK pathway

CRC

PTEN/AKT signaling

GBM

AK2/STAT3 pathway

HCC

ERK1/2 pathway

OSA

CDC4

OC

NF-κB pathway

BC

p38-MAPK pathway

OSA

CDC4

OC

NF-κB pathway

BC

P38-MAPK pathway

GC

Bax/Bcl-2

OSA

CDC4

Viability

Apoptosis

SIRT7

Viability

Metastasis

Carcinogenesis

SIRT, Sirtuin; TC, thyroid cancer; CRC, colorectal cancer; RB, retinoblastoma; BC, breast cancer; PC, prostate cancer; NSCLC, non-small cell lung carcinoma; GC, gastric cancer; HCC, hepatocellular cancer; NB, neuroblastoma; PAC, pancreatic cancer; BLC, bladder cancer; OSA, osteosarcoma; OC, ovarian carcinoma; RCC, renal cell carcinoma; SSCC, skin squamous cell carcinoma; GBM, glioblastoma multiforme.

TABLE 14.3 Name

Activators and inhibitors of sirtuins used in the treatment of various cancers. Modulated targets

Cancer types

Biological actions

(A) Activators of sirtuins Resveratrol

SIRT1, SIRT3, and SIRT5

Leukemia, prostate cancer, colon cancer, lung cancer, and breast cancer

Inducing autophagy and apoptosis

Piceatannol

SIRT1, SIRT3, and SIRT5

Breast cancer, colon cancer, and prostate cancer

Inducing autophagy and apoptosis; inhibiting migration and invasion

SIRT1720

SIRT1

Multiple myelomas

Decreasing invasion, metastasis and tumor growth

(B) Inhibitors of sirtuins UBCS039

SIRT5, and SIRT6

Cervix cancer, colon cancer, and lung cancer

Inducing autophagy-associated cell death

Ex-527

SIRT1

Leukemia, lung cancer

Inducing growth inhibition and apoptosis

Nicotinamide SIRT1, SIRT2, SIRT3, SIRT5, and SIRT6

Laryngeal cancer, leukemia, skin cancer, breast cancer, and prostate cancer

Reducing inflammatory cytokines, preventing recurrences

Tenovin 6

SIRT1, and SIRT2

CML, AML

Inducing p53-dependent growth arrest; inhibiting Wnt/β-catenin signaling

Sirtinol

SIRT1, and SIRT2

Breast cancer, lung cancer, and prostate cancer

Inducing growth arrest with attenuated Ras-MAPK signaling

AC 93253

SIRT1, SIRT2, and SIRT3

Prostate cancer, pancreatic cancer, and lung cancer

Inhibiting cell growth and motility by regulating Src-related pathways

Salermide

SIRT1, and SIRT2

Colorectal cancer, Glioblastoma multiforme

Inhibiting cancer cell proliferation and growth, inducing apoptosis by modulating p53-dependent pathway

SIRT, Sirtuin; AML, acute myeloblastic leukemia; CML, chronic myeloblastic leukemia; MAPK, mitogen-activated protein kinase.

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TABLE 14.4

Functions of SIRT1 in different cellular substrates.

Function

Cellular substrates

Effects of SIRT1

Genomic stability

H3K9Ac, H4K16Ac, H1K26Ac, Suv39h1, TERT

Modification of chromatin through the formation of heterochromatin structure and positive regulation of telomere length

DNA repair γ-H2AX, BRCA1, Rad51, MRN complex, Ku70, Bax

Induction of HR and NHEJ-mediated DNA repair; induction of formation of NBSI (nibrin) foci and direct recruitment to DNA damage sites; induction of Ku70-dependent DNA repair signaling

Stress response

Inhibition of apoptosis and promotion of DNA repair by deacetylase p53; promotion of cell survival during oxidative stress by inducing DNA repair in cooperation with foxo3a

p53, foxo3a, p300, set7/9, MnSOD

DNA DNMT1, DNMT3b methylation

Alteration of enzymatic activities of DNMTs by deacetylating different lysines

SIRT1, Sirtuin1; Suv39h1, suppressor of variegation 3 9 homolog 1; TERT, telomerase reverse transcriptase; γ-H2AX, phosphorylated form of variant histone H2AX; BRCA1, breast cancer type 1 susceptibility protein; Rad51, DNA repair protein Rad51 homolog 1; MRN complex, Mre11-Rad50-Nbs1 complex; Ku70, X-ray repair cross-complementing 6; Bax, BCL2 associated x protein; HR, homologous recombination; NHEJ, nonhomologous end joining; Foxo3a, forkhead box o3 alpha; p300, E1A binding protein p300; Set7/9, set domain-containing lysine methyltransferase 7; MnSOD, manganese-containing superoxide dismutase; DNMT1, DNA methyltransferase 1; DNMT3b, DNA methyltransferase 3 beta.

14.2.1 Sirtuin1 SIRT1 was the first family member to be discovered and has been continuously studied ever since. SIRT1 is localized in both nuclei and cytoplasm and affects their stability, transcriptional activity, and translocation [23,24]. A brief overview of SIRT1 protein functions is shown in Table 14.4. SIRT1 plays a key role in epigenetic regulation of gene expression by changing the structure of chromatin. Deacetylation of histones by SIRT1 induces chromatin condensation, whereas acetylation by HATs causes chromatin decondensation [7]. This balance is crucial for normal cellular functions and any disturbance of it will be related to cancer [3,4]. SIRT1 contributes to carcinogenesis through deacetylation of nonhistone proteins more than that of histone proteins [12]. However, SIRT1 seems to play contradictory roles, and dysregulation of its expression has frequently been reported in many human malignant diseases (Tables 14.1 and 14.2). Moreover, the involvement of SIRT1 in cancers may depend on the tumor type and origin, and the presence of different environments such as stress or cell death stimuli.

14.2.2 Sirtuin3 The peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α), along with estrogen receptor-related alpha (ERR-α), can powerfully regulate SIRT3 gene expression via an estrogen-related receptor (ERR)-binding element mapped to the SIRT3 promoter region [27,28]. SIRT3 indirectly participates in stimulating PGC1-α gene expression through phosphorylation of cAMP response element-binding protein (CREB) [27 29] (Fig. 14.3A). The PGC1-α expression is essential to maintaining proper mitochondrial functions [27]. Under stress conditions, SIRT3 is first expressed as an inactive precursor (44 kDa, 399-amino acid) in the cytoplasm, and is subsequently translocated to the mitochondrial matrix. The mitochondrial matrix processing peptidase (MPP)-protein generates a mature active form of SIRT3 via proteolytic cleavage (28-kDa, 257-amino acids) [29]. The majority of mitochondrial protein functions are mediated by deacetylation activities. SIRT3 is a deacetylase family protein involved in the modulation of different mitochondrial protein functions (Fig. 14.3B). Therefore SIRT3 may be a mitochondrial fidelity protein and acts as a guardian of mitochondria in a manner similar to p53 as the guardian of the genome [30]. Table 14.5 briefly describes the functions of SIRT3 in the mitochondria. SIRT3 plays a significant role in the development of different human cancers. SIRT3 can interact with different mitochondrial proteins and contribute to modulate the levels of cellular reactive oxygen species (ROS) [30]. Altered ROS levels may be one of the predominant risk factors for the induction of cancer [30]. These indicate that SIRT3 is a critical regulator in the pathogenesis of cancers. However, it is still unclear whether SIRT3 acts primarily as an oncogene or as a tumor suppressor gene. The enzymatic effects and involvement of SIRT3 in cancers are summarized in Tables 14.1 and 14.2.

14.3 Involvement of sirtuins in oral cancer SIRTs are involved in many types of cancer and participate in oral carcinogenesis [12 21]. SIRT1 and SIRT3 have been the most extensively studied in oral cancer and are promising strategies against this cancer. These

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FIGURE 14.3 Expression, activation, and functions of sirtuin3 (SIRT3): (A) ERRα and PGC1-α bind to the promoter region of SIRT3 and induce its expression. The expressed SIRT3 further induces PGC1-α through CREB phosphorylation; (B) under stress, SIRT3 is first exported into the cytoplasm and directed to mitochondria via its targeting sequence. There it undergoes cleavage by MPP-protein and generates an active form of SIRT3. The activated SIRT3 deacetylates numerous mitochondrial proteins and modulates myriad biological functions, including metabolism and antioxidant synthesis. ERRα, Estrogen receptor-related alpha; PGC1-α, peroxisome proliferator-activated receptor γ coactivator 1-α; CREB, cAMP response element-binding protein; MPP, matrix processing peptidase. TABLE 14.5

Effects of sirtuin3 on mitochondrial cellular substrates.

