Colon Cancer Diagnosis and Therapy Vol. 3 (Colon Cancer Diagnosis and Therapy, 3) 3030727017, 9783030727017

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Dhananjay Shukla Naveen Kumar Vishvakarma Ganji Purnachandra Nagaraju   Editors

Colon Cancer Diagnosis and Therapy Vol. 3

Colon Cancer Diagnosis and Therapy Vol. 3

Dhananjay Shukla Naveen Kumar Vishvakarma Ganji Purnachandra Nagaraju Editors

Colon Cancer Diagnosis and Therapy Vol. 3

Editors Dhananjay Shukla Department of Biotechnology Guru Ghasidas Vishwavidyalaya Bilaspur, Chhattisgarh, India

Naveen Kumar Vishvakarma Department of Biotechnology Guru Ghasidas Vishwavidyalaya Bilaspur, Chhattisgarh, India

Ganji Purnachandra Nagaraju Division of Hematology and Oncology School of Medicine University of Alabama Birmingham, Alabama, USA

ISBN 978-3-030-72701-7    ISBN 978-3-030-72702-4 (eBook) https://doi.org/10.1007/978-3-030-72702-4 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

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Preface

Colorectal cancer (CRC) is the third most frequently diagnosed cancer and second leading cause of cancer-related mortalities. Colon cancer is often referred as CRC, which combines colon and rectal cancer, which initiates in the rectum. CRC can be treated if it is found in initial stages and localized in the bowel. Estimated new cases by the American Cancer Society for 2020 were 104,610 for colon cancer and 43,340 for rectal cancer with approximately 53,200 mortalities associated with CRC.  Surgery is the primary treatment option with 50% success rate. However, recurrence post surgery is the most important challenge currently, which leads to most CRC-associated fatalities. Despite advanced technologies, the 5-year survival rate is diminishing due to recurrence and delayed diagnosis. Additionally, external factors such as excessive alcohol use, smoking, family history of cancer, and hereditary conditions like FAP, HNPCC, and lynch syndrome are the major challenging factors contributing to CRC occurrence. Therefore, it is essential for the researchers and clinicians to develop novel therapeutic strategies in order to prolong the survival rate of CRC patients. In this third volume, our authors have presented novel advanced therapeutic strategies for the management of CRC. The underlying mechanism of CRC pathogenesis is extensively investigated by researchers associated with cancer research. The molecular-level understanding of the tumor microenvironment and tumor stroma interaction is very much essential as the interaction promotes tumor invasion and metastasis. In this volume, Manisha et al. and Arundhati et al. have outlined the role of chemokines and tumor-associated macrophages, respectively, in tumor microenvironment in relation to colon cancer progression and therapeutic interventions. The general therapeutic regimens included are chemo and radiotherapy. Researchers are now developing novel advances in these conventional therapies. Patel et al. have described the ongoing advances in chemoradiotherapy for the treatment of CRC, which will assist clinicians and researchers and also benefit patients. As mentioned, CRC is a pathogenesis resulting from both genetic and epigenetic alterations, which eventually lead to invasion and metastasis. Additionally, varied signaling cascades that are involved in CRC progression are dysregulated due to the involvement of mutations in genetic and epigenetic alterations. The landscape of epigenetics determines the vii

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conformation of chromatin if the DNA is accessible for the transcription factors to control gene expression. Thus, the epigenetic alterations and their cross talk with DNA methylation and histone modifications are extensively studied to identify the inheritance of cancer cells. Our authors have included necessary mechanisms and therapeutic interventions for epigenetic regulation of CRC. Similarly, the genetic variants and missing heritability associated with the risk for CRC occurrence can be easily discovered through proper understanding of the mechanism of single nucleotide polymorphism. At present, the genome-wide association study (GWAS) platform has led to the identification of multiple genetic variants associated with the occurrence of CRC. Our authors have presented the clinical significance of genetic variants in the detection of colon cancer. Conventional therapies develop chemoresistance, which eventually results in recurrence and metastasis. Additionally, these therapies lead to multiple side effects as the drug is delivered to both healthy and cancer cells. Targeted therapies use aberrant pathways that are involved in CRC progression such as SHH, Wnt, EGFR, Kras, and Notch. Development in gene therapies promotes the identification of dysregulated pathways that are caused due to mutation in genes like Kras and p53. Thus, advanced strategies for safe and effective delivery of drug are essential. Yashwant et al. and Vikas et al. have introduced underlying mechanisms that are involved in developing chemoresistance and therapeutic interventions for targeting the dysregulated signaling pathways for CRC therapy. Additionally, researchers are now focusing on the therapeutic role of bacteria and cyanobacteria as novel anti-cancer agents. Unlike conventional therapies, these targeted therapies are directly targeting tumor cells, leaving the healthy cells unaffected. Bacterial products like peptides, toxins, and bacteriocins are widely encouraged as therapeutic agents for colon cancer. Similarly, cyanobacterial secondary metabolites play a vital role in varied biological activities, including antimicrobial, immunosuppression, and anticancer activities. Thus, use of cyanobacterial and bacterial compounds would be a promising approach for novel drug discoveries. This volume includes chapters related to bacterial cancer therapy and cyanobacterial secondary metabolite as a potential drug candidate against colon cancer. Despite advances in therapeutic strategies, multi drug resistance is always an obstacle for cancer therapy. Thus, the RNA-based therapies that include RNA interference, RNA aptamer, ribozymes, and antisense oligonucleotide are found advantageous for their higher potency, specificity, and reduced toxicity. Our author Ajay has focused on the development of RNA-based drug development for CRC. This volume also includes articles for mechanistic exploration and therapeutic management for CRC metastasis. The mechanistic studies are clinical trials (however, not all) that include understanding of biological, behavioral, and pathological process of a disease in respect to the drug intervention efficiency. These mechanistic studies accelerate evaluating the drug efficacy after administered into the patient that infers the adverse effects and cytotoxicity of the drug. The malignant progression of any cancer is associated with the development of inflammation. Tumor-extrinsic inflammation is caused by bacterial and viral infections. The pathogens, including Helicobacter pylori and hepatitis C virus, are wellstudied pathogens that promote cancer progression. These pathogens induce

Preface

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pathogenesis via epithelial injury and inflammation. TLR4 and IL-23/IL-17 are some mediators that activate inflammation accelerating colorectal tumorigenesis. Therefore, a complete understanding of the relationship between inflammation and microbiota causing tumorigenesis is essential. Julia et al. has have introduced pathogenic inflammation, which must be targeted for therapeutic intervention against colon cancer. Additionally, consumption of some pre- and probiotics may exert beneficial effects for cancer prevention. In this regard, Rajat et al. have outlined the role of food additives and intestinal microflora, which are required for CRC prevention. Furthermore, for the prevention and recurrence of cancer, maintaining healthy weight, daily exercise, and a healthy balanced diet is essential. This volume includes chapters outlining the nutritional intervention for prevention and management of colon cancer. Furthermore, researchers are now focusing on chemo-preventive application of plant-based polyphenols and their derivatives for cancer therapy, such as curcumin. This application and mechanism are widely studied due to its reduced toxicity and heightened efficiency in improving the therapeutic efficacy of chemodrugs or radiotherapy drug when used in adjuvant or combinational therapies against CRC.  In this volume, our authors have described the antineoplastic applications against CRC. Altogether, this volume provides a precise and an in-depth understanding of underlying molecular mechanisms and therapeutic options that are currently available. It is our great pleasure to present this comprehensive summary of novel therapeutic strategies to the science community for the benefit of patients. Bilaspur, Chhattisgarh, India Bilaspur, Chhattisgarh, India Birmingham, Alabama, USA

Dhananjay Shukla Naveen Kumar Vishvakarma Ganji Purnachandra Nagaraju

Contents

 Updates on Clinical Trials in Diagnosis and Therapy of Colorectal Cancer����������������������������������������������������������������������������������������    1 Farhan Ullah, Hariharasudan Mani, Maha Wazir, Sana Hussain, Saeed Ali, and Sarfraz Ahmad  Wnt Signaling-Related Long Noncoding RNAs: Critical Mediators of Drug Resistance in Colon Cancer������������������������������   21 P. Vasudeva Raju and RamaRao Malla  The Triad of Estrogen, Estrogen Receptors, and Colon Cancer ����������������   41 K. R. Sumalatha, Syamala Soumyakrishnan, and M. Sreepriya  Clinical Significance of Genetic Variants in Colon Cancer��������������������������   69 Irina Nakashidze, Nina Petrović, Nino Kedelidze, and Begum Dariya  Role of Nano-immunotherapy in Colorectal Cancer: An Updated Review ����������������������������������������������������������������������������������������   93 Devanabanda Mallaiah and Ramakrishna Vadde  Mechanistic Exploration and Therapeutic Management of Colon Cancer Metastasis����������������������������������������������������������������������������  105 Anupam Kumar Srivastava  Chemoresistance in Colorectal Malignancies: Molecular Mechanisms and Strategies to Overcome.����������������������������������  123 Henu Kumar Verma, Yashwant Kumar Ratre, and Pellegrino Mazzone  Therapeutic Intervention of Signaling Pathways in Colorectal Cancer����������������������������������������������������������������������������������������  143 Vikas Chandra, Ashutosh Tiwari, Rajat Pratap Singh, and Kartiki V. Desai

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 Targeting Pathogenic Inflammation for Therapeutic Intervention Against Colon Cancer����������������������������������������������������������������  173 Julia Fleecs, Eden Abrham, Mikale Kuntz, M. Nadeem Khan, and Ramkumar Mathur  Role of Tumour-Associated Macrophages in Colon Cancer Progression and Its Therapeutic Targeting��������������������������������������  193 Arundhati Mehta, Vivek Kumar Soni, Yashwant Kumar Ratre, Ajay Amit, Dhananjay Shukla, Ajay Kumar, and Naveen Kumar Vishvakarma  Advances in Chemoradiotherapy for Treatment of Colon Cancer ������������  217 V. K. Patel and H. Rajak  Cytotoxic and Chemopreventive Activity of Polyphenols and Their Derivatives in Colon Cancer ��������������������������������������������������������  241 Harit Jha and Ragini Arora  Prevention and Management of Colon Cancer by Nutritional Intervention ����������������������������������������������������������������������������  277 Vibha Sinha, Sapnita Shinde, Vineeta Dixit, Atul Kumar Tiwari, Ashwini K. Dixit, Naveen Kumar Vishvakarma, Sanjay Kumar Pandey, Alka Ekka, Mrinalini Singh, and Dhananjay Shukla  Role of Food Additives and Intestinal Microflora in Colorectal Cancer����������������������������������������������������������������������������������������  307 Vivek Kumar Soni, Ajay Amit, Vikas Chandra, Pankaj Singh, Pradeep Kumar Singh, Rudra Pratap Singh, Girijesh Kumar Patel, and Rajat Pratap Singh Effect of Milk and Dairy Products in Colorectal Cancer����������������������������  325 Sarang Dilip Pophaly, Soumitra Tiwari, Awadhesh Kumar Tripathi, and Manorama  Development of RNA-Based Medicine for Colorectal Cancer: Current Scenario����������������������������������������������������������������������������������������������  339 Ajay Amit, Sudhir Yadav, Rajat Pratap Singh, and Chanchal Kumar  Bacterial Cancer Therapy: Promising Role in the Treatment of Colon Cancer������������������������������������������������������������������������������������������������  361 Rishi Srivastava, Shweta Sonam, Naveen Kumar Vishvakarma, Rajesh Sharma, and Shree Prakash Tiwari  Antineoplastic Effects of Curcumin Against Colorectal Cancer: Application and Mechanisms��������������������������������������������������������������������������  383 Vivek Kumar Soni, Arundhati Mehta, Yashwant Kumar Ratre, Chanchal Kumar, Rajat Pratap Singh, Abhishek Kumar Srivastava, Navaneet Chaturvedi, Dhananjay Shukla, Sudhir Kumar Pandey, and Naveen Kumar Vishvakarma

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 Role of Chemokines in Colorectal Cancer����������������������������������������������������  427 Manisha Mathur, Sonal Gupta, Beiping Miao, Prashanth Suravajhala, and Obul Reddy Bandapalli Index������������������������������������������������������������������������������������������������������������������  441

About the Editors

Dhananjay  Shukla  is an assistant professor in the Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India. Dr. Shukla obtained his MSc in biotechnology from APS University Rewa, Madhya Pradesh. He obtained his PhD in biotechnology from the Defence Institute of Physiology and Allied Sciences at the Defence Research and Development Organisation, and Jamia Hamdard University, Delhi, India. Dr. Shukla did his postdoctoral research work at the Centre for DNA Fingerprinting and Diagnostics (CDFD) Hyderabad, Telangana, under DBT-Postdoctoral Fellowship award. He received advanced research training at the Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, USA.  Dr. Dhananjay uses in vitro, in vivo, and in silico models to explore the role of bioactive compounds against lung diseases and cancer prevention. Dr. Dhananjay’s current research interest is to evaluate phytomedicines against lung pathologies and cancer. He has published over 25 research papers in highly reputed international journals having high impact factors and has presented more than 15 abstracts at various national and international conferences. Dr. Dhananjay has been working as a faculty member since 2013 in the Department of Biotechnology at Guru Ghasidas Vishwavidyalaya.

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About the Editors

Naveen  Kumar  Vishvakarma  is currently Assistant Professor of Biotechnology at Guru Ghasidas Vishwavidyalaya. He earned his master’s degree in microbiology and then undertook his doctoral research in tumor immunology. During his doctoral research, he worked in the area of tumor acidity–mediated immunosuppression. After completing doctoral research work, he worked as a postdoctoral fellow/research associate at Banaras Hindu University, Manitoba Institute of Cell Biology (Canada), and Moffitt Cancer Center and Research institute (USA). During his work at Moffitt Cancer Center, he demonstrated the role of acidic tumor microenvironment in selection of aggressive phenotype with metabolic alterations. In 2013, he joined HNB Garhwal University as assistant professor and later moved to his current position at Guru Ghasidas Vishwavidyalaya in 2014. His current research interest includes modulation of tumor metabolism, evaluating derivative anticancer drugs, and chemosensitization. Ganji  Purnachandra  Nagaraju  is An Assistant Professor in the School of Medicine, Division of Hematology and Oncology, University of Alabama, Birmingham. Dr. Nagaraju obtained his MSc and his PhD, both in biotechnology, from Sri Venkateswara University in Tirupati, Andhra Pradesh, India. Dr. Nagaraju received his DSc from Berhampur University in Berhampur, Odisha, India. His research focuses on translational projects related to gastrointestinal malignancies. He has published over 100 research/review papers in highly reputed international journals and has presented more than 50 abstracts at various national and international conferences. He has trained and continues to train many fellows, residents, medical students, and graduate/undergraduate students. Dr. Nagaraju is author and editor of several published books by Springer Nature and Elsevier. He serves as an editorial board member of several internationally recognized academic journals. Dr. Nagaraju has received several international awards including FAACC. He also holds memberships with the Association of Scientists of Indian Origin in America (ASIOA), the Society for Integrative and Comparative Biology (SICB), the Science Advisory Board, the RNA Society, the American Association for Clinical Chemistry (AACC), American Society for Clinical Pathology (ASCP), and the American Association of Cancer Research (AACR).

Updates on Clinical Trials in Diagnosis and Therapy of Colorectal Cancer Farhan Ullah, Hariharasudan Mani, Maha Wazir, Sana Hussain, Saeed Ali, and Sarfraz Ahmad

Abstract  Colorectal cancer (CRC) is the third most common cancer overall. CRC could be sporadic, which is most common at 70% of CRC, inherited (5%), and familial (25%). The most common genes that are affected in CRC are oncogenes, tumor suppressor genes, and genes related to the DNA repair mechanisms. Structural screening tests (such as colonoscopy, sigmoidoscopy) are superior to stool tests. Studies have shown that colonoscopy has reduced the incidence of CRC by 67% and case fatality rate by 65%. Microsatellite instability (MSI), chromosomal instability (CIN), and CpG island methylator phenotype (CIMP) are the major mutations that occur in CRC. These can also act as biomarker, which could be detected in stool, blood, or tumor biopsy. Based on patient characteristics and tumor features, CRC patients could be divided into four groups (from Group 0 to Group 3). First-­line chemotherapeutic agents for adjuvant chemotherapy are FOLFOX (5-FU/LV/Oxaliplatin) or capecitabine/LV/oxaliplatin. Palliative chemotherapy options involve 5-FU with leucovorin, capecitabine alone, FOLFOX or FOLFIRI regiments (5-FU/LV/irinotecan). Efficacy of combination regimens are enhanced by the use of monoclonal antibodies such as anti-VEGF (bevacizumab) and anti-EGFR (cetuximab, panitumumab). Some of the alternative treatments currently ongoing are discussed in this chapter. These include agarose tumor microbeads where tumor growth inhibitory factor causing a negative inhibitory signal is found to slow the tumor growth. Chronic inflammatory products such as cytokines, chemokines, reactive oxygen species (ROS), reactive nitrogen species, and arachidonic acid derivatives are risk factors for CRC, and COX inhibitors such as NSAIDs could have some effect prophylactically and for treatment against CRC. Probiotics could act on CRC possibly by apoptosis of diseased cells and also possibly by antioxidant effect. Functional foods containing polyphenols could F. Ullah · M. Wazir · S. Hussain Department of Internal Medicine, Khyber Teaching Hospital, Peshawar, Khyber Pakhtunkhwa, Pakistan H. Mani · S. Ali Department of Internal Medicine, University of Iowa, Iowa City, IA, USA e-mail: [email protected]; [email protected] S. Ahmad (*) AdventHealth Cancer Institute, FSU and UCF Colleges of Medicine, Orlando, FL, USA e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_1

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reduce ROS, thereby reducing CRC by maintaining a balance between ROS and antioxidants. Metal-based drugs such as platinum-based agents (like carboplatin, oxaliplatin) and gold-based drugs (such as auranofin) also exerts antitumor effect, possibly through platinum-DNA adducts and ROS, respectively. Keywords  Colorectal carcinoma · Diagnosis · Therapy · Clinical trials · Agarose macrobeads · Biomarkers: RENCA: NSAIDs

Abbreviations BMI Body mass index CAPOX Capecitabine/LV/oxaliplatin CIMP CpG island methylator phenotype CIN Chromosomal instability COX Cyclooxygenase COXibs Cyclooxygenase-2 inhibitor CRC Colorectal carcinoma CT Computerized tomography CVD Cardiovascular disease DNA Deoxyribonucleic acid EGFR Epidermal growth factor receptor FAP Familial adenomatous polyposis FBLN1 Fibulin FOLFIRI 5-FU/LV/irinotecan FOLFOX 5-FU/LV/oxaliplatin GLOBOCAN Global Cancer Observatory GSN Gelsolin KRAS Kirsten rat sarcoma viral oncogene homolog MSI Microsatellite instability NCL Nucleolin NSAIDs Non-steroidal anti-inflammatory drugs PEBP1 Phosphatidylethanolamine-binding protein PEDF Pigment epithelium-derived factor PRDX1 Peroxiredoxin-1 PSAP Prosaposin RENCA Renal cortical adenocarcinoma cell line (mouse) RNA Ribonucleic acid RON Reactive nitrogen species ROS Reactive oxygen species Serbp1 Serpine1 SPARC Secreted protein, acidic, and rich in cysteine TIMP2 Tissue inhibitor of metalloproteinase 2 USPSTF United States Preventive Services Task Force VEGF Vascular endothelial growth factor

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1  Introduction As reported by the Global Cancer Observatory (GLOBOCAN) 2018, the third most common overall and the second most common cancer in women is colorectal carcinoma (CRC). It is the second leading cause of cancer-related deaths per year, with a crude mortality rate of 11.5 (Cancer Today, 2020). Estimated new cases of CRC in the year 2020 are 147,950, which makes 8.2% of all the new cases of cancer. The estimated deaths in the year 2020 from this cancer are 53,200 (8.8% of all cancer deaths) (Cancer of the colon and rectum – Cancer stat facts, 2020). Its 5-year relative survival is 64.6% (Cancer of the colon and rectum – Cancer stat facts, 2020). Like all other metastatic diseases, CRC tumor is more common in elderly patients, diagnosed commonly in the age group of 65–74 years, with a median age of 67 years (Cancer of the colon and rectum – Cancer stat facts, 2020). Because of the increase in the prevalence of the risk factors of CRC (obesity, inactive lifestyle, red meat consumption, exposure to alcohol, tobacco, etc.), its incidence is on a continuous rise, more noticeably in developing countries, where the “Western” lifestyle is getting more common (Rawla, Sunkara, & Barsouk, 2019).

2  Pathophysiology Like all other cancers, CRC initiates with mutations in specific genes. The most common genes that are affected in the CRC are oncogenes, tumor suppressor genes, and genes related to the DNA repair mechanism (Fearon & Vogelstein, 1990). The key molecular pathways involved in colorectal carcinogenesis are shown in Fig. 1. CRC can be classified into the following three types, based on the origin of mutation: sporadic, inherited, and familial. Sporadic CRC is the tumors derived from point mutations. Seventy percent of CRC are sporadic in origin. The mutations can occur in many types of genes in this type of carcinoma, so its pathogenesis is heterogenous (Fearon & Vogelstein, 1990). Around 5% of the CRC are inherited. We can divide the inherited CRC into polyposis and nonpolyposis. Familial adenomatous polyposis (FAP) is the major subtype of the polyposis group (Lynch & de la Chapelle, 2003). Roughly 25% of all the colorectal cancers are familial colorectal cancer, the pathogenesis of which is also inherited mutation, but they are not categorized as inherited cancers as such, because they do not fit in any of the subclasses of the inherited cancer (Stoffel & Kastrinos, 2014).

3  Screening and Diagnosis In order to catch the tumor in its early stage, the current screening tests for colorectal cancer are as follows: stool occult blood test, stool exfoliated DNA test, flexible sigmoidoscopy, colonoscopy, and CT colonography. Structural examination tests

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Fig. 1  Key molecular pathways involved in colorectal carcinogenesis. Mutations affecting the proteins involved in WNT (orange), MAPK/PI3K (green), SMAD/TGF-β (blue), or DNA repair (purple). Abbreviations: MAPK mitogen-activated protein kinase, PIK3 phosphoinositide 3-kinase, SMAD, TGF-β transforming growth factor-β, DNA deoxyribonucleic acid. (Adapted and modified from Mármol, Sánchez-de-Diego, Dieste, Cerrada, & Yoldi, 2017)

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(e.g., sigmoidoscopy, colonoscopy) are superior to stool tests as the former can detect pre-malignant lesions and cancer (Levin, Lieberman, McFarland, et  al., 2008). Studies have shown that the cases of colorectal carcinoma (Brenner et al., 2010; Brenner, Chang-Claude, Seiler, Rickert, & Hoffmeister, 2011) and case fatality rates (Baxter et al., 2009) have been reduced significantly by the colonoscopy screening test. The study done by Kahl et al. (2018) demonstrated that the incidence of colorectal carcinoma reduced by 67% and the case fatality rate by 65% because of the colonoscopy. Once the suspicion arises, either because of the signs and symptoms or screening tests, for confirmation, the gold standard test is the histopathology of the biopsy attained during the structural examination tests or surgery. The majority of the CRC in the histopathologic picture are adenocarcinomas. In the last few years, the rapid expansion of knowledge and progression in molecular biology facilitated us in better understanding the pathogenies and molecular pathways/targets responsible for CRC. As noted earlier, the majority of CRC is due to some genetic alterations, which could be sporadic or familial (Burt, Bishop, Lynch, Rozen, & Winawer, 1990; Winawer, Fletcher, Miller, et  al., 1997). Some molecular tests are expected to be more sensitive and specific than the current diagnostic methods. These molecular tests will also provide the genetic data of the malignant process and thus, will facilitate the management. Such a biological entity is called a biomarker, which can not only be used to detect the presence of carcinoma but also measures the progression and the outcomes of treatment. The desirable properties of biomarkers are as follows: (i) their sensitivity and specificity are high, (ii) they are safe and affordable, (iii) they are easy to measure, (iv) their diagnostic accuracy is high, and (v) they facilitate treatment selection process (Diamandis, 2010). Microsatellite instability (MSI), chromosomal instability (CIN), and the CpG island methylator phenotype (CIMP) are the major mutations that occur in CRC which alters the DNA, RNA, proteins, and/or metabolites. These mutations can act as a biomarker as they can easily be detected in stool, blood, or biopsy of tumors (Ludwig & Weinstein, 2005). As there are limitations in every CRC screening test, over the last two decades, scientists/physicians have studied a number of molecular biomarkers, and the outcomes are somewhat reassuring, but some shortcomings in the study reduced the trustworthiness of the assumption (Wu, Yang, Zhang, et al., 2013). Because of the extensive work done in the field of microbiology and biochemistry, several biomarkers were studied, but MSI and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation are the most commonly used biomarkers in CRC, which help in tumor classification, prognosis, and management (Ludwig & Weinstein, 2005). In Table 1, we provide a summary of the biomarkers that are presently in use or under the investigation for CRC. Despite all the shortcomings of the biomarkers, a significant amount of work are still ongoing. We believe that it is imminent that the biomarkers will be significantly advantageous not only in the diagnosis and prognosis of CRC but also will be useful in the development of new treatment strategies.

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Table 1  Current biomarkers of CRC Molecular marker type Biomarker DNA MSI test. panel of mononucleotide marker (Bat-25, Bat-26, NR-21, NR-24, MONO-27), ≥30% of unstable loci are considered MSI tumors KRAS, NRAS

Contribution to cancer Accumulation of alteration in highly repeated DNA sequences

Sample used for the test Predictive use TumorFor MSI+ based tumors: Prognosis: good samples Aggressively: low Treatment: lack of response to 5-FU, good response to irinotecan

Proliferation enhancement through EGFR-­ signaling activation

If mutated: Prognosis: bad and poor survival (codon 12 and 13) Treatment: limited response to EGFR

BRAF

Proliferation enhancement through EGFR-­ signaling activation

If mutated: Classification of CRC: sporadic Prognosis: poor Treatment: limited response to EGFRtargeted therapy

CpG Island methylator phenotype, e.g., vimentin methylation

Transcriptional regulation which lead to colorectal carcinogenesis

Classification of CRC in CIMP, presence of BRAF mutations

Integrity of cell-free DNA (cfDNA)

Apoptosis

Diagnosis and monitoring

Status In use

References # Bacher, Flanagan, Smalley, et al. (2004), Geiersbach and Samowitz (2011), Iacopetta and Watanabe (2006)

Aprile, Macerelli, Maglio, Pizzolitto, and Fasola (2013), De Roock, Claes, Bernasconi, et al. (2010), Kislitsin, Lerner, Rennert, and Lev (2002) Fransén et al. Tumor- In use (2004), based Sartore-­ samples Bianchi, Martini, Molinari, et al. (2009), Wong and Cunningham (2008) Gonzalez-Pons Tumor- Under evaluation and Cruz-­ based Correa (2015), samples, in tumor Wheeler, samples stool, and in use Loukola, blood Aaltonen, samples for stool Mortensen, and Bodmer (2000) Blood Under Umetani, Kim, sample evaluation Hiramatsu, et al. (2006)

Tumorbased samples, stool

In use for tumor-­ based samples and under evaluation for stool

(continued)

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Table 1 (continued) Molecular marker type Biomarker RNA Gene microarray and gene panels of RNA

Contribution to cancer Unknown

Predictive use CRC diagnosis evaluation of relapse risk

Sample used for the test Tumorbased samples, stool, blood Tumorbased samples, stool, blood

miRNA biomarker panel. e.g., miR-21, miR-106a

Unknown

Diagnosis and prognosis

EGFR ligand biomarker panel (amphiregulin, epiregulin, DUSP6, and SLC26A3)

Proliferation enhancement through EGFR-­ signaling activation

Response to EGFR-­targeted therapy

Tumorbased samples

Protein

Tumor-specific protein determination, e.g., calprotectin, CEA, DAF, CA19-9

Unknown

Diagnosis, prognosis, monitoring

Stool, blood

Other

Circulating nucleic acids, proteins and tumor cells

Unknown

Diagnosis, monitoring

Blood

Adapted and updated from Mármol et al. (2017)

Status References # Clinical Barrier, Boelle, validation Roser, et al. (2006), Wang, Jatkoe, Zhang, et al. (2004) Clinical Link, Balaguer, validation Shen, et al. (2010), Takai, Kanaoka, Yoshida, et al. (2009) Under Baker, Dutta, evaluation Watson, et al. (2011), Jacobs, De Roock, Piessevaux, et al. (2009), Khambata-­ Ford, Garrett, Meropol, et al. (2007) Clinical Holten-­ validation Andersen, Christensen, Nielsen, et al. (2002), Hundt, Haug, & Brenner (2007), Quaye (2008), von Roon, Karamountzos, Purkayastha, et al. (2007), Xing et al. (1996) Clinical Cohen, Punt, validation Iannotti, et al. (2009), Sastre, Maestro, Puente, et al. (2008), Uen, Lu, Tsai, et al. (2008)

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4  Management The first-line treatment of CRC differs from case to case depending on the following: (a) Features of tumor, i.e., the size or the extent of the tumor, number of nodes involved, metastasis, biomarker status, etc. (b) Status of the patient, i.e., age, prognosis, other morbidities, etc. Based on these characteristics, the management of CRC patients are divided into the following classes (groups): • Group 0: No mets or resectable lungs/liver metastasis and good prognosis. For patients in this group, the first-line treatment is resection of the CRC. Chemo­ therapy is not of any significant benefit in this group in terms of overall survival (Van Cutsem et al., 2010, 2014). • Group 1: Those people who have possibly a resectable metastatic CRC.  For these patients, the first-line treatment is the induction of chemotherapy with subsequent surgical resection. The proposed chemotherapy for this group is cytotoxic doublet or triplet, with or without anti-vascular endothelial growth factor (anti-VEGF) or anti-epidermal growth factor receptor (anti-EGFR) in wild-type KRAS strategies (Van Cutsem et al., 2010, 2014). • Group 2: People with unresectable metastatic CRC are classified in this group. For these patients, palliative treatment is more beneficial compared to curative. Rapid regression of the mets is requisite, for which the preferred choice usually comprises a cytotoxic doublet with anti-VEGF or anti-EGFR.  For those who respond to the initial treatment, we may take ablation into account to reduce the risk of progression (Van Cutsem et al., 2010, 2014). • Group 3: People with unresectable disease and absence of treatment (intensive or sequential). The principal goal of treatment in this group is to inhibit the tumor progression and to improve the quality of life. Chemotherapy usually comprises fluoropyrimidines with or without a biological agent (Van Cutsem et  al., 2010, 2014). For palliative therapy, FOLFOX (5-FU/LV/oxaliplatin), CAPOX (capecitabine/ LV/oxaliplatin), or FOLFIRI (5-FU/LV/irinotecan) are the first-line choices (Van Cutsem et al., 2014; Venook, 2005). Second-line treatments are usually prescribed for those cases whose organ function is good. The choice of second-line therapy depends on the pattern of resistance, for example, in a patient who develops resistance to FOLFOX or CAPOX, the second-line treatment is CAPOX and vice versa (Venook, 2005). In order to enhance the effect of chemotherapy on the CRC, monoclonal antibodies such as anti-VEGF (bevacizumab) and anti-EGFR (cetuximab, panitumumab) are used in combination with the first-line treatment (Van Cutsem et al., 2014). With the passage of time and advancement in medicine and pharmacology, studies are ongoing to develop better treatment options for CRC. Many substitutes for the current treatment options are under investigation. Collectively, the goal of all these

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studies is to develop a treatment option that is more useful with fewer side effects and cost-effective. Below we explain some of the most important studies that are ongoing currently in the field. Also, Table 2 illustrates the summary of presently ongoing clinical trials of alternative treatment options for CRC.  The key mechanisms of actions of the alternative medications are shown in Fig. 2.

4.1  Agarose Tumor Macrobeads Some studies suggest that like normal organs of the body, the tumor also follows a growth pattern, which is described by the Gompertzian growth curve. There is initially an exponential growth, which is then pursued by a plateau as the growth slows Table 2  A summary of important ongoing clinical trials for alternate treatments of colorectal cancer Phase of study II

Sponsors Zhejiang University

Study start date July 25, 2020 Oct. 25, 2018 Nov. 30, 2017

Status Not yet recruiting

Clinical trial NCT04131803

Agent Probiotics with standard therapy

NCT03559543

Ocoxin®-Viusid®

II

Catalysis SL

NCT03170115

Induction chemotherapy with aspirin in rectal cancer Aspirin in post-­ surgical metastatic Asian colorectal cancer Aspirin for reduction of colorectal cancer risk Aspirin for Dukes C and high-risk Dukes B colorectal cancer

II

Insituto Nacional de Cancer, Brazil

III

Anhui University

Oct. 2015

Recruiting

N/A

Massachusetts General Hospital

July 6, 2015

Active, not recruiting

III

Cancer Center, Singapore, University of Oxford, Australasian Gastro-Intestinal Trials Group National Cancer Institute, NIH

Dec. 2008

Recruiting

March 2013

Recruiting

Jiangsu Cancer Institute Fred Hutchinson Cancer Center

Sep. 1, Recruiting 2019 Dec. 1, Not yet 2020 recruiting

NCT02607072

NCT02394769

NCT00565708

NCT01349881

NCT04324476 NCT04211766

III S0820, adenoma secondary prevention trail Bevacizumab plus II XELOX/XELIRI I Fiber and fish oil in the prevention of colorectal cancer

Recruiting

Recruiting

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Fig. 2  Causative pathway for alternative treatments. Abbreviations: ROS reactive oxygen species, GSN gelsoin, FBLN1 fibulin, NCL nucleolin, PSAP prosaposin, PEDF pigment epithelium-­ derived factor

down. This similarity of the growth pattern shows that similar to the organ’s growth, the growth of the tumor is also controlled by the positive and the negative growth regulators (Prehn, 1991). A study done by Brown, Malkinson, Rannels, & Rannels (1999) showed that compensatory hyperplasia will often be noticed if we partially remove a tumor. The study also showed that if the tumor mass is not present but the biological signals indicate the presence of tumor mass, then these negative signals will either slow down or cease the tumor growth (Brown et al., 1999). The development of agarose macrobead culture cells is based on these hypotheses. Two concentric layers of agarose, with inner space for cancer cells, create an agarose macrobead. RENCA cells (a mouse renal cortical adenocarcinoma cell line) once encapsulated in these macrobeads develop colonies of a single cell type. In 6–24 months of encapsulation, the growth of these colonies, by following the Gompertzian growth curve, acquires a stable size. These cells undergo a transformation during that period. Minimum two of the cell subtypes create tumor colonies. The conditions in the agarose macrobeads stimulate the production of tumor growth inhibitory factors. These inhibitory factors then reduce or stop the growth of tumor outside the macrobeads both in vitro and in vivo. These inhibitory growth factors are as follows: gelsolin (GSN), fibulin (FBLN1), nucleolin (NCL), prosaposin (PSAP), pigment epithelium-derived factor (PEDF), Serpine1 (Serbp1), secreted protein, acidic and rich in cysteine (SPARC), tissue inhibitor of metalloproteinase 2 (TIMP2), phosphatidylethanolamine-binding protein (PEBP1), and peroxiredoxin-1 (PRDX1). The mechanism of actions of RENCA macrobead is unknown, but it is

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assumed that the above-noted molecules produce several signals, which create a hindrance in the time of the cell cycle of tumor cells, particularly if the time of the S phase is increased, and thus, the number of mitosis decreases (Smith, Gazda, Conn, et al., 2011). At present, the RENCA macrobeads are in phase II–III clinical trials. Like all other medications, these macrobeads although well-tolerated by the patients have some reported side effects. The most common one is fatigue and anorexia, which lasts for days to a few weeks (Ocean, Berman, Parikh, et al., 2015).

4.2  Anti-inflammatory Drugs One of the risk factors for CRC is chronic inflammation. The immune cells and their secretory products are mainly responsible for the inflammatory process. These secretary products are cytokines, chemokines, reactive oxygen species (ROS), reactive nitrogen species (RNS), and arachidonic acid derivatives (COX and lipoxygenase pathway products) (Janakiram & Rao, 2014). As the inflammatory process has a part in the development and advancement of CRC, anti-inflammatory medications can be used prophylactically and also in the management of CRC. For this purpose, the nonsteroidal anti-inflammatory drugs (NSAIDs; which inhibits the COX pathway) are studied well. Over the years, aspirin has been proved to be one of the NSAIDs that really helps in the risk reduction of developing CRC by reducing the inflammatory products (Chan, Arber, Burn, et al., 2012; Nishihara, Lochhead, Kuchiba, et al., 2013; Sutcliffe, Connock, Gurung, et al., 2013). Several systematic reviews and meta-analyses have demonstrated up to a 20–30% reduction in CRC risk with the use of aspirin (Cole, Logan, Halabi, et al., 2009; Dubé, Rostom, Lewin, et al., 2007). The studies done by Giovannucci and colleagues (Chan et al., 2005; Giovannucci et al., 1994; Giovannucci, Egan, Hunter, et al., 1995) demonstrated that for the preventive effect of aspirin, it should be used for a minimum duration of a decade. Even though the results are promising, the United States Preventive Services Task Force (USPSTF) because of the concern of the lag period restricted the long-­ term use of aspirin in cardiovascular disease (CVD) and CRC prophylaxis only for those who have a life expectancy of more than a decade. The daily dose of aspirin and its bioavailability vary, depending on the height, weight, and body mass index (BMI) of an individual. But a relatively low dose (81–100 mg/day) is usually recommended (Petrucci, Zaccardi, Giaretta, et  al., 2019; Rothwell, Cook, Gaziano, et al., 2018). Several studies showed that the higher dose (≥325 mg/day) is presumably required for CRC prevention (Burn, Bishop, Mecklin, et  al., 2008; Burn, Gerdes, Macrae, et al., 2011; Flossmann et al., 2007; Logan et al., 2008; Sandler, Halabi, Baron, et al., 2003). Additionally, some other studies showed that taking a low-dose aspirin every other day is sufficient (Cook, Lee, Gaziano, et  al., 2005; Cook, Lee, Zhang, Moorthy, & Buring, 2013; Rothwell, Wilson, Elwin, et al., 2010).

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Other NSAIDs that have shown some evidence of CRC prevention is sulindac (Rigau et  al., 1991). Sulindac has also an effect on the reduction or cessation of tumor growth when used in combination with atorvastatin (Suh, Reddy, DeCastro, et al., 2011). The newer NSAIDs like COX-2 inhibitors (COXibs) have fewer side effects and better tolerated and their efficacy in the prevention and treatment of CRC has also been demonstrated (Gupta & Dubois, 2001). Newer NSAIDs are being further studied that hopefully will have fewer side effects and that will affect colon cells only; one such example is celecoxib microbeads. These medications are under surveillance in preclinical settings (in vitro studies) only (McDonald, Quinn, Devers, et al., 2015).

4.3  Probiotics The probiotics that are studied most for CRC treatment are the lactic acid bacteria such as Lactobacillus, Streptococcus, Enterococcus, Lactococcus, Bifidobacterium, and Leuconostoc (Chong, 2014; Kahouli, Tomaro-Duchesneau, & Prakash, 2013; Zhong, Zhang, & Covasa, 2014). The mechanisms of action of probiotics are not clear, but its role is supported by the assumption that one of the major risks for CRC is dysbiosis. This hypothesis was assessed by Pala, Sieri, Berrino, et al. (2011) by studying the relationship between yogurt consumption and CRC risk. Chen, Lin, Kong, et al. (2012) studied the mechanisms of action of probiotics in the CRC treatment and prevention and found that these bacteria may induce the apoptosis of diseased cells. The probiotics may also reduce the risk of CRC by reducing oxidative stress in the lumen by producing the antioxidant peptides with free radical scavenging activity (Sah, Vasiljevic, McKechnie, & Donkor, 2014).

4.4  Functional Foods Studies have shown that an excess of reactive compounds (e.g., ROS) can harm any cells in the body by the oxidation of lipids, protein, and DNA (Guimarães, Barros, Carvalho, & Ferreira, 2010). They are involved in the pathogenesis of many diseases, e.g., arthritis or cancer (Ferreira, Barros, & Abreu, 2009). Because of the effects of ROS, sustaining the balance between the ROS and antioxidants is very important in reducing the risk of diseases, including cancers. Keeping this in mind, the polyphenols that are obtained from natural resources can help in reducing the adverse effect of redox. Cereals, legumes, oilseeds, fruits, vegetables, and beverages have ample number of polyphenols (Macheix, Fleuriet, & Macheix, 1990; Velioglu, Mazza, Gao, & Oomah, 1998). Many studies have shown the preventive effects of polyphenols, especially of pro-anthocyanidins, flavonoids, resveratrol, tannins, epigallocatechin-­3-gallate, gallic acid, anthocyanins, and few extracts of plants (Jiménez et al., 2016; Shahidi & Ambigaipalan, 2015).

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4.5  Metal-Based Drugs There are several different types of metal-based drugs that are developed for the treatment of CRC. The most important of them are platinum- and gold-based drugs as described below. 4.5.1  Platinum Among all the metal-based chemotherapeutic compounds, the most valuable one is cisplatin. In the 1960s, the chemotherapeutic effect of the cisplatin was detected by chance (Dasari & Tchounwou, 2014). It causes cell death by binding with the cell DNA, which then produces mono- and bi-functional adducts through inter- and intra-crosslinks (Johnstone, Suntharalingam, & Lippard, 2015; Woźniak & Błasiak, 2002). Although many patients tolerate cisplatin very well, the most common side effects are that they stock up in the proximal tubules of the kidney and cause dysfunction and potential death. It also accumulates in the sensory hair cells of the cochlea and increases the ROS activity, which then results in the apoptosis of the cells (Gonçalves, Silveira, Teixeira, & Hyppolito, 2013; Karasawa & Steyger, 2015; Peres & da Cunha, 2013). Resistance to the action of cisplatin develops once there occurs an enhancement of the repair of cisplatin-DNA adducts (Amable, 2016; Siddik, 2003). Other noteworthy drugs in this group are oxaliplatin and carboplatin. The mismatch repair protein for both cisplatin and oxaliplatin is different; therefore, there is no cross-resistance between these two (Wang & Lippard, 2005). And this is the reason why oxaliplatin has different side effects. We administer oxaliplatin as a part of the FOLFOX regime; this combination increases the response rate by almost 50%. This combination also increases the survival rate in advanced CRC (Ciombor, Wu, & Goldberg, 2015; de Gramont, Figer, Seymour, et al., 2000). 4.5.2  Gold-Based Drugs We use gold in many chemotherapeutic medications, and the most significant gold-­ based anticancer drug is auranofin. This drug inhibits thioredoxin reductase, thus increasing the ROS, which speeds up the process of cell death. As the binding protein differs from that of cisplatin, so we can use it in the tumor’s treatment that developed the resistance to cisplatin (Marzano et al., 2007; Roder & Thomson, 2015).

5  Conclusion and Future Perspectives In order to improve the survival and quality of life of CRC patients, several developments and discoveries are ongoing. Studies are ongoing to develop a sensitive and specific noninvasive screening test. A biomarker is a wonderful discovery in this

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sense, but still more translational works need to be done to improve its efficacy, because these molecules cannot only help us in the diagnosis but also in the determination of treatment and prognosis. Also, there is an intense desire to educate the general population about the risk factors of CRC, and screening, as we all know that prevention is the best treatment strategy. Similarly, advancements in the treatment options will play very vital roles in reducing the prevalence and case fatality rate. Currently, a lot of research works are ongoing in this direction to develop a better treatment with fewer side effects, high efficacy, and cost-effectiveness, which ultimately would improve the quality of life and will increase the survival outcomes. In this regard, agarose macrobeads, NSAIDs, probiotics, functional foods, and metal-­ based drugs are currently under active investigations. Acknowledgments  The authors are grateful to Ms. Kristina K. Greiner for graphics assistance. Conflict of Interest Disclosure  The authors have no conflicts of interest with this research work.

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Wnt Signaling-Related Long Noncoding RNAs: Critical Mediators of Drug Resistance in Colon Cancer P. Vasudeva Raju and RamaRao Malla

Abstract  Colon cancer is the third largest cancer-related death in the world and is increasing in developing countries. Several molecular pathways controlled by multiple molecules and genes are reported to be involved in colon cancer development. Wnt signaling is recognized as a hallmark of colon cancer. It is a critical regulator of the early and late stages of colon cancer metastasis. Emerging studies have suggested that most human genomes have been transcribed as long noncoding RNA (lncRNA). This chapter presents the current status of colon cancer worldwide, therapeutic options for targeting metastasis, and the importance of the Wnt signaling pathway in colon cancer metastasis and describes the downregulated and upregulated lncRNA in colon cancer. This chapter also uncovers the role of Wnt signaling pathway-related lncRNAs in drug resistance and metastasis of colon cancer cells. Finally, this chapter discusses phytochemical therapeutics for targeting Wnt pathway-­related lncRNAs in colon cancer. In conclusion, Wnt pathway-related lncRNAs that modulate the diverse mechanisms of drug resistance and metastatic ability of colon cancer are potential targets of phytochemical therapeutics. Keywords  Colon cancer · Drug resistance · lncRNAs · Metastasis phytochemicals · Wnt pathway

P. V. Raju Department of Biochemistry and Bioinformatics, GIS, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India R. Malla (*) Department of Biochemistry and Bioinformatics, GIS, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India Cancer Biology Lab, Department of Biochemistry and Bioinformatics, GIS, GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_2

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Abbreviations 5-FU 5-fluorouracil ABC ATP-binding cassette APC Adenomatous polyposis coli ATP Adenosine triphosphate B3GALT5-AS1 Beta-1,3-GalTase 5 anti-sense Bax Bcl 2-associated X Bcl2 B-cell lymphoma 2 BRAF B-Raf CASC15 Cancer susceptibility 15 CC Colon cancer CCAT1 Colon cancer-associated transcript 1 CCAT2 Colon cancer-associated transcript 2 CD133 Cluster of differentiation 133 CD44 Cluster of differentiation 44 CK1 Casein kinase 1 CPT Camptothecin CRC Colorectal cancers CRNDE Colorectal neoplasia differentially expressed CUR Curcumin CYTOR Cytoskeleton regulator RNA DNA Deoxyribonucleic acid EGCG Epigallocatechin-3-gallate EGFR Epithelial growth factor receptor EMT Epithelial-to-mesenchymal transition GEN Genistein, quercetin GSK-3 β Glycogen synthase kinase 3 β GWAS Genome-wide association studies HOTAIR HOX transcript antisense RNA HULC Highly upregulated in liver cancer K-ras Kirsten rat sarcoma virus LGR5 Leucine-rich repeat-containing G-protein coupled receptor 5 LIFR-AS1 Leukemia inhibitory factor receptor-antisense 1 lncRNAs Long noncoding RNAs MALAT1 Metastasis-associated lung adenocarcinoma transcript 1 MAP myh-associated polyposis (MAP) MAPK Mitogen-activated protein kinase MDRP 1 Multidrug-resistant protein 1 miRNAs microRNAs MLH1 mutL homolog 1 MMP9 Matrix metalloproteinase9 MRP1 Multidrug resistance protein 1 NATs Natural antisense transcripts

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NF-κB Nuclear factor κ B OX Oxaliplatin PDT Photodynamic therapy P-gp p-glycoprotein PI3K Phosphatidylinositol 3-kinase PTEN Phosphatase and tensin homolog RNAi RNA interference RSV Resveratrol SLAIN2 Slain motif-containing protein 2 SLC Solute carrier STARD13-AS StAR-related lipid transfer domain 13 antisense TCF T-cell factor TEM Transanal endoscopic microsurgery TGF-β Transformation growth factor-β TP53 Tumor protein 53 UCA1 Urothelial carcinoma-associated 1 VEGF-A Vascular endothelial growth factor-A Wnt Wingless-related integration site YAP1 Yes-associated protein 1

1  Introduction Colon cancer (CC) or colorectal cancers (CRC) are related to cancers developed in the large intestine of the gastrointestinal system. The colon is made up of mucosa (the inner lining of the cavity of the intestine), submucosa, muscularis, and serosa. Most colon cancers arise from mucosal cells because they are replaced continuously after shedding in the digestive process. In replacing the degenerated cells with new cells, the cells may lose control over the cell cycle resulting in cancer development. These are continually exposed to toxic substances other than carcinogenic substances that transform the mucosal cells to cancerous cells. Several risk factors like aging, obesity, smoking, modern diet habits, alcohol abuse, family history, etc. are associated with colon cancer development (Dekker, Tanis, Vleugels, Kasi, & Wallace, 2019). The majority of colorectal cases are sporadic, around 5% cases are hereditary, and some cases have a family history (genetic predisposition). The treatment options for colon cancers are surgery, chemotherapy, and radiotherapy, similar to other cancers (Kuipers et al., 2015). Colon cancer is the third largest cancer-related death in the world and is increasing in developing countries. CRC prevalence is more common in men than women and in developed countries compared to developing countries. According to the Cancer Facts and Figures 2020 released by the American Cancer Society, the age-­ adjusted cancer death rates associated with colon cancer in United States of America decrease over the years due to advancements available in the treatment options and

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awareness. The number of new colon cancer cases in America is estimated to be 52,340 and 52,270 for males and females. The estimated deaths are 28,630 and 24,570 in males and females, respectively, according to the report. The incidence of cases and death rates are comprised around 8–9% of the total cancer incidence and deaths. In India, the incidence of colorectal cancers is low compared to the world statistics. The rate of CRC deaths (age-adjusted cancer death rates) in India was found to be 7.2 and 5.1 per 100,000 people in males and females, respectively (Patil et al., 2017; Rawla, Sunkara, & Barsouk, 2019). The patients with symptoms of blood in stools, abdominal pain and bowel habit changes, fatigue, and weight loss are investigated for the presence of cancer tissues. Generally, colonoscopy is performed with video assistance to locate the tumor. By colonoscopy, even biopsy samples can be taken and used for histology for conformation. Biomarkers like APC, PTEN, BRAF, and TP53 are evaluated for the diagnosis of specific colon cancer types. Different treatment options like surgery, chemotherapy, and radiotherapy are employed to treat colon cancer. For nonmetastatic cancers, surgery is the first main option for treating colon cancers by assessing the age, fitness, and stage of tumor of the patients. For the primary CRC type, colon surgery and rectal surgery are performed with or without adjuvant therapy. For early-stage rectal tumors, transanal endoscopic microsurgery (TEM) is performed with minimal invasion. Neoadjuvant and adjuvant treatments are suggested for tumors located deep in the colon and rectum to prevent tumor and metastasis reappearance. Adjuvant therapies include chemotherapy, hormone therapy, radiation therapy, and immunotherapy. For metastatic cancers, multidisciplinary approaches should be employed for the success of the treatment. Chemotherapy is the first-line treatment for metastatic cancers, usually employed as combinational chemotherapy. The combinational chemotherapy includes treatment with leucovorin, 5-­fluorouracil, and oxaliplatin. Combinational chemotherapy is observed to have more beneficial effects than single treatments. Targeted therapies along with combinational chemotherapy are employed for metastatic cancers. The targeted therapies target VEGF-A, EGFR with monoclonal antibodies, and proangiogenic growth factors with fusion proteins (Kuipers et al., 2015). The systemic therapies using neoadjuvant and adjuvant chemotherapeutics show only 50% response in colon cancers. Drug resistance develops almost in all patients, which hinders the therapeutic strategies of chemotherapy. Drug resistance in cancer patients leads to major failures of cancer treatments and finally to death. Drug resistance in cancer may be noncellular (extracellular factors like tumor microenvironment) and cellular (enzymes, transport systems, and drug targets in cancer cells). The transport systems involved in drug resistance are the ATP-binding cassette (ABC) transporters and solute carrier (SLC) transporters. The ABC transporters efflux the drug molecules using the energy obtained by ATP hydrolysis. The ABC transporters like P-gp, MRP1, and BCRP are upregulated in colon cancer and efflux the drug molecules. To improve the chemotherapeutic strategies, agents that downregulate the ABC transporters or inhibit the drug efflux can be employed (Hu, Li, Gao, & Cho, 2016). MicroRNAs (miRNAs) also enhance the therapeutic efforts by reducing drug resistance in cancers. MiR-21 is shown to help the treatments by preventing drug resistance in colon cancers (Wu, Sheng, Zhang, Yang, & Wang,

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2018). Phytochemicals are important molecules that can be used for therapeutic purposes. They confer protection against the disease by different mechanisms. These phytochemicals have been shown to modulate the lncRNAs and thus can be employed to combat cancer in which lncRNAs play a role in pathogenesis. The compounds like curcumin, resveratrol, camptothecin, genistein, quercetin, and epigallocatechin-­3-­gallate are used in cancer treatments.

2  Colon Cancer and Wnt Signaling Several molecular pathways controlled by multiple molecules and genes are reported to be involved in colon cancer development (Fig. 1) (Potter, 1999). The hereditary colon cancers include familial adenomatous polyposis, myh-associated polyposis, hereditary nonpolyposis colorectal cancer, and hamartomatous Colon Cancer / Colorectal cancer (CRC) Hereditary Familial adenomatou s polyposis

Mutation in APC gene

myh associated polyposis

Sporadic

Hereditary non-polyposis Colon cancer

Mutation in myh - gene

Altered Wnt I β - catenin pathway

Cell cycle gene expression changes

Mutation in DNA mismatch repair genes Mutation in K-ras, APC. p53. Wnt genes

Failure of oxidative DNA damage repair

Altered gene expression in cancer related genes

Chromosomal instability

Loss of cell cycle regulated control Dysregulated cell cycle pathways

microsatellite instability

Altered expression of genes involved in DNA repair. apoptosis and cell cycle

Epigenetic modulation

CpG island hypermethylati on in promoter region of genes APC. MLH1 and others

Expression changes in in genes APC. MLH1

Loss of control on cell cycle

Development of CRC

Fig. 1  Molecular pathways leading to the development of colon cancer. Hereditary colon cancers include familial adenomatous polyposis, myh-associated polyposis, hereditary nonpolyposis colorectal cancer, and hamartomatous polyposis syndromes. Familial adenomatous polyposis (FAP) is caused by the germline mutations of the APC gene. APC protein regulates β-catenin degradation. The altered Wnt signaling affects the gene expression of proteins involved in the cell cycle. Myh-associated polyposis (MAP) is due to the mutations in the myh gene involved in the repair of oxidative DNA damage through base excision repair. Sporadic colon cancers include the pathways of chromosomal instability, microsatellite instability, and epigenetic modulation

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polyposis syndromes. Familial adenomatous polyposis (FAP) is caused by germline mutations of APC (adenomatous polyposis coli) gene, which results in the formation of truncated APC gene products. APC protein regulates β-catenin degradation, which is involved in Wnt signaling. The altered Wnt signaling affects the gene expression of proteins involved in the cell cycle. Myh-associated polyposis (MAP) is due to the mutations in myh gene involved in the repair of oxidative DNA damage through base excision repair. The mutations in the DNA mismatch repair genes results in the development of hereditary nonpolyposis colorectal cancer. The sporadic colon cancers include the pathways of chromosomal instability, microsatellite instability, and epigenetic modulation (CpG island methylator). Chromosomal instability pathway describes the deletion or duplication of different chromosomal segments, resulting in mutations in genes related to K-ras, APC, p53, and some tumor suppressor genes. Due to the DNA mismatch repair mechanism’s failure, microsatellite appears in chromosomes, and microsatellite instability (MSI) develops. The microsatellites may alter the expression of genes involved in DNA repair, apoptosis, cell cycle, etc., which lead to the development of colon cancer. Epigenetic modulator substances make the changes in DNA (DNA methylations and histone modification) without affecting the DNA sequence and the gene expressions. The hypermethylation of DNA is observed in genes like APC, MLH1 (mutL homolog 1), etc., which are involved in the development of colon cancer (Aceto, Catalano, & Curia, 2020; Al-Sohaily, Biankin, Leong, KohonenCorish, & Warusavitarne, 2012; Nguyen & Duong, 2018). Wnt signaling is recognized as a hallmark of colon cancer. It is a critical regulator of the early and late stages of colon cancer metastasis (Basu, Haase, & Ben-Ze'ev, 2016). β-catenindependent PI3K, along with Wnt signaling, promotes colon cancer cells’ metastasis by downregulating PTEN expression (Ormanns, Neumann, Horst, Kirchner, & Jung, 2014). Mechanistic studies demonstrated that Wnt signaling promotes metastasis either by direct contact with lncRNAs or through miR-mediated signaling in colon cancer (Sun et al., 2019; Zhang et al., 2018; Zhou et al., 2017). Even β-catenin promotes PI3K and AKT inhibitor resistance and promotes metastasis (Tenbaum et al., 2012). However, lncRNAs counteract stem cells by inhibiting Wnt signaling in colon cancer (Ordóñez-Morán, Dafflon, Imajo, Nishida, & Huelsken, 2015). These studies disclose that Wnt signaling controls metastasis by regulating various lncRNAs.

3  Long Noncoding RNA in Colon Cancer The current scientific literature describes that noncoding RNAs are playing an essential role in gene regulation. The majority of these noncoding RNAs (lncRNA) are short sequences and proved their role in gene regulation. The recent past literature describes the long RNA sequences around 200 pb, and more than 200 bp RNA sequences are discovered and have shown their role in gene regulation. The lncRNAs regulate gene expression both in the nucleus and cytoplasm. In the

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nucleus, the lncRNAs interact with transcriptional machinery to either enhance or repress the transcription. These lncRNAs also modify the DNA architecture epigenetically by guiding chromatin-modifying complexes or regulating nuclear architecture. In the cytoplasm, the lncRNAs either degrade or stabilize the mRNA by different binding positions in the mRNA with associated protein. The lncRNAs and circular RNAs act as binding sites for the microRNAs and sequester the microRNAs, affecting these microRNAs’ regulatory functions in the cytoplasm (Hombach & Kretz, 2016; Mercer, Dinger, & Mattick, 2009). Some of the lncRNAs like colon cancer-­associated transcript 1 (CCAT1), colon cancer-associated transcript 2 (CCAT2), colorectal neoplasia differentially expressed (CRNDE), and urothelial carcinoma-­ associated 1 (UCA1) are shown to be upregulated. These lncRNAs upregulation is associated with tumor progression. Some of them act as blood biomarkers. Genome-­wide association studies (GWAS) in colon cancer revealed that UCA1, CCAT1, RP5-881L22.5, BC005081, and NOS2P3 are upregulated. AK055386, AC078941.1, RP4-800J21.3, RP11-384P7.7, and RP11-628E19.3 are downregulated in colon cancer. Among these, AK055386 is significantly downregulated and UCA1 is significantly upregulated in colon cancer. UCA1 is associated with colon cancer and AK055386 is associated with tumor size (Jing et al., 2017). Recent studies demonstrated the overexpression of HOX transcript antisense RNA (HOTAIR), MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), highly upregulated in liver cancer (HULC), cancer susceptibility 15 (CASC15), and cytoskeleton regulator RNA (CYTOR) and the suppression of B3GALT5-AS1 and STARD13-AS in colon cancer tissues as well as cell lines.

4  W  nt Pathway-Related lncRNAs and Colon Cancer Metastasis Emerging studies have suggested that most human genomes have been transcribed as lncRNAs. The lncRNAs receive increased attention due to the advent of the technologies for sequencing the whole genome and transcriptome. These studies suggest that they function as the intermediary between DNA and protein as well as cellular protagonists. They regulate gene transcription by remodeling chromosomes, processing transcript, and post-transcriptional modifications. Recent studies have recognized that many human diseases are linked to dysregulated lncRNAs, including cancers. The lncRNAs promote cancer metastasis by remodeling chromatin, interacting with chromatin, and acting as ceRNAs and natural antisense transcripts (NATs). LncRNAs interfere with chromatin remodeling machinery by acting as signal lncRNAs or scaffold lncRNAs. Chromatin remodeling impacts the proliferation of cancer cells, progression of tumors, or metastasis via LncRNAs (Table 1). They promote metastasis via different signaling cascades by acting as enhancers, scaffolds, and decoys by physical interaction with RNA molecules or proteins (Sanchez Calle, Kawamura, Yamamoto, Takeshita, & Ochiya, 2018).

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Table 1  Wnt pathway-related lncRNAs in the regulation of colon cancer metastasis Name of the lncRNA HOTAIR

Type of lncRNA Oncogenic

Expression Upregulated

MALAT1

Oncogenic

Upregulated

HULC

Oncogenic

Upregulated

CASC15

Oncogenic

Upregulated

CYTOR

Oncogenic

Upregulated

Functions of lncRNA Mediates colon cancer metastasis by negatively regulating miR-34a Associates with EMT of colon cancer cells High expression related to a higher rate of recurrence Negatively regulates E-cadherin Upregulates vimentin and MMP-9 Regulates TGF-β-induced EMT by reducing the expression of the epithelial marker and enhancing mesenchymal marker Highly expressed in CD133/CD44 positive colon cancer cells Downregulation enhanced the apoptosis by decreasing the expression of Bcl2 and increasing Bax Its gene silencing inhibited metastasis by repressing Wnt/β-catenin signaling YAP1-mediated MALAT1 induced EMT and neovascularization by sponging miR-126-5p Regulates miR-106b-5p by acting as a sponge and competing with SLAIN2-mediated microtubules mobility Promotes metastasis of colon cancer by inducing degradation of β-catenin via negative regulation of GSK-3 β through H3K27-­ dependent trimethylation of the promoter Supports colon cancer development by negatively regulating miR-101-3p Upregulated by cytokines from cancer-­ associated dendritic cells Associate with metastasis of colon cancer Its gene silencing reduced the proliferation and metastasis by miR-613-mediated modulation of RTKN Promotes colon cancer metastasis by activating Wnt/β-catenin signaling Its ablation reduced the colon cancer cell proliferation and metastasis by suppressing leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), target miR-4310 via inhibiting Wnt/β-catenin signaling Promotes metastasis of colon cancer cells by inducing the expression of EMT markers Promotes metastasis by inducing EMT phenotype Induces translation of β-catenin from cytosol to nucleus by inhibiting casein kinase 1 (CK1) Its transcriptional activity is enhanced by β-catenin/TCF complex by forming a positive feedforward (continued)

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Table 1 (continued) Name of the Type of lncRNA lncRNA B3GALT5-AS1 Tumor suppressor STARD13-AS

Tumor suppressor

Expression Functions of lncRNA Downregulated Reduces migration and invasion of colon cancer cells by directly binding to the promoter region of miR-203 Downregulated Overexpression negatively regulates the metastasis by reducing the expression of cyclin E, cyclin D, N-cadherin, and vimentin in the colon cancer cells

HOX transcript antisense RNA (HOTAIR) mediates metastasis through diverse mechanisms. It is reported as an oncogenic promoter and lymph node metastatic mediator in colon cancer (Luo et al., 2016). For example, luciferase reporter gene assay demonstrated that HOX transcript antisense RNA (HOTAIR) mediates colon cancer metastasis by negatively regulating miR-34a (Peng, Zhao, Wei, & Wu, 2019). It is associated with the EMT of colon cancer cells and linked to poor prognosis and can be used as a potent predictor of colon cancer metastasis. In addition, high expression of HOTAIR is related to a higher rate of recurrence. Even HOTAIR can be used as a predictive marker in colon cancer patients’ blood in addition to primary tumor tissues. Its levels in the blood were positively correlated with primary tumor tissue. The hazard ratio of blood HOTAIR was higher, indicating patients’ higher mortality (Svoboda et  al., 2014). Silencing of HOTAIR enhanced the E-cadherin expression and simultaneously reduced the vimentin and MMP9 expression to reduce migration of colon cells (Wu et al., 2014). Also, the expression of HOTAIR and paralogous 13 HOX genes in the colon’s proximal region has significance in predicting colon cancer in patients as both genes were silent in normal tissues but overexpressed in cancer tissues (Tatangelo et al., 2018). It is required for EMT as well as the maintenance of stem cell character in cancer cells. Silencing of the HOTAIR gene abrogated TGF-β-induced EMT of colon cancer cells by reducing the expression of the epithelial marker, E-cadherin, and enhancing vimentin, a mesenchymal marker. Further, the CD133/CD44-positive subpopulation of colon cancer cells expressed higher levels of HOTAIR (Pádua Alves et  al., 2013). These studies suggest that HOTAIR is the most powerful predictive marker of colon cancer metastasis and a poor prognostic marker. MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is a novel class of lncRNA overexpressed in colon cancer tissues and mediates metastasis. The knockdown of MALAT1 by siRNA enhanced the apoptosis by decreasing the expression of Bcl2 and increasing Bax and inhibited metastasis by repressing Wnt/ β-catenin signaling (Zhang, Li, Xue, & He, 2020) in colon cancer cells. However, YAP1-mediated MALAT1 induced EMT and neovascularization by sponging miR-­126-5p (Sun et al., 2019). Further, MALAT1 regulates miR-106b-5p by acting as a sponge to enhance metastasis through competing with SLAIN2-mediated microtubule mobility (Zhuang et al., 2019). Conversely, MALAT1 promotes metastasis of colon cancer by inducing the degradation of β-catenin via the negative

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regulation of GSK-3 β through H3K27-dependent trimethylation of the promoter (Zheng et  al., 2020). A contrasting tumor-suppressive role of the non-canonical PTEN-miR-­MALAT1 axis was reported by Zhi et al. in colon and breast cancers (Kwok, Roche, Chew, Fadieieva, & Tay, 2018). A study by Chunyan et al. demonstrated that MALAT1 supports colon cancer development by negatively regulating miR-101-3p. Interestingly, the knockdown of miR-101-3p counters the antitumor activity of MALAT1 gene silencing (Luan, Li, Liu, & Zhao, 2020). Even MALAT1 is the target of various signaling molecules or regulatory molecules in promoting metastasis in colon cancer. For example, cytokines from dendritic cells associated with tumors promote colon cancer cells’ metastasis by upregulating Snail by enhancing MALAT1 expression (Kan et al., 2015). These studies suggest MALT1 as an oncogene and a promoter of metastasis in colon cancer by modulating different miRs. Recent studies demonstrated that miRNA-mediated lncRNAs regulate metastasis of colon cancers. Highly upregulated in liver cancer (HULC) is one of the oncogenic lncRNAs, associated with the metastasis of colon cancer. It is highly expressed in colon cancer cell lines as well as tumor tissues. Silencing of HULC reduced the proliferation and metastasis of colon cancer cells by miR-613-mediated modulation of rhotekin (RTKN) (Dong, Wei, Lu, & Bi, 2019). Cancer susceptibility 15 (CASC15) is an oncogenic lncRNA, highly expressed in colon cancer tissues, and promotes colon cancer metastasis by activating Wnt/β-catenin signaling. Ablation of CASC15 reduced the colon cancer cell proliferation and metastasis by suppressing leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) target miRNA, miR-4310 via inhibiting Wnt/β-catenin signaling (Jing et al., 2018). Cytoskeleton regulator RNA (CYTOR) or Linc00152 promotes the metastasis of colon cancer cells by inducing the expression of EMT markers. Silencing of CYTOR reduced the metastasis; however, ectopic expression promoted the metastasis by inducing the EMT phenotype in colon cancer cells. This study reported that CYTOR promotes the translation of β-catenin from cytosol to the nucleus by inhibiting casein kinase 1 (CK1). However, the transcriptional activity of CYTOR in the nucleus is enhanced by β-catenin/TCF complex by forming a positive feedforward loop (Yue et al., 2018). B3GALT5-AS1 is a tumor suppressor lncRNA, downregulated in colon cancer tissues metastasized to the liver. Gain-of- and loss-of-function studies suggested that B3GALT5-AS1 negatively regulates the migration and invasion of colon cancer cells by directly binding to the promoter region of miR-203 (Wang et al., 2018). Overexpression of miR-203 suppresses the metastasis by repressing the expression of Wnt (Chen et  al., 2019). Another tumor suppressor, lncRNA STARD13-AS, is a GTPactivating protein for Rho and is poorly expressed in colon cancer patients. However, its overexpression negatively regulates metastasis by reducing the expression of cyclin E, cyclin D, N-cadherin, and vimentin in the colon cancer cells (Yang et  al., 2019). These studies infer that Wnt signaling-­ dependent lncRNAs negatively regulates metastasis in colon cancer.