Cellular substrate

Biological roles

GDH

Induction of amino acid metabolism; induction of glutamate into αKG conversion; induction of energy production

IDH

Induction of isocitrate to αKG conversion; induction of reduced glutathione synthesis

LCAD

Induction of fatty acid oxidation

AceCS2

Increased AceCoA availability for the Kreb’s cycle

CypD

Inhibition of the glycolytic pathway; induction of HKII detachment

MnSOD

Prevention of ROS generation

Complex I, II

Induction of energy production

OGG1

Induction of mitochondrial DNA repair; protection of mitochondrial functional integrity

GDH, Glutamate dehydrogenase; αKG, alpha-ketoglutarate; IDH, isocitrate dehydrogenase; LCAD, long-chain acyl-CoA dehydrogenase; AceCS2, acetyl-CoA synthetase 2; AceCoA, acetyl-CoA; CypD, cyclophilin D; HKII, hexokinase II; MnSOD, manganese superoxide dismutase; OGG1, 8-oxoguanine glycosylase.

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SIRTs are involved in regulating three important processes in oral carcinogenesis: epithelial-to-mesenchymal transition (EMT), invasion, and metastasis [12 21]. Here we summarize current knowledge on the role of these SIRTs in oral cancer and discuss their puzzling dual function as tumor suppressors and tumor promoters.

14.3.1 Sirtuin1 and oral cancer The regulatory role of SIRT1 in oral cancer is vigorously debated owing to the belief that it can have both tumorigenic and antitumorigenic roles [12 18] (Table 14.6). Downregulation of SIRT1 expression is correlated with the metastatic phenotype, whereas upregulation of this protein results in opposite effects [14 16]. SIRT1 aids in maintaining epithelial integrity by inducing the expression of epithelial cadherin, which contributes to the prevention of both invasion and metastasis in oral cancer [14]. According to our previous studies, the expression levels of SIRT1 are positively associated with epithelial epithelial interaction, and downregulation of its expression may lead to malignant transformation of the epithelia [13]. DNA hypermethylation is often associated with transcriptional downregulation of genes [22]. We confirmed that hypermethylation of SIRT1 was followed by its transcriptional downregulation. We further analyzed the DNA methylation status of SIRT1 in oral cancer patients having the habit of betel quid (BQ) chewing, which is one of the main etiological factors for the development of this cancer in South and Southeast Asian countries [13,31]. We confirmed the occurrence of SIRT1 hypermethylation in oral cancer of both BQ chewers and nonchewers, suggesting that SIRT1 is a tumor suppressor [13]. We found that the methylation level of SIRT1 was significantly higher in macroscopically healthy oral epithelia with a BQ chewing habit than in those of nonchewers. This result suggests that DNA hypermethylation of SIRT1 is possibly a predictive biomarker for malignant transformation of the oral epithelium [13]. A previous report demonstrated that expression levels of SIRT1 were significantly lower in cancerous tissues than in noncancerous tissues. The expression levels of SIRT1 were lower in the advanced stages of cancer than in the early stages [17]. These findings indicate that downregulated transcriptional levels of SIRT1 gene expression may contribute to cancer development at advanced stages [17]. Collectively, downregulated expression of SIRT1 could represent a prognostic biomarker in oral cancer. However, it is still unclear how downregulated transcription of SIRT1 is linked to oral carcinogenesis. SIRT1 inhibits transforming growth factor-beta (TGF-β)-mediated malignant transformation, invasion, and metastasis in oral cancer [14]. TGF-β has shown enhanced malignant transformation, invasion, and metastasis in oral epithelial cells by inducing its downstream targets including matrix metalloproteinases, and EMT-related markers slug, snail, and so on [33,34]. Similarly, overexpressed TGF-β increases collagen deposition, leading to the pathogenesis of oral submucous fibrosis (OSF), a precancerous condition in BQ chewers [33,34]. SIRT1 may be inhibited to cause OSF via TGF-β inhibition. Based on these observations, SIRT1 may have the ability to prevent malignant transformation, invasion, and metastasis (Fig. 14.4). Therefore downregulated transcriptional levels of SIRT1 caused by environmental exposures including BQ chewing, cigarette smoking, and alcohol drinking may potentially induce malignant transformation of the oral epithelium via increased expression of TGF-β [13,14]. Overexpressed TGF-β is likely to silence SIRT1 epigenetically by inducing the expression of methyl-CpG-binding protein 2 (MeCP2) [35]. The promoter region of SIRT1 possesses a potential regulator of epigenetic factors, MeCP2 [27]. MeCP2 has been shown to interact with DNA methyltransferase 1 (DNMT1) and recruits the latter to induce SIRT1 promoter methylation [35]. Environmental carcinogens for oral cancer reportedly induce carcinogenesis through pathways involved in TGF-β production [33,34,36]. These findings may help to elucidate the underlying molecular mechanism of SIRT1 DNA hypermethylation followed by its transcriptional downregulation in the process of oral TABLE 14.6

Roles of SIRT1 in the process of oral cancer development.

References

Effects of SIRT1

(A) Tumor suppressor [13,15,16,17,32] Maintenance of epithelial polarity by increases the expression of E-cadherin; suppression of expression of N-cadherin and Vimentin; downregulation of genes involved in migration and invasion, such as CSK2A2, FRA1, ACTB, and Slug; downregulation of TGF-β downstream targets; induction of p21 and G1/S phase cell cycle arrest (B) Tumor promoter [18]

Induction of cisplatin resistance by increasing the expression of annexin A4, stathmin, SOD2, and thioredoxin; induction of growth and survival of cancerous cells

SIRT1, sirtuin1; SOD2, superoxide dismutase 2; CSK2A2, casein kinase II subunit alpha; FRA1, fos-related antigen 1; ACTB, beta-actin; Slug, snail family transcriptional repressor 2; TGF-β, transforming growth factor-beta.

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FIGURE 14.4 Possible regulatory mechanism of sirtuin1 (SIRT1) in oral cancer: (A: a1 3) The TGF-β ligand binds to its receptor and induces phosphorylation of smad2/3. Phosphorylated smad2/3 binds with acetylated smad4 and forms a complex known as the SMAD complex. The SMAD complex translocates to the nucleus, binds with its coactivator CBP/p300, and induces TGF-β downstream targets, which are related to the invasive and metastatic potentiality of oral epithelia. (B) TGF-β and its associated downstream molecules act on fibroblasts, leading to the induction of oral carcinogenesis. (C: c1 2) In the cytoplasm, SIRT1 can inhibit phosphorylation of smad2/3 and remove acetyl groups from smad4 protein. These effects help to prevent the formation of the SMAD complex. In the nucleus, SIRT1 binds to the promoter region of TGF-β and inhibits CBP/p300-mediated acetylation, resulting in transcriptional suppression of TGF-β-mediated malignant transformation, invasion, and metastasis. (D) Environmental carcinogens cause downregulated transcriptional levels of SIRT1 in epithelial cells, leading to enhanced TGF-β-mediated invasion and metastasis. TGF-β, Transforming growth factor-beta; CBP, CREB-binding protein.

carcinogenesis. Further studies will lead to a better understanding of the molecular basis of oral carcinogenesis caused by altered SIRT1 transcriptional levels. Conversely, despite evidence of the tumor-suppressing effects of SIRT1, some studies have demonstrated the promoting effects of this protein [18]. Upregulation of annexin A4 promoted the progression and chemoresistance of numerous tumors [37]. Overexpression of SIRT1 induced cisplatin resistance in oral cancer by

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elevating the level of annexin A4, and chemical inhibitors of SIRT1 significantly abolish this action [18]. Hypoxia within the tumor microenvironment has a well-documented role in promoting tumorigenesis [38]. Recently, SIRT1 promoted tumorigenesis via incorporation with hypoxia-inducible factor-1 alpha (HIF-1α) [38]. From these findings, SIRT1 may function as a tumor-inducer for oral cancer. Thus further studies are warranted to evaluate the precise molecular mechanism of SIRT1 in oral carcinogenesis induced by various environmental exposures.

14.3.2 Sirtuin3 and oral cancer The key role of SIRT3 is to serve as a mitochondrially localized tumor suppressor via its ability to inhibit ROS generation [30,39]. Environmental risk factors are often promoted to oral cancer via ROS-based mechanisms [1,40]. ROS are involved in processes of oral carcinogenesis through induction of genetic and epigenetic alterations [1,40]. SIRT3 decreases the levels of cellular ROS and prevents the malignant transformation of oral epithelia [19,32]. The inhibition of SIRT3 promotes cellular transformation and malignant progression of oral carcinogenesis [19,32]. Stable SIRT3 expression has enhanced the activity of the forkhead box O3 alpha (foxo3a), a well-documented tumor suppressor protein [19,41]. SIRT3-mediated activation of foxo3a in oral epithelium increases the synthesis of antioxidants, and the genes involved in apoptosis [41,42]. These antioxidants decrease cellular ROS production and prevent ROS-induced cellular transformation. These findings may explain the underlying mechanisms by which SIRT3 contributes to preventing cellular alterations and malignant transformation of the oral epithelium. However, other possible mechanisms cannot be ruled out. SIRT3 is linked to metabolic reprogramming, the “Warburg effect,” in cancer cells, which is often associated with glycolytic production of ATP [43]. Increased SIRT3 expression was documented to reverse this process by inducing oxidative phosphorylation [44]. These observations suggest that SIRT3 promotes energy production via oxidative phosphorylation instead of anaerobic glycolysis [45]. HIF-1α mediates the cellular response to hypoxia and activates transcription of genes involved in carcinogenesis [44]. Under hypoxic conditions, SIRT3 decreases ROS levels, which contributes to destabilization and subsequent degradation of HIF-1α [46]. Therefore SIRT3 may inhibit to cause carcinogenesis via HIF-1α inhibition [43]. However, further studies are needed to confirm our hypothesis in oral carcinogenesis. Collectively these findings imply the protective effects of SIRT3 when its expression can contribute to preventing the onset of oral carcinogenesis. Despite the evidence of tumor-suppressive effects, the role of SIRT3 in oral cancer is a matter of dispute. Decreased transcriptional levels of SIRT3 led to induction of apoptosis in oral cancer, whereas an increase of its expression abrogated this function [20,21]. Increased SIRT3 transcriptional levels enhanced aggressive cancerous phenotypes through induction of chemotherapeutic resistance [20,21]. The exact molecular mechanisms via which this occurs are, however, unknown. It is hypothesized that SIRT3-mediated decreased ROS levels may contribute to the process of carcinogenesis. In fact, the underlying mechanisms by which chemotherapeutic agents kill cancer cells are not associated with an increase of antioxidants, but rather the production of more ROS, leading to irreversible oxidative stress [47]. An increasing body of evidence indicates that not only various therapeutic approaches depend on ROS, but further elevation of cellular ROS can also, indeed, effectively kill more cancer cells [47,48]. These observations may explain why SIRT3-mediated decreases in ROS levels contribute to oral carcinogenesis. Meanwhile, it should be noted that SIRT3 is a stress-responsive deacetylase; therefore its expression may protect cancer cells from genotoxic and oxidative-mediated death [20,21], although we cannot rule out other possible mechanisms by which SIRT3 is involved in oral carcinogenesis. Thus further studies are warranted to determine how SIRT3 manages ROS levels to promote oral carcinogenesis. Under suspension conditions, cancer cells aggregate to become anoikis resistant and escape cell death [20]. One of those survival signals is modulated by SIRT3 and promotes cancer progression [20]. These findings suggest tumor-promoting effects of SIRT3 when its expression is correlated with oral carcinogenesis [20,21]. A recent report demonstrated that, although SIRT3 is overexpressed in oral cancer, the enzymatic activities are significantly reduced due to a nonsynonymous point mutation in its coding region [19]. Accordingly, mutation in the coding region was responsible for a 37% reduction of SIRT3 enzymatic catalytic efficacy [19]. These results indicate that a complex relationship may exist between SIRT3 and oral cancer. However, there is strong evidence to support the hypothesis that SIRT3 is a critical regulator in oral carcinogenesis. Further studies are required to elucidate SIRT3’s complicated role in oral cancer and to develop SIRT3-based chemotherapeutic approaches for preventing or treating this cancer. Table 14.7 briefly describes the findings on the relationship of SIRT3 to the development of oral cancer [19 21].

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TABLE 14.7

Roles of sirtuin3 in oral cancer development.

References Effects of SIRT3 (A) Tumor promoter [20,21]

Induction of anoikis resistance; inhibition of sensitivity to ionizing radiation and cisplatin treatment; induction of growth, survival, and proliferation by maintaining ROS at threshold levels

(B) Tumor suppressor [19,32]

Induction of nuclear translocation of Foxo3a; induction of SOD2, CAT and GADD 45 expression; prevention of ROS synthesis; induction of genomic stability

SIRT3, sirtuin3; ROS, reactive oxygen species; Foxo3a, forkhead box 03 alpha; SOD2, superoxide dismutase 2; CAT, catalase; GADD 45, growth arrest and DNA damage.

TABLE 14.8

Resveratrol in the treatment of oral cancer.

Signaling pathways

Modulated targets

Biological actions

Mitochondrial intrinsic pathway; caspase cascade Upregulation of Bak and Bax; downregulation of Bcl-2 pathway; EMT pathway and Bcl-xl; increased protein levels of Apaf-1, procaspase-9, procaspase-3, PARP, and ICAD; increased E-cadherin expression, decreased N-cadherin, Snail and Slug expression

Induction of apoptosis; inhibition of migration and invasion

Autophagy pathway; AMPK pathway; Akt/ mTOR signaling; PI3K class III/Beclin-1/Atgsassociated signals; caspase cascade pathway

Increased expression of autophagic genes, including Atg5, Atg12, Beclin-1, and LC3-II; enhanced phosphorylation of AMPK, Beclin-1, PI3K class III and LC3-II; decreased Rubicon protein level; increased protein levels of caspase-3/-9, cytochrome c, Apaf-1, AIF, Endo G, Bax and Bad, decreased protein level of Bcl-2 and phosphorylation of Bad on Ser136

Induction of autophagy and apoptosis in drug-resistant cell lines

JNK1/2 pathway; ERK1/2 pathway

Downregulation of MMP-9 expression; inhibition of the phosphorylation of JNk and ERK

Inhibition of migration and invasion

Not reported

Downregulation of MMP-9 expression

Inhibition of migration and invasion

EMT-pathway

Downregulation of ALDH1, CD44, OCT4, NANOG, and Inhibition of growth and proliferation; NESTIN expression; decreased EMT-related markers reduction of cancer cell stemness

TGF-β pathway

Upregulation of SIRT1 expression; decreased level of acetylated Smad4; upregulation of E-cadherin expression; decreased N-cadherin and Vimentin expression; reduction of MMP 7 expression

Inhibition of migration, invasion, and metastasis

uPAR pathway; ERK1/2 pathway; EGFR pathway

Downregulation of uPAR downstream proteins; downregulation of integrin β1; decreased in pERK1/2 levels

Inhibition of migration, invasion, metastasis, and tumor growth; induction of chemosensitivity in drug-resistant cells

EMT pathway

Decreased Slug and TWIST1 expression, downregulation of HIF-1α and VEGF expression

Inhibition of migration, invasion, and metastasis

EMT, Epithelial-mesenchymal transition; AMPK, activated protein kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinases; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; TGF-β, transforming growth factor-beta; uPAR, urokinase receptor; EGFR, epidermal growth factor receptor; Bak, Bcl-2 homologous antagonist/killer; Bax, bcl-2-like protein 4; Bcl-2, B-cell lymphoma 2; Bcl-xl, B-cell lymphoma-extra large; PARP, poly (ADP-ribose) polymerase; E-cadherin, epithelial cadherin; Apaf-1, apoptotic protease activating factor 1; Endo G, endonuclease G; BAD, BCL2-associated agonist of cell death; MMPs, matrix metallopeptidases; ALDH1, aldehyde dehydrogenases; OCT4, octamer-binding transcription factor 4; HIF-1α, hypoxia-inducible factor 1-alpha; VEGF, vascular endothelial growth factor.

14.4 Potential therapeutic implications of sirtuins in oral cancer Over the last few years, activators and inhibitors of SIRTs have been tested for potential treatment of various human cancers. Resveratrol, a polyphenol phytoalexin found in the skin of red grapes, possesses therapeutic benefits, including antiinflammatory, antiaging, and anticarcinogenic effects [49 52]. SIRT1 and SIRT3 may contribute to these therapeutic benefits [50,51], although it is still unclear whether these effects are mediated via direct or indirect mechanisms [49 52]. Recently, its direct mechanism was demonstrated by resveratrol-mediated anticancer effects. Activation of SIRT1 in resveratrol inhibited cell growth and proliferation, via negatively regulating oncogenic pathways [53 58]. Table 14.8 briefly