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5  Wnt Pathway-Related lncRNAs in Drug Resistance Expression profiling of lncRNAs in clinical colon cancer tissues has been suggested to be involved in the activation or inactivation of the Wnt pathway to regulate drug resistance (Table 2). The scientific developments showed the evidence for the mediation of lncRNA-miRNA complexes in the functions of lncRNAs in drug resistance. Many lncRNAs bind to miRNA to regulate target genes’ expression instead of colon cancer target mRNAs. Most of the activated Wnt signaling pathways promote drug resistance. For instance, HOTAIR enhances drug resistance in colon cancer via miR-203a-3p-dependent Wnt/β-catenin signaling pathway (Xiao et al., 2018). With constant chemotherapeutic drug treatment (5-fluorouracil, oxaliplatin, and irinotecan), the expression of lncRNA LINC00973 increased, possibly to develop resistance in colon cancer cells (Zinovieva et al., 2018). Kuijie Liu et al. reported that lncRNAs dysregulated in photodynamic therapy (PDT) treated the colon cancer cells. This study demonstrated that LIFR-AS1 is significantly upregulated in PDT-­ treated CC cells, which mediates resistance to PDT by negatively interacting with miR-29a (Liu et al., 2018). LncRNA POU6F2-AS2 mediated resistance in the CC cells. The upregulation of POU6F2-AS2 promoted resistance to 5-FU by increasing Table 2  Wnt pathway-related lncRNAs in the regulation of drug resistance in colon cancer Name of the lncRNA HOTAIR

Type of lncRNA Oncogenic

LIFR-AS1

Oncogenic

POU6F2-AS2

Oncogenic

snaR

Tumor suppressor

SNHG15

Oncogenic

CCAT1

Oncogenic

HT 19

Oncogenic

PVT1

Oncogenic

Mechanism of drug resistance Enhances drug resistance in colon cancer via miR-203a-3p-­ dependent Wnt/β-catenin signaling pathway Mediates resistance to photodynamic therapy by negatively interacting with miR-29a Promotes resistance to 5-FU by increasing the cell proliferation and cell cycle via silencing the expression of miR-377 and enhancing BRD4 expression Silencing of POU6F2-AS2 enhanced the sensitivity of CC cells to 5-FU Downregulated in 5-FU-resistant CC cells Silencing decreases the apoptosis by increasing the viability and cell cycle progression Silencing enhanced the sensitivity of CC cells to 5-FU by decreasing the expression of Myc Mediates 5-FU resistance in CC cells by decreasing the apoptotic rate Silencing using siRNA enhanced the 5-FU sensitivity by increasing the apoptotic rate Overexpression mediates CC cells’ resistance to 1,25-dihydroxy D3 vitamins by inhibiting the expression of vitamin D3 receptor via miR-675-5p Promotes cisplatin resistance in CC cells by enhancing cell proliferation, migration, and invasion as well as promoting escape from apoptosis via increasing the expression of Bcl2-dependent MDR1 and MDRP 1

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the cell proliferation and cell cycle via silencing the expression of miR-377 and enhancing BRD4 expression. However, silencing of POU6F2-AS2 enhanced the sensitivity of CC cells to 5-FU (Xu et  al., 2020). In contrast, lncRNA snaR was downregulated in the 5-FU-resistant CC cells, and siRNA-mediated silencing decreased the apoptosis by increasing the viability and cell cycle progression (Lee et al., 2014). Silencing of SNHG15 enhanced the sensitivity of CC cells to 5-FU by decreasing the expression of Myc (Saeinasab et  al., 2019). Another lncRNA, CCAT1, mediates 5-FU resistance in CC cells by decreasing the apoptotic rate, whereas silencing using siRNA enhanced the 5-FU sensitivity by increasing the apoptotic rate (Yang, Pan, & Deng, 2019). The transfer of exosomal lncRNA, HT 19, from cancer-associated fibroblasts enhanced the stemness as well as chemoresistance in the CC cells by activating β-catenin through sponging with miR-141 (Ren et al., 2018). However, overexpression of HT19 mediates CC cells’ resistance to 1,25-dihydroxy D3 vitamins by inhibiting the expression of vitamin D3 receptor via miR-675-5p (Chen et al., 2017). Abnormally expressed lncRNAs mediate resistance to vincristine in the CC cells (Sun et al., 2015). Plasmacytoma variant translocation 1 (PVT1) promotes cisplatin resistance in the CC cells by enhancing cell proliferation, migration, and invasion as well as promoting escape from apoptosis via increasing the expression of Bcl2-dependent MDR1 and MDRP 1 (Ping et al., 2018). These studies suggest that various Wnt pathway-related lncRNAs mediate the drug-resistant mechanisms in colon cancer.

6  W  nt Pathway-Related LncRNAs in Colon Cancer Treatment Wnt signaling-dependent lncRNAs are known to mediate drug resistance in colon cancer. The deregulated lncRNAs significantly support drug resistance via Wnt/β-­catenin signaling pathway in many tumors, including colon cancer (Hu et al., 2018). Therefore, the association of lncRNAs with a specialized characteristic of colon cancer tissues, especially the Wnt/β-catenin signaling pathway, may contribute as a potential biomarker as well as a therapeutic target (Zhang, Li, Zhou, & Lu, 2020). LncRNAs modulate the gene expression patterns as discussed in the above sections by activating the expression or repressing the expression. The altered gene expression of specific LnCRNAs is associated in the development of cancers. Some of the treatment options include preventing the gene expression changes associated with cancer development. This can be achieved by the using technologies like RNAi, etc., to interfere with the pathogenesis-specific gene changes. But the success of these treatments depends on the selection and design of such interfering agents. In  this regard some phytochemicals have shown to modulate the functions of lncRNAs, which can be employed to combat the disease progression. For example, consider certain lncRNAs may activate genes that lead to development cancer or activate oncogenes. In this condition we can employ naturally available compounds

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Table 3  Wnt pathway-related lncRNAs in colon cancer treatment Phytochemical Cellular/molecular pathway Curcumin Targets HT 19 lncRNA Inhibits pathogenic signaling cascade of tumor development Silencing of curcumin-induced upregulated PANDAR increased apoptosis rate in CRC cells Sensitizes gemcitabine resistant cells by targeting PVT1 Resveratrol Upregulates the lncRNAs, HOTAIR, PCA3, and NBR2 Downregulates the lncRNAS PVT1, H19, and UCA1 Reduces the expression of MALAT1 EGCG Induces the expression of cisplatin transporter CTR1 by targeting NEAT1

from plants, which we use as our food sources to inhibit lncRNA-induced activation. Similarly some lncRNAs may specifically inhibit the tumor suppressor genes and lead to cancer development; some phytochemicals can prevent this. Several phytochemical molecules have been reported to have the potential to work as modulators of lncRNAs (Table 3). Some phytochemicals include curcumin, resveratrol, camptothcin, genistein, quercetin, epigallocatechin-3-gallate, sulforaphane, berberine, taxol, etc. (Mishra et al., 2019). The preventive role of some phytochemicals in modulating the lncRNA involved in the pathogenesis of cancer are discussed below. Curcumin is a polyphenol obtained from the plant Curcuma longa (turmeric plant). Curcumin has several pharmacological applications and widely used in food preparations. Curcumin is reported to have antioxidant, anti-­ inflammatory, neuroprotective, immunomodulatory, anti-proliferative, and hepatoprotective properties (Zielińska et al., 2020). Apart from a wide range of beneficial effectives, curcumin as an anticancer agent is widely reported and proposed to be included in cancer treatment strategies. Curcumin provides these beneficial effects through different mechanisms by interacting with different cancer molecules (Rodrigues, Anil Kumar, & Thakur, 2019; Wang et al., 2019). It has shown to modulate the effects of some lncRNAs which are involved in the development of different cancers. H19 is an oncogenic lncRNA that is overexpressed in breast cancer, esophageal cancer, lung cancer, etc., and involved in tumor development by altering multiple down signaling. Curcumin inhibits the overexpression of H19 oncogene in different cancer models and inhibit pathogenic signaling cascade of tumor development. Similarly curcumin is shown to modulate the lncRNAs like PVT1, HOTAIR, AK056098, UCA1, PANDAR, etc. (Liu et al., 2019; Mishra et al., 2019). In colon cancer, the lncRNAs alter TGF-β, Wnt, and EMT signaling pathways, leading to cancer pathogenesis. Curcumin is shown to modulate Wnt pathway and confer protection against the Wnt pathway-induced tumor development. The events of cancer development, proliferation, senescence, and apoptosis are not affected by the knockdown of lncRNA PANDAR in vitro models of colon cancer. But silencing of curcumin-induced upregulated PANDAR increased apoptosis rate in CRC cells. This indicates that curcumin-sensitized PANDAR and other drugs target the PANDAR responding for the treatment. This complex mechanism still has to be explored to

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establish curcumin’s potential role in modulating CRC-related lncRNAs (Chen, Yang, Wang, & He, 2017). Resveratrol is a naturally occurring polyphenolic phytoalexin in grapes, peanuts, berries, and red wine. The compound has antioxidant, anti-inflammatory, and anticancer properties. Resveratrol modulates cancer-related process like proliferation, invasion, metastasis, and apoptosis and acts as a potential anticancer drug. Resveratrol can target the noncoding RNAs involved in the development of different cancers (Wang, Jiang, Yu, Zhou, & Wang, 2019). The literature reported that resveratrol upregulated the lncRNAs, HOTAIR, PCA3, and NBR2 and downregulated the lncRNAS PVT1, H19, and UCA1. These lncRNAS are reported to be involved in the development of different cancers, and resveratrol can be used to modulate these lncRNAs for treatments. Resveratrol has been shown to modulate these lncRNAs in ovarian cancer cells (Vallino et al., 2020). MALAT1, a long noncoding RNA, is found to be overexpressed in colon cancer tissues and positively correlated with invasion and metastasis. MALAT1 is downregulated by resveratrol, which inhibited the Wnt/β-catenin pathway, thus preventing invasion and metastasis (Ji et al., 2013). Epigallocatechin gallate (EGCG) is an important major catechin found in green tea and is reported to have antioxidant, antiangiogenic, and antitumor properties. It prevents carcinogenesis by modulating the different signaling pathways involved in carcinogenesis like Wnt, MAPK, PI3K/AKT, etc. (Singh, Shankar, & Srivastava, 2011). EGCG affected the several lncRNA expressions in altering the lncRNA-­ induced carcinogenesis (Hu et al., 2019). EGCG is reported to show some beneficial role in combatting colon cancer by modulating the NF-κB/miR-155-5p/MDR1 pathway and kinase pathways (Cerezo-Guisado et al., 2015; La, Zhang, Li, Li, & Yang, 2019). The mechanism of EGCG in preventing colon cancer by modulating lncRNAs needs to be explored. It induces the expression of cisplatin transporter CTR1 by targeting NEAT1 (Jiang, Wu, Wang, Huang, & Feng, 2016). Apart from the above, naturally occurring phytochemicals like genistein, paclitaxel, silibinin, anacardic acid, emodin, gambogic acid, and quercetin are also studied for their potential in preventing tumor development through lncRNA modulation. The modulation of lncRNAs that induce cancer by phytochemicals is an important strategy and can be employed to prevent cancer progression.

7  Conclusion Colon cancer (CC) or colorectal cancers (CRC) are related to cancers developed in the gastrointestinal system’s large intestine. Most colon cancers are arising from the mucosal cell because the mucosal cells are constantly replaced after shedding in the digestive process. In replacing the degenerated cells with new ones, the cells may lose control over the cell cycle, resulting in cancer development. These cells are constantly exposed to toxic substances other than the carcinogenic substances that transform the mucosal cells to cancerous cells. LncRNAs are a diverse group of

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transcripts that regulate the metastasis of colon cancer via modulation of gene expression. LncRNAs target several signaling pathways to modulate cellular biological processes. However, the Wnt signaling pathway is very critical for metastasis and drug resistance. The lncRNAs promote malignant transformation signals via their DNA, protein, and RNA interactions to inactivate or activate the Wnt/β-catenin signaling pathway. Therefore, lncRNAs can be considered as potential targets of colon cancer. Phytochemicals are essential molecules that can be used for therapeutic purposes. They confer protection against the disease by different mechanisms. These phytochemicals have been shown to modulate the lncRNAs and thus can be employed to combat cancer in which lncRNAs play a role in pathogenesis. Acknowledgments  The authors thank the GITAM management for providing the infrastructural facilities. Funding Details  The corresponding author, Prof. RamaRao Malla (receiver of the grant), thanks CSIR, New Delhi, India (file no. 37(1683)/17/EMR-II; dated: 05.05.2017), for providing funding to carry out this work. Conflict of Interest  The authors declare that there is no conflict of interest.

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The Triad of Estrogen, Estrogen Receptors, and Colon Cancer K. R. Sumalatha, Syamala Soumyakrishnan, and M. Sreepriya

Abstract  Colon cancer is one of the common malignancies in the developed world irrespective of the sexes. The incidence of colon cancer is increasing leaps and bounds in the developing world also during the past few decades. Several risk factors including diet, environmental triggers, sedentary life style, and genetic susceptibility are known to predispose an individual for the development of colon cancer. Sex hormone estrogen plays a major role in various metabolic processes in the body, and deficiency in the levels of estrogen is reported to be a primary causative factor in the pathogenesis of many diseases including postmenopausal osteoporosis, cardiovascular disease, immune dysfunction, and many others. On the contrary, estrogens are also proven to exert a proproliferative effect in the reproductive organs, thereby establishing their role in the development of breast and endometrial cancers. Interestingly, hormone replacement therapy with estrogen is proven to reduce the risk of colorectal cancer (CRC) in postmenopausal women. Estrogen exerts its action by binding with nuclear receptors ERα and ERβ and also through binding with the membrane bound G-protein coupled estrogen receptor (GPER). Recent research has proven that the distribution of nuclear estrogen receptors alpha and beta in different organs and the preferential binding of estrogen to either of these receptors might be instrumental in determining the crucial switch between procarcinogenic and anticarcinogenic effects of estrogen. Reduced expression of the nuclear estrogen receptor beta in the colon is reported to enhance the risk of an individual for the development of CRC. This implicates the usefulness of estrogen receptor β expression as a reliable prognostic and diagnostic marker for CRC. In short, clinical endocrine therapies based on the estrogen-regulated expression of estrogen receptors, development of tissue selective estrogen receptor modulators, and phytoestrogen-based diets can all play a pivotal role in the prevention and therapy of colon cancer in the future. This chapter discusses the influence of estrogen and its constituent receptors in the development of colon cancers and as to how these can be exploited in the diagnosis, to monitor prognosis and to plan therapeutic regimes for the management of malignancies of the colon.

K. R. Sumalatha · S. Soumyakrishnan · M. Sreepriya (*) Department of Microbiology and Biotechnology, Bangalore University, Bangalore, Karnataka, India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_3

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Keywords  Colon cancer · CRC · Estrogen · Estrogen receptor · GPER

Abbreviations CIMP CIN CRC ER ERE FPA GPER HNPCC SERD SERM

CpG island methylator phynotype Chromosomal instability colorectal cancer Estrogen receptor Estrogen responsive elements Familial adenomatous polyposis G-protein coupled estrogen receptor Hereditary nonpolyposis colorectal cancer Selective estrogen receptor degrader Selective estrogen receptor modulator

1  Introduction Cancer progression is a multistep process in which there is a complex interplay between molecular targets in the cell signaling pathways that are triggered by alterations in the genetic and environmental factors. Cancer of the colon and rectum is one of the major causes of morbidity and mortality worldwide. Although colon cancer was originally considered to be a cancer of the Western world due to high prevalence owing to dietary habits, the scenario is quite different now with prevalence seen almost in all parts of the world. This is attributed to changing diet patterns, sedentary life style, stress triggers, and changes in the environment. Colorectal cancer (CRC) is the third most common cancer causing over 700, 000 deaths per year globally (Kuipers, Grady, Lieberman, et al., 2015). It is the leading type of malignancy in women (second) and men (third). It is a multifarious disease perhaps amassing mutations of epithelial and preneoplastic cells that predominantly contribute to the molecular pathogenesis (Granados-Romero, Valderrama-Treviño, Contreras-Flores, et al., 2017). The incidence of colorectal cancer has constantly increased in the recent times as compared to the last few decades. CRC survival is poor, with an overall 5-year survival rate of approximately 45% (Dray, Ruault, Sapinhi, Bouvier, & Faivre, 2003). Colorectal cancer is usually not categorized under hormone-related malignancies. But sex hormones androgens and estrogens and their respective receptors have long been accepted as important factors influencing the onset and progression of CRC. In addition to androgen receptor and nuclear estrogen receptors (alpha and beta) which are clearly proven to have a definitive link in the development of several malignancies, a newly identified estrogen receptor called G-protein coupled

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estrogen receptor (GPER) also referred to as GPR30 has been strongly associated with cancer pathogenesis (Jung, 2019). This chapter discusses in detail the role of the sex hormone estrogen and how its preferential binding with nuclear/membrane receptors could play a pivotal role in the prevention or development of colon carcinogenesis.

1.1  Risk Factors for Colon Cancer Gender is associated with CRC incidence and progression in addition to age and other risk factors. Considering the same age, men have a higher prevalence of CRC than women. Right-sided colon cancer is more common in women and rectal cancer more common in men. Furthermore, a better survival is observed in young women compared to young men after CRC surgery, but in older patients the opposite pattern exists (Jensen, Jacobson, Flesher, et al., 1966; Liao, Tzeng, Yu, et al., 2015). Although the role of gender in the occurrence of colorectal cancer remains imprecise, as the age increases, the prevalence for colorectal cancer also increases. Unhealthy diet, lack of physical activity, obesity, smoking, and alcohol consumption are the modifiable risk factors for the development of CRC (Amersi, Agustin, & Ko, 2005; Haggar & Boushey, 2009). Genetic inheritance/family history of colorectal cancer, hereditary nonpolyposis colorectal cancer (HNPCC), and colonic polyps are the common driving factors to CRC (Henrikson, Webber, Katrina, et al., 2015; Lynch, 2017). In addition to these, personal history of inflammatory bowel disease (IBD) is considered as the third highest risk for the development of CRC, behind only familial adenomatous polyposis (FAP) and HNPCC (Kim & Chang, 2014) (Table  1) (Kim & Chang, 2014). It is well established that in women, an increased level of the hormones estrogens and progestins due to pregnancy or exogenous administration is inversely associated with colorectal cancer development. In agreement with these results, the Women’s Health Initiative estrogen plus progestin clinical trial reported an approximately 40% decreased risk for the development of Table 1  The list of modifiable and nonmodifiable risk factors for colon cancer S. No 1 2

Modifiable risk factors Unhealthy diet Sedentary life style

3 4 5 6

Obesity Smoking Alcohol consumption Suppressed immunosurveillance

Non modifiable risk factors Genetic predisposition/family history Hereditary nonpolyposis colon cancer (HNPCC) Familial adenomatous polyposis Colonic polyps Inflammatory bowel disease Gender

The list of modifiable and nonmodifiable risk factors that could play a role in the onset and progression of colon cancer. Diet as a modifiable risk factor and genetic predisposition/family history as a nonmodifiable risk factor are of prime importance in the pathogenesis of colon carcinogenesis.

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colorectal cancer in treatment group versus placebo group (Chlebowski et al., 2004). Interestingly, the reports published by the other Women’s Health Initiative trial did not indicate a low risk for colorectal cancer among women who underwent hysterectomy and treated with estrogen (Anderson et al., 2004). In support of this finding, two observational studies also implicated that there is no decreased colorectal cancer risk in postmenopausal women who were treated with higher levels of estradiol. The endogenous production of sex hormones and their biological effects on the target tissue is dependent on multiple factors like enzymes involved in synthesis, metabolism of these hormones, their interactions with constituent receptors and epigenetic /genetic modifications of the genes involved. All these factors can have a significant influence in determining the risk outcomes of an individual for colon cancer (Gunter et al., 2008). Not much is known with respect to the link between colorectal cancer risk in men and sex hormone levels. One report implicate that low levels of androgen may increase the risk of colorectal cancer in men (Clendenen et al., 2009). Increased CAG repeats in the androgen receptor gene resulted in lower transcriptional activation and hence reduced androgenic effect in tissues which eventually was found to be associated with a high risk for colon cancer (Slattery et al., 2005).

1.2  Diet as a Major Risk Factor for CRC Based on epidemiological studies, diets with high amounts of meat and fat have been hypothesized to increase the incidence of CRC, whereas a diet rich in fiber, fruits, and vegetables has been reported to reduce the risk of CRC.  But recent research has found a poor correlation between diet and CRC incidence. Therefore the influence of diet on CRC risk could be considerably lesser than what was previously reported (Potter, 1996). It was reported by Alexander, Cushing, LowekaSceurman, and Roberts (2009) based on a large meta-analysis which involved data from six prospective cohort studies that there is no correlation between either dietary fats or animal protein and CRC. A meta-analysis by Koushik, Hunter, Spiegelman, et al. (2007) which was based on 14 cohort studies showed that fruit and vegetable intakes were not associated with CRC risk overall but could contribute toward a reduced risk for rectal cancer. There are evidences which strongly suggest that folic acid, calcium, and vitamin D intake could reduce the risk for CRC (Harshman, 2007).

1.3  E  tiology and Molecular Mechanisms Involved in the Development of CRC Colon carcinogenesis is divided into three discernible stages similar to other types of carcinogenesis either sporadic or hereditary/preexposure to inflammatory bowel diseases (IBDs) such as ulcerative colitis and Crohn’s disease (Bye, Nguyen, Parker,

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et  al., 2017). Comparable to other type of cancers, CRC is also categorized into three distinct stages of pathological transformation such as initiation, promotion, and progression (Terzic, Grivennikov, Karin, & Karin, 2010). These changes include the conversion of normal colonic epithelium into hyper proliferative epithelium and eventually into adenoma and carcinoma in situ and finally to invasive and metastatic cancer (Riihimäki, Hemminki, Sundquist, & Hemminki, 2016). Oncogene (KRAS) and tumor suppressor genes such as APC, Smad4, and p53 are the important genes entailed in colon carcinogenesis (Sameer, 2013). CRC is a heterogenous as well as a multifactorial disease. The major molecular mechanisms involved in the development of CRC are (i) genomic instability, such as chromosomal instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP); (ii) genomic mutations including the suppression of tumor suppressor genes and activation of tumor oncogenes; (iii) microRNA; and (iv) epigenetic changes. CRC arises from one or a combination of these mechanisms (Tariq & Ghias, 2016). Statistically, approximately 70% of CRC cases arise sporadically from adenoma to carcinoma state in the alteration to specific morphological traits. About 5% are linked to inherited traits (familial adenomatous polyposis (FAP), hereditary nonpolyposis CRC or Lynch syndrome, and MUTYH-related polyposis), and the remaining 25% of CRC are connected with familiarity, whereas not many cases are associated with high microsatellite instability (MSI) and impairment in DNA mismatch repair (Afrin, Giampieri, Gasparrini, Forbes-Hernández, et al., 2020). Hereditary type of CRC such as familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) represents merely 10% of the total CRC incidence, whereas highest cases of CRC arise sporadically by means of the polyp-cancer sequence, a term that was first ascertained in 1975 (Lechner, Kállay, & Cross, 2005). Invasion and metastases are assisted via the epithelial-­ mesenchymal transition (EMT), with additional genetic alterations during the progression of cancer. Colorectal adenocarcinoma involves the genetic and epigenetic alterations that gather in a sequential manner (Pancione, Remo, & Colantuoni, 2012). The development of tumor in the human body takes a longer time as it involves many stages with lot of episodes to attain the capability of progressive transformations from normal cell to cancerous cell (Sarkar, Horn, & Moulton, 2013). Studies have shown that in CRC development, the initial step involves the reallocation of the proliferative zone in the glandular crypts, along with the formation of atypical crypt foci and adenomatous polyp (Tanaka, 2009). Fearon and Vogelstein, in 1990, demonstrated that CRC development by the classical CIN pathway involves the attainment of mutations in the adenomatous polyposis coli (APC), the mutational inactivation of TP53 (tumor protein p 53) – the tumor suppressor gene  – and the activation of KRAS oncogene. In CIN tumors, genetic instability caused by aneuploidy and loss of heterozygosity (LOH), which amount to sporadic tumors as well as familial adenomatous polyposis (FAP) cases, correlated with germline mutations starting from a biallelic inactivation of APC gene. The mutations in the tumor suppressor or cell cycle genes perhaps cause cellular transformation (Tariq & Ghias, 2016). Epigenetic alterations cause aberrant methylation of tumor suppressor genes (APC, TGFBRII, IGF2R, BAX), impacting

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the inactivation of these genes and consequent advancement of neoplasia (Kasprzak & Adamek, 2019; Pinheiro, Ahlquist, Danielsen, et al., 2010). The formation of CRC generally takes place from the large intestinal glandular epithelial cells. Some cells of the epithelium gain a sequence of genetic or epigenetic mutations that grant on them a choosy lead to develop cancer. Carcinoma development and metastasis advance from a benign adenoma over decades by the anomalously intensified replication and survival that direct the hyperproliferative cells into progression (Ewing, Hurley, Josephides, & Millar, 2014). Reabsorption of water and remaining minerals and nutrients in the chime is the chief function of the colon (Bassottia & Battagliab, 2015). The diverse microflora of the large intestine degrades the excess starches and proteins. The gastrointestinal epithelium is arranged as an axis of crypts and villi so as to enhance the absorption (Thompson, Forest, & Battle, 2018). The pluripotent colon stem cells and progenitor cells were positioned in the lower part of the crypt and exhibit a key role of self-renewal (Umar, 2010). Progenitor cells undergo differentiation into specialized epithelium cells, and they drift out of the crypt and up the villus. The Paneth, enteroendocrine, and goblet cells as well as the enterocytes are the differentiated epithelial cells. These cells endure apoptosis; after a period of 2 weeks, they reach the crest of the villus and are discarded with the feces. These events are extremely controlled by a variety of vital cell signaling proteins such as Wnt, TGF-β, and BMP (Rawla et al., 2019).

1.4  Treatment Strategies Different types of CRC show dissimilar driving mutations, owing to which it is extremely difficult to have a common therapeutic strategy that suits every type. Surgical intervention is the primary recommended course of treatment that mainly depends upon the progression stage, tumor topography, and the existence of other comorbid conditions and health complications (Mastalier, Tihon, Ghiţă, et  al., 2012). In general, the ultimate CRC treatment is to accomplish complete removal of the tumor and metastatic lesions, which predominantly needs surgical intervention. Depending upon the size, surgical treatments such as (a) polypectomy (Zauber, Winawer, O’Brien, et al., 2012), (b) endoscopic mucosal resection (EMR) (Deyhle, Largiader, Jenny, et  al., 1973), and (c) laparoscopic surgery (Cianchi, Trallori, Mallardi, et al., 2015) are recommended. While these are recommended in the case of very early cancer, in advanced cases with cancer invading into the colon, the approaches that were usually followed by surgeons are (a) partial colectomy (PC) (Guan, Zhao, Yang, et  al., 2017) and (b) lymphadenectomy. However, in very advanced stages, if the cancer has spread invasively, surgery is considered noneffective. In patients with advanced disease, the development of drug resistance and cancer recurrence will rapidly emerge and cease the effectiveness of cytotoxic and neoadjuvant therapies. Inspite of tremendous development in screening programs to enhance the reduction in CRC incidence, almost a quarter of CRCs are diagnosed at a progressive stage with metastasis. The options to treat such patients considerably

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are radiotherapy and chemotherapy. Targeted therapy is a novel approach that has promising survival chances for CRC patients. Cetuximab and bevacizumab are the successful anti-EGFR (epidermal growth factor receptor) agent and anti-­ angiogenesis agent, respectively, that are used in colon cancer treatment (Xie, Chen, & Fang, 2020). 5-Fluorouracil-based therapy is generally considered as the first-line treatment in advanced cancer patients. Novel agents such as oxaliplatin, irinotecan, and capecitabine have considerably increased the choices available to deal with advanced colorectal cancer patients and has subsequently improved the survival rate (Gill, Thomas, & Goldberg, 2003). New chemotherapy regimens, the application of prognostic testing, and the combination of new targeted therapies are the areas of vibrant clinical investigation (André & Gramont, 2004).

2  Estrogens: The Multifunctional Hormones Estrogens are the dominant sex hormones present in women, but they are also present in men. These are cholesterol-derived steroid hormones produced via the aromatization of androgens mainly in the ovaries, corpus luteum, and placenta (in women) but also in other tissues, such as adipose, muscle, and nervous tissue, liver, breast (in both women and men), and Leydig cells of the testes (in men) (Mauvais-Jarvis, Clegg, & Hevener, 2013). There are three main forms of circulating estrogens in women: estrone (E1), which is dominant in postmenopausal women and is mainly produced via the aromatization of androgens in the adipose tissue; 17β estradiol (E2), which is the most potent form of estrogen and predominant in premenopausal women; and estriol (E3), which is the weakest form of estrogen and predominant during pregnancy (Cui, Shen, & Li, 2013). Estrogens play a key role in the reproductive function, development of primary and secondary sexual characteristics, and sexual behavior in women. Estrogens are also important for many physiological functions in both men and women, such as cell growth and differentiation, as well as maintaining a normal bone metabolism, cardiovascular, nervous and immune system (Lombardi et al., 2001). Estrogens on the other hand are implicated in various pathological conditions including cancers of reproductive tissues, such as ovary, uterus, and breast cancer in women and prostate cancer in men, but also cancers of nonreproductive tissues including the gastrointestinal tract (Burns & Korach, 2012; Jia, Dahlman-Wright, & Gustafsson, 2015). All these actions of estrogens in the human body are mediated via specific proteins inside the cell known as estrogen receptors (ER).

2.1  Synthesis of Estrogens Each form of estrogen is derived from cholesterol by a series of reactions. The key product from the overall biosynthetic process is E2 which is reported to be the most biologically active and potent form of estrogen in a woman’s life during

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premenopausal stage. E1 is largely produced by the adipose tissue from the dehydroepiandrosterone of the adrenal gland. E1 exerts a major role during postmenopausal period. E3 referred to as the weakest estrogen is derived from E1 by 16α-hydroxylation. It has a key role during pregnancy when the placenta produces high levels of this form of estrogen. The levels of E2 can be regulated by estrogen deactivation that involves a strong control on estrogen metabolism resulting in the conversion of E2 to a less effective E1 or E3 (Birkhauser, 1996) and E2 sulfation mediated through estrogen sulfotransferase forming 17 beta-estra-1,3,5-triene-3,17-diol-3-sulfate that does not interact with the receptors of estrogen (Kotov, Falany, Wang, et al., 1999). Hence the ratio of estrogens in the circulation could indicate an exquisitely delicate balance existing between the synthesis and deactivation of estrogen. The most prevalent mechanism controlling the synthesis of estrogen is through the regulation of aromatase enzyme which is a key enzyme in the final step of E2 synthesis. Aromatase is a member of the cytochrome P450 superfamily and is widely expressed in many sites, including the brain, gonads, blood vessels, liver, bone, skin, adipose tissue, and endometrium (Santen, Brodie, Simpson, et al., 2009). The expression of aromatase enzyme in a specific tissue is determined by three important factors: presence of tissue-specific promoters, availability of required transcription factors, and alternative splicing processes. Aromatase is expressed in the gonads of both sexes. Aromatase expression is found exclusively in luteal and granulosa cells of the ovary. Alternatively in the gonads of male, aromatase is majorly expressed in the accessory glands and testis, thereby facilitating the production of higher levels of E2 required for spermiogenesis, maturation, and motility of the sperms (Carreau, de Vienne, & Galeraud-­ Denis, 2008). Estrogen synthesis differs between reproductive and nonreproductive women. In nonreproductive women, such as young females before puberty or women after menopause, extragonadal sites are the main sources of estrogens, including the kidney, adipose tissue, skin, and brain. Unlike the ovarian-synthesized estrogen, which is mainly released into the bloodstream, estrogen synthesized within these extragonadal compartments mostly acts locally at the site of synthesis and functions as a paracrine and/or intracrine factor to maintain important tissue-specific functions (Inoue, Miki, Abe, et al., 2012). Estrogen synthesis is also occurring in male testes and acts locally to regulate normal male gonadal development and spermatogenesis, in particular spermiogenesis. Estrogen production in all tissues contributes to cellular health. The two most important target sites of E2 production are the ovaries and brain.

2.2  The Nuclear Receptor Superfamily Receptors (proteins) present inside the cells and that possess transcriptional effects are referred to as nuclear receptors. When they get activated by their ligands, they are transported into the nucleus, regulating the expression of their target genes

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(Huang, Chandra, & Rastinejad, 2010). In humans, the nuclear receptor superfamily includes 48 members, which are involved in regulating many important physiological activities such as cell metabolism, reproduction, inflammation, immune response, cell proliferation, and electrolyte balance. The endogenous ligands for nuclear receptors include small and lipophilic molecules such are steroid hormones (estrogen, progesterone, androgens, glucocorticoids, mineralocorticoids), thyroid hormones, and vitamins A and D.  Furthermore, exogenous compounds found in plants or in environment, referred to as xenobiotics, can activate the nuclear receptors, thereby disturbing the normal endocrine functions of the body – a phenomenon described as endocrine disruption (Casals-Casas & Desvergne, 2011). Those nuclear receptors that have an unknown or unidentified endogenous ligand are named as orphan nuclear receptors. Even though the nuclear receptors have different target genes and different functions, they all share a similar structure. They are composed of five domains and have an N-terminal (domain A/B), a DNA-binding domain (C domain), and a C-terminal (E/F) which is lipophilic in nature and holds the binding site of the ligand (ligand-binding domain). The D domain connects the DNA-binding domain with ligand-binding domain. Many diseases such as diabetes cardiovascular disease, rheumatoid arthritis, cancer, allergy, asthma, neurological, and psychological disorders are reported to be associated with nuclear receptors (Mazaira, Zgajnar, Lotufo, et al., 2018). Due to the fact that nuclear receptors bind to small lipophilic molecules that can easily be modified through drug design, they have been in the focus of pharmaceutical field as a possible target for the treatment of different diseases (Sladek, 2003).

2.3  Estrogen Receptors Many crucial signaling events triggered by estrogen are initiated upon binding with receptors. Estrogen receptors belong to the nuclear receptor superfamily, but membrane-­ associated ERs have also been identified (Levin, 2009; Pietras & Marquez-Garban, 2007). Two nuclear receptors – ERα and ERβ – and a membrane-­ bound G-protein coupled estrogen receptor (GPER) have been reported to be the major receptors to which estrogen binds (Fig.  1) (Prossnitz & Barton, 2011). Furthermore, ERs have been identified in cytoplasmic organelles such as the mitochondria and endoplasmic reticulum. It is suggested that mitochondrial ERs are involved in apoptotic/anti-apoptotic signaling; however, their role in human physiology and pathophysiology is not fully understood (Liao et al., 2015).

2.4  Nuclear Estrogen Receptors: Structure and Isoforms Till date, two ERs have been identified, estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). The first ER that was identified in the year 1958 was ERα encoded by the ERS1 gene (cloned in 1985) present in chromosome 6 (Jensen et al.,

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Fig. 1  Schematic illustration of the effects of estrogen on nuclear estrogen receptors alpha and beta and membrane receptor GPER. The influence of estrogen on pathogenesis of various diseases

1966). ERβ was discovered many years later in 1996 from prostate and ovary tissues of rat and is coded by ESR2 gene located on chromosome 14 (Kuiper, Enmark, Pelto-Huikko, et al., 1996). Both ERα and ERβ are members of the nuclear receptor family. Their structure consists of five domains: A/B domain (ligand independent domain), C domain (DNA-binding domain, DBD), D domain (hinge domain), and E and F domains (ligand-binding domain), organized in B which forms three major compartments (Jia et al., 2015). Domain A/B is associated with the N-terminal compartment and exhibits activation function 1 (AF1) in a ligand-independent fashion. The DNA-binding domain (DBD) is formed by C domain and is the compartment which binds to a specific region of the promotor of target genes. The C-terminal compartment, which involves the domains E and F, is called the ligand-binding domain (LBD) and includes the activation function 2 (AF2) that plays an important role in the receptor dimerization after ligand binding (Gronemeyer, Gustafsson, & Laudet, 2004). The D domain, also called the hinge domain, connects DBD with LBD and included amino acid sequences important for posttranslational modification (Nilsson, Koehler, & Gustafsson, 2011). ERα receptor is longer than ERβ; however, the receptors share a high homology between domains, especially in the DNA-binding domain. The original sequence of the ERs can be modified due to alternative splicing of mRNAs. Therefore, several

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isoforms of ERs are identified. In humans, three ERα isoforms exist: ERαΔ3 where the C domain is absent, ERα46 where the AF1 region is absent, and ERα36 with both AF1 and AF2 regions missing. Regarding ERβ, at least four different isoforms were identified. All ERβ isoforms have a modified C-terminal compartment which does not allow them to function in a ligand-dependent way. Even though it is demonstrated that ERβ1 is the only functional isoform (Leung, Mak, Hassan, et  al., 2006), the physiological role of ER isoforms is still unclear (Heldring, Pike, Andersson, et al., 2007).

2.5  Membrane-Associated Estrogen Receptors Apart from the classical nuclear ERs, extranuclear ERs have been identified in the plasma membrane. These receptors are called membrane-associated ERs, and studies have shown that these receptors are identical to the classical nuclear ERs and coded by the same genes. Mice cells with a double knockout of ERα and ERβ also lack the presence of plasma membrane ERs. Membrane-associated ERs comprise approximately 10% of all ERs in a cell (Pedram, Razandi, & Levin, 2006). The activation of these receptors mediates rapid estrogen actions by generating secondary messengers in the cells such as cyclic adenosine monophosphate (cAMP) and calcium, activating protein kinase cascades such as the mitogen-activated protein kinase (MAPK), and the phosphoinositide 3-kinase (PI3K) pathways and can lead to both transcriptional and nontranscriptional effects (Bjornstrom & Sjoberg, 2005). Furthermore, some of the membrane-associated ERs attach to extracellular sites or lipid raft domain in the plasma membrane, thus interacting with other transmembrane receptors such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and insulin-like growth factor receptor I (IGFR1) (Pietras & Marquez-Garban, 2007). Studies suggest that the palmitoylation of ERs, as well as the transporter caveolin-1, are necessary for the localization of ERs at the plasma membrane the cells (Levin, 2009).

2.6  G-Protein Coupled Estrogen Receptor 1 (GPER-1) GPER is a transmembrane protein and does not exert identical mechanisms like nuclear receptors ER alpha and ER beta. Estrogenic action is mediated by GPER in the cardiovascular system, nervous system, skeletal system, immune system, and reproductive system, exerting key control on glucose metabolism in the pancreas, renal function, bone metabolism, and malignant transformation. There has been a lot of research interests with respect to the relevance of GPER in tumor progression. The binding of estrogen to GPER in the plasma membrane activates the receptor. This triggers a phenomena culminating in the activation of signaling cascades leading to cell proliferation and growth and progression of tumors (Jung, 2019).

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GPER is a transmembrane receptor with seven domains that belongs to the group of G-protein coupled receptor family. The G-protein coupled receptors (GPCRs) are the largest family of the cell surface receptors, which pass across the cell membrane seven times and transduce most of the signaling in our body. The heteromeric G protein consists of three main subunits: alpha (Gα), beta (Gβ), and gamma (Gγ) with several subtypes described for each subunit. The β and γ subunits form an inseparable β/γ complex. Upon ligand activation, Gα binds and hydrolyzes guanosine triphosphate (GTP) releasing guanosine diphosphate (GDP), which in its inactive state is connected to the Gα subunit of the receptor. The released GDP binds to the β/γ complex which separates from Gα when the receptor gets activated. All these changes lead to the activation of several cascades of signaling pathways (Rosenbaum, Rasmussen, & Kobilka, 2009; Wettschureck & Offermanns, 2005). The GPER was first discovered as an orphan G-protein coupled receptor localized at the plasma membrane or endoplasmic reticulum, referred as GPR30, which mediated the rapid effects of 17β estradiol in human cells (Revankar, Cimino, Sklar, et al., 2005). The activation of GPER by estrogen or estrogen agonists stimulate the intracellular calcium release, production of cAMP, and cyclin D2 and PI3K activation, together with a transactivation of EGFR (Pedram et al., 2006). Even though it is reported that high levels of 17β estradiol are required to activate this receptor (Maggiolini, Vivacqua, Fasanella, et al., 2004), GPER-mediated estrogen effects are implicated in many physiological responses in different tissues and organs of the human body, such as the reproductive, nervous, immune, and cardiovascular systems, as well as in cancer progression and metastasis (Prossnitz & Barton, 2011).

2.7  Distribution of Estrogen Receptors ERs are widely expressed in reproductive and nonreproductive organs and systems of the human body, regulating various physiological processes. In the majority of these tissues both ER subtypes, ERα and ERβ, are expressed almost to the same extent. However, in some tissues such as the liver, lungs, and colon, only one ER subtype is dominant (Paterni, Granchi, Katzenellenbogen, et al., 2014). Even in those organs where both ERs are expressed, one of the receptors has the prominent role. For example, ERα has a more prominent role in preserving the bone density, while ERβ has a more prominent role in the brain. Additionally, ERβ opposes the ERα hyperproliferative effects in breast and ovary (Bottner, Thelen, & Jarry, 2014). However, there are recent reports that suggest a bifaceted role of ERβ in cancer tissues that have a stable expression of both estrogen receptors. In breast cancer cells which are ERα/ERβ-positive, it has been reported that rather than antagonizing ERα-mediated proliferative effects, ERβ forms heterodimers with ERα both in the presence and absence of 17β-estradiol, culminating in transcriptional changes that does not induce antiproliferative effects (Card & Zeldin, 2009).

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3  Estrogen Signaling Pathways Estrogen receptor activation culminates in downstream nongenomic and genomic effects. These actions can be dependent or independent on ERs and ER-ligands; therefore, four different pathways are identified (Cui et al., 2013).

3.1  E  R-Dependent, Nuclear-Initiated Estrogen Signaling (Genomic Effects) This is the main signaling pathway which leads to genomic effects through the activation of nuclear ERs in a ligand-dependent manner. The activation of the ERs resulting in conformational changes will follow the binding of the ligand. Depending on the type and concentration of the ligand and the number of the receptors present, ERs can form homodimers (ERα-ERα or ERβ-ERβ), heterodimers (ERα-ERβ), or a combination of the two. After ligand binding, the ER-ligand complexes translocate from the cytoplasm into the nucleus, where they bind to the so-called estrogen responsive elements (EREs) and regulate the expression of ERE-containing genes. As the DNA-binding domain of ERα and ERβ exhibit 98% homology, they exert similar affinity and selectivity for binding the EREs.

3.2  ER-Dependent, Membrane-Initiated Estrogen Signaling An alternative mechanism that is dependent on ER is referred as “membrane-­ initiated” pathway, in which signaling events induced by estrogen is triggered either in the cytoplasm or membrane culminating in the downstream effects that are not fully dependent on transcription or translation. This pathway is responsible for the rapid and acute estrogen effects generated by membrane-associated ERs and GPER. Ligand-dependent activation of membrane-associated ERs or GPER leads to nongenomic effects by regulating the secondary messengers such as cAMP, calcium, and potassium, interacting with tyrosine kinase receptors (EGFR and IGFR1) in the cytoplasmic membrane and activating protein kinase cascades such as MAPK and PI3K. Furthermore, the activation of cAMP and protein kinase cascades via this pathway can lead to genomic effects by phosphorylating AP1 and SP1 transcriptional factors of certain target genes. The rapid effects of estrogen, mediated through the ligand activation of membrane-­associated ERs and GPER, play an important role in the liver, bones, and nervous system (Prossnitz & Barton, 2011).

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3.3  ER-Independent Signaling Although it is well understood that the major biological actions of estrogen are mediated and exerted through ERs, the alternative mechanisms of action have been hypothesized. Research reports have indicated that estrogens exert antioxidant effects and inhibit oxidative stress significantly in an ER-independent manner (Haas, Raheja, Jaimungal, et al., 2012). Being a steroid hormone, estrogen can easily cross the cell membrane and enter into the cytoplasm where it can interact with enzymatic activities without binding to ERs. Estrogen has a phenolic -A ring, which allows it to regulate redox activities providing antioxidant effects. Studies report that estrogen reduces oxidative stress by preventing the mitochondrial release of reactive oxygen species (ROS) (Richardson, Yu, Wen, et al., 2012). Additionally, the ER-independent effect of estrogen has been proven in mouse models of breast cancer, where estradiol could promote breast cancer even in mice with an endogenous deletion of ERα and ERβ (Yue, Wang, Li, et al., 2010).

3.4  Ligand-Independent Activation of ERs Recent research implicates that in the normal physiology of animals, ERs can be activated not only by estrogen-dependent pathway but also in a ligand-independent fashion by many factors such as dopamine, epidermal growth factor, insulin-like growth factor (Ignar-Trowbridge, Nelson, Bidwell, et  al., 1992; Klotz, Hewitt, Ciana, Raviscioni, et al., 2002), protein kinase C (Patrone, Ma, Pollio, et al., 1996), protein kinase A (Schreihofer, Resnick, Lin, et  al., 2001), MAPK (Kato, Endoh, Masuhiro, et  al., 1995), and phosphatidylinositol 3-kinase (Martin, Franke, & Stoica, 2000). The ligand-independent pathway activation is mainly associated with cellular protein kinases that are involved in the phosphorylation of ERs.

4  Ligands The endogenous ligand that activates ERs is estrogen. However, ERs can be activated by many other compounds which can fit into their ligand-binding domain. These compounds are grouped into synthetic estrogens, phytoestrogens (natural estrogen coming from plants), and environmental estrogens. These ligands can be categorized as antagonists, agonists, and selective modulators of the estrogen receptors based on their functions (Heldring et al., 2007; Nilsson & Gustafsson, 2011). Agonists bind to ERs and activate them, while antagonists block ERs activation after binding to them.

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4.1  Selective Estrogen Receptor Modulators (SERMS) SERMs are a group of synthetic nonsteroidal compounds that modulate the effect of ERs. SERMs are grouped in two categories: nonselective (classical) and selective SERMs. Classical SERMs bind to both ERα and ERβ with the same affinity but different efficacy. Tamoxifen and raloxifene are the examples for classical SERMs. Tamoxifen has anti-estrogenic effects in the breast and is used for the treatment of ER+ breast cancer, but it has estrogen-like effects in other tissues especially in the bone, where it preserves the bone density, protecting from osteoporosis. Likewise, raloxifene has the same anti-estrogenic effects in the breast and pro-estrogenic effects in the bone as tamoxifen, but its estrogen-like properties in other tissues are weaker (Jordan, 2001). The other class of SERMs bind selectively to only one of ER subtypes. Also, selective antagonists for each ER subtype exist. They can be silent antagonist (full antagonist is another term used) or partial agonists. A silent antagonist is a competitive ER antagonist that has zero intrinsic activity for activating the receptor. An example is the ERβ-selective antagonist 4-(2-phenyl-­ 5,7bis(trifluoromethyl)pyrazolo(1,5-a)-pyrimidin-3-yl)phenol (PHTPP), which possesses full antagonistic properties with 36-fold selectivity for ERβ over ERα. In addition, a partial agonist is a compound that in a presence of a full agonist acts as a competitive antagonist, but if its concentration is too high, it can activate the receptor as well (Neubig, Spedding, Kenakin, et al., 2003). A specific group are the antagonists that belong to the group of selective estrogen receptor degrader (SERD). When they bind to ER, they cause conformational changes which result in receptor degradation and downregulation (Nilsson et al., 2011). An example of SERD are the compounds named fulvestrant and AZD9496 which bind selectively to ERα (Nardone, Weir, Delpuech, et al., 2019). Fulvestrant is an approved medication for breast cancer patient administered with monthly intramuscular injections. AZD9496 is the first oral SERD that has shown higher bioavailability compared to fulvestrant and has been successfully tested in clinical trials of breast cancer patients (Hamilton, Patel, Armstrong, et al., 2018).

5  Estrogen and Colon Cancer The significance of estrogen in the maintenance of homeostasis is evident from several pathophysiological changes that manifests with the deficiency of estrogen (Gruber, Tschugguel, Schneeberger, et al., 2002). High incidence of CRC among nuns along with the high incidence of other hormone-associated cancers including the breast, uterus, and ovary was reported (Cui et al., 2013). Although, there is little overall gender difference in the risk of colon cancer, there is a variation in the age-­ specific colon cancer gender ratio especially in the age groups of 35–54 and above 54 among men and women. Men had higher risk below 35 years of age, while the risk was higher in females between 35 and 54 years of age. After 54 years of age,

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the male risk again becomes higher (Lombardi et al., 2001). Age-specific fertility and exposure to high dose of oral contraceptives during the 1960s were suggested to be the possible reason for this transient decline in women indicating the direct influence of endogenous/exogenous estrogen in colon cancer. Several case-­ controlled studies and cohort studies examining the association between reproductive events, menstrual factors, exogenous hormones, and CRC stratified by age at diagnosis, tumor site, family history, and other potential risk factors also indicated the role of female sex hormones in colon cancer (Burns & Korach, 2012; Jia et al., 2015).

5.1  Location of Estrogen Receptors in the Colon ERα and ERβ may also localize to distinct cellular subtypes within each tissue (Birkhauser, 1996). It was reported that the location of ER is in the stromal cells rather than in the colon epithelial cells (Kotov et al., 1999), and in situ hybridization studies also indicated that ER is present in stromal cells which are located above the muscularis mucosa (Santen et al., 2009). However, it was reported based on immunohistochemistry that ER is present throughout the colon mucosa (Carreau et al., 2008). Controversy remains over the location of these receptors in the stroma rather than in the colon epithelial cells and their role in colon cancer. But a consistent finding is that normal human colon expressed more ERβ than ERα mRNA, and the expression of ERβ is low or selectively lost in human colon tumor cells (Inoue et al., 2012).

5.2  Estrogen Function in Colonic Epithelium The role of estrone and estradiol in colon cancer cells is not well defined. 17βHSD type 2 (estradiol to estrone) was the dominant form in human colon and was downregulated in colorectal tumors with no expression of the 17βHSD type 1 enzyme (estrone to estradiol) (Mazaira et al., 2018). However, it was reported that females with CRC who had higher 17βHSD 2 mRNA expression had a poor survival rate, thus suggesting a low expression of the 17βHSD type 2 as an independent marker of good prognosis in females with distal colorectal cancer (Casals-Casas & Desvergne, 2011). This was contraindicated on the conversion of estradiol to estrone as protective against colon cancer, and the loss of estrogen inactivation (i.e., conversion of active estradiol to estrone and 17βHSD type 2 enzyme) results in colon tumors. These results suggest a reciprocal role of active and inactive estrogens (estradiol and estrone) in the etiology of colon cancer (Bain, Heneghan, Connaghan-­ Jones, et al., 2007). Despite the positive association of endogenous and exogenous estrogens with cancers, plant estrogens are inversely associated with cancer (Pietras &

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Marquez-­Garban, 2007). The association of phytoestrogens with decreased cancer incidence implies a lack of estrogenicity or estrogen antagonism or differential cellular mechanisms. The growth effect of estradiol (1 and 10 nM) to genistein and tamoxifen was compared on cellular proliferation in human colon cancer cell lines (HT-29, Colo320, Lovo, and SW480 cells) and MCF-7 cells (Levin, 2009). Even at higher concentrations (100 and 500 nM), the colon cancer cells were not responsive to estradiol, while growth was stimulated in MCF-7 cells. However, at 10 μM concentrations of genistein at which growth is stimulated in ERα-positive human breast cancer MCF-7 cells, there was a slight inhibition in growth of HT-29, Colo320, and Lovo colon cancer cells. Also, they reported the presence of ERβ in these cells with no ERα. Different biological effects of phytoestrogens and endogenous estrogens as well as the differences in the ratio of ERα and ERβ can be accounted for the differential effects of these compound tissues specifically. Other phytoestrogens have also been effective chemo-protectants as evidenced in in vitro studies. Phytoestrogens trigonelline (Trig) and diindolylmethane (DIM) have demonstrated the regulation of YAMC cell growth via apoptosis and disruption of the cell cycle (Yager & Chen, 2007). These effects were found to be mediated by ERβ, though neither compound bound directly to the receptor-binding pocket of the protein.

5.3  E  strogen Signaling Through Nuclear Receptors (α and β) in Colorectal Cancer The use of hormone replacement therapy (HRT) has a protective role in the prevention of CRC (Gronemeyer et  al., 2004; Kuiper et  al., 1996). The biggest trial to investigate the role of HRT on CRC incidence in postmenopausal women concluded that the short-term use of combined (estrogen + progestin) HRT decreased the incidence of CRC. However, for those women who were assigned to HRT and developed CRCs, the diagnosis was made at a more advanced stage compared to women in placebo group. These findings implicate the potential applications of estrogen signaling in the prognosis of CRC. The normal colon mucosa expresses dominantly ERβ, while ERα expression is very less. But interestingly, the expression of ERβ declines with advanced CRC, and this decreased expression correlates directly with the stage of the tumor and its differentiation. Furthermore, CRC patients with high expression of ERβ in their tumors have significantly better overall and disease-free survival (Revankar et al., 2005). A large body of evidence has reported the antitumor properties of ERβ induction in in vitro experiments and in vivo mouse models (Heldring et al., 2007; Maggiolini et al., 2004). Over expression of ERβ in colon cancer cells reduced cell proliferation and survival rate and induced cell cycle arrest and apoptosis, downregulated the levels of interleukin 6 (IL-6) which is a potent pro-tumorigenic inflammatory mediator (Card & Zeldin, 2009), and reduced colon cancer metastasis by repressing PROX1 oncogene expression (Jonsson, Katchy, & Williams, 2014). Additionally,

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ERβ-deficient mice, where the receptor was knocked down, had changes in their colon architecture characterized by hyperproliferation, dedifferentiation, decreased apoptosis, and disruption of epithelial tight junctions compared with mice that had an intact ERβ (Chang, Frasor, Komm, et  al., 2006; Frasor, Danes, Komm, et  al., 2003). Moreover, the treatment of ApcMin/+ mice with an ERβ-selective agonist diarylpropionitrile (DPN) reduced the number of polyps in the small intestines of both male and female mice (Richardson et al., 2012). The use of dietary phytoestrogens, which have higher affinity for ERβ than ERα, is linked to a reduced CRC incidence among the populations that consume soy products daily (Power, Mani, Codina, et al., 1991; Yue et al., 2010). All these data confirm that sex steroid hormones are involved in CRC development and suggest that ERβ could play an important role in the early phase of carcinogenesis and thereby could be a primary target in the prevention of CRC. Transfection of colon cancer cells to overexpress ERα resulted in the activation of Wnt/β-catenin pathway. The effect was antagonized when blocking ERα (Ignar-Trowbridge et  al., 1992). Few studies have evaluated ERα expression in colon cancer tissue and reported that ERα expression has an inverse relationship with patient’s survival (Patrone et al., 1996).

5.4  Estrogen Signaling Through GPER in Colon Cancer The downstream effects of binding of estradiol to GPER include the activation of adenylate cyclase, resulting in enhanced cAMP which induces calcium mobilization and activation of MAP kinases. Also, the downstream effect involves the phosphoinositide 3-kinase (PI3K)/Akt pathways the hippo/yes-associated (YAP)/ transcriptional coactivator with PDZ-binding domain (TAZ) pathway and epithelial growth factor receptor (EGFR) transactivation. Activated GPER induces the release of calcium from the endoplasmic reticulum that in turn activates Src-like nonreceptor tyrosine kinases (TK). TKs then activate matrix metalloproteinase (MMP) activation and EGFR activation. EGFR phosphorylation activates the STAT 5 and MAPK/extracellular-regulated kinase (ERK) pathways. These pathways are influenced by ER alpha-36 expression, which is regulated and controlled by GPER. Activation and cross-talk between these signaling pathways via GPER contribute to tumor progression in different types of cancer (Zhu, Huang, Wu, Wei, & Shi, 2016). GPER functions as a tumor suppressor in colorectal cancer. It has been observed that in CRC, the promoter of GPER exhibit increased methylation and histone deacetylation. In addition there is an inverse association with GPER expression with cancer progression and lymph node metastasis. Also the high expression of GPER is strongly associated with longer survival in CRC patients. But there are also reports highlighting that estrogen activates the proliferation of CRC because conjugated estrogen is activated by steroid sulfatase via GPER. Also there is a report that GPER controls the expression of fatty acid synthase which induces CAFS and contributes to cancer progression. Interestingly, E2-activated GPER contributes to the

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migration and proliferation in hypoxia-induced CRC cells, and hormone replacement therapy may be associated with CRC progression. But overall, it can be said that the role of GPER in cancer progression may vary depending on stage of tumor, tumor topography, and tumor microenvironment (Gilligan et  al., 2017). In ER-negative cancer, GPER expression is high, and estrogen signaling mediated through GPER contribute to tumor progression. However, in contradiction to these finding, high GPER expression increases the survival rate in ovarian cancer, even though the levels are lower than those in the normal cells. A GPER selective agonist G1 suppresses the proliferation of tumor cells in CRC, adrenocortical carcinoma, and prostate cancer. Thus GPER expression and its activation (through ligands estrogen) along with nuclear receptor ERα and ERβ could play a central modulatory role in the onset and progression of carcinogenesis (Bustos et al., 2017; Santolla et al., 2012).

6  E  strogen and Estrogen Receptors in the Prognosis of Colon Cancers Although historically associated with tumorigenesis and metastatic malignancies of the reproductive tissues, estrogens are also linked with the pathogenesis of other types of cancers found in nonreproductive tissues including the colon. Selective activation of proapoptotic signaling modulated by ERβ was found to be responsible for antitumor effect exerted by estrogens. A recent report involving the analysis of 1262 patients revealed based on immunohistochemistry that ERβ expression in CRC correlated with a better prognosis, whereas reduced expression was observed in high-grade large-sized tumors. The expression levels were observed to be inversely correlated with Duke’s stage. Interestingly the loss of expression or reduced expression of ERβ was found to be associated with an increased risk (54%) owing to CRC and reduced disease-free survival (DFS). Interestingly, another study reported that increased expression of ERβ is associated independently with an improved prognosis in female colorectal cancer patients (Topi et  al., 2017). It is evident that exploiting ERβ as a prognostic biomarker in CRC might not only implicate a better prognosis but also identify CRC patients who would rather benefit from ERβ-targeted treatment strategies (Edvardsson, Strom, Jonsson, Gustafsson, & Williams, 2011). Rawluszko-Wieczorek, Marczak, et al. (2017) reported the association between high ERα mRNA levels together with increased estrone intratissue concentration with an improved disease-free survival in colorectal cancer patients. This study revealed the relevance between estrogen concentration in the tissues and the expression of the estrogen receptors. Also the study indicated the possibility of these factors being used as biomarkers for CRC and how these biomarkers could be exploited in estrogen-targeted antitumor therapies for CRC.  Another study by Ye, Cheng, Zhang, Wang, et al. (2019) showed that the increased expression of ERα implicated poor prognosis of CRC and this could be useful in planning the treatment regimens for CRC patients at high risk.

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7  M  echanisms of Antitumor Effects by Estrogen and Estrogen Receptors in Colon In vitro studies on carcinoma cell lines and in vivo studies on mouse models have proven that estrogen- or progesterone-activated signaling leads to growth inhibition in colon cancer cells. This is reported to be mediated through the upregulation of several proteins involved in the regulation of cell cycle such as p 53, p 21, and p 27 (Carothers, Hughes, Ortega, & Bertagnoli, 2002; Hsu, Cheng, Wu, et  al., 2006). Estrogen is also reported to maintain genomic stability in epithelial cells of the colon by the upregulated expression of genes involved in mismatch repair (Jin, Lu, Sheng, Fu, et al., 2010). As estrogen is a key player in cPG island hypermethylation phenotype (CIMP), epigenetic events may also participate in this control (Issa, 2008). Estrogens and ligands of estrogen play important roles in the control of both innate and adaptive immunities. It is reported that the homeostasis of several lymphoid and myeloid progenitors of dendritic cells are regulated by both estrogen and ligands of estrogen. These ligands and estrogen also play a role in the activation of dendritic cells and the resultant production of inflammatory mediators. Estrogen also influences the action of dendritic cells and enhances dendritic cell-mediated immune surveillance in the colon, increases CD4+ T-cell response, and regulates responses to TLR agonists (Bengtsson, Ryan, Giordano, Magaletti, & Clark, 2004; Kovats & Carreas, 2008). Cvoro and colleagues reported that both selective agonists of ERβ and estradiol decreased the TNF-induced activation of 18 proinflammatory genes such as IL-6, CSF-2, and TNFα (Cvoro, Tatomer, Tee, Zogovic, et al., 2008). This implicated that the activation of ERβ signaling selectively could negatively influence tumorigenesis through downregulated expression of protumorigenic/proinflammatory signaling cascades in sites where tumors are located. This ultimately triggers on the antitumor immune surveillance mechanisms as well. Menon, Watson, Thomas, Alfred, et al. (2013) reported that a diet rich in phytoestrogens containing isoflavones and fiber can augment the composition of intestinal microbiota through ERβ in female mice. This report has considerable research significance as it is known that microbiota are key players in modulating local inflammation and prevent the progression of intestinal tumors (Fig. 2).

8  H  ormone Replacement Therapy as a Potential Anticancer Strategy Hormone replacement therapy in postmenopausal women increased the risk of breast and endometrial cancers but surprisingly did not increase the risk for cancers of the gastrointestinal tract and lungs. Many reports claim the inverse association between sex hormone association and the risk of gastrointestinal cancer. Exogenous administration of hormones as part of HRT and usage of oral contraceptives is linked with an appreciable decrease in CRC risk. This finding was supported by the

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Fig. 2  Binding of estrogen to constituent receptors and the resultant antitumor mechanisms on colon

cohort analysis on postmenopausal women aged between 50 and 79 (Murphy, Xu, Zervoudakis, TE Xue, et al., 2017) and a more recent study that reported that HRT decreases the risk of CRC remarkably in postmenopausal women (Fernandez, La Vecchia, Balducci, et al., 2001; Lin & Giovannucci, 2010). Interestingly, there are also research claiming that progestin-estrogen therapy proved to be more effective than therapy with estrogen alone, thereby implicating that progestins enhance the activity of estrogens. Hence, HRT would be a potential anticancer strategy and can form the basis for targeted endocrine therapies that reduces the enormous burden of CRC.