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FIGURE 14.5 Possible molecular mechanisms of resveratrol in oral cancer: resveratrol enhances autophagy-induced cell death through increased phosphorylation and activates the AMPK pathway. Resveratrol-induced autophagic genes also contribute to autophagolysosomes in cancer cells. Resveratrol-mediated inhibition of AKT, mTOR, JNK, TGF-β, and ERK-pathways causes decreased downstream protein production and results in the inhibition of proliferation, invasion, and metastasis. It also induces proapoptotic protein production in mitochondria and contributes to cellular apoptosis. AMPK, Activated protein kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; TGF-β, transforming growth factor-beta; uPAR, urokinase receptor; Bax, bcl-2-like protein 4; Bcl-2, B-cell lymphoma 2; E-cadherin, epithelial cadherin; Endo G, endonuclease G; Bad, BCL2-associated agonist of cell death; MMPs, matrix metallopeptidases; HIF-1α, hypoxia-inducible factor 1-alpha; VEGF, vascular endothelial growth factor. Modified from Hu F-W, Tsai L-L, Yu C-H, Chen P-N, Chou M-Y, Yu C-C. Impairment of tumor-initiating stem-like property and reversal of epithelial-mesenchymal transdifferentiation in head and neck cancer by resveratrol treatment. Mol Nutr Food Res 2012;56:1247 1258. https://doi.org/10.1002/mnfr.201200150; Chang C-H, Lee C-Y, Lu C-C, Tsai F-J, Hsu Y-M, Tsao J-W, et al. Resveratrolinduced autophagy and apoptosis in cisplatin-resistant human oral cancer CAR cells: a key role of AMPK and Akt/mTOR signaling. Int J Oncol 2017;50:873 882. https://doi.org/10.3892/ijo.2017.3866; Kim S-E, Shin S-H, Lee J-Y, Kim C-H, Chung I-K, Kang H-M, et al. Resveratrol induces mitochondrial apoptosis and inhibits epithelial-mesenchymal transition in oral squamous cell carcinoma cells. Nutr Cancer 2017;70:125 135. https://doi.org/ 10.1080/01635581.2018.1397708; Shan Z, Yang G, Xiang W, Pei-Jun W, Bin Z. Effects of resveratrol on oral squamous cell carcinoma (OSCC) cells in vitro. J Cancer Res Clin Oncol 2014;140:371 374. https://doi.org/10.1007/s00432-013-1575-1.

describes the anticancer effects of resveratrol in oral carcinogenesis. Additionally, Fig. 14.5 elaborates on its anticarcinogenic effects in oral cancer. However, further studies are needed to evaluate the precise anticarcinogenic mechanisms of resveratrol that could possibly be mediated by the activation of SIRT1 and SIRT3. In addition, curcumin is well-documented to act as an anticancer agent in various human malignant cancers [59]. It is commonly obtained as an extract from Curcuma longa (turmeric). There are other compounds present in the extract from C. longa called curcuminoids. About 60% 70% of the turmeric extract consists of curcumin, 20% 27% of demethoxycurcumin, and 10% 15% of bisdemethoxycurcumin [59]. A recent report described that the anticancer effects of curcumin in oral cancer are possibly partly mediated by the activation of SIRT1 [60]. Curcumin-induced SIRT1 transcriptional levels are involved in the inhibition of migration and apoptosis in oral cancer [60]. The anticarcinogenic effects of curcumin that are modulated by SIRT1 activation in oral cancer are shown in Fig. 14.6.

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References

FIGURE 14.6 Effects of curcumin on oral cancer via activation of sirtuin1 (SIRT1): curcumin induces the production of cAMP, PKA, and LKB-1 proteins, and activates the AMPK-pathway through its phosphorylation. Activated AMPK increases PGC-1α gene expression and leads to mitochondrial biogenesis. Activated AMPK further increases SIRT1 transcriptional levels, which are positively associated with mitochondrial biogenesis by elevating the levels of PGC-1α gene expression. AMPK-induced SIRT1 transcriptional levels lead to cellular apoptosis via ATM/CHK2 and caspase-cascade pathways. SIRT1 also negatively regulates oncogenic pathways and molecules that are associated with cellular survival, growth and proliferation, invasion and metastasis. AMPK, Activated protein kinase; AP, activator protein; cAMP, cyclic adenosine monophosphate; COX-2, cyclooxygenase 2; Egr-1, early growth response protein 1; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; LKB1, liver kinase B1; LOX, lipoxygenase; MMP9, matrix metalloproteinase-9; NFκB, nuclear factor kappa light chain enhancer of activated B cells; NOS, nitric oxide synthase; PGC-1α, proliferator-activated receptor gamma coactivator 1-alpha; PKA, protein kinase A; uPA, urokinasetype plasminogen activator. Modified from Tomeh M, Hadianamrei R, Zhao X. A review of curcumin and its derivatives as anticancer agents. Int J Mol Sci 2019;20:1033. https://doi.org/10.3390/ijms20051033; Hu A, Huang J-J, Li R-L, Lu Z-Y, Duan J-L, Xu W-H, et al. Curcumin as therapeutics for the treatment of head and neck squamous cell carcinoma by activating SIRT1. Sci Rep 2015;5. https://doi.org/10.1038/srep13429.

14.5 Concluding remarks SIRT family proteins are involved in different types of cancer and participate in oral carcinogenesis. Of these proteins, SIRT1 and SIRT3 possess both tumor-suppressive and tumor-promoting effects on oral cancer. Therefore unraveling the underlying mechanisms of these SIRTs might provide therapeutic opportunities for the treatment of oral cancer. Further studies on SIRT1 and SIRT3 will significantly advance our understanding of oral carcinogenesis and might give rise to novel therapeutic strategies.

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14. Impacts of sirtuin1 and sirtuin3 on oral carcinogenesis

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Clin Chim Acta 2015;447:72 8. Available from: https://doi.org/10.1016/j. cca.2015.05.016. [38] Laemmle A, Lechleiter A, Roh V, Schwarz C, Portmann S, Furer C, et al. Inhibition of SIRT1 impairs the accumulation and transcriptional activity of HIF-1alpha protein under hypoxic conditions. PLoS One 2012;7:e33433. Available from: https://doi.org/10.1371/journal. pone.0033433. [39] Ren J-H, Chen X, Zhou L, Tao N-N, Zhou H-Z, Liu B, et al. Protective role of sirtuin3 (SIRT3) in oxidative stress mediated by hepatitis B virus X protein expression. PLoS One 2016;11. Available from: https://doi.org/10.1371/journal.pone.0150961. [40] Sardaro N, Vella FD, Incalza MA, Stasio DD, Lucchese A, Contaldo M, et al. Oxidative stress and oral mucosal diseases: an overview. In Vivo 2019;33:289 96. Available from: https://doi.org/10.21873/invivo.11474. [41] Tseng AH, Shieh SS, Wang DL. SIRT3 deacetylates FOXO3 to protect mitochondria against oxidative damage. Free Radic Biol Med 2013;63:222 34. 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II. Sirtuins and cancer

Index

Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Acetyl-CoA synthase 2 (AceCS2), 220 Acetyl-coenzyme A (acetyl-CoA), 91 Acetyl-L-carnitine (ALCAR), 72 Acetylation, 113
114, 259 histone, 28 Lys310-p65, 51 lysine, 34
35 mitochondrial protein, 65 Activator protein 1 (AP-1), 94, 179 Acute inflammation, 91
92 Acute kidney injury (AKI), 67 Acute lymphocytic leukemia (ALL), 236 Acute myeloid leukemia (AML), 31, 236 Acyl-CoA oxidase 1 (ACOX1), 221 “Acylation code”, 106 Adenine nucleotide translocator 2 (ANT2), 220 Adenosine 5’-monophosphate (AMP), 92
93 Adenosine diphosphate ribose (ADP), 25 ADP-ribosylation of GDH1, 240
241 Adenosine monophosphate-activated protein kinase (AMPK), 6, 64, 92
93, 219
220 in diabetic kidney disease, 70 SIRT1 and, 8
9 Adenosine triphosphate (ATP), 79
80, 91, 196 Adipose tissue, 42
43 Adult neural stem cells (NSCs), 26 Adult stem cells (ASCs), 26. See also Cancer stem cells (CSCs) SIRT1 for maintenance of diverse ASC pools, 28
30 Advanced glycation end products (AGEs), 8 Advanced protein oxidation products (APOP), 51 Aerobic glycolysis, 196 Age-related diseases, 40 Aging, sirtuins and, 84
85 AGK2, 162, 166 Agouti-related peptide (AgRP), 7 Aldehyde dehydrogenase 1A1 (ALDH1A1), 31
32 Alpha ketoglutarate dehydrogenase (KGDH), 106
107, 111
112 and regulation of gene expression, 111
112 Alpha-ketoglutarate dehydrogenase (KGDC), 106
107 Alzheimer’s disease (AD), 4, 84 American Diabetes Association (ADA), 61
62

Amino acid metabolism, 91 direct epigenetic control of, 200 direct posttranslational control of, 205
206 indirect control by sirtuins, 211 5-Aminoimidazole-4-carboxamide-1-b-Dribofuranoside (AICAR), 70 5-Aminoimidazole-4-carboximide riboside (AICAR), 72 Anaphase-promoting complex/cyclosome (APC/C), 221, 238 Angiogenesis, sirtuins in, 137 Angiotensin II (ANG II), 66 Angiotensin II type 1 receptor (AT1R), 66 Animal models of diabetes mellitus, 67
68 Antagonistic roles of mitochondrial sirtuins, 114 Antigen-presenting cells (APC), 92 Apoptosis, 6
7, 135 Ataxia telangiectasia-mutated and Rad3related protein (ATR), 221 Ataxia-telangiectasia (ATM), 9 ATP-citrate synthase (ACLY), 238 ATR-interacting protein (ATRIP), 221 Autophagy, 6 in diabetes mellitus and diabetic kidney disease, 69 Autosomic-dominant polycystic kidney disease (ADPKD), 43

B Base excision repair pathway (BER pathway), 138, 243 BCL-2, 135 BCL2-associated agonist of cell death (Bad), 10 BCL-2 family of proteins, 246 BCR-ABL, 31, 165, 236
237 Berberin, 51 β-hydroxybutyrate (PHB), 205 β-naphthol-containing inhibitors, 163
164 Betel quid chewing (BQ chewing), 266 Bile acid receptor. See Farnesoid X receptor (FXR) Biogenesis, 121 mitochondrial, 8, 43, 72, 85, 209 ribosome, 245 transcription regulators, 80
81 Bone marrow transplant (BMT), 249 Bone marrow-derived macrophages (BMDMs), 93

275

Brain, 44 Branched chain ketoacyl-CoA dehydrogenase (BCKDC), 106
107 Breast cancer stem cells (BCSCs), 30

C c-Jun N-terminal kinase (JNK), 93 c-Myc, 208 C-terminal binding protein 1 (CtBP), 205, 222 Calorie restriction (CR), 28
29, 43, 61 Cambinol, 141, 164 Cancer stem cells (CSCs), 26, 223. See also Embryonic stem cells (ESCs) SIRT1 important in maintaining/ promoting stemness and survival of, 30
31 SIRT2 promoting survival of, 31
32 SIRT6 suppressing stemness of, 33 Cancer(s), 129 cells, 132 direct epigenetic control of cellular metabolism, 198
200 direct posttranslational control of cellular metabolism by sirtuins, 200
206 indirect control of cellular metabolism by sirtuins, 206
211 metabolic characteristics of, 195
197 glucose metabolism in cancers, 196
197 lipometabolism in cancers, 197 other kinds of metabolism in cancers, 197 metabolism nuclear and cytosolic sirtuins involvement in, 180
184 sirtuins and, 179
180, 219
221 regulatory modes of sirtuins in controlling cellular metabolism, 197
198 sirtuins and cancer therapy, 141 Carbamoyl phosphate synthase 1 (CPS1), 96
97, 205, 242 Cardiovascular diseases, sirtuins and, 84 CD38, 247
249 CDC20, 221, 238 CDC25A. See Cell division cycle 25A (CDC25A) CDH1, 221, 238 Cell culture studies, 68 Cell division cycle 25A (CDC25A), 223 Cellular metabolism, 91
92 SIRT1 nicotinamide, and, 10
11 Central nervous system (CNS), 5, 44

276 Chemoresistance, 223
224 Cholesterol metabolism, 210 Chronic inflammation, 91
92 Chronic lymphocytic leukemia (CLL), 236 Chronic myeloid leukemia (CML), 31, 236 Cocoa (Theobroma cacao), 51 Combrestatin analog, 141 Conventional DCs (cDCs), 92 Curcumin, 270 Curcuminoids, 270 Cyclic ADP ribose (cADPR), 28
29 Cyclic AMP response element-binding protein (CREB), 63, 264 Cycline-dependent kinase 9 (CDK9), 138, 221, 238 Cyclooxygenase-2 (COX-2), 94 Cyclophilin D, 220 Cytoplasmic iron
sulfur cluster system (CIA system), 105 Cytosolic sirtuins in cancer metabolism and inflammatory cell, 180
184

D Daratumumab, 249 Deacetylase, 79
80 activity, 93 Dendritic cells (DCs), 92 SIRT1 in, 95 Deoxyribonucleic acid (DNA), 6 repair, 221
222 Deoxyribonucleotide synthesis, 119
120 Dextran sulfate sodium (DSS), 96 dextran sulfate sodium-induced colitis model, 50 Diabetes mellitus (DM), 4
5, 61
62 animal models of, 67
68 novel treatment options in, 71
72 SIRT1 roles, 67
69 and sirtuins, 65
70, 83 effects of SIRT6 and SIRT7 in kidney, 66
67 sirtuins and DKD, 66
67 sirtuins in pathogenesis of diabetes mellitus, 66 Diabetic kidney disease (DKD), 66, 70 adenosine monophosphate-activated protein kinase pathway in, 70 autophagy in, 69 novel treatment options in, 71
72 SIRT1 roles, 67
70 sirtuins and, 66
67 Diet-obtained phytochemicals, 44 Diffuse large B cell lymphoma (DLBCL), 235, 237, 246 Dihydrolipoyllysine acetyltransferase (DLAT), 82, 241 1,4-Dihydropyridine (DHP), 50 Dioxygenases, 224 Direct epigenetic control of cellular metabolism, 198
200. See also Indirect control of cellular metabolism amino acid metabolism by sirtuins, 200 glucometabolism by sirtuins, 198
200 lipometabolism by sirtuins, 200

Index

Direct posttranslational control of cellular metabolism, 200
206, 201t. See also Indirect control of cellular metabolism amino acid metabolism control by sirtuins, 205
206 glycolytic enzyme and transporter control by sirtuins, 200
203 lipometabolism control by sirtuins, 204
205 OXPHOS control by sirtuins, 203
204 DNA methyl transferase (DNMT), 68 DNMT1, 266
267 Dnmt3l, 28 DNA-dependent protein kinase (DNA-PK), 222 Dorsomedial hypothalamic (DHA), 44 Double-strand break sensor (DSB sensor), 138
139 Drp1, 209

E 4E-binding proteins (4E-BP1), 70 E-cadherin, 222 E2F1 transcription factor, 220, 235 Electron transport chain (ETC), 220 Elettaria cardamomum, 51 Embryoid bodies (EBs), 32
33 Embryonic stem cells (ESCs), 26. See also Cancer stem cells (CSCs) SIRT2 for differentiation of ESCs in vitro, 31 SIRT6 epigenetically promoting proper lineage commitment of, 32
33 Epithelial-to-mesenchymal transition (EMT), 222, 264
266 Erythropoietin (EPO), 10 Estrogen receptor-related alpha (ERR-α), 264 Estrogen-related receptor (ERR), 264 European Medicines Agency (EMA), 72

F Farnesoid X receptor (FXR), 62
64 FASII mitochondrial fatty acid pathway (mtFAS), 106
107 Fatty acid oxidation (FAO), 79
80, 91 FK-506-binding protein 12 (FKBP12), 9 FKBP12-rapamycin-associated protein (FRAP), 9 FKBP12-rapamycin-binding domain (FRB), 9 Flavonoids, 45 Food and sirtuins, 50
52 berberin, 51 cocoa, 51 green cardamom, 51 indol-3-carbinol, 51 Mediterranean diet, 50 Xanthigen, 52 Food and Drug Administration (FDA), 72 Forkhead transcription factor (FoxO) FOXO1, 7, 63, 220 FOXO3, 235, 238 FOXO3a, 221
222 transcription factors, 188 Free fatty acids (FFA), 64

G GA-binding protein transcription factor (GABPα/β), 196
197, 209 GA-binding protein β1 (GABPβ1), 221, 244 Gallic acid (GA), 48, 49t Genome instability, sirtuins in, 138
139 Genomic stability, 221
222 Glioblastoma stem cells (GSCs), 31
32 Glioma stem cells (GSCs), 30. See also Hematopoietic stem cells (HSCs) Glomerulus, roles of SIRT1 in in diabetic kidney disease, 67
68 results from animal models of DM, 67
68 results from cell culture studies, 68 Glucometabolism, direct epigenetic control of, 198
200 Glucose, 91 metabolism in cancers, 196
197 SIRT1 in, 63
64 SIRT3 in, 65 SIRT4 in, 65 Glucose transporter 1 (Glut1), 220 Glucose-6-phosphate dehydrogenase (G6PD), 221 Glucose-stimulated insulin secretion (GSIS), 44 Glut1. See Glucose transporter 1 (Glut1) Glutamate dehydrogenase (GDH), 65, 220, 240
241 Glutamate dehydrogenase 1 (GLUD1), 205 Glutamate oxaloacetate transaminase 2 (GOT2), 239 Glutaminase (GLS), 220 Glutamine, 241 Glutaminolysis, 91 Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 220 Glycine cleavage system (GCS), 106
107 Glycogen synthase kinase-3β (GSK-3β), 246 Glycolysis, 91 Glycolysis, indirect control of, 209 c-Myc, 208 HIF-1/2, 208 indirect control by sirtuins, 208
209 LKB1-AMPK, 208 p53, 208 Glycolytic enzymes and transporters, direct posttranslational control of, 200
203 Graft versus host disease (GVHD), 249 Granzyme B (GZMB), 96 Green cardamom (GC), 51 Gross Domestic Product, 5

H H4K16, 238 H4K16Ac, 238 Hallmarks of cancer, 129
132 Heart, 43 Hematologic malignancies, 235
244 facets of SIRT1 in cancer biology, 235 oncogenic roles of SIRT1, 235 SIRT1 in, 236
237