9  Conclusion The sex hormone estrogen and its receptors have a pivotal role in the development of CRC (sporadic, genetic, and postinflammatory). The loss of ERβ expression could be a marker of colonic mucosa at increased risk for the development of neoplasms and induction of ERβ with ERβ-selective agonists like phytoestrogens could exert a chemopreventive effect against CRC.  Their targeted use is, therefore, a

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fascinating field of investigation which can help to delineate the underlying mechanisms that contribute to the pathophysiological, prognostic, and therapeutic aspects. The mechanism of the putative protective effect of estrogens and phytoestrogens on colonic neoplasia although not fully understood seems to be distinctly different from the mechanisms that evokes a detrimental effect in breast cancer. The ERα/β balance seems to have a relevant influence on colorectal carcinogenesis, and ERβ appears to parallel apoptosis, thus exerting an anticarcinogenic effect.

10  Future Perspectives The design and development of new SERMs with selective differential estrogen mimicking and estrogen opposing actions based on the target tissue will greatly enhance the clinical development of modulators that can reduce the incidence of estrogen-related tumors in humans. Also, the development of novel therapeutic strategies that exploits the benefits of the SERMS as well as estrogens termed as tissue selective estrogen complex (TSEC) can be developed in the future which could be a promising approach in the therapy of colon cancer. Acknowledgments  The authors thank the University Grants Commission (UGC), Government of India, for the financial assistance provided to the Department of Microbiology and Biotechnology, Bangalore University, Bangalore, in the form of Special Assistance Programme (SAP)-DRS II which provided support for extensive literature survey and procurement of stationery material and to incur expenditure toward printing and photocopying charges for writing this chapter. Dr. SK acknowledges the financial assistance provided by UGC, GOI, in the form of postdoctoral fellowship (Dr. D.S. Kothari-PDF).

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Clinical Significance of Genetic Variants in Colon Cancer Irina Nakashidze, Nina Petrović, Nino Kedelidze, and Begum Dariya

Abstract  Colon cancer (CC) is a serious global health problem detected, among other gastrointestinal (GI) tumors. Several risk factors suggested for the occurrence of CC include modifiable and nonmodifiable risk factors. Notably, the genetic risk factors play a crucial role in developing susceptible to CC. The molecular heterogeneity of CC also remains a significant and crucial aspect for personalized therapy. Therefore, detecting suitable molecular genetic biomarkers related to CC development are essential to support healthcare providers for therapy. However, the identification and characterization of the genetic variants clinically during the diagnosis for appropriate therapy still remain unclear. Determining the cancer susceptibility via genetic variants, including single-nucleotide polymorphism (SNPs), provides a new opportunity in the diagnostics. Thus, in this chapter, we revised the clinically significant genetic variants associated with CC based on the existing data. As suggested from the previous studies, the gene SNPs (APC, ATM, BRAF, BRCA1, DUSP10, MAP2K1, MLH1, MTHFR) are associated with colon cancer. The miRNA SNPs (rs8905 (PRKAR1A), rs8176318 (BRCA1)) and miRNA target SNPs as well (rs8905 (PRKAR1A), rs8176318 (BRCA1), let-7, miR-34b/c/146a/603, miR-149, I. Nakashidze (*) Department of Clinical Medicine, Faculty of Natural Science and Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia Department of Biology, Faculty of Natural Science and Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia N. Petrović Laboratory for Radiobiology and Molecular Genetics, Department of Health and Environment, “VINČA” Institute of Nuclear Sciences-National Institute of the Republic of Serbia, University of Belgrade, Belgrade, Serbia Department for Experimental Oncology, Institute for Oncology and Radiology of Serbia, Belgrade, Serbia N. Kedelidze Department of Biology, Faculty of Natural Science and Health Care, Batumi Shota Rustaveli State University, Batumi, Georgia B. Dariya Department of Bioscience and Biotechnology, Banasthali University, Vanasthali, Rajasthan, India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_4

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miR-192a/608/27a SNPs) were also described that might affect and alter the genetic susceptibility of CC. Keywords  Colon cancer · Gene · SNPs · miRNA · Mutation · Genetic susceptibility

Abbreviations APC Adenomatous polyposis coli ATM Ataxia telangiectasia mutated BRAF B-Raf proto-oncogene, serine/threonine kinase BRCA1 BRCA1, DNA repair associated BRCA2 BRCA2, DNA repair associated CC Colon cancer CI Confidence interval DUSP1 Dual-specificity phosphatase 1 DUSP10 Dual-specificity phosphatase 10 EGFR Epidermal growth factor receptor FANCA FA complementation group A FANCE Fanconi’s anemia complementation group FISH Fluorescence in situ hybridization GWAS Genome-wide association LIG1 DNA ligase 1 LSC The left-sided colon MAP2K1 Mitogen-activated protein kinase 1 MLH1 MutL homolog 1 MMR Mismatch repair MSH2 MutS homolog 2 MSH6 MutS homolog 6 MSI Microsatellite instability MSS Microsatellite stable MTHFR Methylenetetrahydrofolate reductase OOR Ordinal odds ratio OS Overall survival PMS2 PMS1 homolog 2, mismatch repair system component RC Right colon SNPs Single nucleotide polymorphism SULT2B1 Sulfotransferase family 2B member 1 TCGA Cancer Genome Atlas VDR Vitamin D receptor wt Wild-type

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1  Introduction Colon cancer (CC) is a serious global health problem among the other gastrointestinal (GI) tumors (Bailey et  al., 2015; Brenner, Kloor, & Pox, 2014; Haggar & Boushey, 2009; Siegel, Miller, & Jemal, 2020). Several modifiable and nonmodifiable risk factors are known for the occurrence of CC (Geneve et al., 2019; Kruger & Zhou, 2018; O’Keefe, 2016). Additionally, genetic risk factors also play a crucial role in both predisposition and development of CC. Therefore, identifying suitable molecular genetic biomarkers associated with CC development are essential in order to support healthcare providers for early screening, appropriate counseling, precision treatment therapy, and surveillance (Gala & Chung, 2011; Mullany, Herrick, Wolff, Buas, & Slattery, 2016; Tejpar et  al., 2010). Besides the current advances in this “precision medicine” era, the chemotherapy remains as the primary treatment way in most cancers, including CC. However, the tendency of tumor cells to develop resistance against chemotherapy treatment remains a critical problematic issue until now (He et al., 2017). Cancer is a heterogeneous group of diseases with distinct molecular properties that directly drive the clinical outcome of patients. Therefore, the molecular heterogeneity of CC remains a significant and crucial aspect for personalized therapy (Akkad, Bochum, & Martens, 2015). However, the identification and characterization of the clinically useful genetic variants during the diagnosis for appropriate therapy remain unclear (Molinari et al., 2018). Single nucleotide polymorphisms (SNPs) are a type of genetic variations that occur in more than 1% of the population in the world and account for about 80% of interindividual genomic heterogeneity (Palmirotta et al., 2018). SNPs are located in gene promoters, exons, introns, and 5′- and 3′- untranslated regions (UTRs) that affect gene expression by different mechanisms (Deng, Zhou, Fan, & Yuan, 2017). These mechanisms directly connect to the location of the genetic variant in an individual genome. The SNPs are connected to epigenetic regulation (Deng et  al., 2017). Thus, alterations in the related gene SNPs are related with the development of cancer susceptibility. SNPs are implicated in many human diseases; accordingly encouraged for the study of pharmacogenetics. As already known SNPs are conserved during long evolution processes (Flanagan & Jones, 2019) and are considered as unique markers (including the analysis of quantitative trait loci (QTL) and in association studies of the microsatellites place). Moreover, determining cancer susceptibility using genetic variants, including SNPs, is considered a new opportunity in the diagnostic point (Zirwes, 2015; Nakashidze et  al., 2020; Nakashidze and Ahmad, 2019). It should be noted that SNPs are associated with several processes, including gene expression, DNA mismatch repair, regulation of the cell cycle, metabolism, immune response, etc. (Fig. 1). The type of SNPs, determines the genetic susceptibility of cancer (Deng et al., 2017; He et al., 2015; Landau et al., 2015; Schirmer et al., 2016; Ulaganathan, Sperl, Rapp, & Ullrich, 2015). Thus, investigating the mechanisms of SNPs that increase cancer susceptibility is critical to understand the molecular pathogenesis of tumors. Thus, in the clinical perspective, SNPs are considered as potential diagnostic and prognostic parameters in cancer.

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Regulation of the cell cycle Transcription factor binding

SNPs

Can be affect

Gene splicing mRNA splicing RNA degradation Translatio Gene expression Metabolism

Genetic susceptibility

DNA mismatch repair

Colon Cancer

Immunity Fig. 1  SNPs affect several processes, accordingly, and might tend to increase the genetic susceptibility of CC

As already known, the genetic variations show peculiarities of the susceptibility toward the disease, including cancers. Over a hundred million SNPs have already been detected. The human genome includes 3 billion nucleotides and interindividual sequence variations are found with a frequency of 1/300–1000 nucleotides (Syvänen, 2001). The SNPs are found within introns and intergenic regions, but they are implicated in several processes, including transcription factor binding, gene splicing, RNA degradation, etc (Shaw, 2013). However, the functional impact of SNPs and mutations on gene expression is still unclear (Robert & Pelletier, 2018). As suggested by The Cancer Genome Atlas (TCGA), the analysis of different sequence variations between tumor and normal cells drive the cancerous growth variedly (Robert & Pelletier, 2018). Accordingly, SNPs have a direct implication on mRNA splicing, nucleocytoplasmic export, stability, translation, codon recognition, etc. (Fig.  1) (Moreno et  al., 2018; Nicholson et  al., 2010; Robert & Pelletier, 2018). Additionally, there are several studies reported in relation to basic mechanisms to understand CC and accordingly identify novel approaches for chemotherapy. However, the prevalence of CC and the mortality rate remains critical due to the resistance developed against therapy. Therefore, investigating the genetic alterations would be an appropriate way to treat resistance cancer cells for better therapeutic efficiency. Besides this, diagnosing patients with higher risk prior to screening is very much essential. This could be analyzed via familial predisposition and mutation screening of essential genes. Furthermore, the screening of these mutated genes would promote the chemotherapy that is entirely dependent on the new molecular therapies (Corrie, 2011; Sokolenko & Imyanitov, 2018). The genome-wide association studies (GWAS) identified some cancer susceptibility variants present on chromosome 8q24 (128.14–128.62 Mb) (Ahmadiyeh et al., 2010; Tenesa et al., 2008). According to a meta-analysis, there is an association between polymorphisms on chromosome 8q24 and increased risk of CC (Hutter et  al., 2010). Cicek et  al. (2009) showed that there are significant associations

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between CC risk and rs13254738 (OOR, 0.82; 95% CI, 0.072–0.94; P = 0.0037) and rs6983267 (OOR, 1.17; 95% CI, 1.03–1.32, P = 0.013). Thus, based on the previous literature, there is an association between SNPs at 8q24 and CC, but these risk alleles were not correlated with the survival of the patient. According to another study, rs6983267 (on chromosome 8q24) can be used as a marker in a population-­ based case-control study of CC (Tomlinson et al., 2007). Li et al. (2008) analyzed 561 patients with CC and revealed that rs6983267 SNP is associated with an elevated risk of CC. Especially, rs6983267 (G; T) and (G; G) genotypes have an age-­ adjusted odd ratio of 1.39 (CI: 1.03–1.88) and 1.68 (CI: 1.21–2.33), respectively, for the development of that disease compared to (T; T) genotypes. Similarly, genes including FA complementation group A (FANCA) – rs2238526 and rs3743860; the Fanconi’s anemia complementation group (FANCE) – rs6907678 and rs10947550; and DNA ligase 1 (LIG1) – rs1971775 and rs73054038 had multiple htSNPs and are associated with the risk of colon carcinoma (Pardini et al., 2020). Based on the somatic mutation analysis done by comparing the mucinous adenocarcinoma of the colon (67 cases) and non-mucinous adenocarcinoma (463 cases), Reynolds et  al. (2019) identified that the mucinous tumors are characterized as hypermutated as compared with non-mucinous tumors and include an increased rate of SNVs and InDels. Notably, non-mucinous tumors revealed the increased frequency of the TP53 gene mutation, which also correlates with the chromosomal instability pathway (Reynolds et al., 2019). The missense-type p53 mutations, together with the loss of wild-type p53, accelerate the late stage of colorectal cancer progression through the activation of both oncogenic and inflame (Loree et  al., 2018; Merlano, Granetto, Fea, Ricci, & Garrone, 2017), commonly in the right colon (Le et al., 2017; Reynolds et al., 2019). The existing data suggest that the mechanism and characteristics of tumor biology and pathology for right- and left-sided colon tumors vary (Boeckx et  al., 2018; Brulé et al., 2015; Price et al., 2015). Notably, patients with right-sided colon (RSC) tumors have revealed a significantly worse prognosis (Price et  al., 2015). Accordingly, the pathogenesis also varies genetically in both right and left colon tumors (Baran et al., 2018; Boeckx et al., 2018; Gallois, Pernot, Zaanan, & Taieb, 2018; Ke et al., 2020; Kim, Castro, Shim, Advincula, & Kim, 2018; Petrelli et al., 2017; Saffarian et al., 2019). Moreover, based on the meta-analysis, the LSC tumors showed a lower risk for mortality than RSC tumors (HR: 0.82; 95% CI: 0.79–0.84) analyzed among 1.5 million pooled patients (Petrelli et al., 2017). The progression-­ free survival (PFS) analysis has been shown to be notably improved within patients with wild-type KRAS LS tumors who were treated with cetuximab compared to best supportive care (median survival of 5.4 months and 1.8 months) (Brulé et al., 2015). Additionally, MGMT rs16906252 SNPs are also associated with the risk of CC.  Moreover, these affect the mRNA expression levels in the colon transverse. Based on this data, carcinogenesis mechanisms were suggested to have implications in promoting carcinogenesis (Pardini et al., 2020). The altered mechanism in CC, including KRAS, NRAS, BRAF, Her2, mismatch repair, and others, would potentiate in drug development, clinical trials, and individualized therapy for patients with metastatic colorectal cancer (Mody & Bekaii-Saab, 2018).

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Thus, in this chapter, we aimed to revise the clinically significant genetic variants in CC.

2  S  elected Potentially Clinically Significant SNPs in Colon Cancer 2.1  APC Gene Mutation in Colon Cancer The evidence from the previous research studies reveal that adenomatous polyposis coli (APC) tumor suppressor gene is mostly highly mutated in colorectal cancers (CRC). It is suggested that it is directly implicated in colorectal tumorigenesis. Alterations in APC gene causes activation of the Wnt signaling pathway, eventually dysregulating several cellular processes (Zhang & Shay, 2017). APC gene mutations are associated with CRC carcinogenesis; notably, these APC gene alterations are found in 80% of human colon tumors. Heterozygosity for such mutations produces mostly autosomal-dominant (AD) CC predisposition in humans and murine models. APC gene has significant implications in the regulation of genomic stability (Kwong & Dove, 2009). The multistep tumorigenesis of CC, which additionally includes epigenetic alterations, results in a more advanced malignant phenotype (Matano et al., 2015). It is also suggested that the carriers of any APC mutation have a worst prognosis (Ghatak et al., 2017). Thus, existing data about the prognostic role of APC suggests that the sequencing of APC may have a clinical usage in staging and potential therapeutic assignment of patients (Schell et al., 2016). Recently, CC mouse models were constructed with genetic alterations of human cancers by simultaneous introduction of Apc mutation, KRAS G12D mutation, and Trp53 loss of Trp53 missense-type mutation in organoid cells and orthotopic transplantation of these engineered organoids to mice, thereby causing the development of invasive colonic adenocarcinoma (Fumagalli et al., 2018; O’Rourke et al., 2017). Later Zhunussova et al. (2019) based on the analysis of two patients (one male at the age of 26 with FAP and sigmoid CC and one male at the age of 46 with ascending CC) detected one novel frameshift variant each in combination with other previously reported frameshift variants APC c.3613delA.  Moreover, the frequency of APC I1307K mutation is also associated with CC (Prior et al., 1999). Based on the abovementioned, the APC gene might be useful clinically in CC diagnostics.

2.2  ATM Gene SNPs in Colon Cancer The ataxia-telangiectasia-mutated (ATM) gene is involved in several processes, including DNA repair and maintaining genome stability, and directly affects the process of cancer initiation, invasion, and metastasis. This gene also influences the predisposition of the cancers. The observational studies in CRC patients revealed that the germline mutations in the ATM (DNA-repair genes (DRGs)) are classically associated with susceptibility cancers, including CC (Pardini et al., 2020).

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Additionally, the experimental data suggests that the ATM rs189037 G>A polymorphism is correlated with increased risks of several cancers. ATM rs189037 G>A NSPs (case-control investigation) of 345 gastric cancer patients and 467 controls in China were analyzed. This polymorphism was related to a significantly higher risk of GC (AA and GG: OR (95% CI): 1.80 (1.20–2.70), P = 0.04; GG vs. AA + GA: 1.46 (1.08–1.98); A vs. G: 1.34 (1.10–1.64), P = 0.004). The AA genotype of GC with rs189037 SNPs had lower OS. The ATM rs189037 G > A SNP is associated with increased susceptibility and poorer prognosis in patients in the Chinese population (Tao, Mei, Ying, Chen, & Wei, 2020). According to an analysis done in cancer cell lines with solid malignancies having ATM mutations, about 10 colon adenocarcinomas revealed 50 alterations in the sequence detected in 16 cell lines. Moreover, five colon tumor cell lines revealed a high frequency of deletions in microsatellite uncertainty within the intronic mononucleotide tracts, which has 62% of the sequence alterations, whereas splicing variants 497del22 or 1236del372 were connected with two intron deletions at splice acceptor locations preceded with ATM exon8 or exon12 (Maillet et al., 2000).

2.3  BRAF Gene Mutation and Colon Cancer The BRAF gene has several functions that include the development of proteins and transmit chemical signals from the cell’s nucleus to cytoplasm via the RAS/MAPK pathway. The RAS/MAPK pathway participates in the cell growth, division, cell differentiation, cell movement, and apoptosis. Besides this, the BRAF genes are oncogenic and significantly promote normal cells into the cancerous cell (Sameer, 2013). Thus, BRAF investigation leads to significant improvement in the clinical outcomes of cancers, including CC (Galanopoulos et  al., 2017; Mody & Bekaii-­ Saab, 2018; Sinicrope et al., 2015). A somatic missense mutation of V600E in the kinase domain of BRAF has been detected in CC (Yokota et al., 2011). Interestingly, BRAF mutation is common for proximal CC compared to LC (Jakovljevic et  al., 2012). André and colleagues investigated 2246 patients (resected, stages II to III CC) and analyze the MMR status and BRAF mutation within 1008 formalin-fixed paraffin-embedded specimens; The obtained results revealed that 95 patients had MMR-deficient (dMMR) tumors, and 94 had a BRAF mutation (André et al., 2015). The study association of BRAF mutations with the survival of CRC patients treated with chemotherapy revealed that wild-type BRAF might have some effect on patients’ lower risk of relapse following adjuvant chemotherapy (Ntavatzikos et  al., 2019). According to the comparative analysis of the colon and rectum, notably, the incidence of microsatellite instability-high (MSI-high), BRAF mutations, and CIMP-high in tumors decreased from the proximal colon toward the rectum (Yamauchi et  al., 2012). Moreover, MSI-high BRAF mutation and CpG island methylator phenotype-high (CIMP-high) is more common for colon carcinomas (Yamauchi et al., 2012). The stage III of CC is characterized as a highly complex stage. According to the BRAFV600E mutation and MMR status analysis, patients with dMMR stage III CC almost had similar outcomes as those who had pMMR tumors without BRAFV600E

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mutations. Additionally, it also showed better prognosis when compared to the patients who had pMMR tumors with BRAFV600E mutations (Sinicrope et al., 2015). Notably, patients with MMR proficient tumors without BRAF mutations and those with MMR-deficient tumors showed a similar 5-year disease-free survival rate. The tendency for a better 5-year disease-free survival rate showed stage III patients with distal and proximal MMR proficient tumors lacking BRAF mutations and in those with BRAF mutation (n = 45; 66.0% for distal and n = 140; 50.9% for proximal), as divided at the splenic flexure. The BRAF mutation is also connected to the molecular subtypes of colon (CMS1, CMS2, etc.) carcinomas, including genetic instability, signaling pathway, clinical outcomes, etc. (Dienstmann et al., 2014). Retrospective analysis from a cohort study of 435 chemorefractory metastatic CC patients treated with cetuximab in combination with chemotherapy revealed that the patients with BRAF wild-type primary tumor (n = 158; splenic nine flexure, descending, and sigmoid colon) were found to have a more prolonged median progression-­free survival rate (30 weeks, 95% CI: 26–34 week, p = 0.02) than those with a proximal (n = 45; cecum, 11 ascending colon, and hepatic flexure) BRAF wild-type tumor (18 weeks, 95% CI: 12 11–31 weeks (Missiaglia et al., 2014). Gao et  al. (2017) have not found a significant difference between RSCC and LSCC BRAF gene mutation incidences of KRAS. However, a higher number of BRAF mutations in RSCC (8.4–22.4%) and in LCRC (1.3–7.8%) were identified (Ishida et al., 2005). Furthermore, BRAF (exons 15, codon 600) were investigated in 86 CC patients. The study of the frequencies and distribution of BRAF mutations revealed an association existing between BRAF mutations, histopathology, and related immunohistochemical markers. The BRAF mutation rates in CC were 3.5%. Notably, the KRAS/NRAS/BRAF mutation status were also associated with the expression of some immunohistochemical markers (Yang et al., 2018). In another study based on 521 proximal colon cancers (PCC), in 714 distal CC (DCC) (tumor-node-metastasis (TNM) staging were stage II, stage III, and stage IV), 106 tumors had BRAFV600E mutation. In MSI-L/MSS group, OS was significantly shorter in patients with BRAFV600E mutation (HR 1.90, 95% CI: 1.12–3.23, p < 0.01). Furthermore, it was suggested that RSCC had a higher frequency of BRAFV600E mutations (Cheng et al., 2018). Cheng et al. (2018) also showed that the BRAF mutations were significantly associated with CA19-9, RSCC, mucinous histology, poor differentiation, and lymphovascular invasion. An analysis prospectively done via collecting the bio-specimens with stage III CC, with separate analysis of MSI and MSS tumors from patients who received adjuvant FOLFOX +/− cetuximab in two adjuvant therapy trials. Among them, 4411 tumors were evaluated as BRAF mutations and mismatch repair; 3934 were MSS, and 477 were MSI. Notably, MSS patients with BRAF V600E mutations (HR = 1.54, 95% confidence interval [CI] = 1.23–1.92, P < 0.001) were associated with shorter time to recurrence (TTR) and shorter survival after relapse (SAR; HR = 3.02, 95% CI = 2.32–3.93, P < 0.001 and HR = 1.20, 95% CI = 1.01–1.44, P = 0.04, respectively). OS in MSS patients was poorer for BRAF-mutant patients (HR = 2.01, 95% CI = 1.56–2.57, P < 0.001) (Taieb et al., 2017).

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2.4  BRCA1/BRCA2 Gene in Colon Cancer The significance of BRCA1 mutation in CC at present remains unknown (Oh et al., 2018). The BRCA1 gene is responsible for a protein that acts as a tumor suppressor, preventing cells from uncontrolled growing and progression. Besides the abovementioned, BRCA1 gene protein is implicated in repairing damaged DNA.  Researchers suggest that the BRCA1 protein also regulates the activity of other genes and plays an essential role in embryonic development and interacts with many other proteins that regulate cell division and another process. Overall, the BRCA1 protein plays a significant role in maintaining the genomic stability of a cell. BRCA1 and BRCA2 are included in many cancer risks and are necessary to determine the prevalence of BRCA1/2 mutations in CC.  Thus, a clear understanding about the risk assessment is very much essential for clinicians and genetic counselors for appropriate strategies to prevent the disease (Sopik, Phelan, Cybulski, & Narod, 2015). Based on the five cohort studies, the BRCA mutation is associated with an increased risk of CC and is determined as BRCA1 mutation carriers (Oh et  al., 2018). For instance, in a case, a carrier of BRCA1 mutation (c.4302C>T, Q1395X) along with his sister was tested and found to have the same pathogenic BRCA1 variant. He also had a history of CC on his paternal side, at the age 40 years and again at the age 70 years, with an unknown BRCA status. His paternal great-­ grandfather also had colon cancer in his 50s with unknown BRCA status. Based on the targeted variant, the analysis for familial BRCA1 mutations revealed heterozygous for familial, germline, and pathogenic BRCA1 variant, c.4183C>T, p.Gln1395* (Soyano, Baldeo, & Kasi, 2018). Yurgelun et al. (2015) determined that 15 of 1260 patients determined to have hereditary CC syndromes are also considered as the carrier of BRCA1/2 mutations (Yurgelun et al., 2017).

2.5  DUSP1 Gene SNPs in Colon Cancer The DUSP1 gene had a significant implication in the carcinogenesis (Gang et al., 2017; Shen et al., 2016; Zhang, Zhang, Chen, Liu, & Xiang, 2018). Notably, there is an association between the expression of DUSP1 with tumor grade and patient survival. The mentioned gene might be involved in the inhibition of cell proliferation, cell migration, and invasion in some cancers (Shen et al., 2017). Based on the analysis, rs6687758 is possibly associated with gene expression within a tumor along with the gene DUSP10 that is found overexpressed within colon sigmoid tissue in the patients with rs6687758 GG genotype compared to healthy individuals. Accordingly, gene expression-associated analysis showed a higher expression of DUSP10 gene in CRC cases and a higher expression of this gene in colon tissue of healthy controls when they have the GG genotype for rs6687758. Thus, it is a relationship between the higher expressions of the gene in allele G in rs6687758 in the CC tissue (Alegria-Lertxundi et al., 2019).

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Based on the population-based case-control studies (including CC: n = 1555, 1956 controls), analysis of other genes (DUSP1, DUSP2, DUSP4, DUSP6, DUSP7) DUSP1 rs322351 (OR = 1.43, 95% CI = 1.09, 1.88; TT versus CC) revealed an association with rectal cancer (Padj AMLH1 gene associated. increased the risk of sporadic CC (OR = 2.07; 95% CI: 1.11–3.83; p < 0.02) (Campbell et al., 2009). According to another study, MLH1 –93G>A, the G/G genotype, was detected in 74 patients with CC (51.4%), A/G in 45 (31.2%) patients, and A/A in 25 (17.4%) patients, compared to control G/G which was found in 53 subjects (35.1%), A/G in 61 (40.4%), and A/A in 37 (24.5%). According to these findings, G/G genotype was associated with an increased risk of sporadic CC in patients compared to control (OR = 2.07; 95% CI: 1.11–3.83; p < 0.02). In the same study, it the association between tumor location and distribution of the MLH1 –93G>A polymorphisms was also investigated. In left-sided colon (LSC) tumors, the frequency distribution of the A/G genotype was significantly higher compared to the right-sided (RS) tumors (38 (39.6%) and 0.7 (14.6%)), but the A/A genotype was found to be slightly lower in left colon (LC) cancer (16 (16.6%) and 0.9 (18.7%) p = 0.014, respectively). These findings suggest that MLH1-93G>A polymorphism may play a significant role in evaluating the risk of sporadic colon cancer and may be used as an indicator in some groups of patients with left-sided and recurrent tumors (Mik et al., 2017). The significant differences were detected in the occurrence of Gly322Asp of MSH2 with regard to primary and recurrent disease (p = 0.001) (Mik et al., 2017). The MSH6 39Glu allele is associated with increased risk of CC within men (Gly/Glu or Glu/Glu and Gly/Gly, accordingly, OR 1.27; 95% CI: 1.04 to 1.54). Thus, the MLH1-93A allele is associated with CC risk. The MSH6 Gly39Glu and MLH1-93G>A polymorphisms are associated with the risk of overall CC and MSI-positive colon cancers, respectively (Campbell et al., 2009).

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As already known, the defects in MMR gene-specific genetic variations in MLH1 are also crucial in the individual predisposition to sporadic CC. According to the same studies, SNP rs1800734 of MLH1 gene was significantly correlated with colon cancer risk (OR = 1.13, 95% CI = 1.07–1.18, p = 3.5 × 10−6 (Pardini et al., 2020). Furthermore, Pardini et al., (2020) investigated 15,419 SNPs from 185 DNA repair genes of 8178 CC and 14,659 controls as per GWAS data of the Colon Cancer Family Registry (CCFR) and the Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO). Rs1800734 (in MLH1 gene) is associated with CC risk (p value = 3.5 × 10−6). According to this study, it was also suggested that specific genetic variants in MLH1 are also important in the individual predisposition to sporadic colon cancer. Similarly, Liu et  al. (2018) also investigated (341 colon patients) epigenetic silencing of MLH1 in the proximal colon (14%) of CIMP-H tumors which exhibited MLH1 epigenetic silencing and MSI, while 23 of the 29 (79%) patients were microsatellite stable (MSS) and were found with CIN phenotype. MSI tumors with MLH1 methylation were associated with BRAFV600E mutation only in the CC.

2.7  MTHFR Gene SNPs in Colon Cancer The methylenetetrahydrofolate reductase (MTHFR) gene is responsible for making an enzyme-methylenetetrahydrofolate reductase that has a crucial role in chemical reaction involving the vitamin folate. Previous studies showed that the antisense inhibition of MTHFR can be considered as a potential target for increasing the chemosensitivity of colon cancer cells to 5FU-based chemotherapy (Sawyer et  al., 2014). The studies also suggested that the phenotype of the MTHFR valine protein (677 TT genotype) is connected significantly to folate availability (Friso et  al., 2002). The MTHFR C677T polymorphism is also associated with cancer risk for CC (recessive model: OR = 0.84, 95% CI = 0.74–0.96, Ph = 0.057, I2 = 38.0%) and rectal cancer (recessive model: OR  =  0.87, 95% CI  =  0.77–0.98, Ph  =  0.373, I2 = 7.3%) (Xie et al., 2015). We found that colon cancer patients with 1298 A>C CC genotype had shorter PFS compared with AA or AC genotype (log-rank test, P = 0.004;). Female patients with 1298 A>C genotype also had shorter PFS compared with A-allele (log-rank test, P = 0.021; (132 patients)) (Liu et al., 2019). Yeh et al. (2017) analyzed 287 CC patients. They determined that the percentages of severe leukopenia were significantly higher in patients carrying MTHFR 677 TT genotype (7.1%) than in those carrying the CC or CT genotype (0.8 and 0.9%, respectively) among all CRC patients (P = 0.01), as well as among CC patients (P = 0.003). However, the MTHFR A1298C polymorphism was not associated with chemotherapeutic toxicity. As already discussed, MTHFR is significant for the enzyme in folate-mediated 1-carbon metabolism. The studies indicate that the MTHFR activity correlates with the hypomethylation of genomic DNA. Also, notably the methylated cytosines at the CpG sites easily mutated and contributed to G:C → A:T transitions within the p53 gene. According to the study, MTHFR genes, C677T and A1298C, correlate to colon tumor characteristics (including acquired

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mutations of KRAS and p53 and microsatellite instability (MSI)). Also, a homozygous variant of MTHFR genotypes had a reduced risk of G:C  →  A:T transition mutations in the p53 gene. A phenotypic effect of MTHFR C677T genetic variant on the DNA methylation is also suggested. Accordingly, this experimental data underlie the SNPs of MTHFR implication in the risk CC (Ulrich et al., 2005). Levin et al. (2010) showed that 677 TT genotype was associated with decreased risk of MSS tumors and tumors in the distal colon (DC) while associated with an increased risk for MSI-H tumors and tumors within the proximal colon. Thus, based on the available data, C677T polymorphism in the MTHFR gene might be an associated risk factor for developing CC, presumably by DNA hypomethylation, which also increases proto-oncogene expression in the colon cells. Individuals carrying the T allele of the C677T MTHFR gene have an increased risk of developing CRC, whereas individuals carrying the C allele have a protective effect against the development of CRC. toxicity manifestations (diarrhea) in response to FU chemotherapeutic treatment were higher among TT genotype carriers (5-fluorouracil FU) having gastrointestinal tract (GIT) toxicity (Mansoura, El Wahsha, El Hefnawya, El Gayedb, & Albanac, 2019). According to the stratified analysis of the CRC region (based on the study of 169 CC cases and 1536 control), it was suggested that MTHFR rs3753584 T>C and rs9651118 T>C polymorphisms are associated with the high risk of CC. However, MTHFR rs1801133 G>A polymorphism also showed a low risk of CC. Finally, the findings of this study indicated that the polymorphisms in MTHFR gene rs9651118 T>C and rs4845882 G>A were associated with the increased risk of CRC.  However, MTHFR rs1801133 G>A polymorphism confers a decreased risk of CRC. Further studies are still essential with larger sample size to confirm these findings (Zhang et al., 2017).

2.8  MAP2K1 Gene SNPs in Colon Cancer Mitogen-activated protein kinase kinase 1 (MAP2K1) is responsible for a MEK1 protein kinase that is part of the RAS/MAPK signaling pathway, which transmits chemical signals to control the cell growth and division. Several existing data confirm the MAP2K1 gene promotes cancerous growth (Couto et al., 2017). Based on the population-based case-control studies (including CC: n = 1555, 1956 controls), an analysis of 19 genes (DUSP1, DUSP2, DUSP4, DUSP6, DUSP7, MAP2K1, MAP3K1, MAP3K2, MAP3K3, MAP3K7, MAP3K9, MAP3K10, MAP3K11, MAPK1, MAPK3, MAPK8, MAPK12, MAPK14, and RAF1) was performed. MAP2K1 rs8039880 [OR = 0.57, 95% CI = 0.38, 0.83; GG versus AA genotype] and MAP3K9 rs11625206 (OR = 1.41, 95% CI = 1.14, 1.76; recessive model) were correlated with CC. MAPK8 rs10857561 (OR = 1.48, 95% CI 1.08, 2.03; AA versus GG/GA) was associated with rectal cancer (Padj < 0.05). Moreover, other genetic variation in the MAPK signaling pathway influences the colorectal cancer risk and survival rate after diagnosis. Associations may be modified by lifestyle factors that influence inflammation and oxidative stress (Slattery et al., 2012).

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2.9  SULT2B1 Gene SNPs in Colon Cancer Sulfotransferase family 2B member 1 (SULT2B1) is a protein coding gene. Based on the studies, it is suggested that rs3760806 was significantly associated with the expression of SULT2B1 (169 transverse colon samples and 124 sigmoid colon samples) (P = 3.6 × 10−3 and 1.0 × 10−3, respectively). Furthermore, the results of the GTEx database revealed an eQTL of rs3760806 in the colon tissues, indicating that the novel genetic variant of rs3760806 may affect the expression of SULT2B1 accordingly and subsequently change the function of SUTL2B1 (Li et al. 2018).

2.10  VDR Gene SNPs in Colon Cancer As already well known, vitamin D deficiency is associated with several human cancers. VDR SNPs may have some implication in cancers (Rai, Abdo, Agrawal, & Agrawal, 2017). The vitamin D receptor (VDR) gene is involved in most of the biological processes in the cell. Notably, VDR has a significant regulatory effect for proliferation and differentiation, as well in intestinal barrier function, innate immunity, host defense in the gut, etc. VDR has significant implications in gene expression regulation and protein expression in CC. Previous studies indicated that there exist an association between VDR and the disease-free survival (DFS) of rectal cancer patients (P = 0.037) as well between calcium sensing receptor (CASR) and the OS of CC patients (P = 0.014). While based on the haplotype analysis, the linkage blocks of CASR indicated that the G-G-G-G-G-A-C haplotype (rs10222633-rs; 10934578-rs; 3804592-rs; 17250717-A986S-R990G-rs1802757) is associated with a decreased OS in CC (HR, 3.15; 95% CI, 1.66–5.96) (Pint = 0.017) (Zhu, Wang, Zhai, Bapat, & Sevtap, 2017). Ke et al. (2020) suggested that the SNP of rs11064124 in 12p13.31 is correlated with the higher risk of colon adenocarcinoma (OR of 0.87; 95% CI, 0.82–0.92, P = 8.67E-06). Moreover, the protective rs11064124-G weakens the binding affinity to VDR and increases the enhancer’s activity. It was also suggested that VDR also interacts with two target gene promoters, causing the coactivation of CD9 and PLEKHG6 transcription in the colon adenocarcinoma. Thus, this accelerates a better understanding of the novel insight for the genetic mechanism of colon adenocarcinoma. VDR SNPs have been associated with susceptibility to colorectal cancer (Touvier et  al., 2011). A recent study indicates that a sex-specific association between the VDR polymorphisms and risk for adenomatous polyps (AP) would be a benign precursor for colon cancer (Beckett et al., 2016). Thus, vitamin D/VDR might be a therapeutic target for CC. However, a better understanding about the tissue-specific roles of vitamin D/VDR may offer new diagnostic and prognostic opportunities for CC (Beckett et al., 2016; Klampfer, 2014).

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2.11  MiRNA SNPs in Colon Cancer miRNA is a small noncoding RNA that negatively regulates gene expression by incomplete complementary binding to mRNA molecules, thus, altering the rates of their translation into proteins. They regulate the wide spectrum of cellular processes. Their level changes are crucial for balancing between normal and tumor states (Zhang, Pan, Cobb, & Anderson, 2007). Oncogenic miRNA upregulation and tumor suppressor miRNA downregulation are associated with an increased risk of colon cancer progression and response to therapy (Schetter, Okayama, & Harris, 2012). Some of the tumor suppressor miRNA in colon cancer are let-7, miR-34a, miR-342, miR-365, etc. The onco-miRNAs described till now are miR-17-92 cluster, miR-20, miR-21, miR-17–5p, miR-15b, miR-181b, miR-191 and miR-200c, miR-95, miR-155, etc. (Pelletier & Weidhaas, 2010; Schetter et al., 2012). Considering the fact that SNPs are positioned all over the human genome, it is not surprising that they can alter the function of miRNA molecules and their binding affinity. Firstly, SNPs on miRNA genes can influence miRNA-mRNA interaction, thus change the standard ratio of their interactions. SNPs placed in the miRNA gene can change the miRNA sequence. As a consequence, miRNA can now gain a new function to bind and regulate mRNA molecules (Wen, Xu, & Yuan, 2018). Secondly, SNP in the miRNA gene can alter synthesis rates and thus lower or increase miRNA production, which can also influence on miRNA-mRNA interaction affinity as well. Thirdly, SNPs located in genes are predominantly regulated by miRNA molecules can alter site for miRNA binding. For instance, Mullany, Wolff, Herrick, Buas, & Slattery (2015) have shown that SNPs in miRNA genes rs8905 (PRKAR1A) rs8176318 (BRCA1) significantly alter miRNA expression levels, thus increasing the risk for colon cancer. Another research also showed that a site for let-7 miRNA binding at KRAS gene within LCS6 site contain T instead of G, at KRAS 3′UTR was associated with lowered let-7 binding affinity. Thus, this interaction is associated with unfavorable response against the anti-EGFR therapy (epidermal growth factor receptor) used for CRC patients with metastatic disease (Graziano et  al., 2010). Rong et  al. (2017) in his literature review described let-7, miR-34b/ c/146a/603, and miR-149 gene polymorphic sites to be most likely linked with increased susceptibility to CRC. In contrast, miR-192a/608/27a is involved in CRC progression and promote susceptibility to CRC.  This suggest that miRNA gene placed SNPs can be accelerated as predictive markers for cancer development and to determine efficacy to therapy. It is also important to bare on mind that SNPs in pre-miRNAs can affect thermodynamic stability of the stem-loop structure, which might, in turn, affect miRNA processing, thus producing isomiRNAs which might result in aberrant downstream mRNA regulation (Petrovic et al., 2017). MiRNA profiling, whether it is expression profiling or miRNA SNP profiling, might be utilized for classification and sub-classification of human cancers (Petrovic & Ergun 2018), including CC, and as well for the prediction of response to therapy and disease outcome, and the direction of the treatment cause. Thus, this suggest that miRNA molecules, especially GWAS and whole RNA-­ sequencing methodology in the future might develop tests to predict the risk of CC as

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well as response to various drugs. Future research would be based on miRNA or mRNA target SNP validation which are essential, with more subjects enrolled, in order to gain reliable information on their usage as potential parameters, future biomarkers for diagnosis, and therapy efficacy. Furthermore, bioinformatic analysis will integrate all experimental and clinical data in order to be used by clinicians to modulate therapy course as per the increased risk of CC formation to be checked and tested more frequently, so the tumor can be detected at early stages of the disease.

3  Conclusions The field of cancer genomics is now with novel applications, including early detection and appropriate therapy, that directly associated with the survival of patients. Identifying the molecular mechanisms of any ongoing alteration in the cell provides new opportunities for evaluating and developing new targeted therapies for cancer (Fig. 2). The fundamental understanding of CC is essential that includes knowledge about gene and epigenetic changes to identify new therapeutic targets. Therefore, development of appropriate tumor markers is necessitated for early detection of disease and therapeutic decisions accordingly. Besides the abovementioned dysregulations, the mutation in some genes may directly reflect susceptibility to CC therapeutic processes. Understanding the drug resistance mechanisms that are connected to genetic variations are also essential for CC treatment therapy, as it forms the basis for effective individualized therapy (Hu, Li, Gao, & Cho, 2016; Kloosterman et al., 2017; Tejpar et al., 2010). Thus, based on the existing data, we notably conclude that there is an association existing between the SNPs of some genes (APC, ATM, BRAF, BRCA1, DUSP10, MAP2K1, MLH1, MTHFR) and colon cancer, accordingly having implication in the genetic susceptibility of CC (Fig. 3).

SNPs can be used for:

Risk Assessment early screening

Prognostic predictive Especially for making a treatment decision and choosing the precision therapy

Pharmacogenomics pharmacodynamics Considering the treatment, especially consider for choosing the correct dose

BETTER OUTCOMES/SURVEILLANCE Fig. 2  The potential usage of SNPs in colon cancer. SNPs might be necessary for early screening and assessment of the inherited risk of CC; SNPs might provide valuable prognostic/predictive information for CC. SNPs reflect susceptibility to CC therapeutic processes; accordingly, individualized treatment will be more effective

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Fig. 3 Association between some genes SNPs and colon cancer. The SNPs in the APC, ATM, BRAF, BRCA1, DUSP10, MAP2K1, MLH1, and MTHFR genes may affect the genetic susceptibility of CC and accordingly might increase the risk for CC

Conflict of Interest None Funding None

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Role of Nano-immunotherapy in Colorectal Cancer: An Updated Review Devanabanda Mallaiah and Ramakrishna Vadde

Abstract  Colorectal cancer (CRC) has become the leading cause of death among cancer patients in developed countries. Genetic and environmental factors, along with the immune system, play a role in CRC’s development and progression. Its high prevalence and mortality rate demand early detection and efficient treatment strategies. Though traditional therapeutic approaches such as surgery, radiotherapy, and chemotherapy are used in CRC treatment, they are limited by severe side effects. Advanced cancer immunotherapy treatments are limited by the drawbacks and immune evasion mechanisms developed in CRC. Recently, immunotherapy based on engineered nanoparticles (NP) emerged as a promising approach for CRC treatment. Present review deals with the mechanism, immunology of CRC, and advanced immunotherapy based on nanotechnology. Keywords  Colorectal cancer · Immunotherapy · Nano-immunotherapy · Nanotechnology

1  Introduction Colorectal cancer (CRC) is an adenocarcinoma in the colon or rectum. It is considered a lifestyle disease due to its incidence in people with high carbohydrate and animal-fat diets and sedentary lifestyles. Colorectal cancer has a high incidence rate globally, mainly in developed countries, and is represented as the second most common cancer and also the second cause of death from cancers in countries like Europe (Sánchez-Peralta, Bote-Curiel, Picón, Sánchez-Margallo, & Pagador, 2020). CRC occurrence is more in males than females, and >1.4 million new cases are diagnosed every year worldwide. Even though CRC incidences are low in developing countries like India, the absolute numbers of CRC cases are higher than in developed countries due to fewer early diagnosis resources, advanced treatment methods, and socioeconomic factors (Patil et al., 2017). D. Mallaiah · R. Vadde (*) Department of Biotechnology & Bioinformatics, Yogi Vemana University, Kadapa, Andhra Pradesh, India e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_5

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Surgery, chemotherapy, and radiotherapy are standard conventional therapies for CRC, but complete removal of cancer cells is not possible with surgery and nonspecificity; cytotoxicity to normal cells is associated with chemo- and radiotherapies. Thus, it is crucial to develop advanced therapeutic approaches to treat CRC. Cancer immunotherapy is the best alternative method and produced efficient results in some CRC patients. In addition to advantages, drawbacks are also associated with cancer immunotherapy-based monoclonal antibodies, immune checkpoint inhibitors, vaccines, oncolytic virus, chimeric antigen receptor-T (CAR-T) cells, and cytokines (Johdi & Sukor, 2020). Although the immune system plays a role in anticancer activity, tumors adopt specific mechanisms to tackle the host immune response. Cancer immunotherapeutics became advanced alternative approaches to enhance immunity against tumors. However, cancer immunotherapeutics also resulted in off-target delivery and system toxicity by an improved immune response. Nanoparticles (NP)-based cancer immunotherapy approach overcomes those barriers by targeting and reducing the systemic exposure of immunomodulatory agents and enabling nano-immunotherapy (Guevara, Persano, & Persano, 2019). This chapter deals with the origin, immunology, and nano-immunotherapy of CRC.

2  Mechanism of Colorectal Cancer The development of CRC is a multistep process, which includes histological, morphological, and genetic changes accumulated over time. Initially, abnormal epithelial cells aggregate in the intestinal mucosa and protrude into the intestinal lumen called polyps. A small percent of polyps (adenomas and sessile serrated polyps) only takes several years to acquire malignant potential (Janney, Powrie, & Mann, 2020). CRC is a heterogeneous cancer due to its initiation by multiple components such as genetic, epigenetic alterations, and dysregulated immune response (Fig. 1). The mutations in oncogenes and tumor suppressor genes overactivate Wnt/β-catenin signaling, which transforms normal epithelium into aberrant epithelial polyps. Additional genetic mutations such as p53 and K-Ras further alter polyp into adenomas and then to carcinomas (Simon, 2016). CRC risk also is enhanced by other genetic (familial adenomatous polyposis and Lynch syndrome) and environmental factors (diet with low fiber, high red meat, and

Fig. 1  Development of colorectal cancer through mutations

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microbiota). Besides, the host microbiota maladaptation or dysbiosis in CRC promotes well-known cancer hallmarks such as DNA damage, cell death resistance, and immune evasion (Janney et  al., 2020). Additionally, host immune deficiency also involves in the occurrence and progression of CRC.

3  Immunology of Colorectal Cancer CRC is classified into two molecular subtypes based on the status of DNA proofreading and repair machinery. Defects both in mismatch repair and DNA replication machinery increase the load of mutations, which result in frames shifted proteins (neoantigens) with high immune potentials demonstrated by the infiltration of cytotoxic T cells. The type and magnitude of genetic instability determine the tumor microenvironment immune composition and clinical response to immune therapies (IJsselsteijn, Sanz-Pamplona, Hermitte, & de Miranda, 2019). Galon and colleagues developed a tool called immunoscore, which classifies tumors into low, intermediate, and high immunoscore based on the type, the density of T cells at tumor core, and their invasive margin. Recently, immunoscore was also confirmed as an independent prognostic tool in colorectal cancer by international efforts (Pagès et al., 2018). Immunoscore or immune infiltrate determines CRC prognosis and the efficacy of immunotherapy (Guo, Wang, Qiu, Pu, & Chang, 2020). An immune function switches from immunosurveillance to immunosuppression and allows the conversion of premalignant lesions to CRC cells (Cui, 2020). Regulatory T cells (Tregs) infiltrated in the tumor microenvironment (TME) play a role in CRC, but their final effect depends on their tumor localization, subtypes, and phenotype plasticity (Fantini, Favale, Onali, & Facciotti, 2020). Cancer cells escape from the immune system by various mechanisms such as decreasing their immunogenicity and secreting immune-suppressive factors in the tumor microenvironment. Immature myeloid cells like myeloid-derived suppressor cells play an essential role in cancer immune evasion. The heterogeneous myeloid-­ derived suppressor cells (MDSC) inhibit T cells’ anticancer activity and induction of immunosuppressive regulatory-T and Th-17 cells. MDSC also plays a role in the invasion, angiogenesis, and metastasis of cancer. Although both MDSC (G and M) types increased, G-MDSC levels were higher than M-MDSC and played a crucial role in CRC (De Cicco, Ercolano, & Ianaro, 2020).

4  Role of Nanotechnology in Immunotherapy of CRC Different immunotherapy strategies have been used to promote the immune response against cancer cells. Although cancer immunotherapeutic approaches have been proven efficient against many types of cancers, the patients’ overall response rate remains below 30%. Therefore, it is essential to improve current immunotherapeutics or develop other alternatives (Aikins, Xu, & Moon, 2020). Considering the

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therapeutic efficacy and off-target side effects of immunotherapeutics, researchers have focused on immunotherapy-based nanoparticles because of their unique properties, including adjustable particle size, high carrier capacity, and ease of modification for various delivery demands, and feasibility of variable routes of administration. Nano-immunotherapeutics have been used to reduce immune-suppression, enhance immune response, and generate memory immune cells against cancer cells for long-­ term control (Xiong, Wang, & Tiruthani, 2019). The uncertainties in immunity activation and immune-related adverse effects are overcome with a targeted delivery of cancer antigens, adjuvants, tumor microenvironment modulation, development of vaccines, and persistent immune response production by nanoparticles (Selvaraja & Gudipudi, 2020). Nanoparticles enhance the immune response by specific delivery of vaccines to antigen-presenting cells and allow safe and effective stimulation of immunity toward cancers by stimuli-responsive delivery of therapeutics to tumors (Aikins et al., 2020). Nanoparticles also deliver immuno-modulating agents such as peptides, nucleic acids, and immune checkpoint inhibitors, thereby introducing combinational cancer immunotherapy called nano-immunotherapy (Yang, Ma, Zhao, Yuan, & Kim, 2020). Many studies have examined the anticancer effect of nanoparticles per se or drug vehicles against colon cancer using in vitro and in vivo studies. Gold nanoparticles prepared from Albizia lebbeck showed a cytotoxic effect on HCT-116 colon cancer cell lines (Malaikolundhan et  al., 2020). Aptamer-guided albumin nanoparticles loaded with docetaxel enhanced anticancer effect against CT26 colon cancer cells and also prolonged CT26-bearing mice survival (Yu, Li, Duan, & Yang, 2020). There are studies on the combination of nanoparticles and immunotherapeutics against colon cancer. In murine colon adenocarcinoma model, peritumoral injection of TLR7/8a poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) nanoparticles in combination with anti-programed death-ligand 1 immunotherapy showed a reduction in tumor growth, systemic toxicity, and extended survival when compared with free TLR7/8a treatment (Smith et al., 2020). Conventional immunotherapies’ efficacy is enhanced by nanoparticles’ intrinsic immunogenicity, intelligent targeting, controlled surface modification, and multifunctional potential (Wang, Sun, & Hou, 2020). The short-interfering RNA-loaded lipid-based nanoparticles have been used to silence specific genes involved in the M2 polarization of macrophages, which resulted in increased infiltration of CD11+ b macrophages into tumors and tendency to M 1 macrophages polarization (Shobaki, Sato, Suzuki, Okabe, & Harashima, 2020). Antitumor response of chemo-immunotherapy is highly dependent on the simultaneous induction of tumor autophagy. On-demand autophagy cascade amplification nanoparticles showed optimal immune activation and antitumor activity by the simultaneous triggering of immunogenic tumor death by oxaliplatin and autophagy by STF-62247 in CT26 tumor-bearing mice (Wang et al., 2020). Anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) Si-RNA-loaded chitosan-lactate nanoparticles downregulated CTLA-4 on tumor-infiltrating T cells and thereby facilitated T-cell activation by tumor-lysate-loaded dendritic cell vaccine, which resulted in tumor regression and extension of CT26 tumor-bearing mice survival (Esmaily et  al., 2020). The inhibition of immune checkpoint inhibitors (A2AR and CTLA-4)

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simultaneously by SiRNA-loaded polyethylene glycol-chitosan-alginate nanoparticles enhanced antitumor response in CT26 colon cancer-bearing mice (Ghasemi-­ Chaleshtari et al., 2020) synergistically. Synthetic HDL (sHDL) nanodiscs co-loaded with docetaxel (DTX) and cholesterol-modified Toll-like receptor 9 (TLR9) agonist CpG (cho-CpG) oligonucleotide exhibited enhanced anticancer effect by reducing overall tumor size and extended MC-28 tumor-bearing mice survival when compared with free agents (Scheetz et al., 2020). The photothermic therapy by indocyanine green (ICG) liposome with a combination of PD-1/TIM-3 blockade strategy inhibited distant tumors in the MC38 tumor model (Huang et al., 2020). Polymeric micelles loaded with anti-SMC2 antibodies (Ab-SMC2) inhibited tumor sphere formation in HCT116 colon cancer cell lines (Montero et al., 2020). Nanomedicine-based immunotherapy target tumor necrosis factor receptor 2 (TNFR2), including DNA methylation inhibition, showed maximum anticancer potential (Al-Hatamleh et al., 2019). Targeted radionuclide therapy combined with blockade of the PD-1/PD-L1 axis improved mice survival and long-term control of tumors (Chen et al., 2019). Co-silencing of PD-1 or PD-L1 by PLGA nanoparticles inhibits the colon tumor growth and provides efficient strategy based on nano-­ immunotherapy (Kwak et al., 2019). In MC-38 colon adenocarcinoma models, the co-delivery of doxorubicin and immunoadjuvants effectively control tumors by biodegradable nanoparticles (Da Silva et al., 2019). Calcium phosphate nanoparticles loaded with CpG and tumor antigens and immune checkpoint blockers against PD-L1 increase cytotoxic CD8+ T-cell response and control the tumor (Heße et al., 2019). 3-aminopropyltriethoxysilane (APTES)-modified Fe3O4 nanoparticles (FeNPs) loaded with CpG showed antitumor effect by producing a humoral and cellular response in C26 colon cancer (Zhang et  al., 2018). Treatment with DMA-­ based liposomes loaded with pIL-15 inhibited tumor growth significantly and prolonged tumor-bearing mice survival (Liu et al., 2018). The FAT1-specific monoclonal antibody mAb198.3 loaded to superparamagnetic nanoparticles (spmNPs) reduced the tumor growth by improving the bioavailability and pharmacodynamics of anti-FAT mAb198.3 (Grifantini et  al., 2018). The combination of chemo-­ immunotherapy with nanodiscs and anti-programmed death 1 therapy exhibited inhibition colon carcinoma tumors and tumor recurrence in survival-protected mice (Kuai et al., 2018). Slow and continuous release of an anti-PD-1 peptide (APP) by hollow gold nanoshell (HAuNS) into biodegradable poly(d, l-lactic-co-glycolide) nanoparticles through photothermic ablation showed strong antitumor effect against primary and distant tumors (Luo et  al., 2018). Lipopolysaccharide-coated CuS nanoparticles showed anticancer and anti-metastatic effects by immuno-photothermic therapy in CT26 tumor-bearing BALB/c mice (Jang et  al., 2017). The cross-linked lipid-­ polymer adjuvant nanodepots laden on the immunologically dying tumor cells activate antigen presentation by T cells and initiate antitumor response. Combining tumor cell vaccination and immune checkpoint blockade therapy leads to the complete removal of tumor and recurrence (Fan et al., 2017). The combination of gold nanoparticles conjugated to programmed death-ligand 1 antibody (αPDL1) therapy and computed tomography imaging allows prediction of therapeutic response

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48 hours by noninvasive manner (Meir et al., 2017). Antibiotin-coated iron-dextran is conjugated with inhibitory checkpoint PD-L1 and stimulatory 4-1BB and prepared as immunoswitch particles, which delay the tumor growth and extended colon cancer mice survival (Kosmides, Sidhom, Fraser, Bessell, & Schneck, 2017). A nanovaccine delivers tumor antigens to dendritic cells, activates interferon genes (STING), and inhibits tumor growth potential in the colon cancer model (Luo et al., 2017). Modified nanoparticles with pIL12 inhibited CT26 cells ex vivo and significantly reduced tumor growth by promoting apoptosis and hindering angiogenesis and proliferation (Goodwin & Huang, 2017). Lipid calcium phosphate nanoparticle encapsulated with phosphorylated adjuvant and cancer peptide antigen reduced the tumor growth and prevented liver metastasis (Goodwin & Huang, 2017). Nanoparticles encapsulated with chlorin e6 (Ce6) (photosensitizer) and imiquimod (R837) (TLR-7 agonist) in combination with CTLA-4 checkpoint blockade destroyed both primary and distant tumors and also prevented recurrence by immune memory effect (Xu et al., 2017). Lipid nanoparticles coated with TRAIL-activated DR5 crosslinking and showed a robust anticancer effect against colon cancer (De Miguel et al., 2016). Chemo- and photodynamic therapy combined with core-shell nanoscale coordination polymers enhanced the checkpoint blockade cancer immunotherapy (He et al., 2016). Cationic nanoliposomes carrying FL and TRAIL genes showed a significant effect on colon cancer both in vitro and in vivo studies (Sun et  al., 2012). The fractalkine-Fc deliveries by DNA/704 nanospheres efficiently inhibit colon cancer’s lung metastasis and provide a promising approach in cancer immunotherapy (Richard-Fiardo et al., 2011). Polydopamine nanoparticles loaded with tumor cell lysate also played a role in cancer immunotherapy of colorectal cancer (Wang et  al., 2019). Some studies showed that immunologically cold tumors are treated with nanoplatforms, along with photoimmunotherapy (Yu et al., 2020). There are other studies proven by cellular models that nanoparticles and chemo- and immunotherapy play a significant role in treating colorectal cancer (Bauleth-Ramos et al., 2020). Some nanoformulations by PEGylated lipid nanoparticles along with chemo- and immunotherapeutic demonstrated that liver metastasis of colorectal cancer was decreased significantly (Guo, Yu, Das, & Huang, 2020). The use of nanoformulation by spherical polymeric nanoparticles along aminobisphosphonates showed strong immunotherapeutic potential against colorectal cancer (Di Mascolo et al., 2019). Nanoparticles, which induce immunogenic cell death and the PD-L1 checkpoint blockade, are promised as an effective immunotherapeutic strategy for colorectal cancer (Wen et al., 2019). Nanoformulation with quercetin and alantolactone showed excellent immunotherapeutic agents to CRC, resistant to traditional immunotherapy (Zhang et al., 2019). The immunostimulatory chemotherapeutic combination, along with anti-PD-L1 antibody treatment, eradicated the colorectal cancers (Duan et  al., 2019). Lipid-­ protamine-­DNA nanoparticle system loaded with the coding sequence for LPS along with anti-PD-L1 mAb therapy inhibits liver metastasis of colorectal cancer (Song et al., 2018). Black phosphorus nanosheets are also exploited in the chemo-­ immunotherapy of colorectal cancer (Ou et  al., 2018). Some nanoparticles were designed to enhance immunotherapy and simultaneously to modulate tumor

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micro-environment, which prevented metastasis of colorectal cancer (Feng et al., 2018). Liposomal nanohybrid centrosomes conjugated with anti-EGFR antibody and IRDye800CW and MRI contrast DOTA-Gd exploited in the photodynamic therapy of CRC.  The theranostic function, along with PD-L1 immunotherapy, showed a significant antitumor effect against colorectal cancer (Li et  al., 2018). Lipid-protamine-DNA nanoparticle loaded with oxaliplatin and PD-L1 was shown to be an effective and safe immunotherapeutic approach in murine colorectal cancer (Song et al., 2018). The chlorin-based nanoscale metal-organic framework, in combination with synergistic photodynamic therapy and checkpoint blockade immunotherapy, effectively inhibited local and distant tumors in colorectal cancer (Lu et al., 2016). The chitosan-TPP/nanoparticles formulated with IL-12 are exploited liver immunity to prevent liver metastasis of colorectal cancer (Xu et al., 2012).

5  Conclusions Many studies investigated the use of nanoparticles in the delivery of chemo- and immunotherapeutic agents in the control of colorectal cancer and found nanoparticles efficiently and specifically target tumor sites compared to immunotherapeutics alone. Among the nanosystems, lipid-based nanosystems are exploited as the major carriers of chemo- and immunotherapeutics. Nanoparticles with chemo- or immunotherapeutics along with other photothermal or photodynamic combination therapy exhibited synergistic antitumor effect against colorectal cancer and allowed the development of nano-immunotherapy as an alternative advanced therapy for primary or metastatic cancers. Conflicts of Interest  The author declares no conflicts of interest.

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Mechanistic Exploration and Therapeutic Management of Colon Cancer Metastasis Anupam Kumar Srivastava

Abstract  The metastasis of colon cancer is the leading cause of death in patients with colon cancer. Surgery and chemotherapy have long been the first choices for cancer patients. However, the prognosis has never been satisfying, particularly for patients with metastatic lesions. The early clinical symptoms of colon cancer obvious abdominal pain, diarrhea, adenomatous polyps, thrombosis, anemia, blood in the stool, and spleen cyst. Along with this intestinal obstruction for advanced cancer, which seriously disturb people’s life and health. This chapter describes the advantages and disadvantages of various methods of treating colon cancer from the intestinal flora, targeting therapies, Western medicine, traditional Chinese medicine, other aspects of treatment methods for colon cancer, and future trends. As well as it explores some recent studies which found that the intestinal flora may regulate immunity, improve inflammation, and prevent the growth of cancer cells. The treatment of metastatic colon cancer needs a center with advanced imaging technologies and expertise to guide diagnosis and staging. Persistent research on colon cancer has said that left and right colon cancers have unrelated clinical and biological characteristics which require continuous efforts to provide personalized treatments. The chapter also states about the worldwide guidelines with current updates on the recommended targeted drugs on the basis of the increasing number of high-quality clinical trials. Targeted therapy is a new optional approach that has successfully prolonged the overall survival of colon cancer patients. The National Comprehensive Cancer Network (NCCN) guidelines represent the systemic therapy for advanced or metastatic disease including sequencing and timing of therapies, maintenance therapy, and the therapy after progression. This chapter also provides an overview of the existing CRC-targeted agents, humanized monoclonal antibody (mAb), and their underlying mechanisms, as well as a discussion of their limitations. Keywords  Colorectal cancer · Chinese traditional medicine · Chemotherapy · Metastatic colon cancer · Intestinal flora

A. K. Srivastava (*) Department of Biochemistry, All India institute of Medical Sciences (AIIMS), Bathinda, Punjab, India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_6

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1  Introduction In the United States, colorectal cancer is the third most common diagnosed cancer and is the second most deadly malignancy, after lung cancer. In 2020, an estimated 10,4610 people were diagnosed with colon cancer only and 53,200 died due to their disease (seer.cancer.gov, 2017; Siegel & Miller, 2020). The incidence of colorectal cancer in the overall US population is declining, due in large part to an increase in colonoscopic screening (cancer.org, 2017; Doubeni, Corley, Quinn, et  al., 2018; Edwards, Ward, Kohler, et  al., 2010; Nishihara, Wu, Lochhead, et  al., 2013). However, the rate of colorectal cancer is rising among adolescent and young adult (AYA) patients (seer.cancer.gov, 2016; Siegel, DeSantis, & Jemal, 2014; Siegel, Jemal, & Ward, 2009). In fact, 5.7% of patients with newly diagnosed colorectal cancer are under 45 years of age, and 20.5% are younger than 55 years of age (seer. cancer.gov, 2017). The metastasis of colon cancer is the leading source of death in patients with colon cancer. Some clinicopathologic and biologic features like BRAF mutation and hypomethylation demand the evaluation for hereditary colorectal cancer syndromes, unique to AYA patients (Weinberg, Marshall, & Salem, 2017). The treatment of colon cancer can be explored from the area of targeted therapy, traditional Chinese medicine, Western medicine (chemotherapy), intestinal flora, and other aspects. Besides this, the theme from the recent NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for colon cancer will also be discussed here. Metastatic patterns differ remarkably between colon and rectal cancers. Rectal cancer more frequently metastasized into the thoracic organs and the nervous system and normally within the peritoneum. Mucinous and signet ring adenocarcinomas more frequently metastasized within the peritoneum compared with generic adenocarcinoma and less frequently into the liver. Lung metastases occurred frequently together with nervous system metastases, whereas peritoneal metastases were often listed with ovarian and pleural metastases. Thoracic metastases are nearly as common as liver metastases in rectal cancer patients with a low stage at diagnosis. In colorectal cancer patients with solitary metastases, the survival differed between 5 and 19 months, reliant on T or N stage (Riihimäki et al., 2016).

2  Current Treatment of Colon Cancer Currently, the advances in primary and adjuvant treatments has improved the survival of CRC patients. Treatment of mCRC has experienced substantial changes in the last 30  years. New therapeutics and combination regimens have directed to marked improvements in both response rate (RR) and overall survival (OS). Classically, the ideal CRC treatment is to achieve complete removal of the tumor and metastases, which mostly requires surgical intervention. However, despite the arrival of numerous screening programs to reduce CRC incidence, nearly a sector of

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CRCs is diagnosed at an advanced stage with metastases, and 20% of the remaining cases may develop metachronous metastases, which makes curative surgical control difficult and leads to tumor-related deaths. For those patients with unresectable lesions or who are intolerant to surgery, the goal is extreme shrinkage of the tumor and suppression of further tumor spread and growth, and radiotherapy and chemotherapy are the leading strategies for controlling the disease in such patients. Of note, in some cases, chemotherapy or radiotherapy might be functional before or after surgery as neoadjuvant or adjuvant treatment to maximally reduce and steady the tumor (Xie, Chen, & Fang, 2020).

2.1  Choice of First-Line Treatment Generally, the choice of first-line treatment should be predisposed by the tolerability of the medication as well as the patient’s comorbidities, biological age, and favorites. It is important to emphasize that decisions regarding the first-line treatment are critical for the patient’s outcome. First-line treatment has a significantly higher overall response rate (ORR) and longer progression-free survival (PFS) than the consecutive treatment lines (Modest, Hiddemann, & Heinemann, 2014). Finally, the choice of first-line treatment describes the possible consecutive regimens. 2.1.1  Chemotherapy Current chemotherapy includes both single-agent therapy, which is mainly fluoropyrimidine (5-FU)-based, and numerous agent regimens containing one or several drugs, including oxaliplatin (OX), irinotecan (IRI), and capecitabine (CAP or XELODA or XEL). Although studies have contended that first-line single-agent therapy is not inferior to combined regimens in terms of overall survival (OS), the combined therapy regimens FOLFOX (5-FU+OX), FOXFIRI (5-FU+IRI), XELOX or CAPOX (CAP+OX), and CAPIRI (CAP+OX) endure the mainstream approaches in the first-line treatment, while patients with meagre performance or at low risk of deterioration are recommended to receive single-agent therapy. When selecting additive agents, efficacy appears to be similar, and only adverse events may differ amongst different regimens. Emerging evidence does not support stouter efficacy in the multiple-agent regimen FOLOXIRI (5-FU+OX+IRI), which is infrequently applied because of its potentially increased toxicity. Nonetheless, data from research performed in recent eras show that using chemotherapy in patients with CRC, especially those with metastases, has pushed their OS time to almost 20 months, resultant in chemotherapy becoming the backbone of CRC treatment. But chemotherapy is associated with certain boundaries, such as existing systemic toxicity, unsatisfying response rate, unpredictable innate and acquired resistance, and low tumor-­ specific selectivity. Therefore, huge investments have been pledged to develop new approaches to refine or even substitute existing CRC chemotherapy (Xie et al., 2020).

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2.1.2  Therapeutics and Regimens The standard of attention for the majority of patients is the combination of 5-­fluorouracil (FU)/leucovorin (LV) with either oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) together with a monoclonal antibody (mAb) against either vascular endothelial growth factor (VEGF) or epidermal growth factor receptor (EGFR) [6]. Bevacizumab is a mAb against VEGF, whereas cetuximab and panitumumab are focused against EGFR.  All three are approved for first-line treatment in mCRC. FOLFOX and FOLFIRI show a comparable efficacy regarding OS [7]. For clinically fit patients, a higher ORR and a longer OS can be attained with the triple combination of FOLFOXIRI alone [8] or in combination with bevacizumab [9]. Intravenous 5-FU can be replaced by the oral fluoropyrimidine capecitabine either in combination with oxaliplatin (CapOx) [10] or irinotecan (CapIri) [11]. In general, capecitabine should not be combined with cetuximab due to the negative results obtained in the COIN study [12]. Especially for elderly patients, the combination of capecitabine and bevacizumab is actual and well tolerated [13]. Cetuximab and panitumumab are both approved for first-line treatment with either FOLFIRI or FOLFOX (Holch, Stintzing, & Heinemann, 2016). 2.1.3  Targeted Therapy The awareness of molecular-targeted therapy has a quite long history. The concept of a chemical that definitely targets a microorganism was first proposed in the initial 1900s and expanded to cancer treatment in 1988. This concept was renewed and has flourished in the past 20 years. Twenty-three targeted therapies can work on cancerous cells by directly inhibiting cell proliferation, differentiation, and migration. The tumor microenvironment, including local blood vessels and immune cells, might also be altered by targeted drugs to impede tumor growth and enact stronger immune surveillance and attack. Small molecules, such as monoclonal antibodies, are major players in targeted therapies. Many such agents have been developed and brought into preclinical and clinical trials. The list of recommended CRC-targeted agents from guidelines such as those from the National Comprehensive Cancer Network (NCCN) is being updated quickly, given the unprecedented speed of the emergence of large trials. Various pathways mediating the initiation, progression, and migration of CRC, such as Wnt/β-catenin, Notch, Hedgehog, and TGF-β (transforming growth factor-β)/SMAD, as well as those proficient of activating signaling cascades, such as phosphatidylinositol 3-kinase (PI3K)/AKT or RAS/rapidly accelerated fibrosarcoma (RAF), contain ideal sites for targeted therapy (Xie et al., 2020). Besides this, the miRNA may also be used as target for anticancer remedy, and miRNA-based therapies can be an effective method for individuals to treat colon cancer. Luo et al. have explored that the expression of a long-chain noncoding RNA (lncRNA) HOTAIR is closely related to tumor metastasis and plays an important role in cancers such as colon cancer, gastric cancer, esophageal cancer, and pancreatic cancer, especially in the occurrence and metastasis of colon cancer; these can

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be used as a new target for the treatment of colon cancer. A microtubule-associated protein TPX2 is encoded by a gene located on a human chromosome that promotes proliferation and metastasis of the colon cancer cells and aids as a novel prognostic biomarker and therapeutic target for colon cancer. Iron-saturated bovine lactoferrin (Fe-bLf) nanocarriers shows anticancer effects and can be used to target colon and other cancer stem cells. The p38γ-activated ternary complex of Hsp90 and KRAS may be a novel target for the treatment of KRAS-dependent colon cancer. The developmental pluripotency-related 4 gene (Dppa4) plays a major role in the self-­ renewal of the embryonic stem cells. This gene can be re-expressed in several malignant tumors and has been revealed to play a large role in the development of colon cancer. So, it can also be used as a new target for the treatment of colon cancer. Morita et al. have found that DnaJ (Hsp40) homologous subfamily B member 8 (DNAJB8), a member of the heat shock protein (HSP) 40 family, inhibits the accumulation of damaged folding toxic proteins related to the development and metastasis of cancer (Morita et al., 2014). Li et al. found that combining DNAJ8-targeted immunotherapy with other standard therapies can effectively cure cancer and indicated that it can be a new target for colon cancer-targeted immunotherapy (Li et al., 2019). 2.1.4  S  ystemic Therapy for Advanced or Metastatic Disease (NCCN GUIDELINES) When reviewing the NCCN guidelines, clinicians should be aware of several things. The current management of dispersed metastatic colon cancer involves various active drugs, either in combination or as single agents: irinotecan, 5-FU/leucovorin (LV), oxaliplatin, capecitabine, bevacizumab, cetuximab, ziv-aflibercept, panitumumab, ramucirumab, trifluridine-tipiracil, regorafenib, pembrolizumab, nivolumab, etc. The putative mechanisms of action of these agents are wide ranging and include interference with DNA replication and inhibition of the actions of vascular endothelial growth factor (VEGF) and epidermal growth factors (EGFRs). The choice of therapy is based on the consideration of the purpose of therapy, the type and timing of prior therapy, the mutational profile of the tumor, and the differing harmfulness profiles of the constituent drugs. Although the specific regimens recorded in the guideline are designated according to whether they pertain to initial therapy, therapy after the first progression, or therapy after the second progression, it is important to clarify that these recommendations characterize a continuum of care and that these lines of treatment are blurred rather than discrete. As initial therapy for metastatic disease in a patient appropriate for intensive therapy (i.e., one with a good tolerance for this therapy for whom a lofty tumor response rate would be potentially beneficial), the panel recommends a choice of five chemotherapy regimens: FOLFOX (i.e., mFOLFOX6), FOLFIRI, CapeOx, infusional 5-FU/LV, or capecitabine or FOLFOXIRI with or without targeted agents (Benson, Venook, Cederquist, et al., 2017).

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2.2  Sequence and Timing of Therapies Limited studies have addressed the sequencing of therapies in advanced metastatic colon cancer. The results from a randomized study to appraise the efficacy of FOLFIRI and FOLFOX regimens as initial therapy and to determine the effect of using consecutive therapy with the alternate regimen after the first progression showed neither sequence to be significantly superior with respect to progression-­ free survival (PFS) or median overall survival (OS). A combined analysis of data from seven recent phase III clinical trials in advanced CRC provided support for a correlation between an increase in median survival and administration of the three cytotoxic agents, i.e., 5-FU/LV, oxaliplatin, and irinotecan. Generally, the panel does not consider one regimen (i.e., FOLFOX, CapeOx, FOLFIRI, 5-FU/LV, capecitabine, FOLFOXIRI) to be preferable over the others as initial therapy for metastatic disease. The panel also does not designate a preference for biologic agents used as part of the initial therapy like bevacizumab, cetuximab, panitumumab, etc. (Benson et al., 2017).

2.3  Maintenance Therapy Mostly it is the strategy after the initial treatment. This method involves intensive first-line therapy, followed by less intensive therapy until progression in patients with good response to initial treatment. Numerous studies have examined a strategy with de-escalation after an initial treatment phase followed by a maintenance therapy with re-escalation in the case of progressive metastatic CRC. A series of open-­ label phase III trials demonstrated that the quality of life was not affected by maintenance therapy. Especially in the context of oxaliplatin-containing regimens, the duration is frequently limited due to cumulative neurotoxicity. For example, the CAIRO3 study was assessing maintenance therapy with capecitabine/bevacizumab versus observation in 558 patients with metastatic CRC and with stable disease. The GERCOR DREAM trial (OPTIMOX3) was a study that randomized patients with metastatic CRC without disease progression on bevacizumab-based therapy to maintenance therapy with bevacizumab or bevacizumab plus erlotinib. 2.3.1  Bevacizumab Bevacizumab is a humanized mAb that blocks the activity of VEGF, a factor that plays an important role in tumor angiogenesis. Collective results from several randomized phase II studies have shown that the addition of bevacizumab to first-line 5-FU/LV showed better OS in patients with unresectable metastatic CRC compared with those receiving these regimens lacking bevacizumab. A study of earlier untreated patients receiving bevacizumab plus irinotecan/fluorouracil/leucovorin (IFL) also provided support for the inclusion of bevacizumab in the initial therapy

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(Hurwitz, Fehrenbacher, Novotny, et al., 2004). FOLFOXIRI with bevacizumab is also an accepted combination, while no randomized controlled trials have equated FOLFOXIRI with or without bevacizumab. Several meta-analyses have shown an advantage for the use of bevacizumab in first-line therapy for metastatic CRC. A recent analysis of the SEER-Medicare database found that bevacizumab added a modest improvement to the OS of patients with stage IV colorectal cancer diagnosed between 2002 and 2007. Overall, the addition of bevacizumab to first-line chemotherapy looks to offer a modest clinical benefit. No data directly address whether bevacizumab should be used with chemotherapy in the perioperative treatment of resectable metastatic disease. A recent meta-­analysis of randomized controlled trials presented that the addition of bevacizumab to chemotherapy is associated with a higher incidence of treatment-related mortality than chemotherapy alone (relative risk [RR], 1.33; 95% CI, 1.02–1.73; P  =  0.04), with hemorrhage (23.5%), neutropenia (12.2%), and gastrointestinal perforation (7.1%) being the most common causes of fatality (Ranpura, Hapani, & Wu, 2011). Also, the risk of stroke and other arterial events is increased in patients receiving bevacizumab, especially in those aged ≥65 years. When chemotherapy plus bevacizumab or chemotherapy alone was administered before surgery, with a delay between bevacizumab administration and surgery of at least 6 weeks, the incidence of wound healing complications in either group of patients should be low. Preclinical studies suggested that termination of anti-VEGF therapy might be associated with accelerated recurrence, more aggressive tumors on recurrence, and increased mortality. 2.3.2  Cetuximab and Panitumumab These are monoclonal antibodies directed against EGFR that obstruct its downstream signaling pathways. Cetuximab is a chimeric mAb, whereas panitumumab is a fully human mAb. Cetuximab and panitumumab have been studied in combination with FOLFIRI and FOLFOX as initial therapy choices for the treatment of metastatic CRC. The incidence and severity of skin reactions with cetuximab and panitumumab seem to be very similar. Furthermore, the presence and severity of skin rash in patients receiving either of these drugs have been shown to predict amplified response and survival. An NCCN task force addressed the management of dermatologic and other toxicities associated with anti-EGFR inhibitors. Cetuximab and panitumumab have also been associated with a risk for venous thromboembolic and other serious adverse events.

3  The Role of Primary Tumor Sidedness A strong evidence for the predictive value of primary tumor sidedness, and the response to EGFR inhibitors as the first-line treatment of patients was found in the phase III CALGB/SWOG 80405 trial. The study showed that patients with all RAS wild-­type, right-sided primary tumors (cecum to hepatic flexure) had longer OS, if

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treated with bevacizumab than if treated with cetuximab in the first line. Cetuximab and panitumumab are monoclonal antibodies directed against EGFR that inhibit its downstream signaling pathways, but EGFR position as assessed using immunohistochemistry is not predictive of treatment efficacy. Furthermore, cetuximab and panitumumab are only operative in approximately 10–20% of patients with CRC.

4  The Role of KRAS and NRAS Status The recommendation for KRAS/NRAS testing, at this point, is not intended to indicate a preference regarding regimen selection in the first-line setting. Rather, this primary establishment of KRAS/NRAS status is appropriate to plan for the treatment continuum, so that the information may be obtained in a non-time-sensitive method and the patient and provider can discuss the implications of a KRAS/NRAS mutation, if present, while other treatment options still exist. Note that because anti-­ EGFR agents have no role in the management of stage I, II, or III disease, KRAS/ NRAS genotyping of CRC at these earlier stages is not recommended. KRAS mutations are early measures in CRC formation, and therefore a very tight correlation exists between the mutation status in the primary tumor and the metastases. For this reason, KRAS/NRAS genotyping can be performed on archived specimens of either the primary tumor or a metastasis. Approximately 40% of colorectal cancers are characterized by mutations in codons 12 and 13 in exon 2 of the coding region of the KRAS gene.

5  HER2 Overexpression It is a member of the same family of signaling kinase receptors as EGFR and has been successfully targeted in breast cancer in both the advanced and adjuvant situations. HER2 is rarely overexpressed in CRC, but the prevalence is higher in RAS/ BRAF wild-type tumors. Evidence from studies does not support a prognostic role of HER2 overexpression. However, initial results indicate that HER2 overexpression may be predictive of the resistance to EGFR-targeting monoclonal antibodies.

6  Cetuximab with FOLFIRI The use of cetuximab as initial therapy for metastatic disease was investigated in the CRYSTAL trial, in which patients were randomly assigned to receive FOLFIRI with or without cetuximab.

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7  Panitumumab with FOLFIRI FOLFIRI with panitumumab is listed as an option for first-line therapy in metastatic CRC based on extrapolation from data in second-line treatment.

8  Cetuximab with FOLFOX Few trials have assessed the combination of FOLFOX and cetuximab in first-line treatment of metastatic CRC. In a retrospective evaluation of the subset of patients having known tumor KRAS exon 2 status enrolled in the randomized phase II OPUS trial, the addition of cetuximab to FOLFOX was associated with an increased unbiased response rate and a very slightly lower risk of disease progression (7.7 vs 7.2 months [a 15-day difference]) compared with FOLFOX alone in the subset of patients with KRAS exon 2 wild-type tumors.

9  Panitumumab with FOLFOX Panitumumab in combination with either FOLFOX20 or FOLFIRI has also been investigated for first-line treatment.  The combination of FOLFOX and panitumumab leftovers an option as initial therapy for patients with advanced or metastatic disease. Importantly, the addition of panitumumab had a detrimental impact on PFS for patients with tumors characterized by mutated KRAS/NRAS. There are different randomized, open-label, multicenter trials from different countries comparing the efficacy of FOLFIRI plus cetuximab versus FOLFIRI plus bevacizumab or cetuximab or panitumumab versus bevacizumab in the first-line treatment of KRAS exon 2 wild-type, metastatic disease. Economic analyses recommend that bevacizumab may be more cost-effective than EGFR inhibitors in the first-line therapy for metastatic CRC. At this time, the NCCN panel considers the addition of cetuximab, panitumumab, or bevacizumab to chemotherapy as equivalent choices in the first-line, RAS wild-type, metastatic setting.

10  Therapy After Progression Decisions regarding therapy after progression of metastatic disease depend on previous therapies. The panel recommends against the use of interferon alfa, mitomycin, taxanes, methotrexate, pemetrexed, sorafenib, sunitinib, erlotinib, or gemcitabine, either as single agents or in combination, as therapy in patients displaying disease progression after treatment with standard

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therapies. These agents have not been shown to be effective in this setting. Still, no objective responses were observed when single-agent capecitabine was administered in a phase II study of patients with CRC resistant to 5-FU.  The recommended therapy options after the first progression for patients who have received prior 5-FU/LV-based or capecitabine-­b ased therapy may be dependent on the initial treatment regimen. A randomized trial found that the addition of a targeted agent after the first-line treatment recovers the outcomes but also increases toxicity. Some important specific biological therapies are discussed below.

10.1  Cetuximab and Panitumumab in the Non-­First-Line Setting For patients with wild-type KRAS/NRAS colorectal cancer who qualified progression on therapies not containing an EGFR inhibitor, cetuximab or panitumumab plus irinotecan, single-agent cetuximab or panitumumab, or cetuximab or panitumumab with FOLFIRI is suggested. For patients with wild-type KRAS/ NRAS CRC progressing on therapies that comprise an EGFR inhibitor, administration of an EGFR inhibitor is not recommended in subsequent lines of therapy.

10.2  Bevacizumab in the Non-First-Line Setting In the ML18147 (TML) trial, patients with metastatic CRC that progressed on regimens comprising bevacizumab received second-line therapy consisting of a different chemotherapy regimen with or without bevacizumab. This study met its primary endpoint, with patients continuing on bevacizumab having a modest improvement in OS (11.2 vs 9.8  months; HR, 0.81; 95% CI, 0.69–0.94; P = 0.0062). Subgroup analyses from this trial found that these treatment possessions were independent of KRAS exon 2 status. The panel added the extension of bevacizumab to the second-­line treatment options in the 2013 versions of the NCCN Guidelines for Colon and Rectal Cancers. It may be added to any regimen that does not cover another targeted agent. The panel recognizes the lack of data suggesting a profit to bevacizumab with irinotecan alone in this setting but believes that the option is satisfactory, especially in patients whose disease progressed on a 5-FU- or capecitabine-based treatment. When an angiogenic agent is used in second-line therapy, bevacizumab is chosen over ziv-aflibercept and ramucirumab (discussed later), based on toxicity and/or cost. It may also be appropriate to consider adding bevacizumab to chemotherapy after the progression of metastatic disease if it was not used in the initial therapy.

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10.3  Ziv-Aflibercept It is a recombinant protein that has a portion of the human VEGF receptors 1 and 2, fused to the Fc portion of the human IgG1. It is designed to act as a VEGF trap to prevent the activation of VEGF receptors and thus inhibit angiogenesis. The VELOUR trial tested second-line ziv-aflibercept in patients with metastatic CRC that progressed after one regimen containing oxaliplatin. Ziv-aflibercept has only shown motion when given in conjunction with FOLFIRI in FOLFIRI-naïve patients. No data suggested the activity of FOLFIRI plus ziv-aflibercept in patients who progressed on FOLFIRI plus bevacizumab or vice versa, and no data suggested the activity of single-agent ziv-aflibercept. Furthermore, the addition of ziv-aflibercept to FOLFIRI in first-line therapy of patients with metastatic CRC in the phase II AFFIRM study had no advantage and increased toxicity.

10.4  Regorafenib Regorafenib is a small molecule inhibitor of multiple kinases (including VEGF receptors, fibroblast growth factor [FGF] receptors, platelet-derived growth factor [PDGF] receptors, BRAF, KIT, and RET) that are involved with various processes, including tumor growth and angiogenesis. The phase III CORRECT trial randomized 760 patients whose disease progressed on standard therapy to best supportive care with placebo or regorafenib. The trial met its primary endpoint of OS (6.4 months for regorafenib vs 5.0 months for placebo; HR, 0.77; 95% CI, 0.64–0.94; P = 0.005). PFS was also significantly but modestly improved.

10.5  Ramucirumab Another antiangiogenic agent is ramucirumab. It is a human mAb that targets the extracellular domain of VEGF receptor 2 to block VEGF signaling. In the multicenter, phase III RAISE trial, 1072 patients with metastatic CRC whose disease progressed on the first-line therapy with fluoropyrimidine/oxaliplatin/bevacizumab were randomized to FOLFIRI with either ramucirumab or placebo. The primary endpoint of OS in the intent-to-treat population was met at 13.3 and 11.7 months. PFS was also improved with the addition of ramucirumab.

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10.6  Trifluridine/Tipiracil (TAS-102) Trifluridine/tipiracil is an oral combination drug, comprising a cytotoxic thymidine analog, trifluridine, and a thymidine phosphorylase inhibitor, tipiracil hydrochloride, which prevents the degradation of trifluridine. Early clinical studies of the drug in patients with CRC were promising.

10.7  Pembrolizumab and Nivolumab The fraction of stage IV colorectal tumors characterized as MSI-H (mismatch repair-deficient [dMMR]) fluctuated from 3.5% to 5.0% in clinical trials and was 6.5% in the Nurses’ Health Study and Health Professionals Follow-up Study. dMMR tumors have thousands of mutations, which can encode mutant proteins with the potential to be known and targeted by the immune system. However, programed cell death ligands 1 and 2 (PD-L1 and PD-L2) on tumor cells can overwhelm the immune response by binding to programed cell death protein 1 (PD-1) receptor on T-effector cells. This system evolved to protect the host from an unimpeded immune response. Many tumors upregulate PD-L1 and thus evade the immune system. Therefore, it has been hypothesized that dMMR tumors may be sensitive to PD-1 inhibitors.

10.8  C  etuximab or Panitumumab Versus Bevacizumab in Second-Line Setting The randomized, multicenter, phase II SPIRITT trial enrolled patients with KRAS wild-type tumors whose disease progressed on first-line oxaliplatin-based therapy plus bevacizumab to FOLFIRI plus bevacizumab or FOLFIRI plus panitumumab. No difference was found in the primary endpoint of PFS between the arms (7.7 months in the panitumumab arm vs 9.2 months in the bevacizumab arm; HR, 1.01; 95% CI, 0.68–1.50; P = 0.97).

11  Intestinal Flora The microbes present in the intestine are commonly referred to as the “intestinal flora,” which is a complex, large microbial ecosystem. This intestinal flora regulates some metabolic and physiological functions; the metabolism of undigested food, including regulation of lumen pH; the regulation of intestinal movement and stimulation of immune function; etc. This intestinal flora is observed as a unique organ

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with metabolic, immune, and inflammatory central functions in the human body and the pathogenesis of colon cancer. Under normal circumstances, in the human intestinal tract, the intestinal flora preserves a steady state, and the disorder of the intestinal flora activates epigenetic modifications and abnormal intestinal signaling pathways through the 16S rRNA gene. Whole-genome microgenomic sequencing analysis found that intestinal flora is associated with IBD, colon cancer, and other diseases. Researchers also through the study of human patients as well as rodent cancer models found that the changes in the intestinal flora will increase the risk of cancer such as colon cancer. Similarly, the 16S rDNA sequencing platform was used to determine the differences in colony characteristics between cancer and adjacent normal mucosa tissues. The genomic analysis of colon cancer flora and histological analysis of tumor tissues illustrates that Fusobacterium is a key factor in colon cancer. The benefit of these studies is the use of the molecular pathology epidemiology database, but there are certain limitations. For example, depending upon the number of tissue Fusobacterium nucleatum DNA, the distribution of chemotherapy use may not be significantly different. Zhu et al. have noticed that the probiotics include Bifid bacterium strains as beneficial bacteria that control the intestinal flora and its metabolism, and tungstic acid treatment selectively inhibits molybdenum-cofactor-dependent microbial respiratory pathways that are only operable during the onset of inflammation to prevent probiotic expansion and thus helpful in the treatment of colon cancer. In addition, leptin receptor (LPR) and vitamin D receptor (VDR) can induce probiotics to produce anti-inflammatory effects, although there are silent obstacles to the widespread use of probiotics in the clinics. It has been of countless help in the treatment of colon cancer. Li X et al. quote that “the anaerobic fungus Faecalibacterium prausnitzii is one of the main components of the intestinal flora and has been measured as a biological indicator of human health. The increase in the amount of Faecalibacterium prausnitzii may reduce the risk of colon cancer. The use of Faecalibacterium prausnitzii as a potential active ingredient in probiotic preparations is expected to be used to delicacy of colon cancer.” Gao et al. have explored that histamine has a potential antitumor effect, and the intestinal flora can arbitrate inhibition of inflammation-related colon cancer through luminal amine histamine production (Gao et  al., 2017). Intestinal flora is also affected by numerous factors like intestinal inflammation, genetic factors, diet, and environmental factors. It was found that dietary fiber can produce butyrate under the action of intestinal flora; it can inhibit the survival and growth of colon cancer cell lines (Bultman et al., 2016). Dietary fiber can also increase the richness of Prevotella bacteria to improve glucose metabolism and can affect drug resistance. Therefore, governing intestinal flora might be an effective strategy to decrease drug resistance. By studying the dietary components of specific pathogen-free (SPF) and sterile (GF) mice, it has been initiated that the complex nutrient mixtures such as proteins and fibers affect intestinal permeability and have a great influence on the growth of colitis. It has been found that adding walnuts to our food can increase the number of thick-walled bacteria in the intestine and reduce the number of bacteria such as Bacteroides, which indicates that eating walnuts may change the intestinal flora and may provide us with a new mechanism for health. Metabolomics may be used to demonstrate the role of gut microbiota (Li et al., 2019).

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12  Traditional Medicine Traditional Chinese medicine is broadly used in several inflammation-related disorders. The main compounds of the medicinal plant Scutellaria baicalensis are baicalin and scutellarin. The study which used human intestinal bacteria group culture and HPLC analysis initiates that baicalin can be converted to baicalein. Another study shows that baicalin has a limited antiproliferative effect on cancer cells, while baicalein has a significant antiproliferative effect on cancer cells, particularly on HCT-116 human colon cancer cell lines. Therefore, baicalein is an effective anticancer metabolite and has a chemopreventive effect on colon cancer. The Astragalus saponin (AST) gained from the medicinal plant Astragalus can play a role in antitumor and apoptosis elevation in colon cancer cells. Araliaceae ginseng can prevent and treat many chronic diseases. As described by Li X et al., several studies have shown that ginsenosides Rg3 and Rh2  in ginseng have anticancer effects, which can reduce the incidence of colon cancer, while protosan diol (PPD) can increase the anticancer effect of the chemotherapeutic agent 5-FU, thereby improving the overall illness of the patients. Guan Chang Fu Fang (GCFF) is extracted from the three plants of Agrimonia pilosa Ledeb., Patrinia scabiosaefolia, and Solanum nigrum L., which is similar to the original PPD. It can also be combined with 5-FU to treat colon cancer. However, Wang et  al. have indicated that 5-FU can cause intestinal flora disorder and colon damage, and the polysaccharide carboxymethylated sclerotium (CMP) isolated from Poria cocos can regulate the balance of the intestinal flora and alleviate FU-induced colon injury. The main active components of Sophora flavescens are alkaloids and brass, and its different workings have a good inhibitory effect on the activities of cell lines such as SW1116, SW620, and SW480, and the ethanol extract of Sophora also repressed the proliferation of colon cancer HT29 cells. Yang et al. have explored that the consumption of Ganoderma lucidum mushroom can stop the occurrence of colon cancer in rodents, so the mechanism of action was studied, and it was found that Ganoderma lucidum extract regulates secondary bile acids, flora, mucin, and propionate associated with colon cancer. It has an impact on colon health. Some prescriptions such as Shiquan Dabu decoction, Shenqi decoction, Qihuang decoction (botanical name: Radix Astragali), Jiedu Sangen decoction, and Mylabris decoction (common name: blister beetle) can also be used to treat colon cancer, and the mechanism of action can be used in the method of metabolomics of traditional Chinese medicine. Metabolomics based on the symptoms can be used to determine the efficacy of the prescriptions. It uses metabolomics technology to determine and identify the biomarkers of the symptoms and uses the biomarkers of the symptoms as parameters to estimate the overall efficacy of the prescription. Using this method, one can understand the mechanism of action and may provide effective help for the treatment of colon cancer (Li et al., 2019).

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13  Other Therapies For the treatment of colon cancer, a model of colitis-associated colon cancer named azoxymethane/dextran sulfate (AOM/DSS) was recognized in the laboratory, and this model was used to investigate the part of p38γ and p38δ in colon cancer associated with colitis; it was found that p38γ/δ deletion can decrease tumor formation, but p38 γ/δ deficiency does not have plentiful effect on advanced tumors. Iwanaga et al. have evidenced that prostaglandin D2 (PGD2) formed by mast cells can inhibit colitis and subsequent tumor formation and therefore can be used to prevent and treat colon cancer (Iwanaga et  al., 2014). Canine uric acid acts as a tryptophan metabolite to avoid the proliferation of cancer cells like colon cancer and kidney cancer. It is considered to be a possible chemo-preventive agent for colon cancer. A small molecule compound sulindac can treat pre-stage adenoma and prevents colon cancer, but it is toxic for the cardiovascular and renal systems. Small doses of sulindac may be used in combination with other chemo-preventive agents to treat colon cancer, which can increase its effectiveness. Cytochrome P450 2W1 (CYP2W1) is a monooxygenase that is usually spotted in 30% of colon cancers, but it is not expressed in non-transformed adult tissues. It can be used as a new treatment for colon cancer. Bee venom (BV) also has anticancer activity and is a traditional medicine for treating skin diseases, cancerous tumors, and rheumatism. It can induce apoptosis by activating DR4 and DR5 and inhibiting NF-КB, thereby inhibiting the growth of colon cancer cells. Vitamin D can regulate intestinal barrier function and antibacterial peptide synthesis. Epidemiological studies have found that vitamin D supplementation can alleviate the signs and symptoms of colitis and have a protective and therapeutic effect on colon cancer (Li et al., 2019). Laparoscopic surgery has become another alternative treatment for colon-­ related diseases in the last few decades. Laparoscopic colectomy has many advantages in postoperative recovery as compared with conventional surgery, because inflammation can endorse colon cancer recurrence and metastasis. Laparoscopic surgery may also cause postoperative complications. Therefore, surgeons should minimize their postoperative complications and progress their survival rate through their own efforts (Xia et  al., 2014; Zhao et  al., 2014). Postoperative intestinal obstruction (POI) is the most common complication after intestinal surgery, and it is related with dendritic cells (DCs) and macrophages. Modifications in the intestinal flora can prevent inappropriate activation of these cells. It can be used as a new way to prevent POI (Li et al., 2019). During the patient’s hospitalization, for stage III colon cancer patients, the communication and cooperation between the surgeon and the oncologist can punctually recognize postoperative and chemotherapy-related complications. This may reduce some unnecessary mistakes and improve patient care strategies in a timely manner, and appropriate individual treatment strategies can be used to improve patient survival. Treatment of mCRC/colon cancer has undergone large changes in the last two decades. New therapeutics and combination treatments have led to marked improvements in both response rate (RR) and overall survival (OS). Notwithstanding the

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advances so far, life expectancy unfortunately continues to be imperfect in the majority of patients with metastatic colorectal cancer. New strategies are desirable to improve the prognosis. To this end, the identification of a potential target and first experiences with checkpoint inhibition in patients with mismatch repair-deficient tumors are promising, and also further investigation is still required to develop effective approaches for medical intervention.

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Chemoresistance in Colorectal Malignancies: Molecular Mechanisms and Strategies to Overcome. Henu Kumar Verma

, Yashwant Kumar Ratre, and Pellegrino Mazzone

Abstract Colorectal cancer (CRC) remains the third most common cause of cancer-­related deaths in both men and women worldwide. Despite recent advances in clinical practice and effective preventive screening measures, CRC is the leading cause of premature cancer deaths. Chemotherapy is widely used for effective treatment of tumor cells, but the outcome and prognosis of patients are not positive due to chemoresistance. Cancer cells slowly increase resistance to nearly all chemotherapeutic agents through a various mechanisms and pathways. Drug resistance, which may be inherent or acquired, may lead to poor treatment outcomes and relapse of the tumor. In most cases, drug resistance may develop resistance to many other drugs that are not similar in structure and function to the first drug. Various molecular mechanisms indicate that cell deregulation and molecular factors are involved in drug-induced phenotypic CC cell switching. Among them, drug detoxification, apoptosis, autophagy, drug-induced DNA damage, and epithelial-­mesenchymal transition (EMT) are the key predictors. Impressively, CRC drug-resistant phenotypes can be due to the consequence of tumor microenvironment (TME). However, the restoration of TME is evolving as a vital figure in supporting chemoresistance and overcoming the cytotoxic effects of drugs. Understanding the molecular factors associated with the development of resistance can help us develop new therapeutic strategies based on the molecular target and reduce the rate of relapse. In this perspective, this chapter discusses the existing up-­to-­date knowledge of drug resistance mechanisms and their involvement in

H. K. Verma (*) Department of Developmental and Stem Cell Biology, Institute of Experimental Endocrinology and Oncology CNR, Naples, Italy e-mail: [email protected] Y. K. Ratre Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, India P. Mazzone Section of Stem Cell and Development, Istituto di Ricerche Genetiche “Gaetano Salvatore” Biogem, Ariano Irpino, Italy

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_7

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the identification of CRC failures. We also discussed the potential therapeutic approach for overcoming drug resistance. Keywords  Chemoresistance · Colorectal cancer · Molecular mechanism · Pathways · Therapeutics

1  Introduction Colorectal carcinoma (CRC) is the third leading cause of cancer-related mortality worldwide (GBD 2017 Colorectal Cancer Collaborators, 2019). The GLOBOCAN 2018 data indicate that the incidence of CRC is widely heterogeneous by region and is highly frequently demonstrated in developed countries (Bray et al., 2018). At the cellular level, CRC originates initially from colonic mucosal epithelia through the spread, differentiation, and migration of dysregulated colonocytes, which gradually accumulate the formation of an unusual group of cells inside the epithelium. Patients with colon cancer (CC) may be treated with surgery alone, while patients with rectal cancer (RC) are often treated with chemotherapy, including 5-fluorouracil (5-FU), followed by surgery (Millan et al., 2015). Together, after surgery, CRC patients may be treated with chemotherapy or other targeted therapy. Trends in the burden of CRC have undergone significant changes around the world as a result of the development of cancer screening, with broad guidelines for colonoscopy in the late 1990s (Simon, 2016). In the current scenario, radiation therapy, combined immunotherapy, and surgery are promising clinical solutions for CRC. However, these remedies are not beneficial in several patients because of the resistance of chemotherapeutic agents (Van der Jeught et al., 2018). In tumor, the intensive and acquired drug resistance mechanisms make it a main hindrance to the development of effective cancer treatments. In the last few years, a study by the American Joint Committee on Cancer (AJCC) has shown that the overall 5-year survival rate has been significantly reduced to 51% in advanced CRC patients (Crooke et al., 2018). CRC does not occur in such a predictable manner; this inconsistency may be directly related to the significant heterogeneity of the cancer cell that further induces CRC progression through local and systemic spread across the human body. Recently, the impression of the tumor microenvironment (TME) has attracted more attention in these two drug resistance mechanisms in the CRC. In addition, many reports suggest the involvement of TME and molecular signaling molecules in various drug resistance processes in many cancers, including CRC (Russi et al., 2019; Senthebane et al., 2017). Hence, it is important to adopt novel approaches and screening methods to identify a novel therapeutic target in order to achieve an improved treatment and survival of CRC patients. To achieve this, this chapter highlights the molecular signaling pathways associated in the drug resistance of the CRC and discussed the potential therapeutic strategy to provide novel insights and overcome drug resistance.

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2  Chemoresistance in CRC Treatment with fluoropyrimidines (FPs) such as 5-fluorouracil (5-FU)-based chemotherapy is known to increase the overall survival rate of CRC patients, and since 1957, it has played an important role in the treatment of CRC (Chen et al., 2019; Goodwin & Asmis, 2009). Chemodrugs, including oxaliplatin, irinotecan, and capecitabine, have also emerged in the recent years. Western medicine for progressive CRC patients includes a grouping of drugs such as 5-FU and leucovorin with irinotecan or oxaliplatin (Braun & Seymour, 2011). Cancer treatment in the CRC has made significant progress with the development of immunotherapy, including bevacizumab and cetuximab. Despite the significant progress in health outcomes with a number of methods, such as chemodrug-associated immunotherapy, the 5-year overall survival for advanced CRCs is still only 10–15% with 5-FU alone (Xie, Chen, & Fang, 2020). The appearance of drug resistance is the main obstacle to this reflection. Almost 50% of CRC patients are resistant to 5-FU-based chemotherapy (Zhang et al., 2008). Efficacy was first identified in the 1990 US-based clinical trial INT-0035, which clearly shows the positive effects of 5-FU on surgery alone (Moertel et al., 1990). Although chemotherapy has been widely used in adjuvant and metastatic-advanced CRC settings, other several groups of chemodrugs have been studied as described in Fig. 1. 5-FU is a synthetic pyrimidine analog that is administered intravenously and is widely used in cancer treatment. It also ensures the endothelial transition to its secondary metabolites. Various mechanisms are used to develop fluorinated nucleotides that are integrated into DNA instead of thymidine, thus further inhibiting DNA replication and resulting in cell death. An active metabolite is FdUMP

Fig. 1  American Food and Drug Administration (FDA)-approved chemotherapeutic agents in colorectal cancer. VEGFR: vascular endothelial growth factor receptor; EGFR: epidermal growth factor receptor; immune checkpoint inhibitor agent Immune checkpoint inhibitors (ICI)

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(fluorodeoxyuridine monophosphate) which suppresses the enzyme thymidylate synthase (TS). Furthermore, 5-FU increases the TS expression via FdUMP and acts as the primary determinant of resistance (Showalter et al., 2008). TS is encoded by the TYMS gene, which is necessary to convert 5-FU to 5-fluoro-2-deoxyuridine (5-FUDR) and is responsible for several genetic alterations in the TYMS promoter region. Recently lower TYMS expression was seen in CRC patients with significantly improved survival rates compared to those with higher TYMS expression (Jiang et al., 2019). This concept was further validated by Ntavatzikos et al., indicating an inverse association between TYMS gene expression and survival rate (Ntavatzikos et al., 2019). In addition, it was shown that the overexpression of TP is significantly associated with 5-FU response (Panczyk, 2014). Therefore, cells with higher TP levels should be more sensitive to 5-FU due to an increase FdUMP concentration. However, the results are contradictory in response to 5-FU chemotherapy and TP expression. Soong et al. demonstrate that low TS expression is an independent risk factor for worse outcomes for CRC patients treated with surgery alone, while low TS expression is highly predictive of improved outcomes for patients treated with 5-FU chemotherapy (Soong et al., 2008). A large meta-analysis study showed that the effect of 5-FU therapy had a marginally significant risk ratio (RR) of 0.76 for OS, and results showed that TP could be promising and tangible for 5-FU chemotherapy in the CRC prognosis (Che et al., 2017). The suppression of TS by the enzyme of FdUMP makes it possible to establish a cluster among FdUMP and CH2THF. Many proteins also control endothelial folate levels by lowering 5.10 methylenetetrahydrofolate (CH2THF) to 5-­methyltetrahydrofolate (5-CH3THF) by the enzyme methylenetetrahydrofolate reductase (MTHFR) (Zaitsev et  al., 2019). MTHFR stimulates and converts CH2THF to CH3THF, thus decreases the quantity of CH2THF and simultaneously reducing the function of TS. In addition, the reduced enzyme activity of MTHFR stimulates the production of CH2THF and tends to increase the suppression of TS by increasing the concentration of FdUMP-CH2THF.  Furthermore, many single-­ nucleotide SNPs play a vital role in the action of MTHFR. Especially the 677 TT and 1298 CC genotypes may positively be linked with a decreased CRC risk with 5-FU treatment (Etienne-Grimaldi et al., 2010). Although the study shows that this polymorphism is unlikely to predict the effectiveness of CRC adjuvant 5-FU therapy after complete resection, 677 CC polymorphism might be linked with decreased toxicity in 5-FU therapy (Afzal et al., 2009). It appears feasible that the Increased activity of this enzyme will acquire resistance, and findings did not show certain elevated levels or stimulation of the MTHFR are correlated with chemoresistance. However, over the last 20  years, several TYMS and MTHFR genes have been known to contribute to 5-FU resistance and may play a role as potential objects for long-term drug resistance therapy (Panczyk, 2014). Irinotecan (CPT-11) is another FDA-approved chemodrug for the treatment of CRC in 1996. CPT-11 is an artificial compound of camptothecin which suppresses topoisomerase I (Topo I). Inside the cell, CPT-11 undergoes intracellular changes, such as catalysis, and is then metabolized to SN-38, which has a thousand-time

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higher anticancer activity than CPT-11 (Li et al., 2017). Further, both these molecules inhibit the Topo 1 and DNA complexes that reflect the function of DNA replication and transcription. Thus, the higher concentration of Topo I makes the cells more sensitive to irinotecan (Kciuk, Marciniak, & Kontek, 2020). In addition, multiple trials supported irinotecan as a treatment for CRC in the early 1990s (Douillard et al., 2000; Giacchetti et al., 2000). Results showing that single-agent irinotecan had only a moderate rate of success of 14–22% in clinical studies. European trials showed that those who had received only 5-FU drug has similar response rates 22 and 20% in metastatic CRC (Shimada, Rougier, & Pitot, 1996) but had a stronger impact when administered in combination with oxaliplatin/ capecitabine and oxaliplatin/leucovorin/infusional 5-FU (Madi et  al., 2012). Furthermore, this has been implemented as an initial treatment option for metastatic CRC, but not for adjuvant chemotherapy, as no additional benefit to FP monotherapy has been shown (Van Cutsem et al., 2009). Resistance to irinotecan in the CRC tends to develop by several mechanisms, including one with a low intra-tumoral level of the active metabolite SN-38. Overexpression of Topo I alters the SN-38-Topo I-DNA complex activity and also regulates the downstream process including cell death suppression, cell cycle regulation, and DNA repair progression. The results of in vitro experiments of transporter protein genetic variants, such as MTHFR and ABCG2, showed drug toxicity in patients with increased drug resistance to irinotecan and SN-38. However, a combination of other drugs may play a potential role in the selection of first-line therapy for advanced CRC patients (Zhao et  al., 2014). In addition, a study found that sorafenib can inhibit resistance to irinotecan and enhance the efficacy of irinotecan by impeding irinotecan-mediated pathways in the CRC (Mazard et al., 2013). Oxalato-platinum or 1-OHP (OHP) is a third-generation platinum analog with an additional mechanism to block the replication and transcription of DNA, leading to cell death. Oxaliplatin was accepted for use in Europe in 1996 and obtained regulatory approval from the US FDA in 2002 with full permission for the treatment of metastatic CRC patients when administrated in combination with 5-FU and leucovorin (Stein & Arnold, 2012). This drug combination is referred to as FOLFOX and used as a first-line chemotherapy approach for CRC as a treatment. Oxaliplatin has been shown to have a low response rate of around 6.1% in phase II clinical trials in the front-line setting (Suenaga et  al., 2015). Several studies have shown that progression-­free survival (PFS) and overall OS increased in combination with 5-FU and leucovorin (FOLFOX) compared to 5-FU and leucovorin alone with response rates as high as 50% (Maindrault-Goebel et al., 2000; Petrelli & Barni, 2013). Oxaliplatin mechanisms of resistance differ significantly from cisplatin (CDDP) and carboplatin. Actuality, it is shown to be aggressive in the in vitro study that is resistant to other platinum compounds in the first-generation (Mehmood, 2014). It is also well known that enhanced proliferative passage and reduction of MMR are CDDP resistance but not oxaliplatin resistance. A comparative study of CDDP-­ resistant tumor cells exposed to CDDP and oxaliplatin demonstrates that oxaliplatin had a much lower concentration of cytotoxic activity than CDDP and also produced fewer DNA-Pt adducts (Göschl et al., 2017; Seetharam, Sood, & Goel, 2009). These

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additives are likely different from some of those developed by CDDP, simply due to the fact that the imbalance of MMR cells establishes a resistance to CDDP but not to oxaliplatin (Dasari & Tchounwou, 2014). That means that imbalance of MMR cells can distinguish oxaliplatin DNA-Pt adducts and induce apoptosis. Capecitabine is a 5-FU pro-drug, the first oral chemotherapeutic agent approved by the FDA for the CRC. It is excreted and altered into 5′-deoxy-5-fluorocytidine (5′-OFCR) and 5′-deoxy-5-fluorouridine (5′-DFUR). Subsequently, 5′-DFUR is solubilized by TP to 5-FU and will have cytotoxic effects. Most of these study results confer resistance to 5-FU. Especially, TP, a highly crucial enzyme for converting capecitabine to 5-FU in the cancerous cells, plays a vital role in its resistance. The altered function of hnRNP H1/H2  in tumor cells with higher TP expression may have favorable risk-benefit ratio to capecitabine, while abnormal TP splicing confers acquired resistance (Lin et al., 2015; Stark et al., 2011). Evidence on capecitabine and irinotecan combination therapy (XELIRI) with or without bevacizumab as a second-line treatment option for advanced CRC has been provided in multicenter phase II and III studies (Garcia-Alfonso et al., 2015; Kotaka et al., 2016). The success rate of capecitabine treatment in extensive CRC, such as stage III and distant metastases, was initially confirmed by randomized controlled phase III trials attributing capecitabine to weekly 5-FU/leucovorin (LV). Finally, oral capecitabine reached the same efficacy as IV 5-FU/LV. Capecitabine revealed significant clinical safety benefits and the comfort of an oral agent (Van Cutsem et al., 2001). Later, these findings were verified in a large meta-analysis of non-inferiority randomized clinical trials which clearly indicated similar OS rates in single-drug and combination with capecitabine-containing single-agent regimens treated with 5-FU-containing regimens in CRC (Cassidy et al., 2011). In contrast, the factors discussed in the previous section, there is still significant heterogeneity in the CRC. The findings of cancer stem cells (CSCs) and their resistance to treatment, as well as their ability to self-regeneration, have brought attention to these disconcerting cells. This unique small fraction of the tumor cells was shown to be highly predictive in patients (de Sousa et al., 2011). CSCs, therefore, appear to be more chemotherapy-resistant. CSCs have shown the potential treatment challenges including chemotherapy, radiotherapy, and, more recently, immunotherapy (Vermeulen et al., 2010; Zeuner et al., 2014). A number of chemotherapy drugs are currently being used as a therapeutic option for the CRC. However, this disease has underlying mechanisms that result in decreased medical potential (Fig. 2). The clinical outcome will ultimately improve through a detailed research of drug resistance and by targeting the CSC population.

3  Immunotherapy As the understanding of cancer genetics continues to expand, novel therapeutic targets have been identified, and chemodrugs have been developed that affect malignant cells more elegantly and rationally than all active cells. It has resulted in

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Fig. 2  Old and new targets in metastatic colorectal cancer. mAb, monoclonal antibodies; HER, human epidermal growth factor receptor; C-MET, mesenchymal-epithelial transition factor; EGF, epidermal growth factor; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; IR, insulin receptor; VEGF, vascular endothelial growth factor

well-response and often less toxic side effects than other cytostatic treatments. Two major types of therapeutic approaches, including immunotherapy and small molecular inhibitors, are diverse in purpose and route of action. This phenomenon has led the science partner to call immunotherapy a “breakthrough of the year” in 2013 (Couzin-Frankel, 2013). However, the advanced CRC seems to have been a poor immunotherapy candidate in early results (Brahmer et  al., 2012). However, the response of cancer cells to immunotherapy varies depending on the tumor. Indeed, while cells from melanoma or renal cancer were found to be responsive to this therapeutic approach, cells from lung cancer were found to be ineffective. Bevacizumab is a humanized IgG1 monoclonal antibody that targets VEGF and inhibits its binding to the VEGF receptor 2. This signaling protein suppresses endothelial cell reactions related to permeability, spread, mobility, and survival (Strickler & Hurwitz, 2012). The FDA was first approved for advanced CRC treatment in 2004 for use in combination with other first-line chemodrugs. In the United States, bevacizumab is approved with 5-FU infusion, while in Europe and most other countries, it was approved with oral 5-FU or infusion. Later, bevacizumab showed a

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promising CRC activity in a number of studies, and early-stage II bevacizumab findings showed high efficacy and clinical benefit in patients. Further, the 20-day plasma half-life allows dosing every 2 or 3  weeks and improved PFS and OS response rate in CRC (Hurwitz et al., 2004). Phase III bevacizumab plus irinotecan in colorectal Cancer (BICC-C) clinical trial has shown improved PFS in combination with irinotecan-based bevacizumab fusion treatment plan for first-line advanced CRC by comparing infusion 5-FU, LV, and irinotecan plus bevacizumab with IFL plus bevacizumab (Fuchs et al., 2007; Fuchs, Marshall, & Barrueco, 2008). Interestingly, a randomized phase III trial also showed that VEGF inhibition of bevacizumab in the second-line therapy after disease progression setting resulted in improved therapeutic outcomes associated with post-progress chemotherapy alone in patients with advanced CRC (Bennouna et al., 2013). The reasons for this are likely to be linked to different antitumor agent resistance mechanisms, including both intrinsic and acquired resistance (Bergers & Hanahan, 2008). Recent xenograft studies have revealed that plasma VEGF levels are significantly increased when bevacizumab is withdrawn due to heterogenic CSCs that tend to cause tumor growth and increase tumor resistance in the CRC (Becherirat et al., 2018). Another most recent phase I PERMAD trial showed resistance to FOLFOX plus bevacizumab in advanced CRC (Seufferlein et al., 2019). Cetuximab is a hybrid mouse/human IgG1 monoclonal antibody that binds to the outer EGFR membrane. In 2004, the FDA first approved cetuximab for use in the CRC as a single regimen or in combination with irinotecan. Cetuximab has been shown to have a success rate of nearly 10.8% as a single regimen in the irinotecan study with improved progression time from 1.5 to 4.1 months and a median survival time of 8.6 months with toxic effect in the combination therapy group (Hubbard & Alberts, 2013). On the other hand, a study showed an 8% partial response rate and increased PFS with improved quality of life, particularly in highly pretreated mCRC patients compared to best supportive care with a median OS rate of 6.1 months with a grade 3 or higher adverse event of 78.5% in the cetuximab group (Jonker et al., 2007). Surprisingly, the inclusion of cetuximab to irinotecan in refractory disease has shown an increase in both PFS and OS compared to the cetuximab single-­ regimen group, indicating a solution to overcome irinotecan resistance (Cunningham et al., 2004). Only about 10–20% of advanced CRC patients responded to EGFR antagonists in preclinical studies. It is now understood that tumors with a KRAS mutation in the human chromosome 12 gene have a high resistance to cetuximab, resulting in a reduced response to treatment (You & Chen, 2012). Recently, a randomized phase II study of cetuximab versus irinotecan and cetuximab showed that cetuximab-­ based treatment tends to benefit patients with chemotherapy-resistant, refractory CRC-modified KRAS G13D (Nakamura et al., 2017).

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4  Chemoresistance Pathways Associated with Colorectal Cancer CRC is one of the most devastating malignancies responsible for cancer-related deaths in the world. Despite the availability of adequate early diagnostic tools and specific systematic treatment options for neoplasm, the CRC remains a major burden for the majority of CRC patients. Chemoresistance is defined as a series of new events and behavioral patterns of malignant cells to achieve upregulated proliferation and growth in order to ensure their survival in the toxic environment of cancer patients (Mathonnet et al., 2014). All these events increase the severity and promote the low survivability of patients with CRC (Kordatou, Kountourakis, & Papamichael, 2014). However, the failure of chemotherapy is one of the key factors in metastatic CRC. In addition, previous findings have shown that cancer stem cells (CSCs) with a potential for self-renewal, uncontrolled proliferation, and differentiation are crucial to the induction of chemotherapeutic resistance in CRC (Park et  al., 2015). Although the majority of patients with advanced CRC therapy are primarily responsive to combination therapy. However, advanced CRC patients will eventually experience the recurrence of neoplasm due to multidrug resistance, which ultimately results in a drop in the overall survival of nearly 10% at an advanced stage (Dahan et  al., 2009). Unfortunately, the most significant limitation in the treatment of patients with colon cancer is its nonresponsive feature to novel immune checkpoint therapy (Zou, Wolchok, & Chen, 2016). Therefore, it is of much importance to highlight and explore the mechanism of chemotherapeutic resistance in patients with CRC.

5  Tumor Microenvironment In the current scenario, the chemoresistance of many tumor cells, including CRC, is identified as one of the key factors for chemotherapy-associated failure and subsequent cancer progression, ultimately leading to the patient’s death (Verma, 2019; Verma et al., 2020; Zheng, 2017). The development of sporadic CRC takes several years to decades, involving several sequential modulations both in conventional mechanisms and in microenvironment signals. However, the study of chemoresistance in CRC was primarily focused on the mechanisms inherent to cancer cells; substitution views suggest a role for TME in the promotion of chemoresistance. Although a number of studies show that, apart from cancer cells, a wide range of factors could facilitate chemoresistance through a variety of other mechanisms. These are not limited to a variety of microenvironment-originated factors such as signaling from stromal cancer-associated fibroblasts (CAFs), adipocytes, extracellular matrix proteins, and various modified leukocytes along with an aberrant vasculature resulting in inflammation and hypoxia (Mumenthaler et  al., 2015; Ren et  al., 2018). Tumor-associated macrophages (TAMs) also play a key role in

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modulating the immune microenvironment of the tumor. Multiple TAM subpopulations are expressed in malignant tumors in two major phenotypic forms, namely, M1 and M2 macrophages. Researchers are currently targeting TAM to study the frequent evolution of TAM heterogeneity, which plays a key role in tumor microenvironment modulation throughout tumor progression under hypoxia and drug resistance. Recently, Yeldag et al. have shown that the hepatocyte growth factor (HGF) may stimulate the nuclear activity of β-catenin in stromal cells, thereby disrupting the chemoresistance-associated stem cells in the CRC (Yeldag, Rice, & Del Rio Hernandez, 2018). Another finding showed the role of CAFs in drug resistance by transferring exosomal H19, resulting in the activation of the β-catenin signaling cascade by acting as a competing circulating endogenous RNA sponge (Ren et al., 2018). More recently, Deng et al. found that CRC-associated IncRNA expression via CAFs could contribute to CRC oxaliplatin resistance by activating β-catenin signaling (Qiu et al., 2020).

6  Epithelial/Mesenchymal Transition Tumor cells are thought to undergo an epithelial-mesenchymal transition (EMT) in response to a combination of different extracellular signals in their surrounding microenvironment. Cross-talk between normal and cancer cells leads to tumor metastases consisting of sequential, interconnected, and specific steps (Fares et al., 2020), and many of the steps favor the transition between two cell states, namely, epithelial and mesenchymal phenotypes. EMT is a developmental regulatory process that accelerates the spectrum of cell trans-differentiation under homeostasis. Likewise, the mesenchymal-epithelial transition (MET) only reverse of the EMT appears to occur after the spread and subsequent development of the distant EMT (Acloque et al., 2009). Furthermore, EMT is a reversible process, as cells can switch from a mesenchymal to an epithelial phenotype, a mechanism known as mesenchymal-epithelial transition (MET). This occurs when migratory cells, such as metastatic cells, arrive at their destination site and reactivate epithelial gene expression. However, a limited number of published evidence restrict the therapeutic approaches based on MET and need further investigation. In recent years, findings have shown the complex and intricate role of EMT in facilitating tumor invasion and metastasis in epithelial-derived cancer. During EMT, epithelial cells gradually lose their physiological morphology (cell polarity, membrane adhesion, cell-to-cell contact, cytoskeleton remodeling) and invasiveness and dramatically increase the production of ECM components (Tiwari et al., 2012). Tumor cells and the activated EMT network are responsible not only for the invasion and metastases of adjacent tissues but also for increased drug resistance. Previous findings have shown that signaling pathways such as TGF and Wnt cascades were associated in the EMT process and play a significant role in multiple carcinomas, along with CRC progression. In colorectal cancer, EMT may be induced by transforming growth factor B (TGF-β), cytotoxic drugs, and cytokines acting in conjunction with intracellular signaling molecules

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such as phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and the kappa-lightchain nuclear factor enhancer of activated B cells (NF-kB) (Suman et al., 2014). Li et al. reported that doxorubicin treated with colon cancer cell line HCT-116 induces EMT cell phenotypes and TGF-β signaling and expressively promotes multidrug resistance (MDR) in the plasma membrane glycoprotein levels (Li et al., 2015). In particular, the lower regulation of the expression of E-cadherin shows the occurrence of lymphovascular invasion, poor tumor differentiation, and decreased diagnosis in CRC patients (He et al., 2013). These results are consistent with the role of E-cadherin as the gateway of the epithelial state in the CRC (Tsai & Yang, 2013). Observational studies also reported that increasing fat diet or high-fat diet (HFD) is also associated with an increased incidence of colon cancer. The study also suggests HFD-mediated development of EMT, which facilitates tumor inflammation by the upregulation of COX2 and activation of MAPK/ERK and PI3K/AKT/mTOR signal cascades in the mouse xenograft CRC model (Tang, Pai, & Chiang, 2012). The EMT is a very complex process, regulated by many transcription factors, signaling circuits, and noncoding RNAs. The EMT program is also associated with many tumor-associated behaviors, including tumor budding, circulating tumor cells, and drug resistance in the CRC. According to the above studies, as well as the clinical findings, the expression of EMT markers is not only related to the clinical course of CRC but also associated with its aggressiveness (chemoresistance). Therefore, targeting EMT for the therapeutic application can aid some additional effort to cope up with colorectal cancer.

7  Autophagy It is now well established that the malignant transformation of colon cancer involves many risk factors, such as metabolic adaptation, multiple genes, MDR, and various environmental factors. Recent findings provide new evidence on mechanisms related to colorectal cancer. Among these mechanisms, autophagy plays an important role in switching normal to malignant cells and acting as a tumor suppressor at an early stage and tumor promoter at an advanced stage of CRC (Mizushima & Komatsu, 2011). Autophagy is a regulatory catabolic process that ensures the recycling of old, damaged, malfunctioning, or aberrant cytoplasmic components in eukaryotic cells. Autophagy is regulated by a number of autophagy-related genes (ARGs), including ATG12, ATG5, and microtubule-associated light-chain protein 3 (LC3), which regulate the sense and transduce and perform every single step of this autophagy pathway, which was regulated by the mammalian rapamycin target (mTOR) and the PI3K complex (Panda et al., 2015). In addition, autophagy plays a significant role in a variety of mechanisms related to cancer progression, inflammation, chemoresistance, and genome stability. The LC3 family of genes encodes three isoforms, such as LC3A, LC3B, and LC3C. It was assumed that LC3 was the first autophagy marker to be involved in the human CRC. Zhang et al. found that the overexpression of the LC3B-II gene with the CRC cells and autophagy increased

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the aggressiveness of the CRC (Zheng et al., 2012). In addition, there is a sufficient number of data describing the unregulated mechanism of autophagy in colon cancer cells with various stages of cancer progression. It has also been reported that pharmacological (in vitro) or genetic suppression of autophagy has been correlated with increased chemosensitivity in the CRC (Bhardwaj et al., 2018; Ma et al., 2015). In addition, the loss of APC tumor suppressor in mice mode autophagy has been reported to promote the initiation and progression of intestinal carcinoma. In conversely to this, ATC7 deficiency restricts the initiation and progression of antitumor T response and microbiota imbalance induced by APC (Lévy et al., 2015). In current aspects, some findings have shown that the resistance of chemotherapy is significantly correlated with the cytoprotective role of autophagy. In addition, chemotherapeutic drugs also activate autophagy to protect cells from stress-induced damage, thereby promoting neoplasm resistance, which reduces the efficacy of most anticancer drugs (Sui et al., 2013). Some autophagy inhibitors are currently shown to accelerate the efficacy of chemotherapy drugs; for example, 3-­ methyladenine (3-MA)- and hydroxychloroquine (HCQ)-mediated autophagy inhibition can promote 5-FU-induced apoptosis in CRC cells (Zhou et al., 2017). Thus, targeting autophagy and associated pathways might be a hopeful adjuvant therapy especially in the case of advanced stage CRC.

8  Therapeutic Potential for CRC Despite the availability of appropriate treatment and therapeutic options, colorectal cancer remains a major burden on the community worldwide. Since we have observed that different signaling pathways, mechanisms, and other related factors are suppressed in CRC that promotes chemoresistance in CRC and plays a key role in the failure of chemotherapy-based treatment approaches. It has been demonstrated, that several factors are involved in the development of chemoresistance in CRC as they play a key role in the failure of chemotherapeutic approaches. It is now well understood that CRC cells develop chemoresistance due to the involvement of the carcinogenic process itself, along with various treatment-based factors such as MDR, TME, and autophagy. Five-year survival probability depends on the advancement of the disease. Unfortunately, drug resistance is still a major obstacle to the low survival of CRC patients. There is therefore an urgent need for the most effective and well-tolerated therapeutic potions in the third line as well as in the subsequent line of treatment to improve survival in patients with advanced metastatic CRC. Classically, irinotecan (CPT-11) an inhibitor of DNA topoisomerase was previously approved by the FDA as adjuvant therapy for advanced and metastatic CRC. As monotherapy, irinotecan has a very weak response in a clinical trial with 5-FU refractory metastatic CRC patients. However, in combination with 5-FU, FPs, and capecitabine and/or oxaliplatin irinotecan, the result is very effective (Palshof et al., 2017). Some therapeutic drugs, such as capecitabine, mitomycin C, and gemcitabine, are not very effective in this setting. Unlike targeted oxaliplatin or

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epidermal growth factor receptor (EGFR) therapy, some patients may benefit from targeted oxaliplatin therapy (Nielsen et al., 2014). Oxaliplatin is a platinum-based agent approved for metastatic CRC treatment in combination with 5-FU and leucovorin as a first-line chemotherapy drug. This combination is referred to as FOLFOX. Capecitabine is the first-approved oral chemotherapeutic suspension for CRC. A promising result was obtained in the phase III trial of capecitabine in combination with irinotecan, referred to as XELIRI with or without bevacizumab. It is approved as a second-line treatment option for metastatic CRC (Kotaka et al., 2016). Currently, regorafenib and TAS-102 are considered as a combination therapy in phase III trial for the survival of patients with CRC. Regorafenib is a recently approved oral multi-kinase inhibitor for patients who have already received standard chemotherapy anticancer drugs. AS-102 is an orally bioavailable drug prescribed in combination with trifluridine and tipiracil hydrochloride and is currently approved in the United States for the treatment of CRC patients previously treated with fluoropyrimidine, oxaliplatin- and irinotecan-based chemotherapy, anti-­ VEGFR, and, if RAS wild-type, anti-EGFR therapy (Mayer et  al., 2015). Ramucirumab is a monoclonal recombinant antibody designed to block ligand binding (VEGF-A) by binding to the VEGFR2 receptor. It is administered in combination with FOLFIRI for the treatment of metastatic CRC with bevacizumab, oxaliplatin, and FP previously received. As discussed, several chemotherapy drugs have currently been approved for the treatment of CRC. Some of them have adverse cellular cytotoxicity. More evidence-based research is therefore needed to understand the current challenges and limitations of CRC therapy to block oncogenic drivers from chemoresistance that help to produce more potentially effective chemotherapy drugs for CRC prognosis and treatment. A detailed molecular mechanism of drug resistance is given in Table 1.