277

Index

SIRT2 and genomic stability, 237
239 SIRT3, 239
240 SIRT4 and glutamine metabolism, 240
241 SIRT5, 241
242 SIRT6 and age-old Warburg effect, 242
243 SIRT7 and ribosome biogenesis and DNA repair, 243
244 sirtuins regulating the pathways important for, 244
248 therapeutic opportunities, 248
249 tumor-suppressive roles of SIRT1, 236 Hematopoietic stem cells (HSCs), 28 SIRT3 for maintaining the pool and regenerative capacity of, 32 SIRT6 for control of regeneration and stress resistance in, 33 SIRT7 for regulating quiescence and regenerative capacity of, 34 Hemoglobin A1c (HbA1c), 4
5 High-density lipoprotein (HDL), 64 High-fat diet (HFD), 93 HFD-induced metabolic dysfunction, 41 Hippo signalling, 134 Histone acetyltransferases (HATs), 259 Histone deacetylases (HDAC), 259 Histone H3, 222 Homeostasis, 91 Homologous recombination (HR), 138 2-Hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2), 220 5-Hydroxymethylcytosine (5hmC), 31
33 Hypertension, and sirtuins, 71 Hypothalamus, 44 Hypoxia-induced factor (HIF) HIF-1/2, 208 HIF-1α, 92
93, 220, 239
240, 267
268

I Immune cell(s), 91 functions, 92 Immune system, 91 sirtuins in immune system function, 136
137 Immunometabolism, 91
93. See also Lipometabolism role of sirtuins in, 93
97 Indirect control of cellular metabolism, 206
211. See also Direct epigenetic control of cellular metabolism; Direct posttranslational control of cellular metabolism amino acid metabolism, 211 glycolysis, 208
209 lipometabolism, 210
211 OXPHOS by sirtuins, 209 Indol-3-carbinol (I3C), 51 Indole derivatives, 164 Induced pluripotent stem cells (iPSCs), 26 Inflammation, sirtuins involvement in, 179
180 Inflammatory cell, nuclear and cytosolic sirtuins involvement in, 180
184 Inner cell mass (ICM), 26 Insulin, 91
92

Insulin receptor substrate-1 (IRS-2), 62
63 Insulin-like growth factor-1 (IGF-1), 10 Interferon-γ (IFN-γ), 92 Interleukin-4 (IL-4), 92 Interleukin 6 (IL-6), 39
40 Internal tandem duplication in FLT3 (FLT3ITD), 236 Intestinal stem cells (ISCs), 26 Intracellular concentration of organic acids, 112
113 Intracellular NAM phosphoribosyl transferase (iNampt), 63 Invasion, sirtuins in, 137
138 Iron
sulfur cluster (Fe
S cluster), 105 consequences of Fe
S cluster defects in cancer cells, 118
119 Isocitrate dehydrogenase 2 (IDH2), 221 Isoflavone/genistein, 141 IκB kinase (IKK), 93

J Janus kinase (JAK), 179 JAK-STAT signaling, 237 JAK1-mediated SIRT1 phosphorylation, 237

K Ketogenic enzymes, 113
114 Ketone bodies, 205 Kidney(s), 43
44 effects of SIRT6 and SIRT7 in, 66
67 Ku70, 235

L Langerhans cells, 92 Last eukaryotic common ancestor (LECA), 105 Lateral hypothalamic (LHA), 44 Leukemias, 235 Leukemic stem cells (LSCs), 31 Lipid accumulation, 91
92 Lipid metabolism SIRT1 in, 64 SIRT3 in, 65 SIRT4 in, 65 Lipoic acid, 106
107, 109f, 241 Lipoic acid synthase (LIAS), 107
109 Lipometabolism in cancers, 197 direct epigenetic control of, 200 direct posttranslational control of, 204
205 indirect control by sirtuins, 210
211 LKB1, 211 PI3K-Akt, 211 PPARα/γ and PGC-1α, 210 SREBP family, 210 TR4/TAK1, 211 Lipopolysaccharide (LPS), 92 Lipoylation of multienzymatic complexes, 106
109 Liver, 42 Liver kinase B1 (LKB1), 64, 211, 219
220 LKB1-AMPK, 208 Liver X receptor (LXR), 62
63

Liver X receptor-alpha (LXR-α), 64 Liver X receptor-beta (LXR-β), 64 Long-chain acyl-CoA dehydrogenase (LCAD), 220 Long-term hematopoietic stem cells (LT-HSCs), 26 Lymphoid enhancer-binding factor 1 (LEF1), 223 Lymphoma, 235

M Macromolecules, 91 Macrophages, 92 SIRT1 in, 93
96 Malate dehydrogenase 2 (MDH2), 239 Malonyl-CoA decarboxylase (MCD), 220 MCD1, 42 Mammal SIRT1 deacetylation, 41 Mammalian lipoic acid, 109 Mammalian sirtuins, 153
160 SIRT1, 154
155 SIRT2, 155
156 SIRT3, 156
157 SIRT4, 157
158 SIRT5, 158
159 SIRT6, 159
160 SIRT7, 160 Mammalian stress-activated protein kinase interacting protein (mSIN1), 9 Mammalian target of rapamycin complex 1 (mTORC1), 9 Mammalian target of rapamycin complex 2 (mTORC2), 9 MC2494, 166 Mechanistic/mammalian target of rapamycin (mTOR), 6, 70, 92
93 SIRT1 and mTOR pathway in diabetic kidney disease, 70 SIRT1 mTOR, and metabolic disease, 9
10 Mediterranean diet (MD), 50 Mesenchymal stem cells (MSCs), 33, 72 Messenger ribonucleic acid (mRNA), 7
8 Metabolic characteristics of cancers, 195
197 Metabolic disease, 4
5 B-cell function, 5t novel therapeutic strategies with sirtuins for, 6 SIRT1, 6
7 future considerations, 11
13 SIRT1 and AMP-activated protein kinase, 8
9 SIRT1 metabolic function, and obesity, 7
8 SIRT1 mTOR, and metabolic disease, 9
10 SIRT1 nicotinamide, and cellular metabolism, 10
11 Metabolic regulation food and sirtuins, 50
52 gallic acid, 48 nonresveratrol related sirtuin activators, 48
50

278 Metabolic regulation (Continued) nutrition as therapeutic model for sirtuin regulation, 44
46 resveratrol, 47
48 tissue-specific sirtuin-modulated metabolic regulation, 42
44 Metabolism, 234 fuels, 91 Metalloproteinase 9 (MMP-9), 222 Metastasis, 222
223 sirtuins in, 137
138 Metformin, 70, 72 Methionine/choline deficient diet (MCD diet), 42 Methyl-CpG-binding protein 2 (MeCP2), 266
267 Mitochondria, 79
80, 82, 105
106 Mitochondrial biogenesis, sirtuins and, 80
82, 81f Mitochondrial dysfunction sirtuins and, 82
85 aging, 84
85 cardiovascular diseases, 84 diabetes and obesity, 83 neurodegeneration, 84 renal disease, 84 tumorigenesis, 85 Mitochondrial fatty acid synthesis, 116
118 Mitochondrial matrix processing peptidase (MPP), 264 Mitochondrial metabolism, sirtuins and, 82 Mitochondrial sirtuins, 106, 184
187, 239 advantages of possessing mitochondria, 105
106 alpha ketoglutarate dehydrogenase complex and gene expression regulation, 111
112 alternative lipoylation and metabolic consequences, 109
110 antagonistic roles of, 114 consequences of Fe
S cluster defects in cancer cells, 118
119 deoxyribonucleotide synthesis, 119
120 evolutionary implications and new directions in cancer treatment, 120
121 fluctuations of intracellular concentration of organic acids, 112
113 interplay between Sirt3 and isocitrate dehydrogenase in cancer cells, 115 ketogenic enzymes ACAT1 and HMGCS2 as substrates for Sirt3 and Sirt5, 113
114 lipoylation of multienzymatic complexes, 106
109 mitochondrial fatty acid synthesis, 116
118 regulation of pyruvate dehydrogenase complex by mitochondrial sirtuins, 110
111 SIRT3, 184 SIRT4, 185 SIRT5, 185
187 sirtuins regulating iron
sulfur cluster assemblage, 116

Index

tumor-suppressing and tumor-promoting activities of sirtuins, 115
116 Mitogen-activated protein-kinase (MAPK), 179 Mixed lineage leukemia (MLL), 248 MnSOD, 209 MOLT-4 leukemia cell lines, 248 Monocyte chemoattractant protein-1 (MCP1), 39
40 Monocyte-derived DCs, 92 Monomethylation of H4K20 (H4K20me1), 238 Mouse embryonic fibroblasts (MEFs), 239
240 Mouse ESCs (mESCs), 26 mtFAS. See FASII mitochondrial fatty acid pathway (mtFAS) Multidrug resistance 1 (MDR1), 235 Multiple mitochondrial proteins, 106 Multiple myeloma, 235 MYC-driven hematologic malignancies, 244
246 Myelodysplastic syndrome (MDS), 31 Myeloid cell-specific Sirt1 knockout mouse model (Mac-Sirt1 KO mouse model), 93 Myeloid SIRT1, 94 Myeloid-derived suppressor cells (MDSCs), 95 SIRT1 in, 95