9  Conclusion The CRC field has evolved over the last decades. However, drug resistance and non-­ responsiveness to immunotherapy are major challenges in the clinical approach of patients. The chemotherapeutic efficacy of drugs varies in patients with CRC due to genetic variability and other risk factors. A combination of conventional anticancer drugs with ABC transporter inhibitors, EGFR inhibitors, or apoptosis inducers is potentially successful in circumventing drug resistance in CRC.  Several mechanisms were involved in cell and molecular changes that make CRC cells resistant to current chemotherapy strategies. Some chemical agents, including cetuximab and panitumumab, have been approved for use in the CRC. However, most combination therapies failed to reverse multidrug resistance (MDR) in different clinical trials, suggesting that targeting a single molecule is not sufficient to overcome drug resistance in the CRC. Conversely, recent findings suggest that combination therapy will bring enormous benefits to patients in the future. Therefore, it is very important to determine how different signaling molecules, mechanisms, and pathways are

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Table 1  Molecular mechanism of drug resistance against standard chemotherapeutic agents in CRC Chemo-drugs Enzyme/pathways 5-Fluorouracil Thymidylate synthase

Oxaliplatin

Capecitabine

Irinotecan

TAS-102

Mechanism/expression pattern Upregulated expression leading to 5-FU inhibition Thymidine phosphorylase Accelerate salvage pathways expression of nucleotides Low expression leads to an increased response to 5-FU-based treatment Orotate phosphoribosyl Decreased expression transferase (postulated), as high expression correlates with sensitivity Multidrug resistance protein Increased expression leading to increased efflux Glutathione Overexpression of GSH inactivating the platinum and promoting export from cells ERCC1 Higher expression associated with increased nucleotide excision repair Thymidine phosphorylase Lower expression decreases the formation of the active metabolite Elevated expression facilitates Dihydropyrimidine dehydrogenase increased metabolism Multidrug resistance protein High expression leading to increased efflux into cells Uridine diphosphate Elevated expression leading to glucuronosyltransferase increased metabolism of SN-38 Carboxylase Low expression is required to create an active metabolite Topoisomerase-I Decreased duplication of TOP1 gene Alterations in the binding site Thymidine phosphorylase Inhibiting catabolism of thymidine phosphorylase

Reference Zhang et al. (2008) Soong et al. (2008)

Muhale et al. (2011) Zhang et al. (2006) Kelland (1993)

Baba et al. (2012) Petrioli et al. (2010) Koopman et al. (2009) Zhao et al. (2014) Tziotou et al. (2014) Boyer et al. (2004) Nygård et al. (2014) Lenz, Stintzing, & Loupakis (2015)

modulated to support CRC progress. In addition, more evidence-based research is needed to understand the current challenges and limitations of CRC therapy that help to produce more potentially effective chemotherapeutic drugs for CRC prognosis and treatment. In summary, the resistance of cancer cells to drugs remains a significant hindrance to successful chemotherapy. This chapter not only provides insight into the mode of biological action of drug resistance but also the signaling pathway involved in the colorectal cancer chemoresistance molecular network, which facilitates the development of novel therapeutic targets and potential chemosensitive biomarkers to reduce cancer recurrence and improve patient life span.

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Therapeutic Intervention of Signaling Pathways in Colorectal Cancer Vikas Chandra, Ashutosh Tiwari, Rajat Pratap Singh, and Kartiki V. Desai

Abstract  Often cancers are treated with chemotherapeutic agents that attack cell proliferation or DNA repair machinery. Due to their nonspecific nature, noncancerous cells are also eliminated, resulting in systemic, undesirable side effects and poor quality of patient life. Advancement in molecular techniques and massively parallel sequencing strategies has led to better patient stratification and the discovery of unique patient-/cancer-specific molecular markers, gene expression profiles, and mutational profiles. This has supported the development of gene-based or mutation-­ specific therapeutic strategies that are specifically directed against cancer cells but have negligible effects on normal cells. In this chapter, we discuss the outcomes of such targeted therapies in colorectal cancer with special emphasis on those developed against cell signaling molecules. Keywords  Signal transduction · Monoclonal antibody · Inhibitor · Targeted molecules

1  Introduction One of the most frequent cancers observed in recent time is colorectal cancer (CRC). Worldwide this ranks as the second most fatal cancer and the third most predominant malignant tumor (Bray et al., 2018). The world has seen a continuous upsurge in the number of colorectal cancer cases (Siegel, Miller, & Jemal, 2019). CRC was relatively rare till 1950, but now accounting for 10% mortality of cancer and related conditions. Cancer-related death rates have increased by nearly 40% over the last 40 years (Kuipers, Rösch, & Bretthauer, 2013). According to an estimate, the number of new colorectal cancer cases may cross the toll of 2.5 million by the year 2035 (Arnold et al., 2017). However, the improved healthcare infrastructure has enabled the accurate diagnosis and proper treatment of colorectal cancers. Due to V. Chandra (*) · A. Tiwari · R. P. Singh Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India K. V. Desai National Institute of Biomedical Genomics, Kalyani, West Bengal, India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_8

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progressions in primary as well as adjuvant therapies, the survival time in CRC has been improving (Keum & Giovannucci, 2019). The conventional treatment approaches for colorectal cancers include laparoscopic surgery and chemotherapy (Sánchez-Gundín, Fernández-Carballido, Martínez-Valdivieso, Barreda-Hernández, & Torres-Suárez, 2018). However, general cytotoxic agent-based chemotherapy leads to numerous side effects and hence poor quality of patient life (Ragnhammar et al., 2001). Targeted therapy in colorectal cancer was a relatively new intervention. In contrast to general cytotoxic chemotherapy, targeted therapy, owing to its specificity to cancer, gained wide acceptance among clinicians for the treatment of patients suffering from CRC and other cancers (Lee, Tan, & Oon, 2018). Colorectal cancer develops in a stepwise manner and mutations take place over a period of time. These mutations accumulate in due course of time and can activate oncogenes and deactivate suppressor genes (Fearon, 2011). This results in the transformation of normal colonic mucosa into invasive cancers (Fleming, Ravula, Tatishchev, & Wang, 2012). Most of the CRCs cases are sporadic, but some inherited forms are also observed (Burt, 2007). The cancerous cells are generally characterized by uncontrolled replication and metastasis. In the progression of colorectal cancer cells, EGFR/MAPK, TGF-β/SMAD, SHH, Wnt/β-catenin, and Notch signaling pathways are mainly involved (Tiwari, Saraf, Verma, Panda, & Jain, 2018). In colorectal cancers, various changes, including histone modification, DNA methylation of CpG islands, expression of microRNAs/noncoding RNAs, and nucleosome positioning were observed (Ng & Yu, 2015). CpG islands hypermethylation can disturb almost all signaling pathways implicated in carcinogenesis (Bahrami et al., 2017).

2  Alteration in Signaling Pathway Gene In general, signal transduction pathways comprised of ligand and its receptor and several downstream intermediate and effector molecules. Alteration in any member of a pathway may dysregulate the entire pathway resulting in abnormal behavior of cells and the pathway gets out of the track. In colorectal and other cancers, the pathways that govern cell proliferation, growth, survival, and differentiation are dysregulated. For example, various genes belonging to WNT signaling pathway have been found to be altered in colorectal cancer. For example, a very high expression of WNT receptor frizzled (FZD10) has been observed in this malignancy. According to the Cancer Genome Atlas (TCGA), the Wnt signaling pathway has been found to be altered in 93% of all colorectal tumors. This includes biallelic inactivation of the adenomatous polyposis coli (APC) gene or activating mutations of catenin beta-1 (CTNNB1) gene in approximately 80% of cases. Very few mutations in FAM123B/ WTX have been described in colorectal cancer (Cancer Genome Atlas Network, 2012). In addition to the Wnt pathway, mutations in the genes of the PI3K and RAS-­ MAPK pathway are also common in colorectal cancers. Overall, mutation in the rat sarcoma (RAS) has been implicated in 27% of all cancers, and 35–45% of this was

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observed in colorectal cancers alone (Hobbs, Der, & Rossman, 2016; Tan & Du, 2012). Genetic alterations, IGF2 and IRS2 overexpression, and mutually exclusive modifications in PIK3R1, PIK3CA, and deletions in PTEN were found in 4% non-­ hypermutated tumors. In 55% of non-hypermutated tumors, alterations are found in KRAS, NRAS, or BRAF with a pattern of mutual exclusivity. BRAFV600E is mutant version of BRAF where valine (V) is substituted by glutamic acid (E) at amino acid position 600 of the BRAF polypeptide chain. This mutant BRAF does not respond adequately to standard therapies (Douillard et al., 2013). The alteration and co-occurrences of tumors with overexpressed RAS and PI3K pathways were present in approximately 75% of colorectal cancer cases. All these data and results suggest the simultaneous inhibition of the RAS and PI3K pathways is essential to obtain a therapeutic benefit. Apart from these pathways, mutations in the epidermal growth factor receptor like ERBB family of receptors have some translational relevance in colorectal cancers. Mutation or overexpression of this receptor was found in 13% of non-hypermutated and 53% of hypermutated colorectal cancer cases. The dysregulation of TGF-β pathway is known in colorectal as well as other cancers. The TCGA finding suggests genomic alterations in TGFβR1, TGFβR2, ACVR2A, ACVR2B, SMAD2, SMAD3, and SMAD4 in 27% of the non-hypermutated and 87% of the hypermutated tumors. Evaluation of the p53 pathway suggested alterations in TP53 and ataxia-telangiectasia mutated (ATM) genes in 59% and 7% of non-hypermutated cases, respectively (Cancer Genome Atlas Network, 2012).

3  Molecular Targeted Therapy in Treatment of Cancer The therapy that uses drugs or other substances that can target specific molecules (target molecule/molecular targets) to block the proliferation, growth, and metastasis of cancer cells is known as targeted therapy. “Magic bullet” was a term coined by Paul Ehlrich in late nineteenth century to describe certain compounds that can specifically target microbes (Ehrlich, 1906). This historic elucidation is considered as an inspiration to develop the concept of targeted therapy which is now being established and expanded to cancer treatment (Brodsky, 1988). The development of effective molecular targeted therapy in cancer essentially requires an ideal target molecule. One of the primary causes in neoplastic progression is the mutation in the genes that may lead to abnormal expression of some molecules. These specific alterations in genes can help to discriminate cancer cells from normal ones. Such alterations in cancer cells can help in identifying molecular targets against which the drugs can be administered (Røsland & Engelsen, 2015). Growth factor signaling molecules, modulators of apoptosis, cell cycle proteins, angiogenic molecules are some of the most suitable candidates to be targeted in molecular therapeutic approaches. The molecular agents for targeted therapy show diverse functions and can act on cell surface antigens, growth factors, and signal transduction pathways that play essential role in cell growth proliferation and angiogenesis. Some of the targeted therapeutic molecules promote cellular apoptosis (Padma, 2015). For ease in studies, molecular targeted therapy can be classified into gene therapy,

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monoclonal antibodies, immunotherapeutics, small-molecule inhibitors, and cancer vaccines (Padma, 2015). Targeted therapy requires small molecules, preferably   26,000) showed a 22% decrease in the risk of colon cancer in adults who devoured higher amounts of milk and a 16% decrease in people who consumed higher amounts of other milk items (Huncharek, Muscat, & Kupelnick, 2009). Interestingly, a few elements of dairy items, such as calcium, vitamin D, conjugated linoleic acid (CLA), or butyric acid synthesized by probiotic microorganisms, are speculated to diminish the chance of CRC.

6.1  Calcium Dairy items are one of the fundamental sources of dietary calcium, which is hypothesized to inhibit colon malignant growth by restricting auxiliary bile acids (responsible for DNA damage in colon cells), ionized unsaturated fats and consequently diminished their proliferative impacts in the colonic epithelium (Govers & Van der Meet, 1993). Calcium intake of 1200 to 1500 mg/d, or servings of dairy items every day, appears to be the most defensive against colon malignant growth. On a molecular level, calcium has been known to inhibit CRC by affecting intracellular pathways provoking regular colonocyte separation. Calcium has been linked to a lower number of K-ras gene alterations in colorectal cancers in a mouse model (Llor et al., 1991). Calcium also has a protective effect by limiting bile acids and free fatty acids (e.g., deoxycholic and lithocholic acids) (Larsson et al., 2006). As a result of binding of calcium, the degree of toxic concentration of bile acids is diminished, and the interaction of the colonic epithelial cells with these cancer-­causing agents is shortened and weakened as a result of this, bile acids’ proliferative effects in intestinal mucosa cells are now inhibited (Carroll et al., 2010).

6.2  Vitamin D3 Calcitriol, 1α, 25-dihydroxy vitamin D3 (1, 25 (OH)2D3), the most dynamic type of vitamin D, is a pleiotropic hormone with a broad scope of natural activities (Deeb, Trump, & Johnson, 2007). Because of its capacity to manage calcium and

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phosphate digestion, 1,25D3 assumes a significant role in the bone well-being also. 1,25D3 binds to the vitamin D receptor and consequently manages the expression of various genes that control the development, differentiation, and endurance of disease cells (Jacobs et al., 2013). Several studies have found that increasing vitamin D3 reduces the risk of colon cancer and polyp recurrence, and also that having an appropriate level of vitamin D3 is linked to better colon disease patient survival. Vitamin D directs the homeostasis of intestinal epithelium by balancing the oncogenic Wnt signaling pathway and by hindering tumor-promoting inflammation (Klampfer, 2014). Vitamin D3 arrests cell cycle in cancer cells by regulating CDK2, p21, p27, p53, KI67, and E-cadherin and initiates apoptosis by regulating the expression of apoptosis-related genes such as BCL-2, BCL-xL, Mcl-1, and so on (Lamprecht & Lipkin, 2003; Holt, 2008).

6.3  Butyric Acid and Conjugated Linoleic Acid Lipids present in dairy items could be useful, for example, butyric acid and conjugated linoleic acid (CLA). Butyric acid may repress proliferation and activate differentiation in tumor cell lines (Velázquez et  al., 1996). Butyrate is a product of fermentation done by intestinal microflora. Research found that butyrate is inversely correlated with colon cancer (Guarner & Malagelada, 2003). Butyrate is a significant energy source for colon epithelium. The primary function of butyrate in the avoidance of colonic inflammation procedure is that it plays a role in regulating the apoptosis of transformed colonic cells and restrains angiogenesis by managing vascular endothelial growth factor (VEGF) overexpression (Sears, 2005; Prasanna Kumar et al., 2008). Another chemopreventive compound available in dairy items is conjugated linoleic acid (CLA), the most widely recognized omega-6 unsaturated fat (Cesano, Visonneau, Scimeca, Kritchevsky, & Santoli, 1998).

7  Conclusion Malignancy risk factors, such as hereditary qualities and conditions, are out of our control. However, studies suggest that by improving our dietary habits and lifestyle, we can reduce our risk of disease by 70% during our lifetime. Reducing the use of cigarettes, constraining liquor, arriving at a healthy weight, and getting regular exercise are mostly incredible strides for forestalling malignant growth. Adopting a healthy eating regimen can also play an indispensable role. What we eat and do not eat can powerfully affect our well-being, including risk for disease. While research tends to point to associations between specific foods and cancer, rather than solid cause-and-effect relationships, certain dietary habits can have a significant influence on risk. For instance, eating a regular Mediterranean diet rich in fruits, vegetables, and healthy fats like olive oil can bring down the risk of diseases. Alternately, an

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eating routine that incorporates a day-to-day serving of prepared meat builds the danger of colorectal malignant growth. To bring down the risk of some kinds of malignancy, aim to build a diet around an assortment of antioxidants agents, rich foods grown from the ground, nuts, beans, entire grains, and healthy fats. Simultaneously, try to limit the intake of processed and fried foods, as well as unhealthy fats, sweets, and refined carbohydrates for healthy life.

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Role of Food Additives and Intestinal Microflora in Colorectal Cancer Vivek Kumar Soni, Ajay Amit, Vikas Chandra, Pankaj Singh, Pradeep Kumar Singh, Rudra Pratap Singh, Girijesh Kumar Patel, and Rajat Pratap Singh

Abstract  Colorectal cancer (CRC) is one of the most common cancers all around the world with a high mortality rate. Lifestyle differences and environmental factors such as high intake of fat and protein, red meat, and contaminations could increase the risk of CRC. Different food additives are used to improve the taste, flavor, texture, appearance, and preservation of food products. Some of these food additives have negative health impacts on human beings. These food additives can be mutagenic and carcinogenic. Consumption of food additives containing food products increases the risk of cancer including colorectal cancer. The intestinal microflora is also associated with carcinogenesis of CRC. The dysbiosis in gut microbiome due to dietary and environmental changes might be linked with the development and progression of CRC.

The authors Vivek Kumar Soni and Ajay Amit contributed equally as first author. V. K. Soni · V. Chandra · R. P. Singh (*) Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India A. Amit Department of Forensic Science, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India P. Singh Department of Biotechnology, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India P. K. Singh Department of Biochemistry, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India R. P. Singh Department of Environmental Science, Dr. Rammanohar Lohia Avadh University, Ayodhya, Uttar Pradesh, India G. K. Patel Texas Tech University Health Sciences Center, Department of Cell Biology and Biochemistry, Lubbock, TX, USA

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_14

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Keywords  Colorectal cancer · Food additives · Food preservatives · Food dyes · Gut microbiome

1  Introduction Malignancy (cancer) is a lethal disease and leading cause of death around the world (Jemal et al., 2011). Among the different types of malignant tumor, colorectal cancer (CRC) is the third most common type of cancer worldwide (Smith, Renaud, & Hoffman, 2004). CRC is the most common malignancy of the gastrointestinal tract affecting men and women in the same manner (Siegel, Miller, & Jemal, 2015). The risk of developing colorectal cancer is affected by genetic and environmental factors. CRC might be sporadic, inherited, or be identified with a history of inflammatory bowel disease (Zhao, Hu, Zuo, & Wang, 2018). The hereditary CRC includes hereditary non-polyposis colorectal cancer (HNPCC), familial adenomatous polyposis (FAP), and other types of malignancy with a familial history. Majority of colorectal malignancy probably develops sporadically driven by external factors. These external factors from food cause genetic alterations or modulate other processes in the development of CRC. Like most cancers, CRC is driven by an accumulation of genetic alterations in tumor suppressor genes (APC, Smad4, and p53) and oncogenes (K-ras) (Tomasetti, Marchionni, Nowak, Parmigiani, & Vogelstein, 2015). These mutagenic accumulations lead to the development of CRC in a stepwise progression from normal intestinal epithelial cells to adenocarcinoma (Li, Zhang, & Chen, 2018). The increased incidence of CRC in population is influenced by the lifestyle differences which reflect poor and bad dietary habits (high in fats particularly saturated fat), low physical activity, obesity, diabetes, smoking, alcohol consumption, and population aging (Cress, Morris, Ellison, & Goodman, 2006). Our diet contains a variety of carcinogenic and mutagenic substances. Some of these substances are found naturally in the food ingredients, whereas others result from food additives, preparation and processing procedures, environmental pollution, pesticide residues, environmental pollution, and contamination (Carr, 1985). Food additive is a substance or blend of substances, other than a basic foodstuff, which is present in a food as a result of any aspect of production, processing, storage, or packaging. They are utilized to improve and enhance the flavor, taste, quality, appearance, or texture of a product or to extend its shelf life. The different food additives are consistently consumed by large number of population throughout the world, which have both advantages and disadvantages. Some of these food additives have been related with adverse well-being impacts and might be mutagenic or carcinogenic (Mahapatra & Parija, 2018). The gastrointestinal tract in the human body has a noteworthy populace of different microorganisms which play a critical role in the health status of the host (Hooper & Macpherson, 2010). The interaction between the microbiota with various parts of

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the human gut helps in deciding the well-being or disease status of the host (Ursell et  al., 2014). Researchers have found that changes in the intestinal microbiome affect the human physiology and might be a significant risk factor for colorectal cancer (Siegel et al., 2017). Changes in the gut microbiota are influenced by environmental exposure, host genetics, and diet (Turnbaugh et al., 2007).

2  Food Additives Since ancient occasions, chemicals have been added to foods to perform special functions. Basic foods contain no added substances (additives), but when foods are processed to prepare variety of products, an increasing number of food additives are commonly utilized. The human diet contains a large number of structurally diverse chemical substances, mostly of naturally occurring origin, in addition to substances deliberately added, for example, colorants, supplements, etc. Food additives are the natural occurring or chemical substances which are added to the food whether or not they have nutritive value. These food additives are added to food to enhance the taste, color, consistency, texture, flavor, quality, stability, and preservation of foods or to serve some other technological function according to food (Mahapatra & Parija, 2018). These are the consequences of industrialization and advances in the technology of food processing (NRC, 1983). Most food additives may be utilized in limited quantities in specific foodstuffs. Substances added to a food for specific purposes are direct additives, for example, aspartame, low-calorie sweetener, is a direct additive that is intentionally added to soft drinks, puddings, yogurt, and many other foods. An indirect food additive is any substance intended for use as a component of materials used in manufacturing, processing, packaging, or transporting. Indirect additives are unintentionally added to the food in very small amounts and become part of the food. These additives are generally components of food-­ packaging materials, processing aids, pesticide residues, and drugs given to animals. Many chemicals may become parts of food during processing and preparation of food that bring about chemical changes and introduce compounds not ordinarily found in crude items. Our diet additionally contains other undesirable contaminants from natural sources, for example, microorganisms and their metabolites, and the substances that are intrinsic to plants (Pressman, Clemens, Hayes, & Reddy, 2017).

3  Risk Associated with Food Additives Food additives are approved for human consumption after their toxicological studies (acute, subacute, and chronic toxicity) and safety assessment. However, post-­ marketing perception of food additives and their effects should be reserved for a long time (Amin & Al-Shehri, 2018). Information concerning the safety of long-­ term use of such chemicals, their combined effect, and mutability within the

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organism is scant (Moutinho, Bertges, & Assis, 2007). Individual responses vary not only according to dose, age, gender, nutritional status, and genetic factors but also according to long-term exposure to low doses (Hurtado, 1998; Sasaki et al., 2002). Food additives have different beneficial effects; however, they have some adverse toxic effects on health (Fig. 1). Toxicity or benefit relies upon the degree to which the food components interfere with absorption, elimination, or metabolism. Food additives sometimes destroy the nutritional value of food. In nutrition, the likelihood of toxicity of chemical compounds implies that all new compounds ought to be viewed as toxic until their safety is confirmed. However, recent studies demonstrated that consumption of certain food additives increases the risk of cancer in human in spite of the fact that the legal limits of these additives in foods are well respected by the manufacturers. Possible reasons for increased carcinogenicity risk in food additives containing processed foods can be due to the following factors (Gultekin, Yasar, Gurbuz, & Ceyhan, 2015; Mahapatra & Parija, 2018): • Interaction of food additives with some food ingredients or other carcinogenic by-products • Food processing that may change the chemical structure of food additive which might act as carcinogen • Negative synergistic effects of food additives when interacting with other additives • Improper and longer storage conditions of food additive-containing foods • Exceeding the legal limits of food additives that can be added into foods as per acceptable daily intake (ADI) levels

Fig. 1  Possible health risks of food additives

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Food additives are generally safe, but due to the abovementioned possible reasons, the risk of carcinogenicity of some of the food additives used in processed foods might be increased (Gultekin et al., 2015). Different types of food additive and their metabolites have the tendency to cause cancers and tissue injuries (Amin & Al-Shehri, 2018). The food additives nitrite and nitrate used as preservatives in processed meat increase the risk of colon and pancreatic cancer in humans (Bastide, Pierre, & Corpet, 2011). Drinking beverages containing food additives may increase the pancreatic and breast cancer risk (Belpoggi et al., 2006; Mueller et al., 2010). The food additives such as potassium sorbate, ascorbic acid, and ferric or ferrous salts have been shown to have mutagenic and DNA-damaging activities, when combined together. Carcinogenic activity was not detected when these food additives were used separately (Kitano, Fukukawa, Ohtsuji, Masuda, & Yamaguchi, 2002).

4  I nternational Numbering System (E Numbers) of Food Additives To regulate food additives, the European Food Safety Authority of European Union (EU) developed the “E” system that provides a list of several commonly used additives (Hanssen, 1984; Jukes, 2001). It includes those additives that are generally recognized as safe. Each food additive is assigned with a unique number termed as E number which represents the nature of food additives. Nutrients are not included in the E system. The European Union set the criteria as per directives through which food additives are assessed (Jukes, 2001). E numbers for some food additives are as follows: tartrazine (E102), quinoline yellow (E104), carmoisine (E122) amaranth (E123), sodium sulfite (E221), vitamin C (E300), and citric acid (E330) (Jukes, 2001).

5  Categories of Food Additives and Associated Risk Food additives can be divided into five major categories: food preservatives, food coloring agents, flavoring agents, texturizing agents, and nutritional additives (Fig. 2).

5.1  Food Preservatives Food preservatives are naturally occurring or chemical substances that impede the deterioration of food caused by microbes, enzymes, or any other chemical reaction. Salt, sugar, vinegar, etc., are traditionally used as natural food preservatives. Nowadays, the use of chemical food additives has increased tremendously. All the

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Fig. 2  Common categories of food additives

foods have limited shelf life. In order to increase the shelf life and maintain the quality and natural characteristics of foods, certain preservatives are used (Sharma, 2015). These days, generally, all food products contain natural or chemical preservatives. These food preservatives might be associated with negative health effects as well as mutagenic and carcinogenic risk (Table 1). Basically, three types of preservatives are used in foods: antimicrobial agents, antioxidants, and antibrowning agents. 5.1.1  Antimicrobial Agents Antimicrobial agents inhibit the microbial growth which may cause life-threatening illnesses such as salmonellosis or botulism. These agents play a major role in extending the shelf life of various foods by inhibiting the growth of microorganisms. Benzoic acid, propionic acid, nitrites and nitrates, etc., are the examples of antimicrobial agents. These preservatives are generally used in snacks, carbonated drinks, cheeses, bakery products, processed meats, and acidic foods like salad dressings, pickles, fruit juices, and condiments. Some of these antimicrobial agents might be associated with carcinogenicity risk. Benzoates (benzoic acid) can be converted to benzene by decarboxylation reaction. Benzene is a carcinogenic compound when present with ascorbic acid under suitable conditions (Gardner & Lawrence, 1993).

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Table 1  Various food preservatives and associated possible health risks Food preservatives Nitrites and nitrates

Purpose Food products Antimicrobial Snacks, carbonated agents drinks, cheeses, bakery products, processed meats, and acidic foods like salad dressings, pickles, fruit juices, and condiments Benzoic acid, sodium and potassium benzoates

Propionic acid, sorbic acid, etc. Butylated hydroxyanisole and butylated hydroxytoluene

Antioxidants

Soup mixes and cheese spread

Sodium ascorbate

Ascorbyl palmitate, tertiary butylhydroquinone (TBHQ), calcium ascorbate, hydrogen peroxide Sodium sulfite Antibrowning Fruit juices and syrups, agents dried fruits, beer and wine, biscuit doughs Ascorbic acid, citric acid

Associated possible health risks Associated with carcinogenicity risk including colorectal cancer, bladder tumor, thyroid tumor, hepatocellular tumor Associated with carcinogenicity risk including colorectal cancer Associated with some negative health effects, forestomach tumor Development of tumor in the liver, lungs, and gastrointestinal tract, tumorigenic activity in rats, mutagenic Forestomach and urinary bladder carcinoma in presence of butylated hydroxyanisole Associated with some negative health effects

Urticaria, asthma, pruritus, angioedema, mutagenic Associated with some negative health effects, can cause urinary bladder cancer in presence of nitrosamine

Nitrates and Nitrites Nitrates and nitrites are generally used as a preservative in meat industry to provide protection against the bacteria Clostridium botulinum and Staphylococcus aureus and to maintain the flavor and red color of processed meats (Alahakoon, Jayasena, Ramachandra, & Jo, 2015; Cantwell & Elliott, 2017; Lim, Foster, & Riley, 2016). Nitrates and its reduction product, nitrite, occur naturally in plants, meats, and dairy products. Both are the active ingredients of bacon, ham, cured meats, corned beef, hotdogs, sausages, and some cheeses (Yurchenko & Molder, 2007). Nitrates and nitrites from processed meat are the exogenous sources of known potential

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carcinogen, N-nitroso compounds. Nitrate can be reduced to nitrite with the help of intestinal microflora. At high temperature, nitrites get converted into N-nitroso compounds (nitrosamine) by interacting with dietary substrates such as amines or amides which are present in meat proteins (De Mey, De Maere, Paelinck, & Frayer, 2017; Demeyer, Mertens, Smet, & De, 2016; Hur, Yoon, Jo, Jeong, & Lee, 2019). Higher intake of nitrites, nitrates, and nitrosamine from processed meat can cause health problems. These chemicals are toxic and cause cancer in animal and human beings (Demeyer et al., 2016; Omar & Webb, 2014; Sharma, 2015). Excessive consumption of these chemicals is also linked with a higher risk of colorectal cancer (Cantwell & Elliott, 2017). Adu, Sudiana, and Martini (2020) reported that the consumption of nitrite-preserved traditional smoked meat (beef se’i) increased the expression of p53 protein in colon cells of Balb/c strain male mice as an indicator of carcinogenesis. According to the International Agency for Research on Cancer (IARC), the intake of processed meat can increase the risk for colorectal cancer in humans (Bouvard et al., 2015). It was found that the daily intake of 50 g of processed meat can increase the risk of colorectal cancer by 18% (Bouvard et al., 2015). 5.1.2  Antioxidants Antioxidants are used to prevent the oxidation of lipid and vitamin in food products. The oxidation of food is a damaging process which alters the chemical structure and biochemical properties of food resulting in the loss of dietary value of food. The antioxidants slow down the degree of food oxidation (auto-oxidation) and prevent the subsequent development of rancidity and off-flavor. They possess free radical scavenging activity which defends the cells from toxic-free radicals generated during various metabolic processes. Antioxidants are used in food that may be either natural (vitamins C and E) or synthetic chemicals, for example, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (Fiorentino et al., 2008). Some commonly used antioxidants are ascorbyl palmitate, tertiary butylhydroquinone (TBHQ), calcium ascorbate, hydrogen peroxide (H2O2), etc. The antioxidants are useful in preserving dry and frozen foods for long period of time. Certain antioxidants (ascorbic acid and citric acid) function as acidity regulators. Although antioxidants are deemed to confer numerous health benefits, synthetic antioxidants BHA and BHT have shown negative health effects. BHT has been reported to play a role in the development of liver, lungs and gastrointestinal tract tumors (Witschi, 1986). BHA is used in soup mixes and cheese spread. It also has been reported for their tumorigenic activity in rats (Ehrlichman, 1987). 5.1.3  Antibrowning Agents Antibrowning agents are the chemicals which are used to prevent enzymatic and nonenzymatic browning in food products (especially in dried fruits or vegetables). The commonly used antibrowning agents are ascorbic acid, citric acid, sodium

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sulfite, etc. Sulfites are commonly used in fruit juices and syrups, dried fruits, beer and wine, and biscuit doughs. They have been associated with many adverse health effects such as urticaria, asthma, pruritus, and angioedema (Smith, 1991). Sulfites have also been reported for their mutagenic activity in animals (Miller, 1985).

5.2  Food Coloring Agents The color additives are considered as important ingredients of food products. It improves the visual appearance and acceptance of the food products. According to FDA, a color additive is any dye, pigment, or other substance that can impart color to a food product (Pressman et  al., 2017). Food dyes are complex organic compounds that were originally synthesized from coal tar, but nowadays they are derived from petroleum. These synthetic dyes are used in a variety of food products, for example, jam, baked goods, sweets, candies, beverages, dairy products, dry mixes, etc. because they are less expensive and are more steady, and impart intense, uniform color as compared to color pigments derived from natural sources (Singh, Singh, Singh, & Singh, 2019; Tripathi, Khanna, & Das, 2007). The food color additives are categorized in two groups, certified and exempt from certification. The synthetic food dyes are certified color additives, and color pigments derived from natural sources are exempt from certification. Various types of synthetic food dyes are used to provide the color to food products. Some commonly used synthetic food dyes are allura red, tartrazine, sunset yellow, metanil yellow, fast green, erythrosine, indigo carmine, etc. Most widely used dyes in food products are allura red (Red 40), tartrazine (Yellow 5), and sunset yellow (Yellow 6) in the United States. These dyes account for 90% of all dyes used (CSPI, 2010). Synthetic food dyes may have considerable toxicological effects (carcinogenicity, hypersensitivity reactions, and other effects) (Table 2). The use of many food dyes has been banned due to their adverse health effects (IARC, 1978). Most synthetic food dyes are water-soluble azo compounds, but these dyes raise significant health concerns. The azo dyes are also used in various industries such as textile, paper, cosmetic, pharmaceutical, and leather industries (Singh, Gupta, & Singh, 2015; Singh, Singh, Gupta, & Singh, 2019; Singh, Singh, & Singh, 2017a). Different azo dyes have carcinogenic potential by damaging the DNA. The toxic effects of azo dyes may result from the direct action of the dye itself or their metabolic products generated during reductive biotransformation of the azo bond (Singh, Singh, & Singh, 2015). The azo dyes can be metabolized by intestinal microflora to their corresponding aromatic amines with the help of azoreductase enzyme (Umbuzeiro, Freeman, Warren, et al., 2005). The metabolic products of azo dyes can also induce the carcinogenesis in humans as well as animals (Singh, Singh, & Singh, 2014, 2017b). The carcinogenic breakdown derivatives are generally aromatic amines, benzidines, and anilines. Some azo dyes and their metabolic products may be linked to human bladder cancer, splenic sarcomas, colorectal malignancy, hepatocarcinomas, and nuclear anomalies (Choudhary, 1996). Allura red, tartrazine, and sunset yellow contain a potent carcinogen,

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Table 2  Various food dyes, their metabolic products, and associated possible health risks Food dyes Allura red (Red 40) (azo dye)

Food products Confectionery, bakery goods, dessert powders, beverages, candies, cereals, drugs, syrups, soda

Metabolic products Cresidine-4-sulfonic acid and 1-amino-2-naphthol6-­sulfonic acid

Tartrazine (Yellow 5) (azo dye)

Bakery goods, pet foods, beverages, candies, dessert powders, gelatin desserts, cereals, pharmaceuticals, and many other foods commodities

Sulfanilic acid

Sunset yellow (Yellow 6) (azo dye)

Bakery goods, dessert powders, confectionery, gelatin deserts, beverages, candies, sausage and drugs Turmeric powder, cereals

Sulfanilic acid and 1-amino-2-naphthol-6-­­ sulfonic acid

Metanil yellow (azo dye)

Erythrosine (Red 3) (organoiodine compound)

Candies, sausages oral medication, and maraschino cherries

Indigo carmine (anthraquinone dye) Amaranth (azo dye)

Pet food, beverages, candies, and other foods Candies, beverages, sausages, frozen foods

Associated possible health risks Carcinogen, DNA damage in the glands of the stomach, colon, and urinary bladder, colon cancer, intestinal cancer, immune system tumors in mice, allergic reactions Carcinogen, DNA damage in the glands of the stomach, colon, and urinary bladder, intestinal cancer, hyperactivity, hypersensitivity and other behavioral effects in children, allergic reactions Carcinogen, intestinal cancer, allergic reactions

Affects reproductive organs (ovaries and testis), degeneration of the liver, kidney, and stomach, DNA damage in the glands of the stomach, colon, and urinary bladder, mutagenic – Thyroid tumors, induction of chromosome aberrations, kidney or liver disorders Isatin-5-sulfonic acid and Toxic and mutagenic to 5-sulfoanthranilic acid laboratory animals

p-Aminodiphenylamine

–

Cancer in laboratory animals, birth defects, still births, sterility, and early fetal deaths (continued)

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Table 2 (continued) Food dyes Green (fast green 3) (triarylmethane dye) Rhodamine B (amphoteric dye) (banned dye)

Food products Candies, drugs, ice cream, beverages

Metabolic products –

Associated possible health risks Bladder tumors in male rats

3′,6′-Diaminofluoran, and Damages and dysfunctions in the N,N′-diethyl-3′,6′kidney and liver and diaminofluoran retards growth, mutagenic 1-Amino-2-naphthol and Mutagenic, genotoxic Sudan dyes (azo Red chilli powder, effect in the colon and chilli-containing foods 2,4-dimethylaniline dye) stomach of mice such as curry, frozen (banned dye) meats, and spice mixes, sauce, pizza, noodle soup Sweets and confectionery

benzidine. This benzidine is present in the free form or in bounded form. The bound benzidine has been found in higher amounts than free benzidine in food dyes (Lancaster & Lawrence, 1999). The benzidine can be metabolized by gut microorganisms and produces aromatic amines which is responsible for intestinal cancer (Alim, Zahra, & Akhlaq, 2015). The benzidine is also metabolized through N-oxidation which forms electrophilic compounds that can bind covalently to DNA (Choudhary, 1996; Demirkol, Zhang, & Ercal, 2012). This pathway may be involved in benzidine-induced carcinogenesis (Morton, Wang, Garner, & Shirai, 1981). Sudan dyes may also induce DNA damage and genotoxic effect in the colon and stomach of mice (Tsuda et al., 2000).

5.3  Flavoring Agents These additives are added to food products to supplement or enhance the taste, flavor, smell, and consistency. These additives are categorized in two groups, artificial sweeteners and flavor enhancers (Table 3). Artificial sweeteners are added to food products for a strong sweet taste, but they have little or no caloric values (nonnutritive), for example, saccharin, aspartame, sorbitol, sucralose, and acesulfame potassium. These artificial sweeteners are widely used in beverages, juices, sweets, dairy products, marmalade, and chewing gum. Saccharin has been found to be mutagenic and carcinogenic in some animal studies (Wynn & Wynn, 1981). Saccharin can cause urinary bladder cancer in rats and mice (Reuber, 1978). Aspartame is a synthetic dipeptide (l-aspartic acid and l-phenylalanine) (Rangan & Barceloux, 2009). Aspartame and its metabolites, phenylalanine and methanol, can cause brain, prostate, and breast cancers in rats (Olney,

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Table 3  Other food additives and associated possible health risks Food additives Flavoring agents Saccharin Aspartame Monosodium glutamate Texturizing agents Carrageenan, guar gum, xanthan gum Polysorbate 80 and carboxymethyl cellulose

Purpose

Food products

Artificial sweeteners

Beverages, juices, dairy products, sweets, marmalade, and chewing gum Savory foods, snacks, soups, sauces, and meat products

Flavor enhancer

Stabilizers and Ice cream, salad dressings, emulsifiers sauces, soups, and syrups

Associated possible health risks Mutagenic and carcinogenic in animal studies Induce colorectal carcinogenesis Very low toxicity Intestinal inflammation and colorectal cancer

Farber, Spitznagel, & Robins, 1996; Schwartz, 1999). Aspartame can also induce the chromosomal aberrations and DNA fragmentation (Abd Elfatah, Ghaly, & Hanafy, 2012). Aspartame and saccharin were also reported to alter host microbiota and intestinal dysbiosis that can lead to colorectal carcinogenesis (Suez et al., 2014). Monosodium glutamate (MSG) is a non-essential amino acid found in a variety of processed foods like savory foods, snacks, sauces, and canned soups. It is a natural flavor enhancer commonly used to intensify and enhance the flavor of savory dishes (Yamaguchi, 1991). Hata et al. (2012) reported that the MSG-induced diabetic mice are highly susceptible to azoxymethane-induced colorectal carcinogenesis (CRC). The acute metabolic complications of diabetes induce development of CRC (Peeters, Bazelier, Leufkens, de Vries, & De Bruin, 2015; Pietrzyk, Torres, Maciejewski, & Torres, 2015).

5.4  Texturizing Agents These are stabilizer, thickener, and emulsifier agents that are used to add or modify the texture of processed food products (Table 3). Stabilizers are generally natural gums, for example, carrageenan and guar gum, which provide the desired texture in food products. Carrageenan is a natural polysaccharide obtained from edible red seaweed. It is used as texturizing agents in a variety of food products like sauces, soups, and syrups (Necas & Bartosikova, 2013). It has very low toxicity (Necas & Bartosikova, 2013). Emulsifiers are detergent-like molecules which are used as texturizing agents in various processed foods. Emulsifiers can alter gut microbiota that leads to intestinal inflammation and colorectal cancer. Polysorbate 80 and carboxymethyl cellulose are the most common emulsifiers. Both emulsifiers alter the

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intestinal bacteria that induce pro-inflammatory environment and promote the tumor development in mice (Viennois, Merlin, Gewirtz, & Chassaing, 2017).

5.5  Nutritional Additives These additives are added to improve or maintain the nutritional quality of the food products. Nutritional additives include vitamins, minerals, antioxidant amino acids, and fatty acids. The negative health consequences and toxicity of these additives are very low, but these additives can cause toxicity when consumed in higher concentrations than the recommended dietary allowance.

6  Role of Intestinal Microflora in Colorectal Cancer The human intestine is the natural habitat of dynamic and diverse populations of microbial system (Ley, Peterson, & Gordon, 2006). Gut microbiome (microbiota) is a diverse community of microorganisms (bacteria, viruses, archaea, and fungal species) that inhabit the intestine. The human gastrointestinal tract consists of approximately 100 billion microbes, and largest proportions of these microbes are bacteria (Bäckhed, Ley, Sonnenburg, et al., 2005; Savage, 1977). The major proportion of gut microbiome is dominated by three phyla, Firmicutes (30–50%), Bacteroidetes (20–40%), and Actinobacteria (1–10%). Major portion of gut microbiome is strictly anaerobes including Bacteroides, Eubacterium, Fusobacterium, Peptostreptococcus, Bifidobacterium, and Atopobium, whereas facultative anaerobes such as Enterococcus, Streptococcus, and Enterobacteriaceae constitute a lesser portion of gut microbiome (Tlaskalova-Hogenova et al., 2014). The dominant phyla in colon are Bacteroidetes and Actinobacteria which represent approximately 90% of the microbial population of the colon. The composition of gut microbiome is established during the early stages of life and acquired during the first 3 years of childhood (Dominguez-Bello, Blaser, Ley, & Knight, 2011). The development of gut microbiome continues with environmental exposure and physiological processes in the host (Neish, 2009). The composition of the gut microbiota varies along the length of the gut and differs among the people (Neish, 2009). The symbiotic association of gut microbiome is developed after several years of co-evolution and co-­ adaptation leading to a balanced homeostasis in the gut (Blaser & Falkow, 2009). The intestinal microbiome plays an important role in health of the host and gut homeostasis (Goubet, Daillere, Routy, et al., 2018). The microbiome serves immunological, structural, and metabolic functions and maintains a natural defensive barrier to infection (Gagnière, Raisch, Veziant, et al., 2016). Alterations or imbalance in gut microbiome composition due to the dietary or environmental factors is referred to as dysbiosis. Intestinal dysbiosis leads to intestinal barrier damage and impaired function of innate immunity (Ding, Tang, Fan, &

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Wu, 2018; Reinoso Webb, den Bakker, Koboziev, et al., 2018). It can be associated with various intestinal diseases including inflammatory bowel disease (IBD) and colorectal cancer (Arthur & Jobin, 2013; Chassaing & Darfeuille-Michaud, 2011). Intestinal dysbiosis plays a significant role in the colorectal carcinogenesis. The carcinogenesis process is driven by modulated immune response, inflammation, toxic and genotoxic metabolite production, and specific species of bacterial pathogens (Wieczorska, Stolarek, & Stec, 2020). Assessment of changes in the intestinal microbiota during the development and progression of CRC might provide important information for therapeutic strategies (Zou, Fang, & Lee, 2018).

7  Conclusion Colorectal cancer (CRC) is one of the most common malignancies of gastrointestinal tract. The development of colorectal cancer is influenced by genetic and environmental factors. The risk of CRC is increased in worldwide population due to lifestyle changes, dietary habits, and higher consumption of processed foods. Processed foods contain certain food additives. Food additives such as preservatives, coloring agents, and texturizing agents are added to food products for specific purposes which have beneficial as well as harmful effects. Nitrate and nitrite used as preservative in processed meat may increase the risk of CRC and other health problems. The food dye and their metabolic products can also increase the risk of CRC and other types of cancer. The human gut consists of billion of microbes which play a role in intestinal homeostasis. Alteration in gut microbiome composition (dysbiosis) can promote colon carcinogenesis.

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Effect of Milk and Dairy Products in Colorectal Cancer Sarang Dilip Pophaly, Soumitra Tiwari, Awadhesh Kumar Tripathi, and Manorama

Abstract  Colorectal cancer (CRC) is the third most prevalent cancer type worldwide. It is the leading cause of cancer-related deaths in developed countries. Colorectal cancer has multifactorial etiology and can be attributed to various predisposing conditions and diet-related issues. Among many other factors, diet plays a crucial role in the prevention and/or progression of the disease. Dairy foods have long been considered as having beneficial effects with respect to colon cancer. Because of the availability of wide variety of dairy products and myriad of constituent nutrients, the observed beneficial effects are difficult to associate with the exact cause. Most of the correlations between the consumption of dairy products and perceived protective or therapeutic effects have been derived from cohort and meta-­ analyses studies in different population groups. The most documented and widely accepted effect has been observed for milk, calcium, and vitamin D, when consumed both alone or in combination. Casein and whey proteins and their hydrolysates in the form of bioactive peptides offer protection against CRC. Whey proteins and their derivatives are also being used because they improve body composition, muscle strength, and body weight and has reduced chemotherapy toxicity. High fat diet, especially those rich in conjugated linoleic acid, has also been shown to have a strong antitumor activity. Fermented foods and probiotics cultures are also gathering a rich body of evidence due to their beneficial effects on CRC. Keywords  Colorectal cancer · Dairy product · Milk

S. D. Pophaly · A. K. Tripathi · Manorama College of Dairy Science and Food Technology, Dau Shri Vasudev Chandrakar Kamdhenu Vishwavidyalaya, Raipur, Chhattisgarh, India S. Tiwari (*) Department of Food Processing and Technology. Atal Bihari Vajpayee Vishwavidyalaya, Bilaspur, Chhattisgarh, India e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_15

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1  Introduction Food is fundamental to our health and well-being. Last few centuries in the course of human evolution have seen remarkable shifts in eating habits and patterns. With globalization in last few decades, along came the diversity of food on our platter. But along with these changes in food and also in our lifestyle, new forms of diseases with various intensities have encircled our lives. We often tend to forget the role of diet and healthy food in our life, and we focus on dealing with diseases using drugs. Cancer is one such disease, with which humanity has been fighting with all its newly found tools and techniques. Colorectal cancer (CRC) is one of the most prevalent malignancies in the world with approximately 881,000 cancer-related deaths in 2018. It is the third most deadly and forth most commonly diagnosed cancer in the world. The incidence of CRC is high among men than women and is more common in developed than developing countries (Rawla et  al., 2019). Highest incidences of colon cancer are reported in Southern Europe, Australia, New Zealand, and Northern Europe and that of rectal cancer in Eastern Europe, Australia/New Zealand, and Eastern Asia (Rawla et al., 2019). According to the Center for Disease Control and Prevention (CDC) of the USA, risk of getting colorectal cancer increases with age; preexisting conditions such as inflammatory bowel diseases (Crohn’s disease or ulcerative colitis); family history of colon cancer, genetic syndrome such as familial adenomatous polyposis (FAP); and lifestyle-related factors such as sedentary life, low fruits and vegetables, low fiber diet, high consumption of processed and red meat, cholesterol-rich diet, obesity, alcohol consumption, and use of tobacco. A list of major nutrition-related predisposing factors of colorectal cancers is given in Table 1. Nutrition plays a pivotal role in the chances of occurrence, degree of severity, prognosis, and recovery from colon cancer. Dairy products are widely considered to have a protective as well as beneficial effect on colon cancer.

2  Milk Constituents Milk is considered almost a complete food that contains carbohydrates, proteins, fat, minerals, and vitamins. Milk from different species and breeds have the same basic constituents but present in varying concentration. Lactose is a disaccharide that is present as the major carbohydrate in milk and it is often considered a sugar with low carcinogenicity. Protein fraction of bovine milk comprises approximately 80% casein and 20% whey proteins. Casein is present in micellar form with four primary fractions (αs1, αs2, β, and κ casein) and whey proteins are present in soluble form with β lactoglobulin andα lactalbumin as the major fractions. Hydrolysis by proteolytic enzymes, fermentation by certain bacteria, and heating can result in the release of specific peptide fragments from native milk proteins, which have immense biological importance. Milk lipids are chemically very complex in nature

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Table 1  Nutrition-related predisposing factors for colorectal cancer S. No. 1 2 3

Factor/condition Excessive alcohol consumption Moderate alcohol consumption Cholesterol-rich diet

4

Gender

5

Diet deficient in folic acid and vitamin B6 Consumption of selenium, lycopene Pulses consumption Consumption of soya products Consumption of green vegetables Nuts Dried fruits Higher calcium intake High vitamin D Dairy product Meat (red and processed)

6 7 8 9 10 11 12 13 14 15

Elevated /lowered risk Increased risk Reduced risk Increased risk High risk for males High risk

Reference McNabb et al., (2020) Klarich et al., (2015) Azeem et al., (2015), Wang et al. (2017) Rawla et al. (2019) Kune & Watson (2006)

Lowered risk

Kune & Watson (2006)

Lowered risk Lowered risk Lowered risk Lowered risk Lowered risk Lowered risk Lowered risk Lowered risk Increased risk

Azeem et al. (2015)

Ferrer-Mayorga et al. (2019) Aune et al. (2012) Marques-Vidal et al. (2006)

and exist as a unique emulsion. Nearly 98–99% of milk fat is composed of triglycerides. These triglycerides are enclosed in a membrane known as fat globule membrane (FGM). Also, approximately one-third of the fat in whole milk is monounsaturated and small amounts of essential fatty acids are also present. Milk is one of the major sources of conjugated linoleic acid (CLA) in the diet, although it is present in small amounts in milk fat (Davoodi et al., 2013). Several milk constituents such as vitamin D, calcium, casein, whey proteins, CLA, butyrate, saturated fatty acids, and contaminants such as pesticides, estrogen, and insulin-like growth factor I (IGF-I) may be responsible for either a beneficial or a harmful association between dairy products and cancers (Aune et al., 2012; Davoodi et al., 2013) (Fig. 1).

3  Milk Products and Colorectal Cancer Epidemiological data from different studies have pointed toward a correlation between the consumption of dairy products and incidence or prognosis of colorectal cancers. The aqueous extracts of human stool, of a group of individuals who were shifted from a dairy-rich to a dairy-free diet were subjected to cytotoxicity and genotoxicity analyses. Excluding dairy products from diet had a marked effect on several nutrients. The levels of saturated fats, cholesterol, calcium, phosphate, and

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Milk compounds

Vitamins Minerals

Exogenous compounds

Indigenous compounds

of milk

of milk

Contaminants

Calcium

Chemical

CLA

Proteins

Omega 3 Functional enriched compounds

Phytochemical Probiotics Synbiotic

Microbial

Vitamin D Dairy additives Fats

Heating Maillard Irradiation Bioactive peptides

Lactose

Process-produced compound Fermentationproduced compound

IGF-1 Estrogens

Fig. 1  Major constituents of milk and dairy products that might have a beneficial effect on cancer. (Source: Reproduced with permission from Davoodi et al. 2013)

vitamin D decreased significantly as they are present in high quantities in dairy products. This shift resulted in a significant increase in the cytotoxicity of fecal water samples as measured by HT-29 cell assay (Glinghammar et al., 1997). In different cohort studies and meta-analyses, increased consumption of dairy products has been shown to be directly associated with a significant reduction in colon cancer [10]–[15]. In a study, Cho et al. (2004) presented a pooled analysis of cohort data from five different countries and reported that individuals who consumed more than 250 g/day of milk had a 15% reduced risk of developing colorectal cancer, in comparison to those whose consumption level was less than 70  g/day (Cho et  al., 2004). Most of the large-scale cohort studies have demonstrated the inverse association of colorectal cancer with the consumption of dairy foods and calcium in both men and women. Clinical trials, animal trials, and laboratory studies have also complemented the observations of epidemiological data showing the beneficial effects of dairy products and calcium. Intake of calcium at a level of 1200 to 1500 mg/ day seems to have a protective action against colon cancer (Holick, 2008). Dairy products are major sources of calcium in human diet, and consumption of these products is being considered as an important preventive and protective factor for colon cancer. Calcium binds secondary bile acids and ionized fatty acids and thus reduce their proliferative effects in colonic epithelium (Govers & Van der Meet, 1993). Oral supplementation of vitamin D3 and calcium was shown to favorably modulate normal colon tissue and circulating hypothesis based biomarkers of risk for colorectal neoplasms in sporadic colorectal adenoma patients (Bostick, 2015). Higher calcium and vitamin D intake was also reported to increase DNA mismatch repair system activity in the normal colorectal mucosa of sporadic adenoma patients

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(Sidelnikov et  al., 2010). It has also been shown that both the nutrients promote colorectal epithelial cell differentiation and may “normalize” the colorectal crypt proliferative zone in sporadic adenoma patients (Fedirko et al., 2009). As evident from these reports, the effect of calcium is partly related to simultaneous vitamin D intake. Independent vitamin D consumption has also been tested and reported to have strong protective effect against colon cancer. Besides calcium and vitamin D in milk, conjugated linoleic acid (CLA) which is present in high-fat dairy products has also been reported to have anticancer properties. It has been shown that the concentration of CLA in raw milk fat is a major factor that determines the concentration of CLA in milk/dairy products and that heat processing of dairy products does not reduce the level of CLA (Shantha et al., 1995). A wide range of factors affect the levels of CLA in milk, which include breed of cow, age of the animal, and type of feed. The CLA content was found to be the highest in milk from cows grazing on pasture (2.21% of total fatty acid) compared to cows fed a diet containing 50% conserved forage (hay and silage) and 50% of grain (0.38% of total fatty acid) (Stanton et al., 1997). Additives such as rapeseed and soybean to the diet of ruminants also significantly increased the CLA content in milk compared to non-supplemented control (Stanton et al., 1997). Antitumor properties of CLA have been well studied both in vitro and in vivo. Several studies also aimed to explain the potential mechanism of action. Their results showed that CLA affects various fundamental processes that are found altered in tumor cells, including proliferation, differentiation, and apoptosis (Koronowicz & Banks, 2018). The results of a Cohort study in Swedish women showed that women who consumed ≥4 servings of high-fat dairy foods per day (including whole milk, full-fat cultured milk, cheese, cream, sour cream, and butter) had a multivariate rate ratio of colorectal cancer of 0.59 (95% CI: 0.44, 0.79; P for trend  =  0.002) when compared to women who consumed 100 KDa and 50–100 KDa fractions of the CFS, with protein, nucleic acid, and polysaccharide components as the likely candidates responsible for the activity. However, there have been few conflicting reports by certain authors (Norat & Riboli, 2003; Kampman et al., 1994; Ralston et al., 2014) refuting any significant role of cheese and fermented dairy products on colon cancer which could either be attributed to the specific strain of bacterium used or to other compounding factors. Prebiotic compounds such as inulin and fructoligosaccharides (FOS)are also reported to have anticancerous activity, which has been linked to their ability to stimulate beneficial intestinal bacteria; modify gene expression in cecum, colon, and feces; enhance micronutrient absorption in colon; and modulate xenobiotic metabolizing enzymes (Raman et al., 2013). Fermented skim milk extracts derived from Lactobacillus paracasei subsp. paracasei NTU101 effectively reduced CRC cell viability, but they were not cytotoxic to colon epithelial cells and were proposed to have potential application as an anticancer agent (Chang & Pan, 2018). Two

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well-known apoptotic inducers, TNF-related apoptosis-inducing ligand (TRAIL) and short-chain fatty acids (SCFA) synthesized by Propionibacterium freudenreichii, were tested to synergistically induce cell death through apoptosis. It was found that milk fermented with the bacterium induced HT29 cell apoptosis and enhanced TRAIL cytotoxic activity (Cousin et al., 2016). In different cohort studies, higher yogurt intake was significantly associated with decreased CRC risk (Pala et  al., 2011) and weekly yogurt and probiotic consumption was associated with decreased odds of hyperplastic polyp and adenomatous polyp (Rifkin et al., 2020), suggesting that yogurt could be included as a part of diet regimen to prevent colorectal cancer.

5  Conclusion Milk and dairy products are important part of our daily diet, and they not only provide with basic nutrition but also have immense therapeutic value. In last two to three decades, numerous findings on anticancerous effect of milk constituents and milk-derived products have come to light. These products are of great significance with respect to prevention, treatment efficacy, and recovery of patients suffering from colorectal cancer. The major constituents of milk such as lactose, casein, whey proteins, and calcium and minor constituents such as vitamins, CLA, and derived bioactive peptides play a major role in the anticarcinogenic activities of dairy products. Most of the literature supporting a positive role of these compounds in cancer prevention or prognosis have been derived by meta-analyses or cohort studies and these needs to be backed up by robust clinical trials and wet lab data. Cell culture and animal model studies are also used widely, but this again will require clinical testing before any of the product can be prescribed for therapeutic purposes.

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Development of RNA-Based Medicine for Colorectal Cancer: Current Scenario Ajay Amit, Sudhir Yadav, Rajat Pratap Singh, and Chanchal Kumar

Abstract  Colorectal cancer (CRC) is one of the leading causes of death in both men and women worldwide. CRC is the third commonly diagnosed cancer and second leading cause of death worldwide as per the 2018 data. In last decade, standard chemotherapy and target therapy have been improved, but some serious problems have emerged such as multiple drug resistance (MDR) and severe side effect which need to be addressed. RNAs are one of the most abundant molecules present in the living system and are crucial for the proper functioning of essential biological processes including gene regulation and expression which is important for cell division and growth and regulation of oncogenes. There are different treatment modalities based on RNA therapeutics which is available for diseases like cancer which has a genetic basis and CRC is one of them. The RNA-based therapeutics is highly specific and that’s why they have an edge over traditional and routine chemotherapy. In recent times, RNA-based therapeutics based up on RNA aptamer, RNAi (RNA interference), RNA enzyme (ribozyme), and antisense oligonucleotide attracts more study due to its high potential accompanied with flexibility to work against a wide range of tumor. It has been also observed that RNA-based therapeutics is associated with low toxicity and high specificity for target. This chapter focused on the recent advances and clinical study involving RNA-based therapeutics for CRC. Keywords  Colon cancer · RNAi · Ribozyme · Aptamer

A. Amit · S. Yadav · C. Kumar (*) Department of Forensic Science, Guru Ghasidas Vishwavidyalaya, Bilaspur, CG, India R. P. Singh Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, CG, India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_16

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1  Introduction Colorectal cancer (CRC) is one of the leading fatal diseases of the world. CRC is the third commonly diagnosed cancer and second leading cause of death as per the 2018 data (Bray et  al., 2018). Most common CRC treatment of stage I and stage II is surgical resection of the colon. Stage III patients require surgical resection and adjuvant chemotherapy to avoid the risk of recurrence. The main treatment for stage IV metastatic CRC is chemotherapy. However, target therapy can be used as another option for the treatment of stage IV CRC. In recent years, researchers have begun to understand the phenomenon of metastasis, as this is a complex and inefficient process that’s responsible for the leading cause of CRC-related death. Most of the clinic pathological indicators are not sufficient to determine the disease progression for patients diagnosed with advanced CRC stage. More than one third of patients eventually died of systemic disease progression. (Primrose, 2002) Five-year survival rate in case of non-operable metastasis disease is quite poor, ~14% (Siegel et al., 2017). Best effect has been seen in the treatment of early-stage disease, although half of the CRC patients are diagnosed with metastatic stage cancer which later developed into advance-stage-like disease or cancer recurrence appears after the treatment in the following months (Engstrom et  al., 2009). Most commonly used drugs for the treatments include capecitabine, irinotecan, oxaliplatin, fluorouracil (5-FU) plus leucovorin (folinic acid), and trifluridine plus tipiracil (Neugut et al., 2006). Relatively low response rates are observed for targeted therapy (Asghar, Hawkes, & Cunningham, 2010), and it is still unclear whether targeted combination therapy is effective (Kummar, Chen, Wright, et al., 2010). To achieve better clinical outcome usually in most cases, a combination of two or more of these chemotherapy drugs is given during treatment of CRC.  This combination chemotherapy for advanced CRC leads to prolong survival rate and improves life quality (Bustina & Murphy, 2013). However, these drugs frequently showed much broader specificity than initially expected, resulting in systemic toxicity and unexpected side effects (Bray et  al., 2018). Although most of the chemotherapy has its own limitations which include bone marrow suppression, hair loss, vomiting, nausea, diarrhea, neuropathy, and increased risk of infections, multidrug resistance (MDR) is another major limitation of chemotherapy which results in metastasis and cancer relapse (Mansoori, Mohammadi, Davudian, Shirjang, & Baradaran, 2017). 5-FU is the most effective chemotherapeutic agent currently used for the treatment of colorectal cancer; however, almost half of the patient does not respond to this drug (Wilson et al., 2010). When CRC patients receive the same treatment at the same disease stage, the clinical outcome comes out quite different for different patients (Tanaka, Tanaka, Tanaka, & Ishigamori, 2010). The advent of new technology and research showed that cancer is a very complex disease with very high heterogeneity (Lawrence et al., 2013; Tanaka et al., 2010). The CRC progression exhibits various molecular biological changes, epigenetic abnormalities, or genetic mutations (Budinska et  al.,

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2013; Willett et al., 2012). The major obstacle in the treatment of cancer is its ability to evade host’s immune reaction and attain drug resistance. Many studies demonstrate that correcting the abnormal gene expression can eliminate the cancer cells (Dow et al., 2015; Luo, Solimini, & Elledge, 2009). Therefore, the need of the hour is to use new therapeutic strategies directed against the genetic changes and target the multiple cancer biomarkers simultaneously. Ribonucleic acid (RNA)-based therapeutics proved to be highly specific and less toxic and showed high potency, thus showing a huge potential as a new therapeutic approach (Bumcrot, Manoharan, Koteliansky, & Sah, 2006). Many options as RNA therapeutics are available which include antisense oligonucleotides (ASO), aptamers, ribozymes, RNA interference (RNAi), microRNA mimics and inhibitors, long non-coding RNA (lncRNA), and a variety of other RNAs. Due to the availability of a wide range of choices offering more flexible approaches to target disease-causing genes, RNA therapeutics offers competitive advantage over conventional treatments. (Elbashir et al., 2001). The “undruggable” proteins for conventional medicines can be selectively modulated by RNAs. They can even target proteins that have multiple homologous family members. In addition, RNAs can be designed for the targeted genes with a known sequence and easily be synthesized. In present chapter, we will be covering recent research works on various RNA biological functions and its role in gene regulation of CRC progression, which include effects on CRC cell growth, apoptosis, invasion, and metastasis. We will be also discussing RNA-based therapeutic strategies, their advantages, and limitations in CRC treatment.

2  Colorectal Cancer Therapy Based on RNA Use of RNA as therapeutic agent is referred to as RNA therapeutics. Many classes of RNA are used as therapeutic agent which regulates transcription and translation through various mechanisms like antisense oligonucleotides (ASO), small interfering RNAs (short silencing RNA, siRNA), and microRNA (miRNA). These RNA therapeutic molecules are applied to silence the gene expression; however, RNA aptamer blocks the gene function with its very specific binding capacity against target gene (Liu and Guo, 2020). RNA therapeutic agents used in the CRC treatment are summarized in Table 1.

2.1  Antisense Oligonucleotide (ASO) ASOs are short synthetic single-stranded nucleic acid sequences that can specifically bind to cDNA or RNA targets which strictly follow Watson and Crick base-­ pairing rules. Three different antisense mechanisms have been explored for therapeutics: (1) RNase-H-dependent targeted cleavage of mRNA, (2) translation

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Table 1  RNA-based therapeutics used in target therapy of colorectal cancer

RNA agent Hammerhead ribozyme

Targeted cell SW480

Target signaling molecule/ receptor KRAS

Hammerhead ribozyme

HCT-8DDPA

γ-GCS

ASO

MDM2

Ribozyme (Angiozyme) Ribozyme

LS174T and DLD-1 KM12

VEGF-1

HT29

hTERT

miR-143

SW480, LoVo KRAS

miR-143

SW480, 228

DNMT3A

SiRNA

HCT116

KRAS and PIK3CA

miR-143

SW620

MACC1

Aptamer

CEA LS174T, LoVo, SW480

siRNA

SW620

LSD1

ASO

HT29

EGFR

miR-204-5p

LoVo and HCT116

RAB22A

B7-H4 siRNA

LoVo

miR 217

RKO and SW480 HCT116, LoVo, SW480, HCT15, DLD1

CXCL12/ CXCR4 and JAK2/STAT3 signaling MAPK1

Antibody-­ siRNA complexes

KRAS

Functions Induces apoptosis, growth suppression, and expression of angiogenic factor was altered Multidrug resistance (MDR)-associated protein was downregulated Demonstrates antitumor activity in vivo and in vitro Number of metastases was reduced Specifically targets and treats the tumor Suppresses CRC cell growth

Reference Tokunaga et al. (2000)

Nagata et al. (2001)

Wang et al. (2002) Kobayashi et al. (2005) Jeong et al. (2008) Chen et al. (2009) Decreases tumor cell growth Ng et al. (2009) Valentino et al. Synergistic decrease in proliferation and an increase (2012) in apoptosis Inhibits cell growth, Zhang et al. migration, and invasion (2012) Lee et al. Inhibits homotypic aggregation, migration, and (2012) invasion Ding et al. Suppresses proliferation, (2013) migration, and invasion of CRC cells Reduces cell proliferation Najar et al. (2013) Yin et al. Inhibits migration and (2014) invasion, promotes the sensitivity to chemotherapy Inhibits proliferation, Peng et al. invasion, and migration (2015)

Inhibits tumor growth and enhances apoptosis in CRC Deactivates ERK and the MAPK pathway; inhibits tumor growth

Tang et al. (2015) Tao et al. (2015)

(continued)

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Table 1 (continued)

RNA agent ASO

Targeted cell HCT116

Target signaling molecule/ receptor miR-21

siRNA-PEG and SN-38

LS174T

VEGF

siRNA and Dox

HCT116

Snail

ASO AZD4785 SW480

KRAS

Aptamer

HT29

EpCAM

smRNA

HCT116, HT29

P21

Functions Inhibits cell proliferation, reduces invasion and migration Inhibits tumor growth, and enhances the antitumor effect of the chemotherapeutic drug Changes EMT genes, induces cell apoptosis, inhibits migration mRNA inhibits the proliferation of tumor cells Delivers siRNA to cancer stem cells in vivo Inhibits cell proliferation and induces apoptosis

Reference Lee et al. (2016) Mi et al. (2016)

Sadreddini et al. (2017) Ross et al. (2017) AlShamaileh et al. (2017) Wang et al. (2017)

CXCL12: C-X-C motif chemokine ligand 12, JAK2: janus kinase 2, STAT3: signal transducer and activator of transcription 3, LSD1: lysine-specific histone demethylase 1, PEG: polyethylene glycol, VEGF: vascular endothelial growth factor, MAPK1: mitogen-activated protein kinase 1, DHX9: DExH-Box helicase 9, CEA: carcinoembryonic antigen, EpCAM: epithelial cell adhesion molecule, hTERT: human telomerase teverse transcriptase, γ-GCS: gamma-glutamylcysteine synthetase, VEGF-1: vascular endothelial growth factor-1

inhibition by imposing steric block, and (3) RNA splicing modulation of regulated gene expression. In case of RNase-H-mediated cleavage, ASO binds to the complementary region of the target mature mRNA to form a DNA or RNA hybrid. This hybrid then recruits the RNase-H enzyme which selectively cleaves the target mRNA sequence and abolishes the expression of target gene (Chan, Lim, & Wong, 2006). In case of steric block approach, ASO blocks the cellular machinery proteins by binding to motifs like translation initiation site, ribosome- binding site, or 50-cap site, in the mature mRNA (Fig. 1) (Kole, Krainer, & Altman, 2012). Designed ASO can be used to target pre-mRNA and interfere with RNA-processing events, like splicing or nuclear polyadenylation (Arechavala-Gomeza, Khoo, & Aartsma-Rus, 2014; Lee & Rio, 2015). The splice-switching ASO on introduction can manipulate defective splicing by steric blocking of the splicing motifs, it binds to pre-mRNA at position where splicing factors bind, and therefore it alters the subsequent processes, like intron retention, exon skipping, and exon inclusion (Fig. 1). ASOs can also target pre-mRNA on other sites such as the signal sequence of polyA, cleavage stimulation factor (CstF), and cleavage and polyadenylation specificity factor (CPSF), or it can also block the processing of translation arrest or mRNA degradation (Arechavala-Gomeza et al., 2014; Du & Gatti, 2009). ASO was first introduced by Paterson et al. as therapeutic molecule to inhibit translation in 1977 (Paterson,

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Fig. 1  Mechanism of action of antisense oligonucleotide (ASO)

Roberts, & Kuff, 1977), and since then multiple approaches have been employed, viz., different modifications in the structure (e.g., backbone, sugar moiety) to optimize their therapeutic potential. Till date ~50 clinical trials of ASOs have been conducted for the treatment of cancer (https://clinicaltrials.gov). Cell proliferation, cell differentiation, cell cycle, and cell death pathway essential genes were targeted by ASOs for treatment of CRC. Growth factor EGFR plays a very important role in CRC proliferation and progression (Moroni et al., 2005). Najar et al. established EGFR ASO encapsulated within polyamidoamine (PAMAM) nanoparticles. These nanoparticle reduces proliferation of human colon cancer cell line HT-29 (Najar, Pashaei-Asl, Omidi, Farajnia, & Nourazarian, 2013). Mutation in RAS oncogene showed persistent activation of downstream pathways, resulting in several cancers in humans including CRC. Ross et al. employed a high-affinity constrained ethyl-containing therapeutic ASO AZD4785 which targets KRAS mRNA. AZD4785 successfully inhibited KRAS mutant proliferation (Ross et al., 2017). Tao et  al. targeted genes which play an important role in proliferation-­ independent processes in CRC progression such as adhesion, invasion, and migration. Eukaryotic expression vector encoding ASOs against an oncomiR (miR-21) was constructed and transfected to CRC cells. ASO targeting of miR-21 significantly inhibits the invasion and migration of CRC cells (Tao et  al., 2015). Wang et al. inhibited oncogene MDM2 which is a negative regulator of p53, a tumor suppressor gene using ASO. It effectively showed antitumor activity in vitro and in vivo by inhibiting MDM2. This ASO can be used as a potential CRC cancer therapeutic drug (Wang et al., 2002). Abaza et al. explored the role of ASO targeting regulation of proto-oncogene c-MYC. c-MYC ASOs effectively inhibited the growth of CRC cells and make CRC cells more sensitive against several chemotherapeutic drugs including doxorubicin, 5-FU, paclitaxel, and vinblastine (Abaza, Al-Saffar, Al-Sawan, & Al-Attiyah, 2008).

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2.2  Ribozymes RNA molecules that function as enzymes and catalyze biochemical reactions are known as ribozymes. Due to their enzymatic activity, they have a great potential to be used in gene therapy. Natural ribozymes exhibit RNA cleavage and ligation activities (Doudna & Cech, 2002). Tailor-made ribozymes can be designed, synthesized, and delivered in specific cells, to regulate the expression of target gene (Mulhbacher, St-Pierre, & Lafontaine, 2010). Majority of the ribozyme types are tested as therapeutics drugs. However, hammerhead-like ribozymes are used more frequently because they have been extensively studied (Bagheri & Kashani-Sabet, 2004). Hammerhead ribozymes act on target RNA transcript containing NUH triplet (N is any nucleotide and H is any nucleotide except guanosine) and cleave it. Sequences AUC and GUC are the most effective processing sites (Kore, Vaish, & Kutzke, 1998) (Fig. 2). Another class of ribozyme often used as therapeutic molecule is the hairpin ribozyme. Target RNAs possessing the N*GUC sequence (N is any nucleotide) are specifically recognized and cleaved by hairpin ribozyme (Ferre-­ D’Amare, 2004). Many studies reported the use of ribozymes to target various oncogenes and genes responsible for drug resistance in CRC. In colon cancer mouse model, Jeong et al. reported a systemic delivery of trans-splicing ribozyme using adenovirus which recognizes cancer-specific transcripts and effectively reduces tumor burden (Jeong et  al., 2008). A hammerhead ribozyme has been designed against oncogene KRAS mRNA harboring mutations in human colon cancer cell

Fig. 2  Mechanism of action of ribozyme

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lines to preferentially cleave KRAS mRNA transcript. After being employed to cell line, this ribozyme significantly suppresses tumor growth and alters the angiogenic gene expression (Tokunaga et  al., 2000). γ-Glutamylcysteine synthetase (γ-GCS) has been known to play a significant role in both cisplatin and multidrug resistance. A hammerhead ribozyme has been designed against γ-GCS mRNA by Nagata et al. to specifically downregulate γ-GCS gene expression in the human colon cancer cell line HCT-8DDP. Reversal of drug resistance against cisplatin, doxorubicin, and etoposide was observed due to downregulation of γ-GCS expression (Nagata et  al., 2001). Ribozyme RPI.4610 (Angiozyme) was used to target vascular endothelial growth factor receptor 1 (VEGF-1). It has been reported that angiozyme can inhibit metastases in a human KM12 colorectal cancer xenograft model and improved the survival in a murine 4T1 tumor model (Kobayashi et al., 2005).

2.3  RNA Aptamer RNA aptamers are single-strand RNA oligonucleotides with acquired secondly structures and shapes that bind to targets such as proteins, peptides, and small molecules (Fig. 3). High-affinity RNA aptamers can be generated against target molecule through multiple rounds of selection termed as systematic evolution of ligands by exponential enrichment (SELEX) (Gopinath, 2007). RNA aptamers fold in a stable three-dimensional shape which allows the binding to targets with high affinity and specificity (Patel et al., 1997). Aptamers are also known as “chemical antibodies,” and it showed several competitive advantages over protein antibodies. Due to its nature of cell-free assembly it, allows aptamer production cost effective, rapid, and reproducible in bulk or commercial scale (Sun & Zu, 2015). After chemical modifications, these aptamers induce less immunogenicity and are more resistant to nuclease degradation (Keefe, Pai, & Ellington, 2010). Such modification also allows an enhanced tissue penetration due to their smaller size (~100 nucleotide) (Xiang et al., 2015). More functional groups can be attached to the backbone of aptamers due to its nucleic acid nature. As an example, aptamers

Fig. 3  Mechanism of action of aptamers

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can form chimeric structure with siRNA which allows delivery of siRNA to targeted cancer cell population (AlShamaileh et al., 2017). It can be conjugated with chemo drugs like doxorubicin (Bagalkot, Farokhzad, Langer, & Jon, 2006) or combined with nanoparticle to target metastatic CRC cells (Rychahou et al., 2015). A plethora of RNA aptamers have been characterized and identified to bind different targets and have shown great potential as diagnostic and therapeutic tools in various diseases along with CRC. A large number of nuclease-resistant RNA oligonucleotides have screened against tumor-bearing mouse model by Mi et al. These RNA aptamers can localize metastases in hepatic colon cancer. They identified an RNA aptamer which specifically binds to RNA helicase p68, which is upregulated in CRC (Mi et al., 2010). RNA aptamers are isolated against not only cell surface markers but also intracellular key components. Another aptamer was identified by Mi et al. which binds to the DHX9 protein and is also a RNA helicase that is upregulated in CRC. In vivo selective localization of the aptamer in the nucleus of cancer cells indicates that it can be also used for the targeted delivery to the nucleus (Mi et al., 2016). Lee et  al. identified a RNA aptamer which acts against the carcinoembryonic antigen (CEA) domain required for metastases in CRC with high affinity and specificity using SELEX. This aptamer inhibited CEA domain interactions with heterogeneous nuclear ribonucleoprotein M4 and death receptor 5, resulting in inhibition of hepatic metastasis of colon cancer cells in mice (Lee et al., 2012). Sometimes RNA aptamers exhibit antagonistic properties along with its targeting capability. Carcinoembryonic antigen (CEA) overexpression in CRC cells induces cell adhesion, enhances anoikis resistance, and promotes hepatic metastasis (Wirth, Soeth, Czubayko, & Juhl, 2002). This process can be controlled by using RNA aptamers against CEA.

2.4  RNA Interference (RNAi) In the last two decades, RNA interference (RNAi) has rapidly become an innovative and elective tool for studying gene function. RNA interference (RNAi) phenomenon was first reported by Fire and Mello in 1998 in Caenorhabditis elegans (Fire et al., 1998), in Drosophila cell extracts by Hammond et al. and Zamore et al. in 2000 (Hammond, Bernstein, Beach, & Hannon, 2000; Zamore, Tuschl, Sharp, & Bartel, 2000), and in mammalian cells by Tuschl et al. (Elbashir et al., 2001). Later on Fire and Mello won a Nobel Prize for discovering RNAi pathways in 2006. Discovery of siRNA and miRNA opens up a new avenue for treatment of many diseases including CRC.  These technologies have been used for the silencing of oncogenes or proto-oncogenes which are upregulated due to mutation in these genes. Now many siRNA and miRNAs are well characterized and extensively used for the therapeutics purpose.

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Fig. 4  Mechanism of action of RNA interference (RNAi)

2.4.1  siRNA Small interfering RNAs (siRNAs) are 21–25 nt dsRNA molecules with 2–3 nucleotide length overhangs on their 3′ ends (Fig. 4). These RNA sequences were defined as the effectors of the RNAi pathway. These sequences efficiently bind to homologous target mRNAs resulting in cleavage of the RNA transcript near the middle of the sequence pairing. The RNAi pathway has been fully defined and characterized. siRNAs can be produced from long dsRNAs or hairpin-looped RNA which is catalyzed by a RNase III enzyme Dicer, or it can be synthesized artificially and introduced into the cells via electroporation or transfection. Synthetic mature siRNAs can be generated either transient or stable manner from longer dsRNA precursors or from ssRNA molecules which possess complementary dsRNA domain also known as short hairpin RNAs (shRNAs). The RNase III endo-ribonuclease Dicer processed the active siRNAs into a ribonucleoprotein complex, known as the RNA-induced silencing complex (RISC) (Bernstein, Caudy, Hammond, & Hannon, 2001) and which mediate the mRNA cleavage, after the “guide” strand incorporated from the siRNA duplex in cytoplasm (Fig. 1) (Liu, Carmell, Rivas, et al., 2004). siRNA has been extensively used for the induction of cell apoptosis, suppression of CRC cell proliferation, prevention of CRC metastasis, and overcoming multidrug resistance. siRNA technology has been successfully employed to silence COX-2 protein in in  vitro models. Charames and Bapat showed an efficient knockdown of COX-2 using siRNA-mediated tool in HT-29 human colon cancer cells. After knockdown, cancer cell undergoes apoptosis which explains the functional role of COX-2  in colon cancer (Charames & Bapat, 2006). However, using anti-COX-2 shRNAs (shCOX-2) against COX-2 proved more effective compared to RNAi-­mediated COX-2 silencing. Strillacci et  al. used in  vitro strategy for generating stable

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knockdown COX-2  in colon cancer cells (HT-29) (Strillacci, Griffoni, Spisni, Manara, & Tomasi, 2006). The shCOX-2 stable expression in HT-29 (HT-29shCOX-2) cell line induced a strong silencing of COX-2 at mRNA, protein, and end product (PGE2) levels without any toxic effect on cells. Stable COX-2 knockdown does not show any phenotypic variation or affect cell proliferation and cell cycle, but it strongly suppresses the malignant nature of HT-29 colon cancer cells in  vitro (Strillacci et al., 2006). B7-H4 siRNA has been reported by Peng et al. to inhibit invasion, migration, and proliferation of colorectal cancer cell line LoVo. B7-H4 siRNA effectively targets the CXCL12/CXCR4 and JAK2/STAT3 signaling pathway (Peng et  al., 2015). Knocking down of histone demethylase also known as lysine-specific demethylase 1 (LSD1) by siRNA resulted in inhibition of proliferation, migration, and invasion of CRC in vitro (Ding et al., 2013). siRNA against KRAS, used in another study, has been applied to inhibit the proliferation of KRAS-­ mutated CRC cell and to slow tumor growth in a xenograft mouse model (Bäumer et al., 2015). Due to the complex physiological nature of cancer, recently combination therapy has garnered much attention to minimize drug resistance or to push active cancer cells towards apoptosis. Multiple research groups reported combination approaches for siRNA with cancer drugs or small molecule anticancer drugs. Synergistic anticancer effect can be achieved by using a combination of different mechanisms of action. In a LS174T tumor-bearing mouse xenograft model, synergistic effect to suppress the tumor growth using VEGF siRNA and SN-38 (7-ethyl-­10-hydroxycamptothecin) was shown (Lee et al., 2016). Sadreddini et al. conducted a study using the combination of doxorubicin and snail siRNA. The combination regimen effectively induced apoptosis, inhibited proliferation, and reduced migration in human CRC cell line HCT-116 (Sadreddini et al., 2017). Combined siRNA strategy was also used in cancer therapy. A co-targeting strategy was used against RAS pathways and mutated PI3K/AKT/mTOR using siRNAs in CRC cell lines with KRAS and PIK3CA mutations. This combined therapy of siRNA (PIK3CA + KRAS or Akt2 + KRAS) proved to be effective and generated synergistic inhibition of CRC cell proliferation and also activated the apoptosis pathway (Valentino et al., 2012). 2.4.2  MicroRNA (miRNA) miRNAs are short non-coding RNAs of 20–25 nucleotides in length that posttranscriptionally regulate gene expression by making imperfect complementary sequence base pairing to the 3′ untranslated region (UTR) region of mRNAs (Fig. 4). miRNAs regulate multiple cellular functions which include apoptosis, cell development, invasion, and proliferation (Huang et al., 2011). The abnormal expressions of miRNAs are associated with CRC initiation, progression, and metastasis (Luo, Burwinkel, Tao, & Brenner, 2011; Slaby et al., 2007). Multiple enzymes are involved in biogenesis of miRNA like Drosha, Dicer, and DGCR8. Abnormal expression in these proteins due to mutations or deregulated epigenetic changes such as DNA hypermethylation (Toyota et al., 2008), DNA hypomethylation (Hur et al., 2013),

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and histone deacetylation (Humphreys, Cobiac, Le Leu, Van der Hoek, & Michael, 2013) was observed in CRC.  Some of the miRNA showed higher expression in CRC, and these are identified as oncogene miRNAs (or oncomiRs). By inhibiting tumor suppressor genes or genes involved in cell differentiation or apoptosis, OncomiRs regulate cancer progression. Asangani et al. showed that miR-21 negatively regulates tumor suppressor Pdcd4 at the posttranscriptional level which induces invasion, intravasation, and metastasis in colorectal cancer cell lines (Asangani et al., 2008). Fang et al. showed that miR-17-5p induces drug resistance by targeting PTEN, a tumor suppressor gene that plays a dominant role in the P ­ TEN/ AKT/PI3K pathway (Fang et al., 2014). miRNAs showing decreased expression in CRC cells behave like tumor suppressors. Inhibition of RAB22A (a member of the RAS oncogene family) by miR-204-5p which is a tumor suppressor in CRC was demonstrated by Yin et al. (Yin et al., 2014). Zhang et al. demonstrated downregulation of MAPK signaling by miR-217 resulting in inhibition of tumor growth and cell apoptosis in CRC (Zhang, Lu, & Chen, 2016). Downregulation of Wnt/β-­catenin signaling pathways via miR-93 showed suppressed CRC development (Tang et al., 2015). A variety of disease-associated targets can be simultaneously regulated by a single miRNA. It is a major advantage for the treatment of a disease cancer where multiple genes are mutated or dysregulated. Many reports suggest role of miR-143 in various pathways of CRC such as it regulate the oncogene KRAS expression which result in suppression of CRC cell growth (Chen et al., 2009). On other hand, miR-143 repressing DNMT3A which result in its tumor suppressor role in CRC (Ng et al., 2009). miRNA-143 was found to be inhibiting CRC cell invasion and migration by targeting MACC1 (metastasis-associated in colon cancer-1) gene involved in CRC tumorigenesis and metastasis (Zhang et al., 2012). miRNA therapy showed great potential for CRC treatment, but many obstacles need to be addressed including an efficient miRNA delivery system and concern over safety issues related to side effect. 2.4.3  Long Non-coding RNAs (lncRNAs) LncRNAs are a class of RNAs which does not translate into protein having a length of more than 200 nucleotides. The recent discoveries about lncRNA and its role in cancer development are getting more and attention (Yang, Lu, & Yuan, 2014). However, discovery of more number of lncRNAs is going on and their role in cancer progression are still in developing and preliminary stage. lncRNAs are believed to be very important regulators for gene expression. Due to the large size of lncRNAs, it forms complex secondary and tertiary structures. Due to this complexity of the structure enables them to their regulatory function via binding to DNA, RNA, or protein (Table 2). Recent development in the field of lncRNA biology suggests a very strong factor in the CRC progression. LncRNAs can mediate transcription factors and regulate gene expressions and epigenetic changes. For example, it was observed that CRC patients have elevated HOTAIR expression levels in CRC tissue than surrounding

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Table 2  Some of the lncRNAs that act as regulator in colorectal cancer progression lncRNA mechanism Example mRNA interaction SNHG5

miRNA interaction

UCA1

Function Binds target mRNAs to block STAU1-­ mediated degradation Interacts with mRNA 3-UTRs

CRNDE

Represses miR-181a-5p expression

UCA1

Inhibits a tumor-suppressive miRNA miR-204-5p HNF1A-AS1 Regulates SIRT1 by competitively binding miR-34a Protein interaction HOTAIR Acts as a scaffold to assemble PRC2 and LSD1 complexes CCAT1 Promotes long-range chromatin looping at Chromatin interactions the MYC locus

Reference Xue et al. (2014) Xiang et al. (2014) Bian et al. (2016) Tsai et al. (2010) Hu et al. (2012) Yang et al. (2014) Ng et al. (2009)

CCAT: colon cancer-associated transcript 1, HOTAIR: HOX transcript antisense RNA, PRC2: polycomb repressive complex 2, LSD1: lysine-specific histone demethylase 1, CRNDE: colorectal neoplasia differentially expressed, UCA1: urothelial cancer-associated 1, HNF1A-AS1: HNF1A antisense RNA 1, SIRT1: sirtuin 1, SNHG5: small nucleolar RNA host gene 5, STAU1: Staufen double-stranded RNA binding protein 1

normal tissues (Xue et al., 2014). In CRC progression lncRNA HOTAIR acts as a scaffold to assemble lysine-specific demethylase 1 (LSD1) complexes and polycomb repressive complex 2 (PRC2), which is responsible for epigenetic silencing of cancer-related genes (Tsai et al., 2010). CCAT1-L (a lncRNA) transcribed specifically from a locus 515 kb upstream of MYC in human CRC. CCAT1-L plays an important role in promoting long-range chromatin looping and transcriptional regulation of MYC and knockdown of CCAT1-L resulting in reduced long-range interactions between the MYC enhancers and its promoter (Xiang et al., 2014). lncRNAs also play very important roles in posttranscriptional regulation besides the transcriptional and epigenetic regulations. lncRNA SNHG5 binds to target mRNA to block dsRNA-binding protein Staufen homolog 1 (STAU1)-mediated degradation resulting in tumor cell survival in CRC. (Damas et al., 2016). lncRNA UCA1 knows how to control cancer-related pathways due to its interaction with mRNA 3′-UTRs; this interaction prevents them from degradation via miRNA (Bian et  al., 2016). Another study shown by Bian et al. demonstrated that lncRNA UCA1 can also exercise its regulatory function by inhibiting tumor-suppressive miRNA miR-­204-5p (Bian et al., 2016).

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2.4.4  Small Activating RNAs Small activating RNAs (saRNAs) are small double-stranded RNAs that work through an entirely different mechanism called RNA activation (Janowski et  al., 2007; Li et al., 2006). Many research groups demonstrated that saRNAs play a significant role in upregulation of proteins important for tumor. ssRNA induces transcriptional gene activation by targeting gene promoters (Hu et al., 2012; Yang et al., 2013). In vivo study by Wang et al. showed that saRNA p21-saRNA-322 can halt CRC growth by enhancing the expression of p21 protein which is a downstream protein of oncogene P53 (Wang et al., 2017).

3  I mprovement for Enhanced Ability of RNA Therapeutics for Colorectal Cancer RNA-based therapeutics showed great potential for the treatment of CRC; however, these therapies have some shortcomings like delivery to the CRC cells, degradation by nuclease present in cells, and potential immunogenicity. These shortcomings remain to be addressed (Dowdy, 2017). Many reports suggested that some of the issues can be resolved by doing chemical modification of the RNAs (Shukla, Sumaria, & Pradeepkumar, 2010). Recently systemic delivery of RNA therapeutics has improved significantly with the advent of nanotechnology.

3.1  Modification of RNA Chemical Structure Due to the presence of large amount of nuclease in serum and cells, transfected RNAs are very unstable and rapidly degraded in vivo. Modification of coding and non-coding RNAs can affect the RNA structure and function. Various modifications were used to improve stability of RNA without compromising their activities in biological fluids. Some prominent chemical modifications were performed in base, sugar, or phosphate moieties of nucleotides. RNA chemical modifications in RNA moieties may increase specificity and minimize off-target effects (Fluiter, Mook, & Baas, 2009), improve pharmacokinetic (PK) and pharmacodynamic (PD) properties (Geary et  al., 2001), enhance activity (Kawasaki et  al., 1993), and also decrease immunological reactions (Sioud, Furset, & Cekaite, 2007). The most common chemical modifications done in short-synthesized RNA oligonucleotides include 2′-fluoro (2′-F), 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′- MOE), phosphorothioate (PS) backbone modification, locked nucleic acid (LNA) sugar substitutions, and conjugation with cholesterol or polyethylene glycol (PEG) for better delivery. In sPS backbone modification, the non-bridging phosphate oxygen is replaced by a sulfur atom. This simple substitution modification makes RNA more resistant to

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nuclease degradation and improved affinity to plasma proteins with minimal clearance from renal system (Geary, 2009). At 2′ position in the sugar ring, substitutions with 2′- MOE, 2′-OMe, 2′-F, or LNS group drastically improve the stability, potency, and overall pharmacological properties (Gao et al., 2009). Non-nucleotide chemical modification was applied by Kitade et  al. where they added aromatic benzene-­ pyridine (BP-type) analogs to 3′-overhang region of miR-143 has shown better activity and it also improve resistance against nuclease. The modified miR-143 effectively suppressed tumor formation in xenografted tumor of CRC DLD-1 cells (Kitade & Akao, 2010).

3.2  N  anoparticles Mediate Targeted RNA Delivery to Colorectal Cancer Cells In general, RNA has a large molecular weight and is negatively charged, and because of this nature, it can’t pass through CRC cells. To overcome this obstacle, an efficient delivery system is required. Currently a variety of efficient delivery systems are used to improve RNA delivery and efficacy. Generally, two types of methods are used as RNA delivery system: viral vector mediated and non-viral vector mediated. Viral vectors have been used extensively for target delivery of RNA drugs. Viral vectors such as lentiviral, adenovirus, adeno-associated virus, and retroviral showed high transfection efficiency; however, due to its immunogenic response, cytotoxicity has limited potential. Also, these vectors have limited space for insertional mutagenesis (Thomas, Ehrhardt, & Kay, 2003). Non-vector methods like nanoparticle-mediated delivery with the help of liposomes, polymersomes, dendrimers, and inorganic nanoparticles have advantages over viral vector system due to their low cost, bulk production, and lower toxicity to cells, offering a very promising alternate delivery system for RNA therapeutics (Mintzer & Simanek, 2008). Due to smaller size, nanoparticles can easily reach tumor tissue. Nanoparticle also shows EPR (enhanced permeation and retention) effect which helps in the accumulation of RNA drugs within the cancer tissue for longer time to improve the efficacy (Fang, Nakamura, & Maeda, 2011). c-Myc conjugated polyethyleneimine (PEI)-macromolecule polyglycidal methacrylate (PGMA) nanoparticles used by Tanggudu et al. (Tangudu et al., 2015) as an oral delivery against tumor growth suppressed tumor growth efficiently and increased animal survival in a colon cancer model. Poly(lactide-co-glycolide)-based nanoparticles (NP-siDCAMKL-1) encapsulated with siDCAMKL-1 was used by Sureban et al. to treat mice carrying CRC tumor with NP-siDCAMKL-1, resulted in CRC tumor xenograft growth arrest (Sureban et al., 2011). For the targeted delivery of RNA, the conjugated ligands on the surface of nano-carriers widely used for the therapeutics of CRC cells. Survivin siRNA encapsulated in folate-displaying extracellular vesicles (EVs) was specifically delivered to CRC cells, and it efficiently

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inhibited colorectal cancer growth in patient-derived colorectal cancer xenograft mouse model (Pi et al., 2018). However, development of nanocarriers requires more research to be an efficient carrier in CRC therapy, due its several limitations, viz., immunogenicity and toxicity.

4  Pros and Cons in Developing RNA-Based Therapeutics There are two major hurdles in turning RNA into drug: poor pharmacokinetic properties of RNA and rapid degradation by RNases. There is a need to device the method to deliver RNA analogs across the hydrophobic cell membrane into nucleus or cytosol where it has to act. RNA-based therapeutics modulates the internal machinery of cells, changing the expression of targeted genes. RNA molecules offer a broad range of potential applications due to its high degree of structural flexibility, making it suitable to manipulate “undruggable” target sites. RNA therapeutic molecules give the researcher the flexibility to either upregulate or downregulate gene expression within the cells. In addition, many specific protein or protein complexes can be targeted simultaneously. However, DNA-based therapeutics or gene-editing technologies such as CRISPR are used to repair the dysfunctional gene or correct the mutation into the cells. But there is high risk of permanent genome alteration which limits its applications in humans. RNA therapeutics offers advantages as mentioned above for DNA-based therapies, but there are still many challenges that need to be overcome. Major concerns are delivery to specific tissue or organs and its stability inside cells. RNAi has shown an off-target effect in many cases (Ma, Creanga, Lum, & Beachy, 2006). Due to its small size and nature of tolerance against mismatch, these RNAs have a large number of potential targets in genome and could affect many non-intended mRNA targets (Lewis, Burge, & Bartel, 2005). In many cases due to off target affect, phenotype of cells more extensively express then expected (Fedorov et al., 2006). Homogeneous distribution of miRNA and siRNA in tissues throws another challenge. Majority of these RNAs tend to accumulate in the liver, spleen, and lungs that are parts of mononuclear phagocytosis (Park, Park, Pei, Xu, & Yeo, 2016). High distribution of ASOs was observed in the liver and kidney instead of tumor cells (Geary, Norris, Yu, & Bennett, 2015). Finding suitable targets is also a problem with these molecules in complex disease like CRC.  Before developing RNA-based therapeutics, understanding the complex nature of signaling involved in the progression of CRC is required to assess the level of gene suppression and the mechanisms that other genes may compensate to the loss of function of targeted gene.

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5  Conclusion RNA-based therapeutics has gained a lot of attention in the last two decades mainly due to its ability to target varieties of disease genes that were previously nontargetable and its flexibility in modulating a vast number of targets. However, due to challenges in delivery methods and its stability inside cells, more extensive research and time might be required for clinical practice to become a reality. Recent advancement in the field of nanotechnology and chemical technology related to medicine will help solve this problem in RNA-based therapeutics in the coming future, and this RNA therapeutics will be widely adopted. In modern time, it is the need of hour to use this new class of RNA modalities due to its superior outcomes, along with antibodies and small molecule. Currently, researchers well equipped with modern biotechnology and nanotechnology are working on various RNA-based mechanisms and delivery systems to unleash the fullest potential for RNA-based therapeutics.

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Bacterial Cancer Therapy: Promising Role in the Treatment of Colon Cancer Rishi Srivastava, Shweta Sonam, Naveen Kumar Vishvakarma, Rajesh Sharma, and Shree Prakash Tiwari

Abstract  Bacterial colonization and subsequent inflammatory consequences have been associated with the onset of colon carcinogenesis. However, recent shreds of experimental evidence suggest that bacteria and their products can be implemented for therapeutic benefit in malignant disorders including those of colon origin. The use of bacteria or their components for antineoplastic therapy is known as bacterial cancer therapy. Limitations associated with conventional antineoplastic therapeutic approaches like surgery, chemotherapy, and radiotherapy include nonspecific toxicities, chemoresistance, and immunosuppression. Therefore, recently bacterial cancer therapy gained attraction among oncologists. A diverse range of mechanisms has been suggested for underlying antineoplastic activities of bacterial cancer therapy. Direct cytotoxicity to neoplastic cells and preferred colonization in the hypoxic core of tumors are few among suggested. Further, mobility of bacteria makes them independent of blood circulation, which is one of the major limitations for the delivery of chemotherapeutic agents to poorly vascularized regions of malignant tissues. Bacteria and their components also induce an anticancer immune response and elicit protective immunity to reduce the incidences of cancer. The living nature of bacteria makes them a suitable vector for the on-site production of metabolites having anticancer potential. Moreover, through genetic manipulation techniques, bacteria can be engineered to achieve optimal anticancer effects. Besides whole bacteria, their metabolites (enzymes, toxins, and others) also constitute component of bacterial cancer therapy which have a promising role in the therapeutic management of colon cancer. This chapter will discuss the potential application of bacterial cancer therapy in the clinical management of colon cancer along with their mechanisms. Prospects on improving the efficacy and eliminations of associated limitations are also discussed. Collectively, it is

R. Srivastava · S. Sonam · S. P. Tiwari (*) Department of Microbiology, VBS Purvanchal University, Jaunpur, Uttar Pradesh, India N. K. Vishvakarma Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India R. Sharma Department of Biotechnology, VBS Purvanchal University, Jaunpur, Uttar Pradesh, India

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_17

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being suggested that further investigations will shore up the true potential and colon malignancies can be managed through therapeutic regimens including bacterial cancer therapy. Keywords  Bacterial therapy · Bacterial toxins · Colon cancer · Immunotoxins

1  Introduction Cancer is becoming one of the common causes of death worldwide known as a group of diseases consisting of abnormal cell growth that can invade and spread into different organs of the body through metastasis. According to the World Health Organization report, cancer was responsible for 9.6 million deaths in 2018. Globally, cancer causes about one in every six deaths. The most common causes of death due to cancer are as follows: 1.76 million deaths from lung cancer, 862,000 deaths from colorectal cancer, 783,000 deaths from stomach cancer, 782,000 deaths from liver cancer, and 627,000 deaths have been reported from breast cancer (WHO, 2018). Conventional cancer therapies include surgical therapy, chemotherapy, radiotherapy, immunotherapy, hormonal therapy, and bone marrow transplantation. Despite handsome rate of success, each of them is associated with some serious lacuna (Damyanov, Maslev, Pavlov, & Avramov, 2018). Recent decades witnessed the blooms of alternative strategies and exploration of unconventional tools to combat the plethora of malignant disorders. Nature-derived components have shown promising success in anticancer therapies during their preclinical as well as clinical exploration (Rai, Vishvakarma, Mohapatra, & Singh, 2012; Soni, Shukla, Kumar, & Vishvakarma, 2020; Vishvakarma, 2014). Although experimental exploration of bacteria for their beneficial outcomes against malignancies is not new, recent advances in our understanding about microbial physiology, molecular interaction, and cancer biology as cellular entities as well as evolutionary moieties lead to its surge (Mehta, Soni, Shukla, & Vishvakarma, 2020). Owing to their unique characteristics, bacterial strain can be easily manipulated through genetic engineering. They have been serving to mankind as molecular factories and making lifesaving drugs. Now, bacterial components, toxins, and bacterium as whole are being used to cater therapeutic benefit in cancer therapy. Collectively known as bacterial therapy, it utilizes the wild or genetically engineered bacteria, their components, and derivative for antineoplastic agents to improve the efficacy of standard therapeutic strategies. This also serves as promising approach to reduce the limitations of conventional therapies. Due to the unique pathophysiology of solid tumors, the successful treatment of cancer still remains a challenge. Bacterial therapy is a novel approach in cancer treatment either alone or in combination with conventional methods. The positive effect on regression of tumors and inhibition of metastasis has already been shown by bacterial cell itself or by other bacterial instruments. Bacteria-mediated therapy includes use of the bacteria as therapeutic itself or as a delivery vehicle of

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antitumor gene or drug to the cancerous cells. The focus on the bacterial cancer therapy has increased considerably over the past few decades and seems to have a bright future in cancer therapy. Bacterial therapy has several advantages over conventional molecular agents. Conventional small molecular drugs depend on blood circulation to reach target site; however, the motile bacterial strains can swim to the tumor site on their own. Chemotactic movement of bacteria will provide them edge in locating and encroaching the tumor microenvironment. Moreover, colonization ability of few bacteria in low oxygen or hypoxic environment makes them suitable for targeting large solid tumors with hypoxic core. Ease in genetic manipulation provides help in removing the genes responsible for bacterial pathogenesis. Nevertheless, genetically engineered bacteria can serve as on-site production factory for antineoplastic agents, right into tumor microenvironment. A number of bacterial products including deadly toxin can also aid in strategies against cancer. Their ability to kill target cell can be exploited through their targeted deliveries through designing their conjugates with specific monoclonal antibodies or designed small molecular ligands. Malignant disorders are invariably accompanied with suppression of immune response. Various strategies to improve immunity in cancer patients are linked with augmented success rate in therapy. Immunostimulatory abilities of bacteria or their components also provide additional encouragement for their use in therapy of malignant disorders. A multifaceted exertion driven by bacterial therapy makes it fort running candidate in anticancer therapy. This chapter will discuss the various dimensions of bacterial therapies and their benefits in management of colon cancer.

2  Bacteria as Therapeutics in Cancer Treatment Bacteria act as a double-edged sword in cancer (Yaghoubi et al., 2020). On the one hand bacterial toxins and enzymes and oncogenic peptides released during microbial infections can contribute significantly in tumor development (Abdel-Fattah, Hafez, Zaki, & Darwesh, 2017) and thus could be potentially carcinogenic, but on the other hand some of them have a great therapeutic potential to treat cancer. It is difficult to interpret the exact mechanism or causes of cancer; these bacteria mechanism pathways are hard to interpret, and different bacteria use different mechanisms of pathogenesis and accordingly they can promote tumor formation in various regions of human body; for example, Campylobacter jejuni promotes small intestinal lymphomas (Mesnard, De Vroey, Maunoury, & Lecuit, 2012), Citrobacter rodentium promotes human colorectal cancer (Umar, 2012), Helicobacter pylori promotes gastric cancer (P. Correa, 2003), Mycobacterium tuberculosis promotes lung cancer (P.K. Gupta et al., 2016), and Salmonella typhi are reported to promote hepatobiliary carcinoma (C. Caygill et al., 1994). In the 1890s, William Coley was the first to use the bacteria in cancer therapy. He injected two heat-inactivated Serratia marcescens to his cancer patients that caused tumor regression. The heatinactivated bacteria used in this therapy are known as Coley’s toxin (Hoption Cann,

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van Netten, & van Netten, 2003). Bacterial cells have some specific components that act as “pathogen-­associated molecular patterns (PAMPs)” like LPS, peptidoglycan, flagellin, etc. which are recognized by toll-like receptors (TLRs) present on the cells of innate immune system that helps in activation and maturation of tumor antigen-loaded dendritic cells (Fessler, Matson, & Gajewski, 2019; Ludgate, 2012). But sometimes it is difficult to control a bacterial infection during the bacterial cancer therapy; thus, genetically modified bacteria have been taken into consideration for their use as therapeutics in cancer treatment. During the development of cancer, hypoxic regions are created in the core of solid tumors. Some bacterial strains of Salmonella, Clostridia, and Bifidobacteria have ability to colonize in the hypoxic regions of tumors and can invade solid tumors. As a consequence of their ability to colonize in the hypoxic regions, these bacteria can also serve as an ideal tumor-­ targeting vector for targeted drug delivery (Chang et al., 2015; Ganai, Arenas, Sauer, Bentley, & Forbes, 2011; Hetz, Bono, Barros, & Lagos, 2002; Pawar et al., 2014; Wei et al., 2007; Yan, Kanada, Zhang, Okazaki, & Terakawa, 2015). Mycobacterium bovis BCG, Streptococcus pyogenes OK-432, Clostridium novyi, Salmonella typhimurium VNP20009, Magnetococcus marinus, Bifidobacterium longum, and Listeria monocytogenes LADD strain are some of the most promising genera of bacteria for cancer therapy because of in  vivo experiments on cancer models. Mycobacterium bovis BCG is found to be effective against bladder cancer. It stimulates the immune system and increases the proinflammatory cytokine activation and phagocytosis of cancer cells (Biot et al., 2012; Droller, 2017; Felgner, Kocijancic, Frahm, & Weiss, 2016; Herr & Morales, 2008; Kamat et al., 2016). Streptococcus pyogenes OK-432 is effective in oral cancer. It acts by sensitizing the immune system as a consequence of which destruction of neoplasm takes place by activated immune cells (Deweerdt, 2013; Kono et al., 2017; Ohta, Fukase, Suzuki, Ishida, & Aoyagi, 2010; Ohta, Fukase, Watanabe, Ito, & Aoyagi, 2010; Olivieri, Nanni, De Gaetano, Manganaro, & Pintus, 2016). Clostridium novyi is capable of producing specific enzymes and toxins that can destroy cancer cells and is effective in the treatment of leiomyoma, a type of uterine cancer (Felgner et al., 2016; Liu et al., 2014; Paton, Morona, & Paton, 2012; Staedtke, Roberts, Bai, & Zhou, 2016). Salmonella typhimurium VNP20009 produces specific proteins that are effective in the treatment of melanoma when used with some specific chemotherapeutics (Bereta et al., 2007; Felgner et al., 2016; Schmitz-Winnenthal et al., 2018). Magnetococcus marinus can be used as tumor targeting vector that can be used in delivering chemotherapeutic drugs (Felfoul et al., 2016; Martel, 2017). Bifidobacterium longum is one of the normal microflorae in the human gut. Besides having several probiotic activities, the bacteria can biotransform the antitumor agents, secrete therapeutically active compounds, and modulate the expression of cancer-associated genes and cytokines. It is reported to be effective in colorectal cancer (Bergmann et al., 2013; Ewaschuk et al., 2008; Lee et al., 2004; Sivan et al., 2015; Uccello et al., 2012). Listeria monocytogenes LADD strain can be used as vaccines for cancer prevention by inducing antigen-specific T-cell responses and more targeted cancer cells’ elimination. This strain is reported to be effective in a number of cancers including cervical, oropharyngeal, pancreatic, and lung cancer (Bolhassani, Naderi, & Soleymani,

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Fig. 1  Positive and negative aspects of bacteria in relation to cancer

2017; Flickinger Jr, Rodeck, & Snook, 2018; Tangney & Gahan, 2010; Wood, Guirnalda, Seavey, & Paterson, 2008) (Fig. 1). One of the major drawbacks of the conventional cancer therapy is the lack of specificity to the malignant cancerous cells. So, these bacteria can be made even more effective in cancer therapy, since the bacterial strains can be attenuated by genetic modification that can increase their therapeuticability Gardlik & Fruehauf, 2010. A novel methodology is to use the modified bacteria to deprive cancerous cells for oxygen that causes death of cancerous cells. Many strains of obligate and facultative anaerobic bacteria have been identified to have an oncolytic potential agent due to their potential to proliferate in oxygen-deprived or hypoxic conditions (Dang, Bettegowda, Huso, Kinzler, & Vogelstein, 2001; Marth & Möse, 1987). As the highly hypoxic tissues are usually not present in most organs of the body, present only in tumors, thus these bacterial strains can be used to specifically target the tumor tissues. The bacterial strains such as Bifidobacterium, Salmonella sp. (Low et al., 1999), E. coli (Min et al., 2008), and Clostridium (Bettegowda et al., 2004) are shown to have the ability to colonize in the hypoxic areas of tumors. These bacteria can be engineered to express cytotoxic proteins or reporter genes to selectively destroy tumor cells (Dang et al., 2013) and can multiply up to 1000-fold faster in tumors as compared to the normal tissues (Low et al., 1999). Genetic modifications can also improve internalization, colonization, and multiplication of these bacteria in tumor tissues (Gardlik & Fruehauf, 2010). Genetically modified strains of Corynebacterium diphtheria M55 (Marth & Möse, 1987) and Salmonella typhimurium VNP2000 have already been proved to be a useful vector because of their ability to colonize selectively in the tumors (Nemunaitis et al., 2003; Schlechte & Elbe, 1988; Thamm et al., 2005).

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3  P  robiotic Bacteria as a Tool in Cancer Prevention and Therapy Gut normal microbiota has already been proved to have probiotic potential that can protect against various diseases. These probiotic bacterial strains can play a significant role in immunomodulation and display antitumor properties. Lactic acid bacteria have an immunomodulatory effect and can play a significant role in regression of carcinogenesis. By increasing and decreasing the production of anti-­inflammatory cytokines, these probiotic bacteria can play an important role in prevention of carcinogenesis. They can activate the phagocytes that can eliminate cancer cells at an early stage. A list of these probiotic bacterial strains that have been proved to have properties of cancer prevention and treatment is given in Table 1.

4  Bacterial Toxins as Therapeutics in Cancer Immunotherapy Bacterial toxins have a high efficiency and specificity for destruction or inactivation of some vital cellular molecules. Therefore, these toxins can be used to manipulate specific signalling pathways of mammalian cells. An artificial stimulation of immune system that leads to improvements in the ability of immune system to attack cancer cells is called cancer immunotherapy. Bacterial toxins can play a significant role in this regard. Bacterial toxins are artificially modified for specific targeting of cancer cells. These modified toxins usually have two components: the Table 1  Probiotic bacterial strains with anticancer potential Genus Bifidobacterium Enterococcus Lactobacillus

Bacillus

Pediococcus Clostridium

Species/strain having probiotic and anticancer potential Bifidobacterium adolescentis SPM0212 Bifidobacterium lactis Bb12 Enterococcus faecium RM11 Lactobacillus fermentum RM28 Lactobacillus rhamnosus GG Lactobacillus paracasei IMPC2.1 Lactobacillus salivarius FP25/FP35 Lactobacillus plantarum A7 Lactococcus lactis NK34 Lactobacillus casei ATCC 393 Lactobacillus pentosus B281 Lactobacillus plantarum B282 Bacillus polyfermenticus HT-29 Bacillus subtilis ATCC 9398 Bacillus polyfermenticus KU3 Pediococcus pentosaceus FP3 Clostridium butyricum ATCC

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Fig. 2  Basic concept behind the immunotoxin

immune component and the toxin component. Thus, they are called as immunotoxins. In these artificial molecules, the binding site of the bacterial toxin (specific for their natural receptor) is replaced by a ligand that can bind specifically to a cell surface receptor expressed strongly on tumor cells. If the specific antibodies directed against tumor cells are fused with bacterial toxins, the immunotoxins are formed in such a manner they can be redirected to kill the cancer cells (Fig. 2). The toxin component of immunotoxin usually has catalytic activity and works as an enzyme (Thorpe et al., 1978). This part can be genetically engineered to improve its toxic activity that will improve its efficiency to kill the cancerous cells and to decrease the antigenicity so that they can avoid their recognition by immune system (Kreitman, 2006). On the other hand, the antibody part (i.e., the immune component) is shortened to antigen binding domain that will decrease the immunogenicity (Kreitman, 2006). The antibody part of the immunotoxin binds specifically to their target cell and endocytosed. The release of toxic part of the immunotoxin into the cytosol is mandatory in order to kill the target cell. Many toxins have an inbuilt ability to be released in the cytosol just after acidification of the endosomal vesicles or after pore formation in the membrane of endosomal vesicles (Binz & Rummel, 2009). Some others may acquire an endocytic path and reach the cytosol through the Golgi body and endoplasmic reticulum (Lord, Smith, & Roberts, 1999). After reaching the cytosol, the toxin modifies its target by its catalytic activity and initiates a cascade of reaction that ultimately causes cell death. The most commonly used bacterial toxins are diphtheria toxin and Pseudomonas exotoxin A. Both can act on mammalian elongation factor 2 and inhibit protein synthesis to kill their target cell (Gill, Pappenheimer, Brown, & Kurnick, 1969).

4.1  Diphtheria Toxin Diphtheria toxin is the primary virulence factor produced by Corynebacterium diphtheriae and is one of the first bacterial toxins used for cancer treatment. The two distinct subunits form the single-chain protein diphtheria toxin (Choe et al., 1992).

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The enzymatic subunit A acts on mammalian elongation factor eEF2 at diphthamide (a modified histidine amino acid of eEF2) for ADP-ribosylation, leading to its inactivation. It also puts a blockage for the aminoacyl-tRNA at A site of the ribosome. ADP-ribosylation of eEF2 blocks protein synthesis and results in cell death (Strauss & Hendee, 1959). The subunit B contains two domains, namely, transmembrane domain and receptor binding domain. Interaction of receptor binding domain of subunit B with specific receptor present on target cell surface follows a pH-­ dependent insertion to the membrane triggered by transmembrane domain of subunit B. Hence subunit B assists the passage of catalytic subunit A into the cytosol via the translocation domain (Choe et al., 1992) (Fig. 3). The first immunotoxin that was synthesized using the diphtheria toxin is Denileukin diftitox. The reason behind the selection of diphtheria toxin is its extremely toxic nature, as presence of even a single molecule in the cytosol is sufficient to kill a eukaryotic cell (Masaru, Mekada, Uchida, & Okada, 1978). Denileukin diftitox is the first immunotoxin that has been approved by the Food and Drug Administration (FDA) for their use in treatment of cutaneous T-cell lymphoma (CTCL) (Kiyokawa et al., 1989). This immunotoxin is formed by the combination of catalytic subunit of diphtheria toxin with recombinant human interleukin-2 (IL-2). IL-2 specifically targets the IL-2 receptor which is highly expressed on malignant T cells (Waldmann, 1987; Wasik et al., 1994). But IL-2 receptor is most exclusively expressed by malignant T cells; therefore, the use of the targeted toxin produced some severe side effects including blurred vision, nausea, diarrhea, development of skin rashes, and muscle pain. Sometimes, flu-like symptoms and vascular leak syndrome (VLS) may occur (Avarbock et al., 2008) (Fig. 4).

Fig. 3  Components of diphtheria toxin and summary of their action

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Fig. 4  Denileukin diftitox (a) structure: binding domain in the original toxin is exchanged by the human IL-2 and (b) mechanism of action that leads to inhibition of protein synthesis and causes cell death

4.2  Pseudomonas Exotoxin A (PE) Exotoxin A produced by the Pseudomonas aeruginosa is also highly toxic for mammalian cells like diphtheria toxin. Exotoxin A of Pseudomonas aeruginosa is a typical AB type of toxin formed by two domains, namely, domain A and domain B. Domain A possesses catalytic activity, while domain B is responsible for receptor binding activity through interaction with the low-density lipoprotein receptor-­ related protein 1 (LRP1) (Li, Dyda, Benhar, Pastan, & Davies, 1995). Similar to diphtheria toxin, PE also has an ADP-ribosylation activity that modifies the

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elongation factor eEF2 and blocks the protein synthesis that ultimately results in cell death (Iglewski & Kabat, 1975). But the PE needs activation by cell surface serine proteases, called furins. Furins are widely distributed in the human body. Indeed, it is the same enzyme that is needed for the processing of SARS-CoV-2 spike proteins before their binding to ACE2 receptors (Soni et al., 2020). This furin also serves for the pre-activation of bacterial toxins. Extensive analytical strategies revealed that a 38 kDa fragment of the PE toxin has higher degree of cancer cell toxicity without any prerequisite for pre-activation by furin (Kreitman, Sieqall, Chaudhary, FitzGerald, & Pastan, 1992). Thus, PE38-based immunotoxins were formed by exchange of fused antibodies or receptor ligands to target different types of tumor cells. On the other hand, it reduces immunogenicity of the immunotoxin so that the immunotoxin can be used repeatedly to a cancer patient.

4.3  α-Hemolysin of Staphylococcus aureus α-Hemolysin of Staphylococcus aureus is a pore-forming toxin which is different from single diphtheria toxin and Pseudomonas exotoxin A, as they do not have a catalytic domain. Hexamers of the pore-forming alpha toxins bind to their target receptors after oligomerization generates pores of 1–2  nm in diameter into the plasma membrane. It creates an osmotic imbalance leading to unhindered exchange of ions as well as low-molecular-weight molecules across membrane. As all the biological membranes maintain a specific membrane potential across them, these pore-forming toxins short-circuit the membrane potential. Alpha-hemolysin has a single hydrophilic chain that has to be activated by proteolytic cleavage mediated by A disintegrin and metalloprotease 10 (ADAM10) before its insertion into the membrane (Inoshima et al., 2011). The protease recognition site of the toxin is modified by mutagenesis to enhance its specificity against tumor cells. The matrix metalloproteases (MMPs), secreted by tumor cells and responsible for extracellular matrix remodelling, cleave the mutated toxin with modified protease recognition (Pollack, 1983; Walker & Bayley, 1994). Pore-forming immunotoxins may be used more effectively in combination with other chemotherapeutic drugs. The pore-forming ability of these toxins will improve the bioavailability of other drugs by providing them an unrestricted channel in the membrane. Therefore, these toxins have the potential to be used as an adjuvant in standard antineoplastic therapeutic regimens.

4.4  Anthrax Toxin Modification of toxins for the recognition of cancer-specific receptors has proved to be a successful strategy. Anthrax toxin is a two-chain, AB-type toxin secreted by Bacillus anthracis. The toxin contains three non-linked compounds. Part of the anthrax toxin, protective antigen (PA), interacts with target cells and subsequently

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promotes pore formation in membrane. The capillary morphogenesis gene 2 (CMG2) or the tumor endothelial marker 8 (TEM8) serves as docking port for the PA. These CMG2 and TEM8 are also known as anthrax receptors 1 and 2, respectively. The four domains of PA have distinctive roles to play. One domain serves receptor binding domain and interacts with cell surface receptor, while the other domain is cleavage site for furin required for its pre-activation. After proteolytic cleavage by furin and pre-activation, another domain of PA triggers the oligomerization of PA monomers, while the last one acts as endosomal membrane insertion site essential for cytosolic release of effector molecules. After binding of PA and subsequent events, other component of anthrax toxin, lethal factor (LFn), is released into cytosol. The LFn disrupts the cell cytoskeleton of target cell through ADP-ribosylation of actin. Among all the four domains, the receptor binding domain can be modified to direct the specificity of the toxin toward various tumor cells. Different modifications done in anthrax toxin to form the immunotoxin are summarized in Table 2. Table 2  Different immunotoxins formed by modifications of anthrax toxin

Component of Immunotoxin anthrax toxin (mPA-EGF) Mutated PA

(mPA-­ ZHER2)

(LFN-DTA)

mPA-EGF

Mutated PA (N682A/D683A)

Component attached to increase specificity for cancerous cells Epidermal growth factor (EGF)

ZHER2, an affibody with high affinity to human epidermal growth factor receptor 2 (HER2) EGF and Anthrax lethal factor (LFn) linked ZHER2 to the catalytic part of the diphtheria toxin (DTA) LFN-C3 (anthrax EGF receptor lethal factor fused on the cell surface to the catalytic domain of Photorhabdus luminescens (TccC3 toxin)

Targeted surface receptor overexpressed on cancerous cells EGF receptor (EGFR)

HER2 (overexpressed in breast and ovarian cancers)

High specificity into EGFR orHER2-­ overexpressing cells and induce death of the tumor cells EGFR-overexpressing mid-esophageal cells OE21 via the mPA-EGF transporter and in the HER2-overexpressing low esophageal OE33 cells via the mPA-ZHER2 transporter, selectively eliminating the targeted cells

References Mechaly, McCluskey, and Collier (2012) Nicholson, Gee, and Harper (2001)

English, Roque, and Santin (2013)

McCluskey, Olive, Starnbach, and Collier (2013)

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4.5  L  imitations of Immunotoxins for Their Successful Use in Cancer Therapy Immunotoxins have several limitations for their use as anticancer therapeutic agents. Foremost, the toxin component of immunotoxin are foreign proteins; therefore, antibodies are formed in the patient after the first dose. However, removal of T-cell epitopes from the immunotoxins can increase its efficiency for retreatment of cancer patients. Target biomarkers for immunotoxin or any agents with targeted deliveries are carefully selected to minimize the collateral damage to healthy cells during therapeutic interventions. Although the immunotoxins are often highly specific for the cancer cells, the same receptor is sometimes expressed on the surface of healthy cells. Therefore, harmful consequences for healthy cells cannot be omitted completely. Similarly, solid tumors have a pool of cells with different properties and with different surface molecules in close vicinity. Even a targeted delivery of toxins will also trigger undesirable consequences. Therefore, they cannot be delivered to all types of solid tumors. Strategies to overcome these limitations through interdisciplinary approaches are underway in laboratory or clinical investigations.

5  Bacteriocins as Therapeutic in Cancer Treatment Bacteriocins are bacterial proteins or peptides that are ribosomally produced by bacteria to kill or inhibit the growth of similar or closely related bacterial strains. Bacteriocins are a large group of antibacterial compounds with different structures and functions that can be used as narrow-spectrum antibiotics and food preservatives, and some of them also have anticancer activity. Bacitracins are classified in many ways (Yaghoubi, Khazaei, Avan, Hasanian, & Soleimanpour, 2020; Yaghoubi et al., 2020). A classification of bacteriocins produced by Gram-negative and Grampositive bacteria is given in Fig. 5.

5.1  S  everal of These Bacteriocins Are Effective Against Colon Cancer and Other Cancer Cell Lines 1. Pediocin is obtained from Pediococcus acidilactici, induces apoptosis by DNA fragmentation, and has been found to be effective against colon cancer cell line HT-29 (Marshall, 2008; Roberts et al., 2014). 2. Nisin A is obtained from Lactococcus lactis and causes cell cycle arrest and reduction in cell proliferation causes apoptosis due to alteration in calcium influx. It has been found to be effective against colon cancer cell lines LS180, SW48, HT-29, and Caco2, head and neck squamous cell carcinoma, breast adenocarcinoma, liver cancer, and leukemia cell lines (Fujimori, 2006).

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Fig. 5  Classification of bacteriocins and their characteristics: (a) Gram-negative bacteriocins and (b) Gram-positive bacteriocins (Yaghoubi, Khazaei, Avan, et al., 2020)

3. Colicin E1 is obtained from E. coli and is effective against colon cancer cell line HCT116; osteosarcoma cell line HOS; fibrosarcoma cell lines MRC5 and HS913T; leiomyosarcoma cell line SKUT-1; lung cancer cell lines A-549, PC-14, and RERF-LC-AI; various ovarian carcinoma cell lines; and breast cancer cell lines (Danino, Prindle, Hasty, & Bhatia, 2013; Yang, Lin, Sung, & Fang, 2014). 4. Plantaricin A is obtained from Lactobacillus plantarum C11. It induces apoptosis along with necrosis and disintegrates cell nuclei and plasma membrane of cancer cells (Song, Zhu, & Gu, 2014; Zhao et al., 2006). 5. Pyocin S2 is obtained from Pseudomonas aeruginosa and breaks down the DNA by endonuclease activity to kill the cells (Abdi-Ali, Worobec, Deezagi, & Malekzadeh, 2004).

6  Nonribosomal Bacterial Peptides as Therapeutic in Cancer The nonribosomal peptides are a wide range of peptides ranging from antibiotics to biosurfactants which are produced by complex biosynthetic enzymes called nonribosomal peptide synthetases (NRPSs). These peptides can be further modified by posttranslational modification to insert D-amino acids, heterocyclic elements, fatty acids, and other moieties into their peptide backbone in order to gain their specific chemical structure. Some bacterial strains that produce nonribosomal peptides and contribute to their anticancer action are given in Table 3.

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Table 3  Bacterial strains producing nonribosomal peptides and their anticancer action Producing bacterial species Salinispora arenicola

Nonribosomal peptides Arenamides A and arenamides B

Anticancer action Blocks or inhibits theactivation of TNF Moderately cytotoxic to human colon carcinoma cell lineHCT116 Nocardiopsis lucentensis Lucentamycins A Peptides containing Cytotoxic CNR-712, and B 3-methyl-4-ethylideneproline activity for human colon carcinoma cell line HCT116 in vitro Bacillus sp. Mixirins Mixirins are cyclic Inhibits the acylpeptides growth of human colon tumor cell Mixirins A C48H75N12O14 line HCT116 Mixirins B C45H59N12O14 Mixirins C C47H73N12O14 Urukthapelstatin A A cyclic thiopeptide Mechercharimyces This peptide antibiotic C34H30N8O6S2, asporophorigenens demonstrates an YM11-542 anticancer activity against human colon carcinoma cell line HCT116 Improve the Pseudomonasaeruginosa Azurin A small globular intracellular metalloprotein that can penetrate into the tumor cells levels of p53 Enterococcus sp. Entap An antiproliferative peptide G1 arrest of cell cycle, induces apoptosis Effective against human colon adenocarcinoma cell line HT-29 Inhibits the Streptomyces sp. Ohmyungsamycins Cyclic peptides; contain growth or A and B unusual amino acids (N-methyl-4-­methoxytrytoph development of metastaticcells an,hydroxyphenylalanine, Antitumor and N, N-dimethylvaline) activity against colon cancer cell line HCT116 Properties Cyclohexadepsipeptide, cytotoxic NF-kappa-B inhibitors Inhibits the production of nitric oxide (NO) and prostaglandin E2 (PGE2)

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7  Bacterial Enzymes as Therapeutic in Cancer Treatment Cancerous cells are rapidly growing cells, and in order to maintain their rapid growth, they need more number of amino acids than normal cells. Thus, if the supply of essential amino acids is reduced, then it will affect the cancerous cells more adversely in comparison to normal cells. Bacterial cell is the factory for production of a wide range of enzymes. Some of these enzymes can reduce the supply of some essential amino acids to cancerous cells. Arginine deiminase is a bacterial enzyme with anticancer potential, obtained from Mycoplasma hominis or M. arginine which reduces arginine concentration in tumor environment that leads to tumor growth inhibition (Ni, Schwaneberg, & Sun, 2008). Another important bacterial enzyme with anticancer property is l-asparaginase obtained from Escherichia coli which reduces concentration of asparagine in blood that selectively inhibits the growth of 145 sensitive malignant cells (Pritsa, Papazisis, Kortsaris, Geromichalos,, & Kyriakidis, 2001).

8  Combination of Bacteriotherapy with Conventional Therapy The bacteria or the genetically engineered bacteria are being used in combination with conventional therapeutic approaches. This approach of combination therapy is termed as combination bacteriolytic therapy (COBALT).

8.1  Bacteriotherapy-Radiotherapy Combination Radiotherapy is one of the promising approaches in the treatment of many different cancers. Radiotherapy has a major limitation when used for treatment of solid tumors. Solid tumors have a hypoxic or poorly vascularized zones that are resistant to radiation and a nonspecific killing of the healthy tissues along with the cancerous cells. But this limitation could be used as an advantage for bacteriotherapy, because facultative or obligate anaerobic bacteria have the ability to colonize in these hypoxic spaces (Gardlik & Fruehauf, 2010), and therefore they can be used as a vector for targeted delivery of drugs within the solid tumors. Further, radiotherapeutic doses could be lowered to decrease the healthy tissue damage done as a result of radiotherapy. Genetically engineered Salmonella can be used as an antitumor vector that can proliferate selectively within the anaerobic spaces of solid tumors. This Salmonella can be genetically engineered to express the effector components such as the herpes simplex thymidine kinase. They can mediate tumor growth suppression from a site distant from their inoculation site (Pawelek, Low, & Bermudes, 1997). Clostridium novyi-NT spores have been used in combination with radiation

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therapy to treat transplanted tumors in mice (Bettegowda et al., 2003). Although the combination of bacterial therapy with conventional radiation doses is reported to have toxic consequences on organs like the liver, a combination of radioactive iodine with C. novyi-NT has been reported to treat the patients with lower doses of radiation that can limit the toxicity of radiotherapy to normal tissues (Bettegowda et al., 2003; Nuyts et al., 2001).

8.2  Bacteriotherapy-Chemotherapy Combination Chemotherapy is still the method of choice for the treatment of cancer wherever surgical operations are not possible. Cancer treatment with chemotherapeutic agents can lead to some serious side effects that include hematological, gastrointestinal, alopecia, heart, and skin toxicity. Hematological toxicity results in neutropenia which leads to an increased risk to infectious diseases. Chemotherapy also causes mucosal damage and alterations in natural host microflora that leads to gastrointestinal toxicity (Klastersky, 1989; Marshall, 1999; Mego et al., 2013).The tumor cells that remain alive in hypoxic tumors after cancer chemotherapy usually have an increased invasiveness with high probability of metastasis (Davis & Tannock, 2000, 2002). Combination of chemotherapy and bacteriotherapy could be used to target the hypoxic spaces which serve as reservoir for cells that tolerated the chemotherapy (Jia et al., 2007).

9  Conclusion A number of conventional treatment strategies are being used to treat the cancer. Although many times the traditional therapies fail in successful treatment of cancer, but still they are the method of first preference in cancer treatment (Denny, 2004; Mehta et al., 2011). The leading cause of failure of conventional treatment methods is their failure and inefficiency to reach the hypoxic condition within the tumor. Recent investigations suggest that using bacteria as therapeutic for cancer treatment is now being considered as a promising tool because of the unique features of these bacteria to selectively colonize and proliferate in the hypoxic spaces of cancer. Due to lack of the specificity, the chemotherapeutic agents have multiple side effects in the patient. On contrary to this, the bacterial therapeutics have a specific internalization into the tumor cells, and they have specific toxicity for tumor cells with less or no side effects against normal cells. Tumor cells also develop multidrug resistance in many cases (Johnstone, Gelmon, Mayer, Hancock, & BallY, 2000; Yamada, Das Gupta, & Beattie, 2016). Despite many advantages the bacterial therapeutics are still not in much use to treat the cancer because of the lack of well-designed clinical trials, innate bacterial toxicity, short half-life, and DNA instability (Karpiński, Szkaradkiewicz, & Gamian, 2013; Ludgate, 2012; Punj et al., 2004). There is a need

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for further research to precisely modify the bacterial agents with the help of genetic engineering. D-Amino acid substitution, cyclization, and replacement of labile amino acids result in enhanced half-life and stability of bacterial proteins or peptides for their sustainable use in cancer therapy (Riedl, Zweytick, & Lohner, 2011; Tørfoss et al., 2012). Most of the studies on bacterial anticancer agents are used to end the in vitro stage. There is a need of more clinical trials to approve these bacterial agents to be used as drugs for the cancer treatment. Overall, the bacteria-­ mediated cancer therapy is still a promising area for successful cancer treatment. The combination therapy in combination with chemotherapy and radiotherapy can be proved as a better alternative in cancer treatment.