N

NAD1 boosting molecule (NBMs), 248 Neurodegeneration, sirtuins and, 84 Nicotinamide (NAM), 63, 141, 162
163 SIRT1 cellular metabolism, and, 10
11 Nicotinamide adenine dinucleotide (NAD1), 63, 91, 259 NAD1-dependent deacylases, 79, 234 Nicotinamide adenine dinucleotide phosphate (NADPH), 195, 221, 238 Nicotinamide adenine dinucleotide phosphate (NADP1), 92
93 Nicotinamide mononucleotide (NMN), 43 Nicotinamide mononucleotide adenylyltransferase (Nmnat), 189 Nonalcoholic fatty liver disease (NAFLD), 91
92 Noncommunicable diseases (NCDs), 4 Nonesterified fatty acids (NEFAs), 39
40 Nonhomologous end joining pathway (NHEJ pathway), 138, 243 Nonresveratrol related sirtuin activators, 48
50 Notch signalling, 134 NOXA, 246 Nuclear factor erythroid 2-related factor 2 (Nrf-2), 224 Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), 7, 93, 179, 236 deacetylation, 43 inhibition, 96 sirtuins and NF-κB signaling, 246
247 Nuclear respiratory factor 1 (NRF1), 34

Nuclear sirtuins in cancer metabolism and inflammatory cell, 180
184 Nucleotide excision repair (NER), 138 Nutrients, 91 sirtuins as nutrient sensors, 79
80, 80f Nutrition as therapeutic model for sirtuin regulation, 44
46 natural and synthetic molecules applied on sirtuins metabolic activation or inhibition, 45f polyphenols, 45
46

O

O-3,5,40 -Trihydroxystilbene. See Resveratrol Obesity, 5, 39
40, 44 and metabolic disorders, 40f SIRT1 metabolic function, and, 7
8 sirtuins and, 83 Oct4, Sox2, Klf4 and cMyc (OSKM), 26 Oncogene, 224 Oncoprotein SIRT1, 155 SIRT2, 156 SIRT3, 157 SIRT4, 158 SIRT5, 159 SIRT6, 159
160 SIRT7, 160 Oral cancer, 259 SIRTs involvement, 260
268 therapeutic implications, 269
270 sirtuins1 and, 266
268 sirtuins3 and, 268 Oral submucous fibrosis (OSF), 266
267 Organic acids, intracellular concentration of, 112
113 Orlistat, 44 Ornithine transcarbamylase (OTC), 220 Oxidative damage, sirtuins and, 221 Oxidative phosphorylation (OXPHOS), 91, 195 direct posttranslational control of, 203
204 indirect control by sirtuins, 209 Drp1, 209 GABPα/GABPβ complex, 209 MnSOD, 209 PGC-1α, 209 OXPHOS/TCA cycle, 196
197 Oxidative stress, 6, 68 Oxoadipate dehydrogenase (OADC), 106
107 Oxoglutarate dehydrogenase, 241 2-Oxoglutarate dehydrogenase (OGDH), 220 8-Oxoguanine glycosylase 1 (OGG1), 221

P p53, 208, 235 Pancreas, 44 PDH phosphatase 1 (PDP1), 239 Pentose phosphate pathway (PPP), 91, 196 Peripheral blood mononuclear cells (PBMCs), 237

Index

Peripheral neuropathies assessments, 5 Peroxiredoxin-1 (Prx1), 221, 239 Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC1-α), 7, 62
63, 95, 209
210, 219
220, 264 Peroxisome proliferator-activated receptoralpha (PPAR-α), 62
63 Peroxisome proliferator-activated receptorgamma (PPAR-γ), 62
63 PGC-1 related coactivator (PRC), 7 Phosphatase and tensin homolog (PTEN), 71, 133 Phosphatidylinositol biphosphate (PIP2), 70 Phosphatidylinositol-3 kinase (PI3K), 70 Phosphatidylserine (PS), 6
7 Phosphoenolpyruvate carboxykinase (PEPCK), 220 Phosphofructokinase (Pfk1), 220 Phosphoinositide-dependent kinase 1 (PDK1), 9 Phosphotidylinositide 3-kinase (PI3-K), 7 PI3K-Akt, 211 Plasmacytoid DCs (pDCs), 92 Pluripotency genes, 27, 32
33 Pluripotent ESCs through multilevel mechanisms, 27
28 Pluripotent stem cells (PSCs), 26 Poldip2. See Polymerase δ-interacting protein 2 (Poldip2) Poly (ADP-ribose) polymerase (PARP), 234 Polymerase δ-interacting protein 2 (Poldip2), 109 Polyphenols, 45
46 Posttranslational modifications of sirtuins, 85
86 PRDX1. See Peroxiredoxin-1 (Prx1) Pro-opiomelanocortin (POMC), 7, 44 Prolyl-4-hydroxylase proteins (PHD proteins), 92
93 Prostaglandin (PG), 94 Protein kinase A (PKA), 223 Protein kinase B pathway, 222 Protein observed with Rictor-1 (Protor-1), 9 Protein tyrosine phosphatase (PTP), 7
8 PTP1B, 63
64 Proximal tubular cells (PTCs), 66 PRSET7, 238 Pyrroline-5-carboxylase reductase 1 (PYCR1), 220, 240 Pyrrolo [3,2-b] quinoxaline, 50 Pyruvate dehydrogenase (PDC), 106
107 Pyruvate dehydrogenase A1 (PDHA1), 239 Pyruvate dehydrogenase complex (PDC), 220 regulation of, 110
111 Pyruvate dehydrogenase kinase 1 (Pdk1), 220 Pyruvate kinase M2 (PKM2), 92
93, 220
221

Q Quiescence of HSCs, 34

R Rapamycin, 9 Ras homologue enriched in brain (Rheb), 9

Reactive oxygen species (ROS), 27, 79
80, 91
92, 221, 264 Regenerative capacity of HSCs, 34 Regulated transcription coactivator 2 (CRTC2), 63 Renal disease, sirtuins and, 84 Renin
angiotensin aldosterone system (RAAS), 66 Replicative immortality, sirtuins in, 138
139 Reprogramming energy metabolism, sirtuins in, 139
141 Resisting cell death, sirtuins and, 135
136 Resveratrol (Rsv), 47
48, 141 in treatment of oral cancer, 269t Reticuloendotheliosis viral oncogene homolog B (RELB), 80
81 Retinoic acid (RA), 27 Retinoic acid receptor (RAR), 28 Ribonucleotide reductases (RNR), 105 RNA polymerase I (PolI), 243
244 Runt-related transcription factor 2 (RUNX2), 220

S S-adenosylmethionine (SAM), 26, 107
109 Saccharomyces cerevisiae, 6, 9, 79, 234 Satellite cells (SCs), 26 Serine hydroxymethyltransferase (SHMT2), 220 Serine-threonine liver kinase B1 (LKB1), 8
9 Short-term hematopoietic stem cells (STHSCs), 26 Signal transducer, and activation of transcription pathways (STAT pathways), 179 STAT3, 63, 223 Silent information 2 (Sir2), 61, 234 Silent mating type information regulation 2 homolog 1, 6
7 SIRT1 metabolic function, and obesity, 7
8 SIRT1 and AMP-activated protein kinase, 8
9 SIRT1 metabolic function, and obesity, 7
8 SIRT1 mTOR, and metabolic disease, 9
10 SIRT1 nicotinamide, and cellular metabolism, 10
11 Silibinin. See Slybin Sirtinol, 141 Sirtuin 1 (SIRT1), 40
41, 62
63, 154
155, 219
222, 237 and adenosine monophosphate-activated protein kinase pathway, 70 and autophagy in diabetes mellitus and diabetic kidney disease, 69 in dendritic cells, 95 facets of SIRT1 in cancer biology, 235 in glucose metabolism, 63
64 in hematologic malignancies, 236
237 impacts on oral carcinogenesis, 264 involvement in metabolism of cancer and inflammatory cells, 180
181