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Antineoplastic Effects of Curcumin Against Colorectal Cancer: Application and Mechanisms Vivek Kumar Soni, Arundhati Mehta, Yashwant Kumar Ratre, Chanchal Kumar, Rajat Pratap Singh, Abhishek Kumar Srivastava, Navaneet Chaturvedi, Dhananjay Shukla, Sudhir Kumar Pandey, and Naveen Kumar Vishvakarma

Abstract  Curcumin is one of the major bioactive metabolites of turmeric, a spice component common in southeast Asia. Curcumin is known for its anti-­inflammatory and antineoplastic activities against malignancies of a variety of origins. Colorectal cancer is one of the major deadly malignancies. Various strategies have been implemented to treat colorectal cancer; however, they have their own limitations, ranging from nonspecific toxicities to onset of chemoresistance. Drugs of herbal origins have some advantages over conventional therapeutic approaches. Colorectal cancer is no exception; herbal drugs (including curcumin) have proven to be effective for therapeutic applications. Curcumin can alter the molecular expression profile and decreases the rapid pace of cell division in colon malignancies. Metabolic alterations driven by curcumin modulate cellular physiology in neoplastic cells. Curcumin can alter the cell cycle and expression of cell death regulatory molecules. Curcumin has been shown to have a chemosensitizing action in colorectal cancer. Epigenetic modifications by curcumin in colon cancer cells can lead to inhibition of colon canV.K. Soni and A. Mehta contributed equally to this work as first authors

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V. K. Soni · A. Mehta · Y. K. Ratre · R. P. Singh · D. Shukla · N. K. Vishvakarma (*) Department of Biotechnology, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India C. Kumar Department of Forensic Science, Guru Ghasidas Vishwavidyalaya, Bilaspur, Chhattisgarh, India A. K. Srivastava Department of Biotechnology, Mohammad Hasan Post Graduate College, Jaunpur, Uttar Pradesh, India N. Chaturvedi Department of Molecular and Cell Biology, University of Leicester, Leicester, UK S. K. Pandey (*) Department of Botany, Guru Ghasidas Vishwavidyalaya, Bilaspur, India e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_18

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cer cell growth and progression. Moreover, immunomodulation by curcumin can also aid anticancer activity. Diverse approaches have been undertaken to improve the bioactivity and bioavailability of curcumin. This chapter summarizes the ­applications and mechanisms of the antineoplastic activities of curcumin against malignancies of colon origin. Further examination of curcumin for its therapeutic benefits against colon cancer and mechanistic exploration of them will aid exploitation of curcumin for clinical management of this lethal disorder. Keywords  Colon cancer · Curcumin · Anticancer · Immunostimulation

1  Introduction Cancer is one of the primary causes of death in industrialized countries (World Health Organization, 2005). In recent years, early diagnosis and an increase in therapeutic options have reduced the death rate. However, the growth of drug-resistant cancers necessitates a search for innovative and more effective drugs (Barone et al., 2018). Abnormal growth (noncancerous growth) of tissues known as polyps occurs slowly over time in the inner parts of the rectum or colon, eventually forming a tumor in the colorectal region, which is referred to as colorectal cancer (CRC). Later, it has the ability to spread and grow into the lymph system and blood vessels, which is quite difficult to address at the initial stages (Marley & Nan, 2016; Valastyan & Weinberg, 2011). CRC incidence and mortality rates vary markedly around the world. Globally, CRC is the third most commonly diagnosed cancer in males and the second in females, with 1.8 million new cases and almost 861,000 deaths in 2018, according to the World Health Organization (WHO) GLOBOCAN database. The rates are substantially higher in males than in females (Global Burden of Disease Cancer Collaboration et al., 2017). These geographic differences appear to be attributable to differences in dietary and environmental exposures, imposed upon a background of genetically determined susceptibility. Screening of natural metabolites, small-­ molecule inhibitors, and repurposing of standard drugs are being explored to identify candidates for use in effective and safe therapeutic regimens (Kumar, Vishvakarma, Bharti, & Singh, 2012; Mehta, Soni, Shukla, & Vishvakarma, 2020; Vishvakarma, 2014). An increasing database of evidence shows that the polyphenolic compound curcumin (CUR), an active compound of turmeric, has great potential to treat several types of cancer, including CRC (Aggarwal, Kumar, & Bharti, 2003; Kasi et  al., 2016; Kunnumakkara, Anand, & Aggarwal, 2008). It is now understood that curcumin exhibits highly pleiotropic effects, including anticancer, anti-inflammatory, and anti-infective properties. Given its dietary role, it is also well tolerated and safe in humans at dosages of up to 12 grams per day (Gupta et al., 2013). Safe consumption of curcumin is evident in its centuries-long use in traditional medicines, as well

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as its investigation in nearly 70 completed clinical trials to date (clinicaltrials.gov). Despite an abundance of promising preclinical studies in many fields of medicine, there has been a lack of clinical success in regulated trials of curcumin treatment. Although direct contact of curcumin with the gastrointestinal tract potentially lessens the requirement for systemic bioavailability of it, the results of the abovementioned research still indicate a need for improvement in the pharmacokinetics of curcumin. Fortunately, several promising strategies are now being investigated, including use of adjuvants that impede curcumin metabolism, curcumin-containing nanoparticles (NPs), and structural analogues of curcumin (Adiwidjaja, McLachlan, & Boddy, 2017; Kumar, Mittal, Sak, & Tuli, 2016).

2  Colon Cancer Any kind of growth, mass of cells, or tumor in the colorectal region is considered colon cancer or rectal cancer. In 2018, the WHO reported that all cancers combined accounted for about 9.6 million deaths worldwide (one in every six deaths from all causes) (World Health Organization, 2018). CRC is the third most common cause of cancer deaths worldwide. It has been estimated that about 8.1% of all new cancer cases and 8.3% of all cancer deaths are due to CRC (National Cancer Institute, 2018). Colon cancer is a type of adenocarcinoma and is subdivided into low and high grades. Adenosquamous carcinoma, mucinous adenocarcinoma, medullary carcinoma, and signet cell carcinoma are histologically rarer subtypes of CRC. Multiple factors contribute to CRC occurrence, but the exact cause of development of CRC is not well understood. According to experts, diet and heredity play major roles in development of colorectal cancer. Age and genetic factors are common nonmodifiable factors in occurrence of CRC. The observed incidence of CRC starts to rise from the age of 40 years, and about 90% of patients diagnosed with CRC are older than 50 years. Genetic mutation is another cause leading to development of CRC. People with a family history of CRC in a close relative have a greater risk of CRC than those with no family history. The risk in people with a family history of CRC at a young age is six times greater than that in the general population. Around 20% of CRC cases occur in people with a family history of it in first- and/or second-degree relatives (Butterworth, Higgins, & Pharoah, 2006; Lynch & de la Chapelle, 2003), and 5% of patients with CRC have genetic syndrome diseases (Jasperson, Tuohy, Neklason, & Burt, 2010). Patients who suffer from Crohn’s disease, ulcerative colitis, irritable bowel syndrome (IBS), and other chronic diseases have a higher risk of developing CRC (Axelrad, Lichtiger, & Yajnik, 2016). Other risk factors also contribute to the risk of colon cancer, such as obesity, type 2 diabetes mellitus, physical inactivity, smoking, and alcohol consumption. Consumption of processed food, junk food, and processed meat can increase the risk of CRC, whereas a diet high in fruit, vegetables, and fiber is linked to a lower risk of colon cancer (Chan & Giovannucci, 2010; Edwards et al., 2010).

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Up to 20% of patients with CRC have a family history of CRC or adenomatous polyps. Furthermore, inherited genetic conditions such as familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC) are responsible for about 5–10% of CRC cases. Genetic mutations are especially implicated in these inherited conditions: mutations in the tumor suppressor gene APC (APC regulator of WNT signaling pathway) are associated with FAP, and mutations in the MLH1 (mutL homolog 1) and MSH2 (mutS homolog 2) genes in the DNA repair pathway are associated with HNPCC (Haggar & Boushey, 2009). The most common tumor location in CRC is the proximal colon, followed by the rectum and the distal colon. Different tumor sites in CRC have different clinical and biological presentations, prognoses, and responses to treatment (Siegel et al., 2017).

2.1  Initiation of Colorectal Cancer A small mass or localized growth in the inner lining of the colon or rectum is called a polyp and is the start of CRC. Most polyps have the ability to metastasize, grow, and progress as cancer. Such a malignant character is found in adenomas, and 96% of CRC cases arise from adenoma polyps (American Cancer Society, 2017). Because of their favorable environment and proliferation of cells, polyps increase in size over time, providing raw material for genetic mutation and epigenetic changes in the polyps, which promote the ability to invade other tissues and protrude into the rectal area. Over time, polyps increase in size continuously and because they can induce angiogenesis, they are more vascularized than other tissues. Via the lymph and circulatory systems, tumor cells start to spread to other distant metastatic sites (American Cancer Society, 2017; Simon, 2016). Thus, it is very important to identify precancerous polyps so that steps can be taken to prevent their development and progression to cancer and CRC.  The American Cancer Society recommends that individuals with a moderate risk of CRC should be monitored from the age of 50 years and that high-risk individuals should be screened before the age of 50 years. Visual examination and stool-based tests are used in prescreening for CRC, and colonoscopy diagnosis is recommended when prescreening tests turn out to be positive (American Cancer Society, 2017). Nowadays, different treatment strategies—such as surgery, chemotherapy, and radiotherapy—are available, but exactly what measures to take depends on the current stage, size, and location of the tumor, and even the health status of the patient. At the primary stage of CRC, surgical removal of the tumor is one of the most common choices of treatment. Surgical removal must not be considered a permanent cure; it merely minimizes symptoms by removing most of the tumor tissue. In CRC, surgical removal is performed mainly by colectomy, but this can lead to a few critical problems (blood loss, ulceration, thrombosis, infection, etc.). Patients may also suffer from severe pain, swelling, mental and physical stress, etc. as a result of this treatment. Another strategy for treatment of CRC is radiotherapy, in which high-­ energy radiation is delivered to the tumor site to destroy the cancer cells, but it too can cause severe pain, stress, blood loss, ulceration, constipation, etc.

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For metastatic cases, the most frequently practiced treatment is chemotherapy (LaValle et al., 2010). When CRC is detected only at a late stage, the recommended treatment is surgical removal of the tumor along with chemotherapy, but relapse is a major issue, occurring in half of these patients (Garcea et al., 2005; Mudduluru et  al., 2011). The chemotherapeutic drugs 5-fluorouracil (5-FU), oxaliplatin (L-OHP), irinotecan, panitumumab, cetuximab, bevacizumab, regorafenib, capecitabine, etc. are commonly used in CRC treatment. Mostly, drug-sensitive cells are killed by chemotherapeutic drugs but drug-resistant cells remain unaffected. Late-phase CRC also tends to develop multidrug resistance, which makes the medication much less effective. Moreover, one major issue with anticancer drugs is the major side effects they cause (toxicity, hair loss, rashes, development of resistance, etc.) (Chaithongyot et al., 2015).Thus, it is very important to develop a novel strategy or/and therapy that overcomes the drawbacks of current therapies such as surgical resection, radiation, and chemotherapy (Subramaniam et al., 2008).

2.2  Treatment Strategy for Colorectal Cancer CRC is categorized into different stages, which are based on the extent of cancer cell invasion of different tissues. At the fourth stage (Duke stage D), the cancer cells become metastatic and invasive in nature, using the lymph system to travel from their origin to other sites, where they colonize other tissues and form secondary lesions even after treatment. Because of their predominantly metastatic nature, cancer cells can neutralize the current strategies used to treat cancer, and they can thus cause a recurrence. The different stages of CRC and the treatment strategies used for them are described in Sects. 2.2.1–2.2.5. 2.2.1  Stage 0 Colorectal Cancer and Its Treatment This is an early stage of CRC in which a small mass of cells or a tumor is still confined to the inner lining of the colon. It is benign and noninvasive in nature. At this stage, simple excision of the CRC lesion during colonoscopy or polypectomy is usually sufficient. 2.2.2  Stage I Colorectal Cancer and Its Treatment At stage I of CRC, the cancer extends through the mucosa and has invaded the muscular layer of the colon or rectum, but it has not yet spread into other neighboring tissues or lymph nodes. Stage I adenocarcinoma of the colon is generally uncommon, and careful excision of the malignant growth is typically viable. At this stage, tumor excision is performed using both endoscopy and transanal endoscopic microsurgery. If the malignancy is situated in the upper part of the rectum, low anterior resection (LAR) is recommended. At that point, adjuvant chemoradiotherapy with 5-FU or capecitabine is advisable (Carrara et al., 2012).

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2.2.3  Stage II Colorectal Cancer and Its Treatment At stage II of colon malignancy, the infection is somewhat more progressed than at stage  I and has extended beyond the mucosa and the submucosa of the colon. Stage II colon cancer is subclassified into stages IIA, IIB, and IIC. At stage IIA, the disease has not yet spread to lymph nodes or neighboring tissue. It has penetrated the external layer of the colon but gone no further. At stage IIB, the malignancy has not yet spread to lymph nodes but has reached the visceral peritoneum. This is the layer that supports the visceral organs. At stage IIC, the disease has not yet spread to lymph nodes but has grown into neighboring organs or structures. The efficacy of adjuvant chemotherapy during stage  II CRC remains unclear. Careful intervention should focus on wide resection of the tumor along with removal of nearby lymph nodes. Partial colectomy may be the main therapeutic approach in low- and moderate-risk stage II CRC cases. High-risk cases should also be managed with adjuvant chemotherapy. Some chemotherapeutic drugs (for example, 5-FU, leucovorin, L-OHP, and their combination (FOLFOX4)) can be effective when used alone or adjuvantly to treat stage II CRC (Dotan & Cohen, 2011). According to the Johns Hopkins Colorectal Cancer Research Center, radiotherapy can also be utilized adjuvantly in cases of stage II CRC in which the lesion is confined, and this approach reduces the risk of recurrence. The efficacy of radiotherapy has been demonstrated in cases in which the CRC tumor had the capacity for invasion. 5-FU or capecitabine are also utilized together with radiotherapy to increase tumor cell susceptibility to radiation. 2.2.4  Treatment of Stage III Colorectal Cancer Stage III colon malignancy is defined as the presence of a tumor of any size (stage T1–T4 cancer, according to the Tumor–Node–Metastasis (TNM) cancer staging system) with metastasis to local lymph nodes. In stage III CRC, it is important to excise the affected parts of the colon, as well as the nearby lymph nodes, and then administer chemotherapy within 2 months after the surgery. In the advanced phase of stage III CRC (T3–T4, with an invasive nature), neoadjuvant chemotherapy is applied along with radiation therapy, and adjuvant chemotherapy is then used to decrease the risk of recurrence. The European Society for Medical Oncology (ESMO) guidelines on the spread of malignancy recommend use of magnetic resonance imaging (MRI) to help tailor the chemotherapy and decrease the risk of CRC recurrence (Benson 3rd et  al., 2017). Some of the chemotherapeutic drugs commonly used in stage III CRC are L-OHP, FOLFOX, capecitabine, irinotecan, bevacizumab, and cetuximab (Labianca et al., 2010).

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2.2.5  Treatment of Stage IV Colorectal Cancer Stage IV CRC is the most advanced stage of colorectal cancer. It means that the cancer has spread from its origin and infected other tissues and organs. The liver is the organ most commonly affected in CRC, and this occurs in 50–60% of cases. The lungs, peritoneum, brain, ovaries, bones, etc. are less commonly affected in stage IV CRC. In stage IV CRC, primary treatment starts with surgical removal of the segment of the colon containing the malignant growth and the affected lymph nodes. This is essential to decrease the risk of recurrence. Chemotherapy and radiotherapy are commonly used too, either prior to or potentially after the surgical procedure. Sometimes, hepatic artery infusion is utilized if the disease has spread to the liver. Chemotherapy is applied directly in cases of nonresectable tumors. Fluoropyrimidines (5-FU and capecitabine) are widely used for chemotherapy in stage IV CRC. Other chemotherapy agents that may be used, depending on the patient’s condition, include irinotecan, L-OHP, bevacizumab, cetuximab, panitumumab, Ziv-aflibercept, regorafenib, ramucirumab, nivolumab, pembrolizumab, trifluridine, and tipiracil, which all have different therapeutic efficacies and modes of action.

3  Biological Activity of Curcumin For thousands of years, natural products have been utilized in traditional medicine and have provided a wellspring of components for refinement of new medications. Turmeric (Curcuma longa Linn.), a member of the Zingiberaceae family, originates from India, southeast Asia, and Indonesia, and is cultivated in tropical and subtropical areas around the globe. Turmeric is known as a medicinal plant and has received considerable scientific attention for its bioactivity and antineoplastic actions (Abrahams, Haylett, Johnson, Carr, & Bardien, 2019; Aggarwal et  al., 2003; Rastegar, Akbari Javar, Khoobi, et al., 2018; Soni et al., 2020; Soni, Mehta, Shukla, Kumar, & Vishvakarma, 2020; Soni, Shukla, Kumar, & Vishvakarma, 2020; Vishvakarma, 2014; Vishvakarma et  al., 2012; Vishvakarma, Kumar, & Singh, 2011). The yellow color of turmeric is due to the polyphenolic compound curcumin—also known as diferuloylmethane or (1E,6E)-1,7-bis(4-hydroxy-3-­ methoxyphenyl)-1,6-heptadiene-3,5-dione [its International Union of Pure and Applied Chemistry (IUPAC) name]—which has shown antitumor activity against malignant growths of a variety of cancer origins (Aggarwal et al., 2003; Notarbartolo et  al., 2005; Vishvakarma, 2014; Vishvakarma et  al., 2011). Numerous scientific investigations have recognized the potential and properties of curcumin (Adiwidjaja et al., 2017; Aggarwal et al., 2003; Notarbartolo et al., 2005; Vishvakarma, 2014; Vishvakarma et al., 2012). Additionally, curcumin has been shown to be selective and nontoxic in the body (Aggarwal et al., 2003; Vishvakarma, 2014; Vishvakarma et al., 2012). Important targets/focuses of curcumin research are its anti-­inflammatory action and its abilities to limit cell development and modulate controllers of cell proliferation (Adiwidjaja et al., 2017; Notarbartolo et al., 2005; Vishvakarma, 2014;

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Vishvakarma et al., 2011). The bioactivities of curcumin have been shown in both laboratory and preclinical studies (Jantan, Ahmad, & Bukhari, 2015; Vishvakarma, 2014). Interdisciplinary methodologies are now being utilized to overcome limitations in clinical exploitation of curcumin—for example, its low bioavailability (Adiwidjaja et al., 2017; Jantan et al., 2015). Curcumin has likewise been shown to influence different signs of disease. Previous studies have shown that use of curcumin can help rebalance the metabolic dysregulation that occurs in cancer (Siddiqui et al., 2018; Vishvakarma et al., 2011). The WHO has endorsed daily consumption of curcuminoids in the range of 0–3 mg/kg as a food additive. The average consumption of turmeric in the Indian diet is around 2–2.5 g for a 60 kg person, which translates into a daily consumption of roughly 60–100 mg of curcumin (Mahmood et al., 2015). Over the past 30 years, curcumin has been shown to have beneficial impacts on malignancies, immune system ailments, metabolic sicknesses, neurological illnesses, cardiovascular diseases (CVDs), lung infections, liver ailments, and an assortment of other inflammatory ailments (Aggarwal & Harikumar, 2009; Kannappan, Gupta, Kim, Reuter, & Aggarwal, 2011). Curcumin modulates different biological molecules—such as cytokines, growth hormone and factors, receptors, and metastatic and apoptotic molecules—thereby regulating biological processes (Baliga et  al., 2012; Prasad, Gupta, Tyagi, & Aggarwal, 2014; Shehzad & Lee, 2010). Some of the biological activities of curcumin are discussed in Sects. 3.1–3.6.

3.1  Anticancer Activity Curcumin has potent anticancer activity in various types of cancer. Curcumin modulates the mitochondrial membrane potential and enhances apoptosis of cancer cells by suppressing B-cell lymphoma (Bcl)-xL protein (Balasubramanian & Eckert, 2007). The activity of death receptors (DRs) on cells positively correlates with activation of tumor necrosis factor (TNF), leading to apoptosis. It has been reported that curcumin increases the activity of DR4 and DR5, and promotes tumor apoptosis (Ashour et al., 2014; Lee, Li, Tsao, Fong, & Tang, 2012; Moragoda, Jaszewski, & Majumdar, 2001). Many intracellular transcription factors—nuclear factor (NF)-кB, cyclooxygenase (COX)-2, activator protein-1 (AP-1), matrix metalloproteinase (MMP)-9, signal transducer and activator of transcription (STAT)  3, nitric oxide synthase, etc.—are mechanistic tools of cancer and have been shown to be inhibited or downregulated by curcumin in many cell lines (Hahn et al., 2018; Soni, Shukla, Kumar, & Vishvakarma, 2020; Vishvakarma, 2014; Vishvakarma et  al., 2011). Recent work has demonstrated that the anti-Warburg effect of curcumin is mediated by a decrease in glucose consumption and lactate production through modulation of glucose transporters (GLUTs), monocarboxylate transporters (MCTs), lactate dehydrogenase (LDH), hypoxia-inducible factor (HIF) 1α, and pyruvate kinase isoenzyme (PK) M2 (Siddiqui et al., 2018; Soni, Shukla, Kumar, & Vishvakarma, 2020; Vishvakarma, 2014). It has been suggested that deregulated metabolism and the

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tumor microenvironment potentially play roles in the progression of cancer (Vishvakarma & Singh, 2011a). This deregulated metabolic alteration was demonstrated to be alleviated in response to curcumin in a murine model of cancer (Vishvakarma et  al., 2011). Many other targets of curcumin have also been suggested, which directly or indirectly involve the antineoplastic activity of this yellow pigment in turmeric.

3.2  Anti-inflammatory and Antioxidant Activities Curcumin has exhibited promising anti-inflammatory and antioxidant activities in both in  vitro and in  vivo tests. These properties of curcumin are due to its structure and the methoxy and hydroxyl groups it contains (Rahman, Biswas, & Kirkham, 2006). The Janus kinase and signal transducer and activator of transcription (JAK/STAT) signaling pathway is negatively regulated by curcumin, which decreases expression of the proinflammatory cytokines interleukin (IL)-1, IL-2, IL-6, IL-8, and IL-12 (TNFα) and monocyte chemoattractant protein (MCP)  1). Moreover, curcumin exerts anti-inflammatory effects by inhibiting expression of NF-kB, which also negatively regulates COX-2, inducible nitric oxide synthase (iNOS), xanthine oxidase, and lipoxygenase activity (Rahman, Biswas, & Kirkham, 2006). Curcumin effectively hinders inflammatory signals, cell proliferation, metastasis, and angiogenic signals by targeting various regulatory events (Shehzad, Lee, & Lee, 2013; Soni, Mehta, Ratre, et al., 2020). Curcumin modulates the inflammatory cascade, including overexpression of inflammatory biomarkers, free radicals, and lipid peroxides. Inflammation may cause many severe diseases. CVDs, obesity, type 2 diabetes, neurodegenerative diseases, and some cancer-like critical diseases are associated with acute and chronic inflammation (Soni, Mehta, Ratre, et  al., 2020; Soni, Mehta, Shukla, et al., 2020; Vishvakarma, 2014). Curcumin has been used effectively to treat various diseases such as diabetes, obesity, neurological disease, and inflammatory bowel disease (IBD) in traditional Ayurvedic medicine, as well as in modern pharmacological practice (Shehzad et  al., 2013; Prasad et  al., 2014; Deogade & Ghate, 2015). Curcumin exhibits effective antioxidant effects via its free-radical-scavenging action (Deogade & Ghate, 2015). It has also been suggested that use of curcumin could help to prevent harmful consequences of viral infections and could aid management of the COVID-19 coronavirus disease (Soni, Mehta, Ratre, et al., 2020; Soni, Mehta, Shukla, et al., 2020). Analogues of curcumin exhibit stronger antioxidant activity than curcumin itself; for example, Dolai et  al. (2011) demonstrated that a synthetic sugar derivative of curcumin was more effective as an antioxidant. It was concluded that whereas curcumin inhibited tau peptide accumulation and amyloid-β at the micromolar level, the sugar– curcumin conjugate had these inhibitory effects even at nanomolar concentrations (Dolai et al., 2011).

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3.3  Immune-Regulatory Activity Curcumin demonstrates tremendous immunomodulatory activity through molecular regulation of immune components in a systematic manner. Curcumin helps to prevent immune-related disease by interacting and modulating immune cells such as diverse forms of T lymphocytes, B lymphocytes, macrophages, dendritic subsets, and natural killer (NK) cells (Abdollahi, Momtazi, Johnston, & Sahebkar, 2018; Vishvakarma et  al., 2012). Decreases in the numbers of neutrophils and eosinophils and increases in lymphocyte numbers have been observed with curcumin treatment (Shakeri & Boskabady, 2017). TNFα, interferon (IFN)  γ, and IL-1β are pro-­ inflammatory molecules produced by T  helper (Th)  1 cells. Transforming growth factor (TGF)-β, IL-4, and IL-10 are anti-inflammatory molecules produced by Th2 cells. IL-17 (produced by Th17 cells) and immunosuppressive T regulatory (Treg) cells are effective against autoimmune disease. Balance between different T cell subtypes is essential for treatment of immunerelated disease (Han et  al., 2014). M1 and M2 macrophages interact with Th1 and Th2 cells, and share their role in the balance of immune homeostasis (Yang et al., 2018). The tumor microenvironment can influence phenotypic activation of macrophages (Vishvakarma & & Singh, 2010). Diverse mechanisms have been suggested to account for this tumor-induced alteration in immunity. It was reported that in ovalbumin-sensitized rats, curcumin showed a bracing effect on Th1 cells and suppressing action on Th2 cells. Curcumin shows a protective effect against immunotoxicity even in the presence of low lymphocyte counts (Afia, Alshehri, Alfaifi, & Shakor, 2017). Wang et al. (2016) reported that curcumin effectively worked against myasthenia gravis by regulating different immune cells, especially through downregulation of T  cell expression and an increase in numbers of NK  cell receptor protein  1 (NKR-P1) cells. Curcumin also upregulated B cell differentiation and increased the population of B10 cells (Wang et al., 2016). An in vitro study showed that curcumin increased the number of bone-marrow-derived mesenchymal stem cells, modulated the M2 macrophage population, and created an immunogenic microenvironment for cutaneous wound healing (Yang et  al., 2018). An in  silico study also suggested that curcumin showed strong binding affinity for CD4 and CD8 receptors, and strong antioxidant activity. This suppressed deltamethrin-­regulated thymic apoptosis (Kumar, Sasmal, Jadav, & Sharma, 2015). Moreover, curcumin was shown to skew the polarization of macrophage activation toward the antitumor M1 phenotype (Vishvakarma et  al., 2012) in tumor-bearing murine hosts. Aeromonas hydrophila-infected fish fed with curcumin displayed increases in their immune response, lysozyme activity, and immunoglobulin (Ig) G and  IgM levels (Mahmoud, Al-Sagheer, Reda, Mahgoub, & Ayyat, 2017). Curcumin was also shown to increase secretory immunoglobulin (sIgA) levels in weaned piglets (Xun et al., 2015). Moreover, curcumin was found to modulate the levels of other immunomodulatory mediators such as IL-1β, IL-4, IL-6, IL-17A, and TNFα (Lee et al., 2018; Shakeri & Boskabady, 2017; Xun et al., 2015).

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3.4  Cardiovascular Protection CVDs are the number one cause of death worldwide. Curcumin intake is helpful for CVD prevention (Hernández, Wicz, & Corral, 2016; Yu et al., 2016). Curcumin works as a cardioprotectant and protects cardiomyocytes. For example, curcumin prevents induction of hypertrophic tension, which inhibits increases in collagen content and balances extracellular matrix remodeling. Curcumin also inhibits overexpressed activity of gelatinase B and gelatinolytic in H9c2 cardiomyocytes (Kohli et al., 2013) and has been shown to stop norepinephrine-induced apoptosis in cardiomyocytes and to help them regain their normal physiological status (Manghani, Gupta, Tripathi, & Rani, 2017). The venom of Hemiscorpius lepturus restricts adenosine triphosphate (ATP) production by mitochondrial respiration and leads to apoptosis, but this can be prevented by curcumin administration (Naserzadeh et al., 2018). Moreover, curcumin administration increases the viability of H2O2-exposed H9c2 cardiomyoblasts by increasing the activity of heme oxygenase (HO)-1 protein, inhibiting caspase-3 activation, and increasing the ratio of Bcl-2 to Bcl-2-­associated X-protein (Bax) (Yang, Jiang, & Shi, 2017). Furthermore, curcumin inhibits reduced nicotinamide adenine dinucleotide phosphate (NADPH)–mediated reactive oxygen species (ROS) stress by inhibiting the phosphoinositide 3-kinase/protein kinase  B (PI3K/Akt) signaling pathway and prevents apoptosis of cardiomyocytes induced by hyperglycemia (Yu et al., 2016). Bai et al. (2018) reported that curcumin has the ability to restore cardiac function and increase Na+/Ca+ exchanger expression. Use of curcumin has been reported to reduce diastolic and systolic dimensions in the left ventricle and to enhance the activity of the left ventricle in vivo (Bai et al., 2018). In other reports, cardiomyocyte hypertrophy induced by high insulin and glucose levels was found to be attenuated by curcumin administration, which reduced expression of atrial natriuretic factor (ANF) messenger RNA (mRNA), total protein content, and surface area (Chen et al., 2015). Curcumin also suppressed the hypertrophic response via inhibition of the zinc finger transcription factor GATA4 and functional proteins including intrinsic histone acetyltransferase and p300 (Katanasaka, Sunagawa, Hasegawa, & Morimoto, 2013). Moreover, curcumin was found to increase Dickkopf-related protein  3, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and apoptosis signal-regulating kinase 1 (ASK1), preventing chronic heart failure (Cao et al., 2018). It was demonstrated that curcumin suppressed expression of NiemannPick C1-like 1 (NPC1L1) and sterol regulatory element-binding protein-2 (SREBP-2), and decreased absorption of cholesterol in Caco-2 colorectal adenocarcinoma cells (Feng, Zou, Zhang, Li, & Lu, 2017). In summary, it can be suggested that curcumin shows tremendous therapeutic and protective effects on cardiomyocyte injury, chronic heart failure, cardiac hypertrophy, and cholesterol absorption.

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3.5  Antidiabetic Effects Diabetes mellitus is a very severe disorder in both developing and developed countries. Much of the diabetes research to date has focused on development of an effective antidiabetic drug with minimal side effects. Numerous studies have suggested that curcumin shows promise as an antidiabetic agent, which can also minimize the risk of development of type 2 diabetes in prediabetic individuals (Chuengsamarn, Rattanamongkolgul, Luechapudiporn, Phisalaphong, & Jirawatnotai, 2012). Moreover, curcumin can be an important option for treatment of diabetes complications such as diabetic retinopathy (Li, Wang, Ying, Chen, & Yu, 2016). Curcumin controls hyperglycemia by suppressing the activity of α-amylase and α-glucosidase, reflecting its antidiabetic activity. Additionally, the liver, adipose tissue, and skeletal muscle are insulin-producing and regulating tissues, which benefit from curcumin (Wojcik, Krawczyk, Wojcik, Cypryk, & Wozniak, 2018). Molecular docking suggests that curcumin shows a significant inhibitory effect on α-amylase, in comparison with other natural compounds such as berberine and quercetin (Jhong, Riyaphan, Lin, Chia, & Weng, 2015). Akolade et al. demonstrated that curcumin significantly reduced blood sugar levels in rats (Akolade, Oloyede, & Onyenekwe, 2017). Moreover, curcumin treatment increased glucose tolerance and insulin sensitivity in a murine model of diabetes (Gutierres et  al., 2015) and increased Akt phosphorylation and Glut4 levels in skeletal muscles. In diabetic rats treated with curcumin for 12  weeks, it was found that exposure to curcumin increased the numbers of small islets of Langerhans without lymphocyte infiltration in pancreatic islets (Chanpoo, Petchpiboonthai, Panyarachun, & Anupunpisit, 2010).

3.6  Neuroprotection Curcumin shows antioxidant activity and anti-inflammatory potential, giving it a new dimension as a neuroprotective agent to potentiate regulation of the brain through chemical balance. Some investigations have suggested that curcumin potentially decreases H2O2-induced neurotoxicity by suppressing caspase activity, DNA damage, poly (ADP-ribose) polymerase (PARP) cleavage, dysregulation of the MAPK and Akt pathways, and accumulation of ROS. These properties of curcumin make it a potent neuroprotective agent for treatment of several neurodegenerative disorders in humans (Fu et al., 2016). Its neuroprotective potential was also shown in an SH-SY5Y human neuroblastoma cell line, in which increased cell viability and a reduction in H2O2-induced damage were observed (Szczepanowicz et  al., 2016). Research on the antineuroinflammatory potential of curcumin has revealed its ability to prevent secretion of cytokines and proinflammatory mediators. Curcumin upregulates HO-1 transcription and translation, modulates expression patterns and signaling pathways (including those of NF-κB and MAPK), and suppresses inflammation in microglial cells (Jin et al., 2018). Through suppression

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of inflammation, curcumin reduces astrocyte hypertrophy, which contributes to microglial activation in the hippocampus, memory induction, and mood regulation (Kodali et al., 2018). Moreover, curcumin has been found to be effective for prevention of strokes, neurotrophic pain, reperfusion injury, and ischemia, and mitigate axonal injury (Dong et  al., 2018; Liu, Li, Liu et  al., 2016; Liu, Li, Zhang et  al., 2016; Tegenge et al., 2014). In  vitro and in  vivo studies have suggested that use of curcumin is effective against Alzheimer’s disease (AD), Parkinson’s disease (PD), and other neurodegenerative diseases. In neurodegenerative disorders, curcumin facilitates homeostasis and heat shock system regulation by binding proteins or limiting the scattering of other proteins (Hu et al., 2015). It has been reported that accumulation of amyloid-β protein and hyperphosphorylated tau protein is a marker of AD.  Curcumin was shown to induce the transcriptional and translational activity of peroxisome proliferator–activated receptor (PPAR)  γ protein, which attenuated neuroinflammation induced by amyloid-β and restored neuronal condition in a rat model of AD (Liu, Li, Liu et al., 2016; Liu, Li, Zhang, et al., 2016). In another rat model of AD, spatial learning and memory damage by amyloid-β peptide via the JNK signaling pathway was repaired by curcumin treatment (Wang, Ghosh, & Ghosh, 2017; Wang, Li, et al., 2017). Curcumin was also found to improve locomotor activity and protect against neurodegeneration in a mouse model of AD (Eghbaliferiz, Farhadi, Barreto, Majeed, & Sahebkar, 2020). Much of the evidence on PD shows that it mainly involves aggregation of α-synuclein protein (Jiang et al., 2013). Curcumin has the ability to attenuate mechanistic target of rapamycin (mTOR)/ribosomal protein S6 kinase (p70S6K) signaling and reconstruct macroautophagy to decrease A53T α-synuclein and its subsequent accumulation (Sharma & Nehru, 2018). Curcumin prevents iron aggregation on dopaminergic neurons and suppresses astrocytic activation. Moreover, in a study of mice with PD, curcumin improved cognitive function by modulating the level of acetylcholine esterase enzyme and accumulation of motor deficits (Khatri & Juvekar, 2016). Curcumin was also shown to decrease expression of p-p28, activation of caspase-3, and construction of quinoprotein in an SH-SY5Y cell line treated with 6-hydroxydopamine (Meesarapee et al., 2014).

4  Use of Curcumin Against Colon Cancer Colorectal cancer is one of the leading causes of death worldwide and affects men and women equally. Because of its recurrence and lethal characteristics, patients rarely become free from cancer. There is evidence that curcumin could serve as a potent CRC antineoplastic agent by arresting the cell cycle and stimulating programmed cell death. Various in vitro and in vivo investigations have reported that curcumin significantly inhibits colon cancer cell growth, metastasis, recurrence, and multidrug resistance (MDR) phenotypes by targeting different molecular targets and modulating several distinct signaling pathways. The different targets and

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mechanisms described in this section indicate that curcumin is a promising and effective drug for CRC treatment. Apoptosis is one of the main mechanisms by which curcumin exerts its anticancer effects and blocks cell growth. For execution of apoptosis, curcumin targets multiple molecular pathways and enzymes, including Bcl-2 family members (Bcl-2, Bax, and Bcl-xL), protease enzymes (caspases 3 and 8), death receptors (DR5 and Fas), transcription factors (NF-κB and β-catenin), COX-2, and ROS. Histotypes of many cancers, including CRC, are related to defective molecular expression of Bcl-2 family members (Watson, 2004). It has been reported that curcumin increases Bax expression, suppresses Bcl-2 levels in colon adenocarcinoma through phosphorylation of ser15, and stimulates p53 expression (Song et al., 2005). Increased Bax expression could affect Bcl-2/Bax or Bcl-2/Bcl-xL ratios and stimulate cancer cells toward apoptosis. Bcl-2 and Bax-related curcumin-induced suppression have also been reported in HCT116 (Moragoda et al., 2001) and COLO 205 (Su et al., 2006) colon cancer cell lines. To sustain the tumor microenvironment and regulation of cancer metastasis and invasion, the microenvironment of CRC comprises a unique type of cells: CRC stem cells (CSCs) (Boral & Nie, 2012; Schiavoni, Gabriele, & Mattei, 2013). It has been reported that CSCs modulate self-immune cell populations within the tumor microenvironment (Schiavoni et al., 2013), extracellular matrix protein regulation, epithelial–mesenchymal transition (EMT), and factors such as TGF-β, Wnt signaling, and PI3K/Akt (Gulhati et al., 2011). Curcumin has been observed to suppress CRC by hindering cross-talk between CSCs and stromal fibroblasts in the tumor microenvironment, demonstrating potential as a therapy for CRC (Buhrmann et al., 2014). Phase I and II clinical trials of curcumin have shown that oral intake of curcumin is quite effective in suppressing precancerous lesions (Carroll et al., 2011; Cheng et al., 2001). Some investigations have suggested several potent targets for suppression of CRC, such as adenosine monophosphate–activated protein kinase (AMPK)– COX-2 (Lee, Lee, & Kim, 2009; Lee, Park, et al. 2009), transcription factor E2F4 (Kim & Lee, 2010), cyclin D1–CDK4 (cyclin-dependent kinase 4 homolog) signaling pathways (Mukhopadhyay et al., 2002), and JNKs (Collett & Campbell, 2004). These were found to be attenuated by curcumin administration. Furthermore, it has been reported that expression of cyclin E–dependent kinase and CDK2 is upregulated in colorectal carcinoma (Cam et al., 2001). Curcumin facilitates suppression of CDK2 activity through binding of the ATP pocket. CDK1 and CDK2 activity, which is known to maintain cell cycle progression, is also inhibited by curcumin in CRC (Lim et al., 2014). Agarwal et al. (2018) found that curcumin attenuated cell cycle progression in the S and G2/M phases by ROS generation in Smd4 and p53–mutated HT-29 colon cancer cells. Moreover, curcumin-promoted ROS generation made HT-29 cells susceptible to apoptotic cell death through alteration of the mitochondrial membrane potential. This was the first investigation to report that in Smad4 and p53–mutated CRC, ROS-mediated cell cycle arrest by curcumin mediated the modulated mitochondrial-dependent pathway.

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Many investigations have shown that development and proliferation of most cancers (including CRC) are related to constitutive execution of many signaling pathways that boost proliferation, suppress apoptotic cell death, and encourage metastasis. A lot of evidence suggests that molecular members of the epidermal growth factor receptor (EGFR) family—especially ErbB-2/HER-2 and ErbB-3/ HER-3—play an essential role in controlling a few pathways that influence tumor cell endurance, motility, angiogenesis, invasiveness, etc. (Messa, Russo, Caruso, & Di Leo, 1998). Insulin-like growth factor (IGF) and IGF receptors are involved in CRC cell progression and proliferation (Adachi et al., 2002). Additionally, EGFRs are also crucial for resistance against chemotherapeutic agents (Dallas et al., 2009). A review of the literature indicates that curcumin not only hinders development of chemotherapy-resistant colon cancer cells but also increases EGFR inhibition by gefitinib and IGF 1 receptor (IGF-1R) restriction through IGF-1R small interfering RNA (siRNA). Therefore, it can be accepted that the capacity of curcumin to adequately downregulate survival signals executed by multiple growth factors plays a basic part in its ability to hinder chemotherapy-resistant colon cancer (Patel et al., 2010). MDR phenotype formation is an intricate process. P-glycoprotein (P-gp) expression is considered a classic mechanism of MDR (Xie et al., 2016). P-gp, which is encoded by the ABCB1 (ATP binding cassette subfamily B member 1) gene (previously known as MDR1), is considered a membrane transport protein with an active (ATP-dependent) process. P-gp protects tissue by expelling all foreign toxic substances from the inside to the outside of the cell (Leonard, Fojo, & Bates, 2003; Van der Holt et al., 2006). Lu et al. demonstrated that curcumin reduced MDR activity in HCT-8/VCR colon cancer cell lines (Lu, Qin, Yang, Li, & Fu, 2013), and it was observed that curcumin significantly decreased MDR activity by increasing aggregation and holding rhodamine 123 (Rh123) inside the cells. An in vivo xenograft study also demonstrated that curcumin significantly reduced the activity of ABCB1/ P-gp in control cells as well as VCR cells. Thus, curcumin acts as an active chemosensitizer to reduce the effect of MDR activity in CRC (He et al., 2019; Lu et al., 2013). Curcumin has been demonstrated to affect the metabolic wiring of cancer cells (Soni, Shukla, Kumar, & Vishvakarma, 2020; Vishvakarma, 2014). A unique metabolic state and tumor microenvironment—including an upregulated glycolytic phenotype, availability of glucose, the stage of progression, and extracellular physical parameters—govern the resistance phenotype of cancer cells (Vishvakarma, Kumar, Singh, & Singh, 2013). Curcumin can mitigate cancer metabolism and a hyperglycolytic phenotype (Vishvakarma et al., 2012) and can circumvent the chemoresistance of tumor cells (Soni, Shukla, Kumar, & Vishvakarma, 2020). Curcumin also prevents lactate-induced elevation of HIF 1α expression (Soni, Shukla, Kumar, & Vishvakarma, 2020). Elevated lactate levels correlate with progression and aggressive behavior of cancer cells (Vishvakarma & & Singh, 2010; Vishvakarma & Singh, 2011a). A small-molecular inhibitor of the transporter V-ATPase causes tumor retardation (Vishvakarma & Singh, 2011a, 2011b) and supports antitumor immunity in the host (Vishvakarma & & Singh, 2010; Vishvakarma & Singh, 2011b). In a murine model of cancer, curcumin was found to inhibit expression of

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V-ATPase (Vishvakarma et  al., 2011) and promote activation of the antitumor immune response (Vishvakarma, 2014; Vishvakarma et al., 2012). Tumor-specific metabolic behavior, along with the tumor microenvironment—including a hyperglycolytic phenotype, transporter expression, the stage of progression, and nutrient availability—determine chemoresistance in cancer cells (Soni, Shukla, Kumar, & Vishvakarma, 2020; Vishvakarma et al., 2013). Curcumin can mitigate the hyperglycolytic phenotype of tumor cells through modulation of the expression profile (Vishvakarma et al., 2012) and can prevent the onset of chemoresistance provoked by autocrine stimulation (Soni, Shukla, Kumar, & Vishvakarma, 2020). Psychoneuroimmunomodulation by curcumin (Soni, Mehta, Shukla, et  al., 2020) can have implications for tumor retardation and potentiation of the anticancer activity of standard chemotherapeutic drugs. It has been reported that ornithine decarboxylase (ODC) and polyamine synthesis are inhibited by curcumin in CRC (Berrak et  al., 2016; Mehta, Pantazis, McQueen, & Aggarwal, 1997). The action of ODC is frequently utilized as a biomarker for tumor proliferation in animal models of carcinoma. The ability to hinder enlistment of ODC in these models is connected with chemotherapy (Thomas & Thomas, 2001). In a gastrointestinal cancer cell line, curcumin reduced biosynthesis of polyamine and maintained intracellular levels of polyamine while inducing spermine oxidation. It has been shown clinically that curcumin, in association with α-difluoromethylornithine (DFMO) (an ODC inhibitor), significantly reduces growth of cancer and can be used as a substitute for nonsteroidal anti-inflammatory drugs (NSAIDs) to prevent side effects of chemotherapy (Murray-Stewart et al., 2018). A meta-analysis reported that CRC was strongly associated with sirtuin-1 (SIRT1) expression (Zu, Ji, Zhou, & Che, 2016). Higher levels of SIRT1 increase tumor invasion and metastasis in lymph nodes (Lv et  al., 2014; Yu et  al., 2016). SIRT1 has also been found to significantly correlate with KRAS (KRAS proto-­ oncogene, GTPase) and BRAF (B-Raf proto-oncogene, serine/threonine kinase) mutations and malignancy (Kriegl, Vieth, Kirchner, & Menssen, 2012). A liquid chromatography–mass spectrometry (LC-MS) study revealed that the electrophilic α,β-unsaturated carbonyl moiety of curcumin could directly bind with SIRT1, especially with its cysteine 67 (cys67) residue, facilitating ubiquitin-dependent proteasomal degradation of overexpressed oncogenic SIRT1  in an HCT116 cancer cell line. This finding suggested that curcumin could limit progression of CRC (Lee et al., 2018). The Wnt/β-catenin signaling cascade plays an important role in progression, proliferation, invasion, metastasis, and chemoresistance of CRC. In CRC, at least one mutation has been found in Wnt signaling regulator genes (Bahrami et al., 2017; Dou et al., 2017). In most cancers—including colon cancers—microRNA (miR)130a has been found to be dysregulated (Zhang, Tang, et al. 2017; Dou et al., 2017). Additionally, miR-130a is associated with the Wnt pathway, which is suppressed by naked cuticle  2 (Nkd2) inhibition. miR-130a promotes resistance to chemotherapeutic drugs such as cisplatin through overexpression of runt-related transcription factor 3 (RUNX3) in hepatic cancer cells (Xu et al., 2012). Duo et al. found that

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curcumin significantly suppressed expression of miR-130a, which modulated the Wnt signaling pathway and prevented proliferation and progression of colon cancer both in vitro and in vivo (Dou et al., 2017). Huang et al. (2017) first reported that homeobox protein CDX-2 (CDX2) works as a regulator protein in the gut homeostasis, development, and intestinal phenotype (Huang et al., 2017). A recent investigation revealed that CDX2 was also associated with the Wnt signaling pathway (Tóth et al., 2018). Suppression of CDX2 in colon cancer patients resulted in a more adverse prognosis and a diminished response to chemotherapy (Wang, Li, Li, Liu, & Han, 2018). Jiang et al. (2019) demonstrated that curcumin treatment significantly reduced the viability of SW620 human colonic cancer cells via upregulated expression of CDX2 and reduced Wnt signaling, which was established by inhibited expression of c-Myc (MYC proto-oncogene, bHLH transcription factor), survivin, β-catenin, Wnt3a, and cyclin D1. The master regulator p53 has great importance in cell cycle regulation and apoptosis (Sullivan & Lu, 2007). As a tumor suppressor protein, p53 plays an anticancer role through stimulation of expression of genes engaged in the apoptotic cascade and cell cycle regulation, inhibiting cancer progression and antineoplastic transformation (Kim & An, 2016; Lu et al., 2016). In a p53-mutated COLO 320DM (Dukes’ type C stage–derived) colon cancer cell line, curcumin was found to exert an antineoplastic effect through suppression of cell growth and proliferation, and induction of apoptotic cell death. Moreover, curcumin arrested the cell cycle in the G1 phase and reduced cell viability in the S phase (Dasiram, Ganesan, Kannan, Kotteeswaran, & Sivalingam, 2017). Many studies of curcumin have investigated the important role of the Hippo signaling pathway in maintaining the size of organs and tumor development (Wang & Wang, 2016). Sudol et  al. revealed a link with Yes-associated protein (YAP) and expression patterns in carcinogenesis (Sudol, 1994). YAP is associated with malignancy, activates cell proliferation, suppresses apoptosis, aids disturbance of cell contact inhibition, and promotes oncogenesis. A literature review demonstrated that patients with gastric cancer or liver cancer showed greater YAP protein expression and activity (Wang & Tang, 2015). Zhu et  al. found that curcumin actively suppressed YAP protein expression and upregulated autophagy and cell death. It can be concluded that curcumin could serve as a promising therapy for colon cancer patients (Zhu et al., 2018). Literature reports have suggested that oxidative decarboxylation of pyruvate occurs in prostate, lung, and breast malignancy cells, and they have additionally proposed that suppressed regulation of pyruvate dehydrogenase kinase 4 (PDK4) provides raw material for a drug resistance phenotype (Sun et al., 2014). Breast cancer cell resistance to tamoxifen and colon cancer cell resistance to 5-FU correlate with greater expression of PDK4  in comparison with naive cells (Walter et  al., 2015; Zhang et al., 2016). In CRC, an αvβ3 receptor (an integrin family member) has been found to be overexpressed (Bohanes et al., 2015). Curcumin was shown to suppress expression of αvβ3 integrin and elevate expression of the PDK4 gene in SW480 colon cancer cells (Javadi, Rostamizadeh, Hejazi, Parsa, & Fathi, 2018), which facilitates switching to the Krebs cycle and apoptotic cascade activation (Sun et al., 2014).

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Curcumin has also been shown to have potential for prevention of cancer through destruction of malignant cells via suppression of chemotherapy-induced resistance, sensitization to therapeutic drugs and/or radiation, and induction of apoptosis. For treatment of colon cancer, radiotherapy alone or a combination of radiotherapy and chemotherapy are preferred, but resistance to individual chemotherapeutic drugs can develop. The mechanism behind the development of resistance is unclear, but it has been hypothesized that it may involve NF-κB and its regulatory gene products. Curcumin has been shown to increase sensitivity to radiation in CRC-affected xenograft nude mice and to potentially suppress NF-κB activity, its regulatory gene products, angiogenesis, and the Ki-67 proliferation index (Goel & Aggarwal, 2010; Kunnumakkara et al., 2008).

5  U  se of Curcumin Analogues in Colon Cancer Prevention and Therapy Various in  vivo and in  vitro studies of synthetic or semisynthetic curcumin analogues have demonstrated that they have increased tumor-suppressive effects by efficiently targeting various biosynthetic and regulatory pathways in different cancers, including colon cancer, prostate cancer, skin cancer, liver cancer, and stomach cancer. Curcumin analogues definitely have improved viability as chemopreventive and remedial agents in colon malignancy. Recent studies on the efficiency of curcumin analogues are described in this section. In a study by Zhou et al. (2014), a curcumin analogue containing inden-2-one exhibited anticancer effects on prostate cancer PC-3 cells, colon cancer HT-29 cells, pancreatic cancer BxPC-3 cells, H1299 lung cancer cells, and nontumorigenic RWPE-1 human prostate epithelial cells. Inden-2-one has an aromatic ring that exhibits antiproliferative activity. Another analogue of curcumin, IND-4 [(1E,3E)-1,3-bis(3,4,5-trimethoxybenzylidene)-1,3-dihydroinden-2-one], showed potent antineoplastic activity against several cancer cell lines, and its cytotoxic activity was about 20 times greater than that of conventional curcumin (Link et al., 2013). IND-4 facilitated increased interaction with DNA and decreased steric hindrance (Link et al., 2013). Derivatives of the curcumin analogue 3,5-bis(benzylidene)-4-piperidone showed anticancer activity in an HCT116 colon cancer cell line, increasing intracellular ROS levels and diminishing oxygen consumption and mitochondrial membrane potentials. These properties make these derivatives excellent antiproliferative agents for use against most cancer types (Helal et al., 2013). 3,5-Bis(arylidene)-4-piperidones, which are dimeric analogues of curcumin, showed cytotoxic effectiveness against HCT116 and HT-29 colon cancer cell lines. Because they contain amidic carbonyl groups, a low concentration of 3,5-bis(arylidene)-4-piperidones (in the nanomolar to molar range) was found to be sufficient to show cytotoxic activity in comparison with curcumin. It has also been suggested that the presence of strong electron-withdrawing groups in addition to an

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aryl ring increases the efficiency of these curcumin analogues (Das, Michel, Gorecki, & Dimmock, 2013). Kim et al. (2014) synthesized curcumin analogue curcuminoids that exerted their antiproliferative effects via the histone demethylase Jumonji domain (JMJD) 2C in colorectal carcinoma HCT116 cells. The researchers reported that curcuminoid treatment increased expression of JMJD2C, which induced apoptosis and suppressed growth and proliferation of colon cancer cells. Lin et al. (2011) reported that a new curcumin analogue, GO-Y030, potentially suppressed growth of colon cancer. GO-Y030 suppressed phosphorylation of STAT3 by executing cleavage of PARP and caspase-3, inducing the apoptosis cascade, reducing cancer cell survival, and suppressing tumorsphere formation. Anthwal et al. (2014) investigated C-5, another curcumin analogue with cytotoxic properties. They compared the cytotoxic efficiency of conventional curcumin and C-5 in an HCT116 colorectal carcinoma cell line and a KBM-5 chronic myeloid leukemia cell line. It was found that at a low concentration of 5 μM, C-5 had significantly greater cytotoxicity than curcumin. These researchers also reported that substitution of the methoxy group in the aromatic ring increased the activity of C-5. Chen, Liu, Chen, and Xu (2011) synthesized a curcumin analogue, C086, and demonstrated its cytotoxic efficiency against HT-29, SW1116, SW480, KM12, WiDr, and murine colon-26 cancer cell lines. They reported that C086 exerted a cytotoxic effect, in a dose- and time-dependent manner, against SW480 and KM12 colorectal cancer cell lines. They found that this novel curcumin analogue exerted anticarcinogenic effects by suppressing inhibitor of nuclear factor κBα (IκBα) phosphorylation, which prevented activation of NF-κB transcription factor, culminating in induction of apoptosis. The cytotoxic efficiency of C086 was 5–7 times that of curcumin. Lai et al. (2011) reported that the curcumin metabolite tetrahydrocurcumin was more effective than curcumin. Tetrahydrocurcumin showed antineoplastic activity by efficiently inhibiting dysregulated expression of Wnt-1, β-catenin, and glycogen synthase kinase (GSK)-3β phosphorylation in colorectal cancer. Moreover, tetrahydrocurcumin suppressed connexin-43 protein levels and prevented colonic polyps, as well as gap junction formation. Wichitnithad, Nimmannit, Wacharasindhu, and Rojsitthisak (Wichitnithad, Nimmannit, Wacharasindhu, & Rojsitthisak, 2011) developed succinyl curcuminoid derivatives as prodrugs and demonstrated their antiproliferative activity against Caco-2 cells. These prodrugs exhibited cytotoxic activity at a lower concentration than curcumin, with a cytotoxic inhibitory concentration (IC50) value of 1.8–9.6 μM. Yogosawa et  al. (2012) synthesized a curcumin analogue, dehydrozingerone, which showed effective anticlonogenic cancer activity against an HT-29 human colon cancer cell line. Dehydrozingerone exerted its antiproliferative effect by arresting the cell cycle in the G2/M phase, activated apoptosis by increasing intracellular ROS levels, and elevated expression of p21. Basile et  al. (2013) studied the curcumin analogue bis-dehydroxy-curcumin. They noted that it was cytotoxic, induced apoptosis in colon cancer, exhibited an antiproliferative mechanism in G2/M  phase cell cycle arrest, and decreased the mitochondrial transmembrane potential.

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Difluorinated curcumin (CDF) was studied by Roy, Yu, Padhye, Sarkar, and Majumdar (2013), using HCT116 and HT-29 colon cancer cell lines, and significantly reduced miR-21 expression, increased phosphatase and tensin homolog (PTEN) levels, and inhibited Akt phosphorylation. The mechanisms of action of CDF make it an efficient anticancer agent. Srimuangwong, Tocharus, Tocharus, Suksamrarn, and Chintana (Srimuangwong, Tocharus, Tocharus, Suksamrarn, & Chintana, 2012) evaluated a hexahydrocurcumin analogue, which exerted an antiproliferative effect against HT-29 colorectal cancer. It efficiently reduced COX-2 mRNA transcription and translation, preventing cancer cell growth and proliferation. A study performed by Devasena, Menon Venugopal, and Rajasekaran (2005) analyzed the effect of a bis-demethoxy-curcumin analogue (BDMC-A) against colon cancer induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats. Rats treated with BDMC-A showed reduced levels of stress induced by DMH, and BDMC-A showed chemopreventive effects and exerted more efficient modulatory effects than curcumin against colon carcinoma. Many studies have reported that growth, proliferation, and metastasis of colon cancer is regulated by various microRNAs, such as miR-21 (Roy et  al., 2013), miR-­27a, miR-20a, and miR-17-5p (Gandhy, Kim, Larsen, Rosengren, & Safe, 2012). The curcumin analogues CDF (Roy et al., 2013) and RL-197 (Gandhy et al., 2012) efficiently inhibited expression of all of these miRNAs and induced apoptosis in HCT116, HT-29, and SW480 colon cancer cell lines. CDF treatment restored PTEN expression, while RL-21 treatment decreased levels of transcription factors (Sp1, Sp3, and Sp4) and increased intracellular ROS levels. Zhang, Feng, et al. (2017) formulated a curcumin derivative compound named 1-(4-hydroxy-3-methoxyphenyl)-5-(2-­nitrophenyl)penta-1,4-dien-3-one (WZ35), which showed promising antineoplastic activity against a CT26 colorectal cancer cell line, as well as in a CT26 xenograft mouse model. They reported that WZ35 induced the apoptotic cascade by inducing cell cycle arrest in the G2/M phase. WZ35 significantly increased intracellular ROS levels and endoplasmic reticulum (ER) stress in colon cancer. The curcumin analogue EF24, which showed active antitumor activity, was assessed by He et al. (2016). EF24 induced ROS-dependent antitumor activity in HCT116, SW620, and HT-29 colon cancer cell lines. Treatment with EF24 efficiently arrested the cell cycle in the G2/M phase and induced accumulation of intracellular ROS. This activity triggered the apoptotic cascade and efficiently decreased the survival of colon cancer cells in comparison with conventional curcumin even at low concentrations/doses. Sesarman et  al. (2018) studied the CURC-DOX (PEGylated long-circulating liposomes codelivering curcumin and doxorubicin) analogue for its modulatory effects on angiogenesis, apoptosis, and inflammation in a CT26 colon cancer cell line. CURC-DOX efficiently exerted cytotoxic effects through modulation of many regulatory genes, such as the genes encoding vascular endothelial growth factor (VEGF), TNF, IL-12 p40, and IL-6. It also inhibited expression of protein IL-1α, MCP1, and tissue inhibitor of metalloproteinase (TIMP)-2, which is associated with proinflammatory/proangiogenic proteins.

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Sufi et al. (2017) synthesized the curcumin analogue ICA [(1E,6E)-1,7-di(1H-­ indol-­3-yl)hepta-1,6-diene-3,5-dione] and reported its anticancer activity against three human cancer cell lines: a SW480 colon cancer cell line, a K562 leukemia cell line, and an A549 lung cancer cell line. They reported that ICA actively suppressed the cells’ viability. An in silico predictive analysis revealed that ICA actively interacted with GSK-3β kinase in SW480 colon cancer cells. Furthermore, ICA modulated Bcl-2 expression, which induced apoptosis. A cell cycle study also suggested that ICA induced arrest of the cell cycle in both the G0/G1 and G2/M phases. It can therefore be said that ICA is an effective anticancer derivative of curcumin. Recently, Ozawa-Umeta et  al. (2020) formulated a new curcumin analogue, curcumin-β-D-glucuronide (CMG), which has a water-soluble property. CMG showed a cytotoxic effect by reducing NF-κB activity in a CRC xenograft mouse model with resistance to L-OHP. A pharmacokinetic study also suggested that CMG had greater bioavailability and better distribution in malignant tissue than in the blood and major organs. Prabhat et al. (2019) reported that 4,4′-disulfonyldiarylidenyl piperidone (DAP) had novel and efficient antitumor activity consistent with those of other curcumin analogues. DAP showed promising anticancer activity against colon cancer (Prabhat et al., 2019) and activated oxidative stress through formation of free radicals. DAP also protected healthy cells by a scavenging process in which cancerous cells underwent apoptotic death. Meiyanto, Septisetyani, Larasati, and Kawaichi (2018) found that the curcumin analogue pentagamavunon (PGV)-1 was a strong antiproliferative agent. They reported that PGV-1 effectively inhibited growth and proliferation of tumor cells, including those of colon cancer origin. PGV-1 induced cell cycle arrest in the G2/M phase, while coadministration with 5-FU showed synergism and induced cell cycle arrest in the G2/M and S phases. Moreover, PVG-1 attenuated expression of NF-κB and COX-2, and activated apoptosis in WiDr colon cancer cells (Meiyanto et al., 2018). Table 1 summarizes the different curcumin analogues and their possible mechanisms of action. Akt protein kinase B, Bcl B cell lymphoma, BDMC-A bis-demethoxy-curcumin analogue, CDF difluorinated curcumin, CMG curcumin β‐D‐glucuronide, COX cyclooxygenase, CURC-DOX PEGylated long-circulating liposomes codelivering curcumin and doxorubicin, DAP 4,4′-disulfonyldiarylidenyl piperidone, DMH 1,2-dimethylhydrazine, ER endoplasmic reticulum, GSK glycogen synthase kinase, ICA (1E,6E)-1,7-di(1H-indol-3-yl)hepta-1,6-diene-3,5-dione, IκBα inhibitor of nuclear factor κBα, IL interleukin, IND-4 (1E,3E)-1,3-bis(3,4,5-­ trimethoxybenzylidene)-1,3-dihydroinden-2-one, JMJD Jumonji domain, MCP monocyte chemoattractant protein, miR microRNA, MMP matrix metalloproteinase, mRNA messenger RNA, NF nuclear factor, PARP poly (ADP-ribose) polymerase, PTEN phosphatase and tensin homolog, ROS reactive oxygen species, STAT signal transducer and activator of transcription, TIMP tissue inhibitor of metalloproteinase, TNF tumor necrosis factor, VEGF vascular endothelial growth factor, WZ35 1-(4-hydroxy-3-methoxyphenyl)-5-(2-nitrophenyl)penta1,4-dien-3-one

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Table 1  Different curcumin analogues and their effects on colon cancer signaling pathways Curcumin analogues Inden-2-one IND-4

Mechanisms of action Antiproliferative activity Potent antineoplastic activity through increased interaction with DNA 3,5-Bis(benzylidene)-4-­ Inhibition of cell proliferation, increases in piperidone intracellular ROS levels, decreases in MMP levels 3,5-Bis(arylidene)-4-­ Increase in cytotoxicity piperidone FLLL‑7, ‑8, ‑24, ‑32, Increase in JMJD2C histone demethylase ‑59, and ‑60 expression, induction of apoptosis, growth suppression GO-Y030 Suppression of STAT3 phosphorylation through cleavage of PARP and caspase-3, induction of apoptosis C-5 Increase in cytotoxicity C086 Suppression of IκBα phosphorylation, inhibition of NF-κB activation and protein binding, inactivation of TNFα-induced activation Tetrahydrocurcumin Inhibition of dysregulated expression of Wnt-1 and β-catenin; inhibition of GSK-3β phosphorylation; suppression of connexin-43, prevention of colonic polyps and gap junction formation Succinyl curcuminoid Increase in cytotoxicity derivatives Dehydrozingerone Cell cycle arrest in the G2/M phase, activation of apoptosis through increases in intracellular ROS levels, increase in p21 expression Bis-dehydroxy-­ Cell cycle arrest in the G2/M phase, decreases curcumin in MMP levels CDF Inhibition of miR-21 expression, restoration of PTEN expression, inhibition of Akt phosphorylation, increases in intracellular ROS levels Hexahydrocurcumin Reductions in COX-2 mRNA transcription and translation BDMC-A Chemoprevention, reduction in DMH-induced oxidative stress RL-197 Inhibition of miR-27a, miR-20a, and miR-­ 17-­5p expression; decreases in transcription factors (Sp1, Sp3, and Sp4); induction of apoptosis WZ35 Cell cycle arrest in the G2/M phase, increases in intracellular ROS levels and ER stress

References Zhou et al. (2014) Link et al. (2013) Helal et al. (2013)

Das et al. (2013) Kim et al. (2014)

Lin et al. (2011)

Anthwal et al. (2014) Chen et al. (2011)

Lai et al. (2011)

Wichitnithad et al. (2011) Yogosawa et al. (2012) Basile et al. (2013) Roy et al. (2013)

Srimuangwong et al. (2012) Devasena et al. (2005) Gandhy et al. (2012)

Zhang, Feng, et al. (2017) (continued)

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Table 1 (continued) Curcumin analogues EF24 CURC-DOX

ICA

CMG DAP Pentagamavunon-1

Mechanisms of action Cell cycle arrest in the G2/M phase, induction of intracellular ROS accumulation Modulation of VEGF, TNFα, IL-12 p40, IL-6 regulatory genes; inhibition of IL-1α, MCP1, and TIMP-2 protein expression Inhibition of GSK-3β effect, modulation of Bcl-2 expression, cell cycle arrest in the G0/G1 and G2/M phases Reduction in NF-κB activity, increases in bioavailability and distribution Formation of free radicals Cell cycle arrest in the G2/M and S phases, inhibition of NF-κB and COX-2 expression

References He et al. (2016) Sesarman et al. (2018) Sufi et al. (2017)

Ozawa-Umeta et al. (2020) Prabhat et al. (2019) Meiyanto et al. (2018)

6  C  olon Cancer Prevention by Curcumin Through Epigenetic Modifications Recent research on epigenetic changes has provided breakthroughs in our understanding of the mechanism of progression and proliferation of cancer. The results of many research investigations suggest that curcumin has the ability to modulate epigenetic changes and evolve into a promising therapeutic medicine to cure cancer, including colon carcinoma. Recent reports have suggested that DNA CpG methylation strongly correlates with progression of malignancy in colon cancer (Shinde et al., 2020). Curcumin has been shown to have great ability to modulate epigenetic modifications. Curcumin suppresses CpG methylation and exerts antiproliferative effects in HT-29 colon cancer (Guo, Shu, Zhang, Su, & Kong, 2015). Reduced expression of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) correlates with demethylation, which has also been observed in a DNMT1- and DNMT3B-silenced HCT116 colon cancer cell line (Ying et  al., 2009). In colon cancer tissue sections, hypermethylation was observed in MLH1 (Boland & Goel, 2010; Westwood et al., 2019). Jiang et al. reported that curcumin arrested the cell cycle in the G2/M phase and induced apoptosis in MLH1-silenced HCT116 and RKO cell lines (Jiang, Jin, Yalowich, Brown, & Rajasekaran, 2010). Furthermore, curcumin was shown to activate hypermethylation in the MLH1 promoter and to induce microsatellite instability (MSI) disruption (Boland & Goel, 2010; Jiang et al., 2010). Guo et al. (2018) revealed that changes in DNA via CpG methylation are associated with colorectal cancer, which can be reverted by curcumin treatment. Differentially expressed and differentially methylated genes in pairwise comparisons and various pathways—such as lipopolysaccharide (LPS)/IL-1–mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, NO and ROS production in macrophages, and IL-6 signaling—are likely to be impacted by the antitumor actions of curcumin.