279 KO mice, 94 in lipid metabolism, 64 in macrophage, 93
95 in MDSCs, 95 and mTOR pathway in diabetic kidney disease, 70 in normal physiology, 62
65 oncogenic roles, 235 as oncoprotein, 155 and oral cancer, 266
268 roles in glomerulus in diabetic kidney disease, 67
68 results from animal models of DM, 67
68 results from cell culture studies, 68 roles in tubulointerstitium in diabetic kidney disease, 68
69 results from animal models of diabetes mellitus, 68 results from cell culture studies, 69 SIRT1 in T cells, 95
96 SIRT3 in glucose metabolism and lipid metabolism, 65 SIRT4 in glucose and lipid metabolism, 65 in stem cell biology, 26
31 important in maintaining/promoting stemness and survival of CSCs, 30
31 maintains pluripotent ESCs through multilevel mechanisms, 27
28 for maintenance of diverse ASC pools, 28
30, 29f for normal embryogenesis and animal development, 26
27 as tumor suppressor, 154
155 tumor-suppressive roles, 236 Sirtuin 2 (SIRT2), 155
156, 219, 221
222 and genomic stability regulation, 237
239 in immunometabolism, 96 involvement in metabolism of cancer and inflammatory cells, 181 as oncoprotein, 156 in stem cell biology promotion of differentiation of ESCs in vitro, 31 promotion of survival of CSCs, 31
32 as tumor suppressor, 156 Sirtuin 3 (SIRT3), 40
41, 79
80, 156
157, 184, 219
222 in hematologic malignancies, 239
240 in immunometabolism, 96
97 impacts on oral carcinogenesis, 264 interplay between Sirt3 and isocitrate dehydrogenase in cancer cells, 115 ketogenic enzymes ACAT1 and HMGCS2 as substrates for, 113
114 maintaining the pool and regenerative capacity of HSCs during aging, 32 as oncoprotein, 157 and oral cancer, 268 as tumor suppressor, 156
157 Sirtuin 4 (SIRT4), 79
80, 157
158, 185, 219
220 and glutamine metabolism regulation, 240
241 in immunometabolism, 96
97

280 Sirtuin 4 (SIRT4) (Continued) as oncoprotein, 158 as tumor suppressor, 158 Sirtuin 5 (Sirt5), 158
159, 185
187, 219
221, 224 deficiency, 97 in immunometabolism, 96
97 ketogenic enzymes ACAT1 and HMGCS2 as substrates for, 113
114 oncogenic desuccinylase, 241
242 as oncoprotein, 159 as tumor suppressor, 158 Sirtuin 6 (Sirt6), 159
160, 219
220, 222
223 and age-old Warburg effect, 242
243 effects in kidney, 66
67 in immunometabolism, 97 involvement in metabolism of cancer and inflammatory cells, 182
183 as oncoprotein, 159
160 in stem cell biology control of regeneration and stress resistance in HSCs and MSC, 33 epigenetically promoting lineage commitment of ESCs and animal development, 32
33 suppressing the stemness of CSCs, 33 as tumor suppressor, 159 Sirtuin 7 (SIRT7), 160, 219, 221
223 and DNA repair, 243
244 effects in kidney, 66
67 in immunometabolism, 97 involvement in metabolism of cancer and inflammatory cells, 183
184 as oncoprotein, 160 and ribosome biogenesis, 243
244 in stem cell biology, 33
34 regulation of embryogenesis and life span, 33
34 regulation of quiescence and regenerative capacity of HSCs, 34 as tumor suppressor, 160 Sirtuin inhibitors (SIRTi), 161f, 162
166 AGK2, 166 β-naphthol-containing inhibitors, 163
164 indole derivatives, 164 MC2494, 166 MHY2256, 166 NAM, 162
163 SirReal2, 166 suramin, 165 tenovin, 165 thioacyllysine-containing compounds, 165 toxoflavin, 166 Sirtuin-activating compounds (STACs), 247 Sirtuin-rearranging ligands 2 (SirReal2), 166 Sirtuins (SIRTs), 25, 40, 61, 79, 129, 153, 219, 234, 259 activators, 166
167 in angiogenesis, 137 and cancer metabolism, 219
221 and cancer stem cells, 223 and cancer therapy, 141 cancer-related sirtuin-activating compounds, 162f and chemoresistance, 223
224

Index

diabetes mellitus and, 65
70 direct epigenetic control of cellular metabolism by, 198
200 direct posttranslational control of cellular metabolism by, 200
206 dual role in cancer process, 130t expression in organs, 41f food and, 50
52 function in immnue cells, 94t metabolic control of, 98f in genome instability and replicative immortality, 138
139 genomic stability, and DNA repair, 221
222 hypertension and, 71 indirect control of cellular metabolism by, 206
211 in invasion and metastasis, 137
138 involvement in inflammation and cancer metabolism, 179
180 linking metabolism, inflammation, and cancer, 188
190 mammalian sirtuins, 153
160 in metabolic disease, 4
5 NCDs, 4 novel therapeutic strategies with sirtuins for, 6 SIRT1, 6
7 and metabolic physiological regulation, 41f and metastasis, 222
223 mitochondrial, 184
187 and mitochondrial biogenesis, 80
82, 81f and mitochondrial metabolism, 82 nuclear and cytosolic sirtuins involvement in metabolism of cancer and inflammatory cells, 180
184 SIRT1, 180
181 SIRT2, 181 SIRT6, 182
183 SIRT7, 183
184 nutrient sensors, 79
80, 80f in oral cancer, 260
270 and oxidative damage, 221 posttranslational modifications of sirtuins and mitochondrial function regulation, 85
86 regulation of iron
sulfur cluster assemblage, 116 regulatory modes of sirtuins in controlling cellular metabolism, 197
198 in reprogramming energy metabolism, 139
141 and resisting cell death, 135
136 role in immunometabolism, 93
97 sirtuin activators, 166
167 sirtuin inhibitors, 161f, 162
166 β-naphthol-containing inhibitors, 163
164 indole derivatives, 164 NAM, 162
163 SIRTi, 166 suramin, 165 tenovin, 165 thioacyllysine-containing compounds, 165

sirtuin modulators, 160
167 sirtuins and mitochondrial dysfunction in human diseases, 82
85 stem cells and, 26 in sustaining proliferative signaling and evading growth suppressors, 132
134 tumor suppressors or promoters, 224
225 in tumor-promoting inflammation and immune system function, 136
137 tumor-suppressing and tumor-promoting activities of, 115
116 Skeletal muscle, 43 Slybin, 72 Small lymphocytic lymphoma (SLL), 246 Snail, 222 Splitomicin, 163 SRT1720, 248 SRT2104, 249 Stanniocalcin-1, 72 Staphylococcus aureus AW7, 96 Stem cell biology SIRT1 in, 26
31 SIRT2 in, 31
32 SIRT3 in, 32 SIRT6 in, 32
33 SIRT7 in, 33
34 Stem cells and sirtuins, 26 Sterol-regulatory-element binding protein (SREBP), 62
63, 210 Streptomyces hygroscopicus, 9 Streptozosin (STZ), 70 Stress-activated kinases (SAPK), 222 Succinate dehydrogenase (SDH), 96
97, 220 Sulfur mobilization systems (SUF systems), 105 Superoxide dismutase 1 (SOD1), 221 Superoxide dismutase 2 (SOD2), 221 Suramin, 165

T T cells, 92 SIRT1 in, 95
96 Tenovin, 165 Tenovin-1, 141 Tenovin-6, 141 Tetrahydrofolate (THF), 107
109 Theobroma cacao.. See Cocoa (Theobroma cacao) Thioacyllysine-containing compounds, 165 Tissue-specific sirtuin-modulated metabolic regulation, 42
44 adipose tissue, 42
43 brain, 44 heart and skeletal muscle, 43 kidneys, 43
44 liver, 42 pancreas, 44 Toxoflavin, 166 TR4/TAK1, 211 Transforming growth factor-beta (TGF-β), 222, 266
267 Tricarboxylic acid (TCA), 91, 196 TrxR2, 224 Tubulin, 237 Tubulointerstitium in diabetic kidney disease, 68
69

281

Index

results from animal models of diabetes mellitus, 68 results from cell culture studies, 69 Tumor metabolism, 219 Tumor necrosis factor alpha (TNF-α), 6
7, 39
40, 93, 247 Tumor suppressors, 224
225 SIRT1, 154
155 SIRT2, 156 SIRT3, 156
157 SIRT4, 158 SIRT5, 158 SIRT6, 159 SIRT7, 160 Tumor-promoting activities of sirtuins, 115
116 Tumor-promoting inflammation, sirtuins in, 136
137

Tumor-suppressing activities of sirtuins, 115
116 Tumorigenesis, sirtuins and, 85 Twist, 222

U

W Warburg effect, 26, 139, 179
180, 268 Werner ATP-dependent helicase (WRN), 222 White adipose tissue (WAT), 39
40 browning, 42
43 World Health Organization, 4

U937 leukemia cell lines, 248 Uncoupling proteins (UCPs), 7, 48 UCP-2, 62
64

X

V

Y

Vacuolar-type H1 ATPase (V-ATPase), 236 Vascular endothelial growth factor A (VEGF A), 243 VEGF growth factors and their receptors (VEGFRs), 137 Vitamin nicotinamide, 6

Xanthigen, 52

YC8
02, 240

Z Zinc finger E-box-binding homeobox 1 (Zeb1), 222