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Heat shock proteins (HSPs) are associated with most cancers’ endurance and facilitate their growth, proliferation, and drug resistance (Trieb, Sulzbacher, & Kubista, 2016). In particular, HSP27 is involved in MDR activity (Trieb et al., 2016; Xu, Yang, Fang, Xu, & Chen, 2014). Research has revealed that HSP27 induces expression of the drug-expelling ABC transporter in cancer cells (Kanagasabai, Krishnamurthy, Druhan, & Ilangovan, 2011; Xu et al., 2014). In resistance to doxorubicin (Adriamycin) in human breast cancer cells, inhibition of HSP27 switched off drug efflux driven by P-gp protein and ABCB1 gene expression (Kanagasabai et al., 2011). Recent research has shown that in DLD-1 and HT-29 colon cancer cell lines, the effect of curcumin (ROS production, apoptosis, phagocytic activity) was suppressed by HSP27 silencing (Liang et al., 2018). Furthermore, curcumin treatment with or without coadministration of 5-FU significantly decreased survival of an HCT-8 CRC cell line by reducing HSP27, and it was found that HSP27 and P-gp were interdependent (He et al., 2019). In colon cancer, many genes have been shown to have altered expression; for example, MIR491 (microRNA 491) shows decreased expression, whereas PEG10 (paternally expressed 10), CTNNB1 (catenin beta 1), and WNT1 (Wnt family member 1) show increased expression (Li, Shi, Li, Zhao, & Wang, 2018). In an HCT116 colon cancer cell line, curcumin induced expression of miR-491 and suppressed PEG10 activity. Curcumin also inhibits the Wnt/β-catenin signaling pathway, which facilitates induction of apoptosis and suppression of cancer cell proliferation (Li et al., 2018). Administration of curcumin upregulates mRNA expression of the NFE2L2 (nuclear factor, erythroid  2 like  2), HMOX1 (heme oxygenase  1), and NQO1 (NAD(P)H quinone dehydrogenase 1) antioxidant genes, whereas it downregulates the expression of the HDAC1, HDAC2, HDAC3, and HDAC4 genes involved in epigenetic histone deacetylation in leucocytes (Gibbons, 2005). Curcumin significantly reduces expression of HDAC genes and inhibits HDAC more effectively than valproic acid and sodium butyrate (Bora-Tatar et  al., 2009). Moreover, curcumin administration inhibits expression of HDAC1, HDAC3, and HDAC8 proteins and induces H4 histone acetylation (Bora-Tatar et al., 2009).

7  Curcumin Drug Delivery in Colon Cancer After oral intake, curcumin is quickly digested in the intestine and hepatic tissue, and 60–70% is then excreted in the feces. Therefore, substantial intake of curcumin is required to achieve a therapeutic effect. Administration of curcumin via novel drug delivery systems involving liposomes, micelles, and nanoparticles has been recommended and could increase the therapeutic actions of curcumin against growth of colorectal malignancies. Singh, Sharma, and Gupta (2015) demonstrated that curcumin–silica nanoparticle complexes were more cytotoxic than curcumin. The same type of curcumin– silica nanoparticle complex conjugated with hyaluronic acid was found to be more

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effective than curcumin in a COLO 205 colon cancer cell line. Both nanoparticle complexes significantly decreased the survival of cancer cells and increased curcumin uptake into cells, as well as into tumor spheroids. Hyaluronidase facilitated separation of curcumin from the nanoparticles in the target system. Alizadeh et al. (2012) formulated polymeric nanocarrier curcumin (PNCC) and compared its anticarcinogenic efficiency with that of free curcumin. PNCC reportedly achieved 76.7% suppression of tumor size, elevated expression of the proapoptotic regulatory protein Bax, and suppressed expression of the antiapoptotic regulatory protein Bcl-2. PNCC was also significantly more effective in decreasing the Bcl-2/Bax ratio than free curcumin. Administration of thiolated chitosan nanoparticles loaded with PNCC and 5-FU showed greater distribution efficiency, a longer half-life, and therapeutic action in a time- and concentration-dependent manner in tumor tissues. Combination therapy, using nanoparticles loaded with 5-FU and curcumin, demonstrated greater efficiency in suppressing cancer cell survival via induction of cell cycle arrest in the G2/M and S phases (Anitha, Deepa, Chennazhi, Lakshmanan, & Jayakumar, 2014). Another combination of curcumin and 5-FU loaded into N,O-­ carboxymethyl chitosan nanoparticles inhibited survival of an HT-29 colorectal cancer cell line. This combination exerted cytotoxic effects by modulating the central pathway of p53 and cytotoxic metabolite pathways (Anitha, Sreeranganathan, Chennazhi, Lakshmanan, & Jayakumar, 2014). Curcumin-loaded dendrosome nanocarriers (DNCs) have increased solubility in water, enabling the curcumin to reach the target tissue. Curcumin-loaded DNCs were shown to suppress the survival of colon cancer SW480 cells and their adhesion of to a matrix (Dehghan Esmatabadi et al., 2015). DNCs incorporating curcumin also exerted cytotoxic effects in a time- and concentration-dependent manner and downregulated expression of the CLDN1 (claudin 1), NEDD9 (neural precursor cell expressed, developmentally down-regulated  9; previously known as HEF1), and ZEB1 (zinc finger E-box binding homeobox 1) genes. Curcumin-loaded poly(ethylene glycol methyl ether methacrylate) (PEGMEMA)–based micelles were formulated using the reversible addition−fragmentation chain-transfer (RAFT) polymerization technique (Chang, Trench, Putnam, Stenzel, & Lord, 2016). It was reported that these micelles had the capacity to work in a dose-dependent manner and directly interacted with cancer cells. The micelles were thermodynamically stable, allowing efficient cellular internalization, and inhibited cell growth and metastasis in a WiDr colon cancer cell line (Chang et al., 2016). Another study on curcumin conjugates with PLGA [poly(lactic-co-glycolic acid)], linked via ester linkage, reported that they had good ability to suppress colon cancer survival (Waghela, Sharma, Dhumale, Pandey, & Pathak, 2015). It was also shown that the PLGA–curcumin ester-linked conjugates exerted antiproliferative effects more efficiently than free curcumin through increased cellular internalization and balanced release, in a dose- and time-dependent manner. This efficiently suppressed growth and proliferation of HCT116 and HT-29 colon cancer cell lines.

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Li et al. (2014) synthesized curcumin-loaded PLGA–lecithin–PEG nanoparticles by using a slightly modified nanoprecipitation technique. These nanoparticles were then bioconjugated with aptamers (Apt), which were linked with an amide linkage to form Apt-CUR-NP bioconjugates. The characteristics of the Apt-CUR-NP bioconjugates (accessible release and improved bioavailability and cellular internalization) meant they had good ability—64 times that of free curcumin—to suppress an HT-29 colon cancer cell line. One more study on curcumin-loaded nanoparticles, formed by incorporation of curcumin with camptothecin CPT/CUR-NPs using emulsion–solvent evaporation techniques, was done (Xiao et al., 2015). The CPT/CUR-NPs showed improved cellular internalization and inhibited malignant cell proliferation by modulating expression of Bcl-2 mRNA in a colon-26 cancer cell line (Xiao et al., 2015).

8  C  urcumin as a Modulator of the Gut Microbial Environment The role of the intestinal microbiota has received great attention in the last decade for modulation of the conditions of various diseases, especially those of the gastrointestinal tract. In brief, the intestinal microbiome consists of a diverse microbial community, which consists mainly of different classes of bacteria, fungi, viruses, and other rare species (Cani, 2018). It has been established that intestinal microbial species play a significant role in the host’s physiology, performing many basic metabolic, fundamental, and defensive functions that contribute to the host’s well-­being. Likewise, any disturbance in the microbiome profile or dysbiosis may have important ramifications for people at risk of developing different diseases, including colorectal cancer (Gopalakrishnan, Helmink, Spencer, Reuben, & Wargo, 2018). It has been seen that the intestinal microbiota and its metabolic products greatly influence immune regulatory responses; hence, they also influence chronic inflammation. For example, butyrate (a short-chain unsaturated fatty acid) has the ability to inhibit HDAC activity and exert antiproliferative effects by modulating different malignancy proliferative signaling pathways, including the VEGF and JAK/STAT3 pathways. Moreover, butyrate produced by gut microbes effectively attenuates the inflammatory response induced by T lymphocytes and downregulates expression of NF-κB, as well as the STAT3 pathway (Chen, Zhao, & Vitetta, 2019). In the context of the influences of the intestinal microbiome, curcumin can play a significant role. Research on intake of curcumin by animals and humans has demonstrated that curcumin stabilizes the balance between pathogenic and beneficial microbes. It has been established that curcumin attenuates inflammation by regulating intestinal microbes. A study on the effect of curcumin treatment in a dextran sulfate sodium (DSS)–induced colitis model observed inhibition of NF-κB expression and upregulated expansion of CD4+ Foxp3+  regulatory T  cells, leading to a

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reduction in the inflammatory response (Ohno et al., 2017). Interestingly, the proportions of the butyrate-producing bacteria Clostridium cluster IV and subcluster XIVa were increased by curcumin, as were the numbers of Treg cells and fecal butyrate levels (Ohno et al., 2017). It has been reported that some members of the microbiome, such as Ruminococcus-­ like species, are strongly associated with the occurrence of CRC, but the exact mechanism of this association remains unknown. A dietary study demonstrated that use of curcumin as a dietary supplement may increase the proportions of beneficial microbes such as Clostridium and Enterobacter while hindering the growth of Blautia and Ruminococcus-like species (Mori et al., 2019; Peterson et al., 2018). McFadden et al. (2015) investigated the correlation between the gut microbial profile and CRC incidence, and reported that the effect of curcumin on intestinal microbes mediated inhibition of colon cancer cell survival. They found that Lactobacillus abundance was very low in mice treated with the mutagenic agent azoxymethane (AOM), but curcumin treatment significantly increased the Lactobacillus population in the gut microbiota (McFadden et  al., 2015). These examinations confirmed that dietary curcumin can promote advancement of exceptional microbial species within the digestive system to improve their cancer suppressive behavior in humans.

9  Immunomodulatory Effects of Curcumin in Colon Cancer Colon cancer and immune cell modulation are closely associated with each other. In patients with colon cancer, many immune cells, molecules, and their signaling are modulated and either upregulated or downregulated. Several research investigations have reported that curcumin has potential modulatory effects on differentiation and functional activation of immune cells. These effects may hinder colon cancer growth and prevent colon malignancy. Inflammation is viewed as one of the fundamental causes of the genesis of CRC. Inflammatory cells routinely invade tumors in a process called tumor-elicited inflammation and advance tumor development. In research conducted in recent decades, curcumin has perhaps been one of the most impressive natural remedies that has been investigated, in that it can prevent inflammation through its effects on signaling pathways. The findings of this research largely support its role in prevention and treatment of CRC. Various examinations have shown that hyperactivity of Toll-like receptor (TLR) 4 is strongly associated with development of colorectal cancer (Yesudhas, Gosu, Anwar, & Choi, 2014). In these specific circumstances, an innate immune response is promoted by TLR4 receptors, further regulating inflammatory events. Moreover, TLR4 is known to recognize LPS. TLR4 regulates the MyD88, NF-κB, and AP-1-­ type transcription factors. These altered molecular partners of TLR4 have differential expression in CRC (Lu, Yeh, & Ohashi, 2008). Recent investigations have confirmed that curcumin inhibits inflammation induced by LPS by suppressing the

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signaling of TLR4/MyD88/NF-κB (Fu et al., 2014). Additionally, curcumin acts as a barrier to prevent nuclear translocation of the NF-κB subunit, which triggers other proinflammatory genes. Moreover, curcumin suppresses the kinase activity of IκB, which is essential for NF-κB expression (Jobin et  al., 1999). Curcumin has also been shown to have inhibitory effects on TNFα, IL-1β-induced p38, and activation of JNK, which induces cytotoxic activity in HT-29 cells (Moon et al., 2006). During inflammatory responses, COX-2, which is also known as prostaglandin G/H synthase 2, increases synthesis of prostaglandin. Different studies have suggested that COX-2 is closely associated with CRC progression (Cherukuri et al., 2014; Goel et al., 2001), and inhibition of COX-2 expression by curcumin is mediated by interruption of NF-κB expression in colon cancer. Macrophages can create numerous inflammatory cytokines or chemokines to trigger and advance inflammation. Curcumin suppresses TLR4 pathways in macrophages and attenuates expression of inflammatory cytokines, including IL-6, IL-12, and TNFα (Gao et al., 2015; Mohammadi et al., 2019). Additionally, macrophages treated with curcumin have an increased capacity for antigen processing and presentation through the mannose receptor. Likewise, repolarization or depolarization of macrophages facilitates the M2 phenotype in response to curcumin treatment, significantly inhibiting mucosal inflammation (Gao et  al., 2015; Mohammadi et al., 2019). Bereswill et al. (2010) observed that curcumin administration increased active T  cell populations by 20–30% and significantly reduced cancer proliferation, accompanied by increases in levels of the anti-inflammatory cytokine IL-10 in the colon region. Curcumin also significantly reduced the levels of pro-inflammatory cytokines (IL-23 p19, IFNγ, TNFα, IL-6, and MCP1). Curcumin intake was also shown to increase IgA levels in the colon region (Okazaki et al., 2010). Moreover, curcumin administration was found to upregulate expression of IL-1β and reduce elevated expression of IL-6, TNFα, and chemokine ligand (CCL) 2 (Murphy, Davis, McClellan, Gordon, & Carmichael, 2011). Xu, Yu, and Zhao (2017) found that curcumin treatment markedly inhibited the Treg population while significantly augmenting the Th1 cell population in the peripheral system. Findings from various investigations revealed that curcumin reduced expression of Foxp3 in Tregs and IFNγ in CD4+ T cells. Th1 cells are considered antitumor entities. Inhibition of Tregs by curcumin was reported to disrupt the immunoevasion strategy of the tumor and induce apoptosis in colon cancer (Lai et al., 2015; Xu et al., 2017). Another study found that curcumin caused generation of superoxide anions in HCT116 colon cancer cells and affected checkpoint kinase (Chk) 2 phosphorylation at threonine (Thr) 68, culminating in apoptotic cell death (Watson et al., 2010). A recent investigation by Wang et al. (Wang, Ghosh, & Ghosh, 2017; Wang, Li, et al., 2017) indicated that curcumin treatment significantly inhibited LPS-induced IL-1β levels in colon cancer cell lines (Caco-2 and HT-29 cells) and a THP-1 leukemia cell line. Cytokine IL-1β secretion induced by LPS activated the p38 and MAPK signaling pathway and increased myosin light chain kinase expression. This activated the junction protein and disrupts the normal regulatory arrangement.

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10  Conclusion Colon cancer continues to be a major public health burden. Several factors—from genetics to epigenetics to diet—contribute to the incidence of this malignancy. Although there have been significant advances in development of targeted therapies, the 5-year prognosis for metastatic CRC is about 15% (Howlader, Noone, Krapcho, et  al., 2017). With our increased understanding of the molecular and epigenetic/ epigenomic changes that occur during CRC development (Shinde et al., 2020), we will be able to develop new and more precise therapies, such as relatively nontoxic dietary cancer chemopreventive regimens that include curcumin alone or in combination with other relatively nontoxic drugs, such as NSAIDs, to prevent and/or treat new inflammation-mediated colon cancers in humans. Considering the diverse roles of curcumin in tumor biology—ranging from metabolic alteration to genetic regulation, phenotypic modulation, immunoregulation, chemosensitization, and aggressive behavior—there are promising prospects for clinical utilization of curcumin in antineoplastic therapeutic schemes. Established evidence supporting the anticancer activity of curcumin, even in colon malignancies, provides a path for its exploitation for human well-being. Although a few limitations impede the efficacy of curcumin (such as its low bioavailability, low solubility in water, and poor biodistribution), strategies are being tested to improve the usefulness of this golden spice component. The absence of any side effects of curcumin utilization also makes this wonderful pigment of turmeric a forerunner in the list of candidate compounds with anticancer activities. Its potential to rectify other nonmalignant disorders also contributes to its suitability for therapeutic applications. As malignancies often give rise to other physiological and immunological ailments, the multiple efficacies of curcumin will come in handy. Colon cancer patients are known to suffer from physiological upsets and associated illnesses. Utilization of curcumin alone or as an adjuvant will provide benefit to patients with malignant disorders. The effectiveness of curcumin in helping to maintain a state of wellness also makes it a promising prophylactic agent for prevention of malignancies, even those of colorectal origin. The immunostimulatory and antimicrobial activities of curcumin also help to prevent undesirable inflammatory events. The antimicrobial activity of curcumin should reduce the frequency and magnitude of infection-driven inflammation. It has been established that gastrointestinal infections can drive carcinogenic events through the onset of inflammation. Thus, curcumin could blunt the driving force behind oncogenesis by limiting events of microbial infection. The immunoregulatory effect of curcumin should also help to clear any established infection and thereby contribute indirectly to prevention of tumor initiation. Molecular targets of curcumin confirm its anti-inflammatory activity and make this yellow chemical useful against several inflammatory disorders, including malignancies. Collectively, it can be argued that curcumin has proven benefits in curative as well as preventive management of malignant disorders. Its abilities can be exploited against colorectal hyperplastic and malignant disorders. Ongoing clinical evaluation of curcumin for its utility as a standalone anticancer agent and as a chemosensitizing agent will provide further support for its applications. Furthermore, curcumin can ameliorate the side effects associated with colon malignancies.

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Role of Chemokines in Colorectal Cancer Manisha Mathur, Sonal Gupta, Beiping Miao, Prashanth Suravajhala, and Obul Reddy Bandapalli

Abstract  Colorectal cancer (CRC) is one of the most common causes for cancer-­ related deaths worldwide. Chemokines, structurally related cytokines, are present in organs such as the lymph nodes, bone marrow, liver, and lungs. These are upregulated when normal colonic mucosa becomes cancerous and the cancer becomes more malignant as in the case of CRC. In the last few decades, it has emerged that chemokine and their receptor system represent a potential target for immunotherapy of CRC. Keywords  Colon cancer · Chemokine and receptors · Cancer immunotherapy · Potential biomarker

M. Mathur Advance Milk Testing Research Laboratory, Post Graduate Institute of Veterinary Education and Research (RAJUVAS), Jaipur, Rajasthan, India S. Gupta Department of Biotechnology and Bioinformatics, Birla Institute of Scientific Research (BISR), Jaipur, Rajasthan, India Department of Paediatrics, Sawai Man Singh Medical College, Jaipur, Rajasthan, India B. Miao Hopp Children’s Cancer Center (KiTZ), Heidelberg, Germany Division of Pediatric Neuro Oncology, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany P. Suravajhala Department of Biotechnology and Bioinformatics, Birla Institute of Scientific Research (BISR), Jaipur, Rajasthan, India O. R. Bandapalli (*) Hopp Children’s Cancer Center (KiTZ), Heidelberg, Germany Division of Pediatric Neuro Oncology, German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Heidelberg, Germany Medical Faculty, University of Heidelberg, Heidelberg, Germany e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4_19

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Abbreviations COX-2 Cyclooxygenase-2 CRA Colorectal adenoma CRC Colorectal adenocarcinoma CRC Colorectal cancer CRLM Colorectal liver metastases GPCR G-protein-coupled receptors MCP-1 Monocyte chemotactic protein 1 MMP Metalloproteinases MSCs Mesenchymal stem cells TEM Tumor microenvironment TRIM47 Tripartite motif 47

1  Introduction More than 1 million new cases of colorectal cancer (CRC) are diagnosed worldwide each year (Tenesa & Dunlop, 2009). Colorectal cancer is abnormal growth of cells that originates in the colon or the rectum. Most colorectal cancer begins as noncancerous growths called polyps that originate on the innermost layer of the colon or rectum. Some, but not all, polyps become cancerous tumors through inappropriate epithelial proliferative and antiapoptotic activity and spread through other layers (Jones, Chen, Parmigiani, et al., 2008). Eventually the tumor invades through the submucosa and muscularis propria, reaches nearby blood and lymph nodes or other parts of the body, and finally metastasizes (Jones et al., 2008). The progression from normal epithelium through adenoma to colorectal carcinoma is characterized and driven by multiple accumulated mutations of cancer genes (Bozic, Antal, Ohtsuki, et al., 2010). The patients diagnosed with CRC have gastrointestinal bleeding, confirmed by a positive fecal occult blood test, which often results in iron-­ deficiency anemia and frequently produces symptoms such as overt bleeding from the lower intestine and a change in defecation rhythm, for example, diarrhea or constipation (Gajewski & Szczeklik, 2018).

2  Mechanisms of Colorectal Cancer Colorectal cancer forms when the DNA in cells in the colon or rectum develops mutations that may make them unable to control growth and division. In many cases, these mutated cells die or are attacked by the immune system. But some mutated cells may escape the immune system and grow out of control, forming a tumor in the colon or rectum (Fig. 1).

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mutations in Smad-4,

Mutations in adenomatons polyposis coli (APC)

carcinoma

Intermediate adenoma Early adenoma

Late adenoma mutations in p53

Mutations in K-ras

Normal epithelium

Fig. 1  Mechanism of colorectal cancer (CRC). CRC caused by accumulation of mutations in oncogene (K-Ras) and tumor suppressor genes (APC, DCC, Smad-2, Smad-4, and p53)

Genetics

Family history Inherited Syndromes Racial and Ethnic Background Diet Inactive Lifestyle Smoking Alcohol Use

Lifestyle

General

Age History of colorectal cancer Obesity Type II diabetes

Fig. 2  Risk factors associated with the colorectal cancer

3  Potential Causes Associated with CRC Several risk factors in CRC development have been investigated. They have been divided into two groups: modifiable and nonmodifiable (Simon, 2016) (Fig.  2). While these risk factors are strongly linked to the diseases including poor diet, tobacco smoking, heavy alcohol use, and lack of physical activity, can also be

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considered as a cause. People with certain hereditary cancer syndromes or a family history of CRC and the presence of type 2 diabetes have nonmodifiable factors that have a high risk of developing the disease. An early diagnosis of CRC gives the best chance of curing it. Fecal testing, blood testing, sigmoidoscopy, colonoscopy, X-ray, and CT scan are the common screening tests for colorectal cancer.

4  Chemokines and Chemokine Receptors Chemokines are a family of small (8–10  kDa), secreted, and structurally related cytokines with a crucial role in inflammation and immunity (Griffith, Sokol, & Luster, 2014). These small peptides are classified, according to the position of their conserved cysteine residues near the N termini of these proteins, into four groups: C(2), CC(28), CXC(17), and CX3C(1) (https://en.wikipedia.org/wiki/Chemokine; Zlotnik & Yoshie, 2012) (Fig.3). Functionally chemokines can be divided into three groups: 1. Homeostatic: Control the migration of cells during the normal development and maintenance of tissues and lymphoid organs. 2. Inflammatory: Produced in response to infection or injury and directed the migration of leukocytes into the infected or damaged site. 3. Angiogenic: Some chemokines promote the development of blood vessels (pro-­ angiogenic), while others prevent the development of blood vessels (antiangiogenic). Over 48 chemokines and 19 chemokine receptors have been identified so far (https://en.wikipedia.org/wiki/Chemokine). Chemokine receptors are a group of seven-transmembrane G-protein-coupled receptors (GPCR). These are divided into four major groups according to the chemokine ligands with which they interact:

Fig. 3  Chemokines (small peptides) are classified according to the position of their conserved cysteine residues near the N termini of these peptides into four groups: C(2), CC(28), CXC(17), and CX3C(1)

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CC-chemokine receptor (CCR), CXC-chemokine receptor (CXCR), CX3C-­ chemokine receptor (CX3CR), and XCR (https://en.wikipedia.org/wiki/Chemokine; Murphy, Baggiolini, Charo, et al., 2000). Recent studies revealed that they play an important role in promoting cancer cell metastasis in many types of cancers (Balkwill, 2004; Koizumi, Hojo, Akashi, Yasumoto, & Saiki, 2007; Muller, Homey, Soto, et al., 2001; Tanaka et al., 2005).

5  Chemokine Receptors Work in Metastasis Chemokines are present in organs such as the lymph nodes, bone marrow, liver, and lungs. Cancer cells migrate through the body, sharing many similarities with the movement of white blood cells (leukocyte trafficking) in an immune response (Zlotnik, 2006). Chemokines have the ability to attract white blood cells by using chemokine receptors present on their surface (Fig. 4). They work like a chemical beacon by mobilizing white blood cells where they are needed to fight infection or destroy foreign agents; unfortunately, many invasive cancer cells share the same receptors as white blood cells and give the tumors the potential to migrate to distant organs rich in chemokine concentration. Some cancer cells break away from the primary site and travel via the lymphatic system or blood vessels toward sources of chemokines such as the lymph nodes liver lungs, or bone marrow. These cells can begin to grow into a new tumor similar to the primary cancer but in new location, and this migration process is called metastasis, which is a critical aspect of the aggressiveness of cancer.

Other organs like liver, Bone marrow, lymph nodes Cancer cell having G-proteincoupled-receptor Chemokines

Tumor tissue Fig. 4  Chemokine receptor expressed on cancer cells in cancer metastasis

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6  Chemokines and Their Receptor in Colorectal Cancer Tumor cells not only accumulate genetic mutations in themselves but also affect their surrounding cells in order to survive in the chaos of the tumor microenvironment. Chemokines are upregulated when normal colonic mucosa becomes cancerous and the cancer becomes more malignant (Itatani, Kawada, Inamoto, et al., 2016). Proof from various investigations illustrates that chemokine receptors are involved in the development and metastasis of colorectal cancer (Li et al., 2014; Rubie et al., 2006). Pączek et  al. (Pączek, Łukaszewicz-Zając, & Mroczko, 2020) summarize the potential role of chemokines as biomarkers in the diagnosis and prognosis of CRC (Pączek et al., 2020).

7  CC Family of Chemokines and Their Receptors CCR 7.1  CCL2 and CCR2 Monocyte chemotactic protein 1 (MCP-1), also called CCL2, is released in CRC to enhance the accumulation of macrophage and cyclooxygenase-2 (COX-2) expression which leads to colorectal tumorigenesis (Tanaka et al., 2006). Myeloid cells promote development of colorectal cancer liver metastasis in a mouse allograft model via CCL2-CCR2 signaling, so that these cells potentially work for anti-metastatic therapy (Zhao et al., 2013). In addition to myeloid cell recruitment, tumorderived CCL2 has been shown to have direct effects on the tumor vasculature. CCL2 binding to CCR2+ endothelium enhances vascular permeability in a p38/MAPK pathway, which in turn increases colon cancer cell extravasation and metastasis to the lungs (Wolf et al., 2012).

7.2  CCL5 and CCR5 Expression of CCR5 increases in CRC as the tumor size increases and expression patterns appear, leading to “patchiness” in liver metastases (Suarez-Carmona, Chaorentong, Kather, et al., 2019). CCL5 released from CRC cells recruits CCR5+ T-reg, which leads to TGF-mediated apoptosis of CD8+ CTL and tumor growth (Chang et al., 2012). Most of the CD8+ CTL and type-1 helper T cells (Th1) at the invasion front of CRC tissues express CCR5 and CCL5 in the tumor microenvironment which is observed in the stromal CD8+ T lymphocytes at the invasion front (Musha et al., 2005). Mesenchymal stem cells (MSCs) extensively interact with cancer cells and other stromal cells in the tumor microenvironment. MSCs promote the upregulation of CCL5 and proliferation and progression of colon cancer cells in in vivo model of nude mice (Chen, Liu, Tsang, et al., 2017).

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7.3  CCL15 and CCR1 SMAD4 is a transcription factor that plays an important role in tumor suppressors of CRC (Zhao, Mishra, & Deng, 2018). Tripartite motif 47 (TRIM47), a member of the TRIM family proteins, interacts with SMAD4 increasing its degradation. Downregulation of SMAD4 led to upregulation of CCL15 expression and caused growth and invasion in human CRC cells through the CCL15-CCR1 signaling (Liang et al., 2019). CCR1 plays a key role in colon cancer metastasis in mouse model, and CCR1+ bone marrow (BM)-derived cells are recruited to the microenvironment of disseminated colon cancer cells and produce metalloproteinases MMP9 and MMP2, helping metastatic colonization (Hirai, Fujishita, Kurimoto, et al., 2014).

7.4  CCL19/21 and CCR7 CCL19 may play a suppressive role in proliferation, migration, invasion, and pro-­ angiogenesis CRC (Lu et al., 2014). CCL19 is upregulated in lymphoid tissues isolated from colorectal cancer as activated helper NK cells to promote the production of the DC- and memory T cell-attracting chemokine CCL19 in lymph nodes (Lu et al., 2014). The chemokine CCL21 attracts CCR7-bearing cells, especially T and dendritic cells but also colorectal cancer cells. The different levels of CCL21 in the rectum and colon may reflect divergent mechanisms in colorectal carcinogenesis (Wong, Muthuswamy, Bartlett, & Kalinski, 2013). Matrix metalloproteinase-9 is upregulated by CCL21/CCR7 interaction promoting lymph node metastasis in human colon cancer (Mumtaz et al., 2009). CCR7 expression on CRC cells themselves plays an important role in the mechanism of cancer progression. CCR7 expression at the invasion front is strongly correlated with lymph node metastases and decreased survival rate (Li, Sun, Tao, & Wang, 2011).

7.5  CCL20 and CCR6 CCR6 and CCL20 may play a vital role in proliferation and migration of colorectal cancer through autocrine or paracrine mechanisms. So disruption of CCL20-CCR6 signaling is a promising strategy in the treatment of cancer (Günther et al., 2005). CCL20/CCR6 expression is elevated with the induction of colorectal adenoma (CRA), colorectal adenocarcinoma (CRC), and the development of colorectal liver metastases (CRLM) (Ghadjar, Rubie, Aebersold, & Keilholz, 2009). In CRC tissues, the main source of CCL20, a ligand for CCR6, is considered to be TAM, and CCL20 secreted from TAM recruits CCR6+ T-reg to the tumor site, which synergistically promotes tumor progression in mouse models (Frick, Rubie, Kölsch, et al., 2013).

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7.6  CCL24 and CCR3 CCL24 shows accumulation in cancer cells in the course of CRC, as its elevated levels were found in biopsy samples of primary colorectal cancer and adjacent liver metastases (Cheadle et al., 2007). In CRC the number of eosinophils decreased significantly and the expression of CCL24 in glandular cells decreased with tumor progression, whereas the stromal expression of CCL24 appeared to increase. Thus, the eosinophils are antitumor effector immune cells in case of CRC (Cho et al., 2016). High expression in CRC tissues leads to a decrease in chemotaxis of eosinophils, which are effector immune cells with antitumor activity (Zajkowska & Mroczko, 2020).

8  CXC Family of Chemokines and Their Receptors CXCR 8.1  CXCL1/5/8 and CXCR2 Chemokines CXCL1, CXCL5, and CXCL8 with an ELR (Glu-Leu-Arg) motif bind to CXCR2and promote chemoattraction, inflammatory responses, tumor growth, and angiogenesis. CXCL1 involves tumor cell transformation, growth, and invasion; inhibition of CXCL1 in CRC cells causes decrease in cell migration resulting in the prevention of tumor growth in mouse models (Itatani, Kawada, Inamoto, et al., 2016). There is a significant association between CXCL1 and CXCL5 expression with CRC, and both chemokine ligands have a potential role in the progression of CRC (Rubie, Frick, Wagner, et al., 2008). The CXCL1-CXCR2 axis is also important for the formation of the pre-­metastatic niche of CRC liver metastasis, and TSU68, an antiangiogenic receptor tyrosine kinase inhibitor, suppressed CXCL1 expression in the pre-metastatic liver, resulting in suppression of the homing of CXCR2+ neutrophils and subsequent liver metastasis in a mouse model (Bandapalli et al., 2012). In addition, small molecule antagonists for CXCR1 and CXCR2 like SCH527123 (which blocks both CXCR1 and CXCR2) or SCH479833 (which preferentially blocks CXCR2) inhibited CRC by decreasing tumor angiogenesis and enhancing apoptosis in a mouse model (Varney et al., 2011).

8.2  CXCL9/10 and CXCR3 CXCL9/10, ligands of CXCR3, are expressed at high levels in lymph nodes, where CRC cells promoted their metastases by expressing this receptor (Kawada et  al., 2007). CXCL10, secreted by CRC cells, which leads to the predominant accumulation of T helper type 1 (Th1) and Th2 cells, expressing CXCR3 mainly. These CXCL10 recruit the Th1 at the invasive margin front of CRC and contributes to antitumor immunity or favorable prognosis (Musha et al., 2005). Polyphenols

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induce CXCR3 expression on regulatory T cells and increase CXCR3 ligands in the tumor microenvironment, which act together to suppress colorectal cancer through a differential mechanism (Abron, Singh, Murphy, et al., 2018). CXCL10/CXCR3 co-expression is a predictor of metastatic recurrence and poor overall survival in CRC (Murakami et al., 2013). In Treg-cell-­depleted tumor, selective increase of the CXCL9 leads the accumulation of CXCR3(+) T cells and increased IFN-γ mRNA expression. This indicates that targeting Treg cells could be a possible antitumor immunotherapy, which not only affects T-cell effector functions but also their recruitment to tumors (Akeus et al., 2015).

8.3  CXCL12 and CXCR4/CXCR7 CXCL12, stromal cell-derived ligand (SDF-1), and its receptor CXCR4 are one of the representative pairs of chemokine signaling involved in cancer metastasis (Muller, Homey, Soto, et al., 2001). CXCL12 is secreted by Kupffer cells, endothelial cells, and smooth muscle actin-positive myofibroblasts in the liver (Wightman et al., 2015). CXCR4 expressed by CRC cells frequently metastasizes to the liver and makes it the most frequent site of CRC metastasis (Akeus et al., 2015). But this process is controversial as CXCL12 expression in CRC cells appears bidirectional, tumor promotive (Wendt et  al., 2006), and tumor suppressive (Romain, HatchetHaas, Rohr, et al., 2014). Under hypoxia-inducible factor 1-alpha (HIF-1) activity, expression of CXCR4 in CRC cell lines was also upregulated (Kim et al., 2005). CXCR4 expression in CRC patients increases the risk for recurrence and for poor survival (Balabanian et al., 2005). CXCR7 is another novel receptor for CXCL12 (Tripathi et  al., 2009). This membrane-associated receptor, CXCR7, is highly expressed on activated endothelial cells of tumors and rarely expressed on normal somatic cells (Miao et  al., 2007). Many studies revealed the roles of CXCR7  in tumorigenesis (Yang, Dai, Xue, et al., 2015). In CRC there is a positive correlation between CXCR7 expression and tumor development and poor prognosis of patients (Ohta, Tanaka, Yamaguchi, et al., 2005). The binding of CXCL12 to CXCR7 can activate colorectal cancer progression via regulation of the p-ERK and β-arrestin pathways (Li et al., 2014).

9  CX3C Family of Chemokines and Their Receptors CX3CR 9.1  CX3CL1 and CX3CR1 Generally, in colorectal cancer patients, the number of tumor-infiltrating lymphocytes is low. Higher density of tumor-infiltrating immune cells in patients with colorectal cancer correlates to a higher level of expression of fractalkine (CX3CL1) and results in a better prognosis than in those with a weak expression. Thus, the

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CX3CL1 is considered to be an essential biomarker for predicting prognosis in the case of colorectal cancer (Erreni, Siddiqui, Marelli, et al., 2016). Higher expression of the CX3CL1-CX3CR1 chemokine axis functions in CRC patients act as a retention factor, increasing homotypic cell adhesion and limiting tumor spreading to metastatic sites. In contrast low levels of CX3CL1-CX3CR1 by tumor cells identify CRC patients at increased risk of metastatic progression (Zheng et  al., 2013). CX3CR1 upregulation in TAMs was correlated with poor prognosis, and the liver metastasis of colon cancer cells was significantly inhibited in a microenvironment lacking CX3CR1 (Mollica Poeta, Massara, Capucetti, & Bonecchi, 2019).

10  C  hemokines and Their Receptors as Potential Biomarker in Cancer Immunotherapy Immunotherapy is a type of cancer treatment that boosts the body’s immune system to fight against cancer. In the last decades, it has emerged that chemokine and their receptor system represents a potential target for immunotherapy. Chemokines and their cognate receptors induce cell proliferation, tumor growth, and cancer metastasis in all the stages of the disease. By altering and inhibiting the expression of the chemokine system in cancer, we can use them as a potential biomarker in cancer immunotherapy (Cheng, Ma, Wei, & Wei, 2019). CXCLs/CXCR2 signaling activates many pathways, like PI3K, p38/ERK, and JAK pathways, which regulate cell survival and recruiting neutrophils to inflammatory sites. Therefore, CXCLs/ CXCR2 inhibition through many drugs has been considered a promising antitumor treatment (Zhang, Wang, Sun, et  al., 2020). The CXCL5/CXCR2 and CXCL12/ CXCR4 axis can act as a bridge between tumor cells and host cells in tumor microenvironment. Blocking the transmission of CXCL5/CXCR2 and CXCL12/CXCR4 signals can increase the sensitivity and effectiveness of immunotherapy (Chen, Xu, Zong, et  al., 2019; Gong & Ren, 2020; Kumar, Cherukumilli, Mahmoudpour, Brand, & Bandapalli, 2018). CXCL8 may also serve as a potential therapeutic target for the treatment of colorectal liver metastasis, as ShRNA downregulates its expression CXCL8, resulting in significantly decreased cell proliferation, migration, and invasion (Kumar, Cherukumilli, Mahmoudpour, Brand, & Bandapalli, 2018). There are more studies necessary to explain the roles of chemokines and their receptors as potential biomarkers in cancer immunotherapy.

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Index

A Acid sphingomyelinase, 264 Activator protein-1 (AP-1), 390 Adenocarcinoma, 385, 387 Adenomatous polyposis coli (APC), 26, 45, 74, 144 Adenomatous polyps (AP), 81, 386 Adenosine monophosphate–activated protein kinase (AMPK)–COX-2, 396 Adenosine triphosphate (ATP), 393 Adenosquamous carcinoma, 385 Adipocytes, 131 Adjuvant chemoradiotherapy, 221 Adjuvant chemotherapy, 388 Adolescent and young adult (AYA) patients, 106 Advance tumor development, 409 Agarose tumor macrobeads, 9–11 Agent azoxymethane (AOM), 409 Aging, 177 Agonist monoclonal antibodies, 207 Alcohol consumption, 385 Allergic diseases, 185 Allium genus members, 285 Alpha-hemolysin, 370 Alzheimer’s disease (AD), 395 American Institute of Cancer Research, 295 3-aminopropyltriethoxysilane (APTES), 97 Amyloid-β peptide, 395 Amyloid-β protein, 395 Anaerobes, 319 Androgen receptor, 42 Angiogenesis, 150, 198, 199 Angiogenesis inhibitors, 162, 230 Angiogenic, 430

Angiopoietin 2 (ANG-2), 199 Anthrax toxin, 370, 371 Anti-angiogenesis agent, 47 Antibody siltuximab, 204 Antibody-dependent cell-mediated cytotoxicity (ADCC) reactions, 148 Antibrowning agents, 314 Anticancer, 384, 387, 390, 391, 396, 398–400, 402, 403, 411 Anticancer activity apoptosis, 260 chemokines, 255 chemotherapy, 255 COX-2 expression, 260 inflammation, 255 TNF-α and IL-6 development, 259 Anticancer agents, 229 Anticarcinogenic efficiency, 407 Anticolon cancer activity apigenin, 250 CA, 250 carnosic acid, 253 CGA, 249 curcumin, 249 EGCG, 248 flavone, 252 genistein, 251 gingerol, 253 luteolin, 254 mitochondria, 252 quercetin, 251 resveratrol, 251 silibinin, 253 tangeretin, 254 Anti-COX-2 shRNAs (shCOX-2), 348

© The Author(s), under exclusive license to Springer Nature Switzerland AG 2022 D. Shukla et al. (eds.), Colon Cancer Diagnosis and Therapy Vol. 3, https://doi.org/10.1007/978-3-030-72702-4

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442 Antidiabetic agent, 394 Antidiabetic effects, 394 Anti-EGFR (epidermal growth factor receptor) agent, 47 Anti-EGFR inhibitors, 111 Anti-EGFR therapies, 135, 178 Anti-infective properties, 384 Anti-inflammatory, 384, 391, 394 Anti-inflammatory action, 389 Anti-inflammatory activity, 411 Anti-inflammatory cells, 187 Anti-inflammatory cytokines, 183 Anti-inflammatory drugs, 11, 12, 185 Antimicrobial activities, 411 Antimicrobial agents, 312, 395 Antioxidants, 314, 391, 394 Anti-PD-1 peptide (APP), 97 Antiphagocytic communications, 182 Antisense oligonucleotide (ASO) AZD4785, 344 clinical trials, 344 CRC cancer therapeutic drug, 344 CRC treatment, 344 growth factor EGFR, 344 mechanisms, 341, 344 proto-oncogene c-MYC, 344 short synthetic single-stranded nucleic acid sequences, 341 target pre-mRNA, 343 therapeutic molecule, 343 Anti-SMC2 antibodies (Ab-SMC2), 97 Antitumor properties, CLA, 329 Anti-VEGF therapy, 201 Anti-VEGF treatments, 178 Anti-Warburg effect, 390 Apatinib, 162 Apigenin anti-proliferation and cell cycle, 250 Apoptosis, 260, 396 Apoptosis mitochondrial pathway, 261 Apoptosis signal-regulating kinase 1 (ASK1), 393 Apoptotic signalling pathway, 261 Apples bioactive materials, 282 consumption, 282 flavonoids, 282 Aptamers (Apt), 346, 347, 408 Apt-CUR-NP bioconjugates, 408 Arachidonic acid derivatives, 11 Arginine deiminase, 375 Aromatase, 48 Artificial sweeteners, 317 Aspartame, 318

Index Aspirin, 185 Ataxia-telangiectasia mutated (ATM) genes, 74, 75, 145 ATP-binding cassette (ABC) transporters, 24 Atrial natriuretic factor (ANF), 393 Autocrine stimulation, 398 Autophagy, 133–134 Autosomal-dominant (AD), 74 Azo dyes, 315 Azoxymethane/dextran sulfate (AOM/ DSS), 119 B B7-H4 siRNA, 349 Bacillus anthracis, 370 Bacteria, 362 Bacteria, cancer treatment bacteria mechanism pathways, 363 bacterial cells, 364 bacterial strains, 364 Bifidobacterium longum, 364 Campylobacter jejuni, 363 cancer, 365 Citrobacter rodentium, 363 Clostridium novyi, 364 double-edged sword, cancer, 363 genetic modifications, 365 Helicobacter pylori, 363 Listeria monocytogenes LADD strain, 364 Magnetococcus marinus, 364 Mycobacterium bovis BCG, 364 Mycobacterium tuberculosis, 363 Salmonella typhi, 363 Salmonella typhimurium VNP20009, 364 Serratia marcescens, 363 Streptococcus pyogenes OK-432, 364 Bacterial anticancer agents, 377 Bacterial cells, 364, 375 Bacterial enzymes, 375 Bacterial strains, 365 Bacterial therapy advantages, 363 bacteria, 363–365 bacterial enzymes, 375 bacterial toxins (see Bacterial toxins, cancer immunotherapy) bacteriocins, 372, 373 cancer treatment, 362 COBALT, 375, 376 conventional small molecular drugs, 363 conventional therapies, 362 genetically engineered bacteria, 362, 363 nonribosomal bacterial peptides, 373, 374

Index probiotic bacteria, 366 Bacterial toxins, cancer immunotherapy α-Hemolysin, Staphylococcus aureus, 370 anthrax toxin, 370, 371 antibody part, 367 artificial stimulation, immune system, 366 bacteriocins, 373 cancer cells, 366 cytosol, 367 diphtheria toxin, 367, 368 immunotoxins, 367, 372 Pseudomonas exotoxin A, 367 Pseudomonas Exotoxin A, 369, 370 signalling pathways, mammalian cells, 366 Bacteria-mediated cancer therapy, 377 Bacteria‐mediated therapy, 362 Bacteriocins antibacterial compounds, 372 bacterial proteins/peptides, 372 classification, 372, 373 Colicin E1, 373 Nisin A, 372 pediocin, 372 Plantaricin A, 373 Pyocin S2, 373 Bacteriotherapy, 375 Bacteriotherapy-chemotherapy combination, 376 Bacteriotherapy-radiotherapy combination, 375, 376 Banana phenolics, 284 TNF-alpha, 284 Basal cell carcinoma (BCC), 154 Bax-dependent condition, 262 Bax expression, 396 B-cell lymphoma (Bcl)-xL protein, 390 Bee venom (BV), 119 Benzidine, 317 Benzidine-induced carcinogenesis, 317 Berberine, 394 Berry bioactive components, 282 Bevacizumab, 8, 110, 111, 114, 129, 156, 157, 233, 387, 389 Bifidobacterium, 12 Bifidobacterium longum, 364 Bioactive peptides, 330 Bioinformatic analysis, 83 Biologic cooperation, 227 Biological activity, CUR anticancer, 390, 391 antidiabetic effects, 394

443 anti-inflammatory, 391 and antineoplastic actions, 389 antioxidant, 391 anti-Warburg effect, 390 cardiovascular protection, 393 diferuloylmethane, 389 immune-regulatory activity, 392 laboratory and preclinical studies, 390 medicinal plant, 389 neuroprotection, 394, 395 potential and properties, 389 WHO, 390 Biological signals, 10 Biomarkers, 6–7, 13, 24 Biosurfactants, 373 3,5-Bis(arylidene)-4-piperidones, 400 3,5-bis(benzylidene)-4-piperidone, 400 Bis-demethoxy-curcumin analogue (BDMC-A), 401, 402 Body mass index (BMI), 11 Bone-marrow-derived clusters (BMDCs), 200 Bovine lactoferricin (LfcinB), 330 Brachytherapy, 220 Brivanib, 162 Butylated hydroxyanisole (BHA), 314 Butylated hydroxytoluene (BHT), 314 Butyrate, 408 C Caco-2 cells, 401 Caffeic acid (CA) β-catenin/T-cell factor, 250 phosphorylation, 250 Caffeic acid phenethyl ester (CAPE), 250 Calcium, 328, 329 Calcium phosphate nanoparticles, 97 Calcium sensing receptor (CASR), 81 Canakinumab, 186 Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS), 186 Cancer ageing, 245 breast, 245 CRC, 246, 247 mortality, 245 Cancer-associated fibroblasts (CAFs), 131 Cancer cells suppression, 230 Cancer cellular immunotherapy limitations, 183, 184 microenvironment, 179 myeloid cells, 181–183 T cells, 179–181

444 Cancer immunotherapy, 94, 154, 436 Cancer progression, 42, 52, 58, 59, 185 Cancer stem cells (CSCs), 128, 130, 131 Cancer susceptibility 15 (CASC15), 27, 30 Cancer therapy, 218 Cancerous cells, 144, 375 Canertinib (CI-1033), 148 Canine uric acid, 119 Canonical NF-πB pathway, 264 Capecitabine (CAP), 107–110, 114, 125, 128, 134–136, 231, 387, 389 Capecitabine-based therapy, 114 Capillary morphogenesis gene 2 (CMG2), 371 CAR T-cell therapy, 180 Carcinoembryonic antigen (CEA), 347 Cardiovascular diseases (CVDs), 11, 390 Cardiovascular protection, 393 Carnosic acid, 253 Casein, 326, 329 Casein kinase 1 (CK1), 30 Categories of food additives coloring agents, 315–317 flavoring agents, 317–318 food preservatives, 311–315 nutritional, 319 texturing agents, 318, 319 C-C motif chemokine precursor 2 (CCL2), 197 CCL2 and CCR2, 432 CCL5 and CCR5, 432 CCL15 and CCR1, 433 CCL19/21 and CCR7, 433 CCL20 and CCR6, 433 CCL24 and CCR3, 434 CD45+ monocytic cells, 201 CDK activation, 268 Cediranib, 162 Cell-free supernatants (CFS), 332 Cell proliferation, 229 Cell signaling pathways, 42 Cellular protagonists, 27 Cellular toxicity, 155 Cetuximab, 8, 73, 112, 130, 157, 158, 233, 387, 389 in non-first-line setting, 114 with FOLFIRI, 112 with FOLFOX, 113 Cetuximab (Erbitux), 147 Checkpoint inhibitors, 154 Chemical antibodies, 346 Chemical technology, 355 Chemodrug-associated immunotherapy, 125 Chemodrugs, 125, 128, 129 Chemokine and receptors

Index angiogenic, 430 CCL2 and CCR2, 432 CCL5 and CCR5, 432 CCL15 and CCR1, 433 CCL19/21 and CCR7, 433 CCL20 and CCR6, 433 CCL24 and CCR3, 434 classification, 430 CRC, 432 CX3CL1 and CX3CR1, 435 CXCL1/5/8 and CXCR2, 434 CXCL8, 436 CXCL9/10 and CXCR3, 434, 435 CXCL12, 435 CXCR4/CXCR7, 435 homeostatic, 430 immunity, 430 immunotherapy, 436 inflammation, 430 inflammatory, 430 metastasis, 431 potential biomarker cancer immunotherapy, 436 Chemokines, 11 Chemopreventive agents, 268 Chemoradiotherapy chemotherapeutic drugs (see Chemotherapeutic drugs, radiation therapy) concurrent, 221 CRC treatment, 221 drug-radiation interactions, 228–230 drugs, 220 locally advanced cancers treatment, 234 neoadjuvant, 221 palliative, 221 postoperative, 221 preoperative, 221 sequential, 221 standard treatment alternative, 220 therapeutic index enhancement (see Therapeutic index enhancement approaches) Chemoresistance, 32 advanced CRC therapy, 131 capecitabine, 128 CDDP resistance, 127 definition, 131 efficacy, 125 FOLFOX, 127 5-FU, 125 MTHFR, 126 oxaliplatin, 127 oxaliplatin mechanisms, 127

Index TME, 131 treatment with FPs, 125 Chemosensitizing agent, 411 Chemotherapeutic agents, 376 Chemotherapeutic drugs, 230, 388 Chemotherapeutic drugs, radiation therapy bevacizumab plus radiotherapy, 233 capecitabine plus radiotherapy, 231 cetuximab, 233 5-FU plus radiotherapy, 230, 231 Gemcitabine plus radiotherapy, 233 irinotecan plus radiotherapy, 232 mitomycin C, 233 oxaliplatin plus radiotherapy, 231, 232 Chemotherapeutic methods, 230 Chemotherapeutic resistance anticancer medications, 201 cancer therapy, 201 clinical evaluation, 202 Gas6 ligands, 202 haematological tumours, 202 IL6R/STAT3/miR-204-5p, 202 sustainable treatment strategies development, 201 TME, 202 tumour agents, 202 Chemotherapy, 8, 24, 72, 107, 144, 147, 340, 376, 377, 386–389, 399 classification, 218 CRC treatment, 218 durations, 218 patients with CRC, 107 side effects, 219 single-agent therapy, 107 synergistic effect, 218 therapeutics and regimens, 108 Chemotherapy-resistant colon cancer cells, 397 Chimeric antigen receptor (CAR), 180 Chlorogenic acid (CGA), 249 Chromosomal abnormalities, 266 Chromosomal instability (CIN), 5, 45, 177, 185 Chromosomal instability pathway, 26 Chromosomes, 26 Chronic inflammation, 11, 185 Chronic inflammatory responses, 194 Cisplatin (CDDP), 127, 128 Citrobacter rodentium, 363 Citrus fruit (CF), 281 juice, 281 lemon and orange juices, 281 roles, 281 c-Jun N-terminal kinase (JNK), 393 Classical therapeutic strategies, 194

445 Classically activated (M1-type), 182 Clodronate, 205 Clostridium botulinum, 313 Clostridium novyi, 364 Clostridium novyi‐NT spores, 375 c-MYC ASOs, 344 Colicin E1, 373 Colon cancer (CC) APC, 74 ATM, 74, 75 bioactive compounds, 247 (see also Colorectal cancer (CRC)) biomarkers, 24 BRAF gene, 75, 76 BRCA1/BRCA2 gene, 77 cancer development, 23 cancer-related death, 23 carcinogenesis mechanisms, 73 chemotherapy, 24, 72 drug resistance, 24, 31, 32 DUSP1 gene, 77, 78 gastrointestinal system, 23 genetic risk factors, 71 genetic variants, 71 genetic variations, 72 heterogeneous group, 71 human genome, 72 introns and intergenic regions, 72 lncRNAs, 25, 27, 29, 30 long noncoding RNA, 26, 27 MAP2K1, 80 mechanisms, 71 miRNA, 82, 83 MLH1 gene, 78, 79 modifiable and nonmodifiable risk factors, 71 molecular genetic biomarkers, 71 MTHFR, 79, 80 mucosal cells, 23 neoadjuvant and adjuvant treatments, 24 nonmetastatic cancers, 24 non-mucinous tumors, 73 pathology, 73 pharmacogenetics, 71 precision medicine, 71 SNPs, 71, 83 somatic mutation analysis, 73 SULT2B1, 81 symptoms, 24 transport systems, 24 treatment, 32–34 treatment options, 23, 24 tumor biology, 73 VDR, 81 wnt signaling, 25, 26

446 Colon Cancer Family Registry (CCFR), 79 Colon cancer-associated transcript 1 (CCAT1), 27 Colon cancer-associated transcript 2 (CCAT2), 27 Colon malignancy, 388 Colonoscopic screening, 106 Colonoscopy, 387 Colony-stimulating factor-1 (CSF1), 197 Colorectal adenocarcinoma, 45 Colorectal cancer (CRC), 93, 143, 278, 384 agarose tumor macrobeads, 9–11 age and genetic factors, 385 anti-inflammatory drugs, 11, 12 autophagy, 133–134 biomarkers, 6–7 blood vessels, 384 cancer deaths, 385 cancer-related death rates, 143 cancer-related deaths, 3 carcinogenesis, 246 cetuximab with FOLFIRI, 112 chemoresistance (see Chemoresistance) chemotherapy, 47, 107, 340, 386 classical CIN pathway, 45 clinical trials, 9, 11 colonic mucosal epithelia, 124 colonocytes, 246 components, 246 conventional treatment approaches, 144 CSCs, 246 vs. CUR (see Curcumin (CUR)) developing countries, 326 development, 385 development and metastasis, 46 diagnosis, 3, 5 diet, as major risk factor, 43, 44 DNA repair stimulation, 248 drug resistance against standard chemotherapeutic agents, 136 drugs, 340 estrogen signaling through GPER, 58–59 estrogens role (see Estrogens) etiology and molecular mechanisms, 44, 45 family history, 386 fatal diseases, 340 first-line treatment, 107 5-FU, 340 functional foods, 12 gastrointestinal bleeding, 428 genetic alterations, 308 genetic inheritance/family history, 43 genetic mutation, 385, 386 genomic change, 247

Index healthcare infrastructure, 143 HER2 overexpression, 112 hereditary type, 45 hormone-related malignancies, 42 HRT, 57 human gastrointestinal tract, 279 immunotherapy, 128–130 incidence, 106, 279 incidence and mortality rates, 384 inhibition, 279 KRAS/NRAS testing, 112 lymph system, 384 maintenance therapy, 110 malignancy, 42, 246, 320 management, 8, 9 mechanisms, 428, 429 metastasis, 340 metastatic diseases, 3 modifiable and nonmodifiable risk factors, 43 molecular pathways, 4 mortality, 176, 218 multifactorial disease, 45 multiple factors, 385 mutations, 144 NCCN guidelines, 109 non-operable metastasis disease, 340 nutrition-related predisposing factors, 326 pathophysiology, 3, 176 polyps, 386, 428 population, 308 potential treatment challenges, 128 predominance, 246 prevention and therapy, 400–403 probiotics, 12 radiotherapy, 386 repercussion, 247 risk factors, 3, 43–44, 385, 429 risks, 308 RNA-based therapeutics (see Ribonucleic acid (RNA)-based therapeutics) screening, 3, 5 surgery, 386 surgical treatments, 46 targeted therapy, 108–109, 144, 340 therapeutic potential, 134–135 therapy, 8 treatment, 9 treatment of stage, 340 treatment strategies, 46–47 treatment strategy, 387–389 tumor location, 386 tumours and types, 246

Index visual examination and stool-based tests, 386 WHO GLOBOCAN database, 384 worldwide prevalence, 175 Colorectal neoplasia differentially expressed (CRNDE), 27 Combination bacteriolytic therapy (COBALT), 375, 376 chemotherapy, 376 radiotherapy, 375, 376 Combination therapy, 130, 135 Combined immunotherapy, 124 Combined-modality therapy, 218 Concurrent chemoradiotherapy, 221 Conjugated linoleic acid (CLA), 328, 329 Conventional cancer therapies, 362 Conventional treatment strategies, 376 Corynebacterium diphtheriae, 367 COX-2 inhibitors (COXibs), 12 COX-2 production, 259 CpG island methylator phenotype (CIMP), 5, 177, 178 CpG island methylator phenotype-high (CIMP-high), 75 CRC BRAF mutation, 179 CRC stem cells (CSCs), 396 CRC-targeted agents, 108 Crohn’s disease, 44, 194, 385 CRYSTAL clinical trial, 147 CSF-1/CSF-1R, 204 CSF-1R small-molecule antagonist, 204, 205 CURC-DOX (PEGylated long-circulating liposomes codelivering curcumin and doxorubicin) analogue, 402 Curcuma longa, 33 Curcumin (CUR) analogues, 400–403 biological activity (see Biological activity, CUR) vs. colon cancer antineoplastic agent, 395 apoptosis, 396 CDX2, 399 cell cycle progression, 396 clinical trials, 396 CSCs, 396 DFMO, 398 EGFR family, 397 Hippo signaling pathway, 399 histotypes, 396 hyperglycolytic phenotype, 397 IGF receptors, 397 LC-MS, 398 MDR phenotype formation, 397

447 metabolism, 397 microenvironment and regulation, 396 miR-130a, 398 murine model, 397 nonsteroidal anti-inflammatory drugs (NSAIDs), 398 NSAIDs, 398 ODC, 398 p53, 399 PDK4, 399 polyamine synthesis, 398 psychoneuroimmunomodulation, 398 RUNX3, 398 SIRT1, 398 Wnt/β-catenin signaling, 398 YAP, 399 drug delivery, 406–408 epigenetic modifications, 405, 406 immunomodulatory effects, 409, 410 metabolism, 385 microbial environment, 408, 409 nanoparticles (NPs), 385 pharmacokinetics, 385 safe consumption, 384 structural analogues, 385 treatment, 385, 410 Curcumin (diferuloylmethane), 249 adenomatous polyposis, 249 colon carcinogenesis, 249 Curcumin analogues, 400–403 Curcumin-loaded nanoparticles, 408 Curcumin–silica nanoparticle complexes, 406 Curcumin-β-D-glucuronide (CMG), 403 Cyclin E–dependent kinase, 396 Cyclo-oxygenase-2 (COX2), 255, 390 Cytochrome P450 2W1 (CYP2W1), 119 Cytokine-release syndrome (CRS), 184 Cytokines, 11 Cytoskeleton, 250 Cytoskeleton regulator RNA (CYTOR), 27, 30 Cytosol, 368 Cytotoxic chemotherapeutic drugs, 155 D Death receptors (DRs), 390 Dehydrozingerone, 401 Demographic factors, 175 Dendrosome nanocarriers (DNCs), 407 Denileukin diftitox, 368, 369 Dextran sulfate sodium (DSS)–induced colitis model, 408 Diabetes mellitus, 394 Diabetic retinopathy, 394

448 Dickkopf-related protein 3, 393 Diet, 43, 44, 308, 326 Dietary polyphenols, 269 Diferuloylmethane, 389 Difluorinated curcumin (CDF), 402 Diphtheria toxin, 367, 368 Direct additive, 309 Disease-free survival (DFS), 81 Distal CC (DCC), 76 Distal colon (DC), 80 4,4′-disulfonyldiarylidenyl piperidone (DAP), 403 DNA-based therapeutics, 354 DNA-binding domain (DBD), 50 DNA CpG methylation, 405 DNA-damage inducible genes activation, 250 DNA hypomethylation, 80, 349 DNA methylation, 26, 266 DNA methyltransferases (DNMTs), 405 DNA-repair genes (DRGs), 74 DNA replication, 125 DNMT3A and HDAC3 degradation, 266 Docetaxel (DTX), 97 Doxorubicin, 97 Drug delivery, 406–408 Drug-radiation interaction mechanisms DNA repair prevention, 228, 229 hypoxia, 230 radiation damage intensification, 228 repopulation, 229, 230 Drug resistance, 24, 31, 32 Drug-resistant cancers, 384 Dysbiosis, 319, 408 E E-cadherin, 133 ECM-educated macrophages, 201 EF24 induced ROS-dependent antitumor activity, 402 EGFR, RAS/RAF/MEK/ERK signaling, 147–149 Ellagic acid hexahydroxydiphenic acid, 252 Emulsion–solvent evaporation techniques, 408 Endocavitary radiation therapy, 220 Endocrine disruption, 49 Endogenous ligand, 54 Endoplasmic reticulum (ER) stress, 402 Enterococcus, 12 Epidemiological studies, 119 Epidermal growth factor receptor (EGFR), 108, 109, 111, 112, 125, 130, 135, 178, 233, 248 Epidermal growth factor receptor (EGFR) family, 397

Index Epidermal growth factor receptor (EGFR)/ EGFR-associated pathways, 146, 147 Epigallocatechin gallate (EGCG), 34 Epigallocatechin-3-gallate (EGCG), 248, 268 compounds, 248 COX-2 and inflammatory cytokines, 249 DNMT3A and HDAC3, 248 Epigenetic alterations, 45 Epigenetic mechanism cell cycle regulation, 268 cell growth and activity, 268 dietary polyphenols, 266 DNA methylation, 266 DNMTs, 266 EGCG, 269 metastasis, 269 MiRNA, 266 quercetin and luteolin, 269 Epigenetic modifications CUR, 405, 406 Epithelial-mesenchymal transition (EMT), 45, 132–133, 396 developmental regulatory process, 132 epithelial-derived cancer, 132 HFD-mediated development, 133 program, 133 ER-independent signaling, 54 Erlotinib (Tarceva), 148 erpine1 (Serbp1), 10 Escherichia coli, 375 Essential amino acids, 375 Estrogen and colon cancer age-specific fertility, 56 ERs (see Estrogen receptors (ERs)) function in colonic epithelium, 56–57 nuclear receptor superfamily, 48–49 synthesis, 48 Estrogen receptor alpha (ERα), 49–53, 55, 57, 58 Estrogen receptor beta (ERβ), 49–53, 55–61 Estrogen receptors (ERs) DBD, 50 distribution, 52 GPER, 49, 51–52 mechanisms of antitumor effects, 60 membrane-associated, 51 nuclear estrogen receptors, 49–51 nuclear receptor superfamily, 49 nuclear receptors, 49 proapoptotic signaling, 59 Estrogen signaling pathways ER-independent signaling, 54 genomic effects, 53

Index ligand-independent activation, ERs, 54 “membrane-initiated” pathway, 53 Estrogens 17β estradiol (E2), 47 estriol (E3), 47 estrone (E1), 47 GPER (see G-protein coupled estrogen receptor (GPER)) in pathological conditions, 47 role, 47 sex hormones, 47 synthesis, 47–48 European Food Safety Authority of European Union (EU), 311 European Medicines Agency (EMA), 148 European Society for Medical Oncology (ESMO) guidelines, 388 Evidence-based research, 135, 136 External beam radiation therapy (EBRT), 219 Extracellular matrix (ECM), 194 collagen IV and laminins, 199 components, 199 degradation, 200 fingerprint, 200 related proteins, 200 remodelling and organization, 200 Extracellular matrix protein regulation, 396 F FA complementation group A (FANCA), 73 FADD-dependent pathway, 261 FAM123B/WTX, 144 Familial adenomatous polyposis (FAP), 3, 25, 26, 43, 45, 308, 326, 386 Fanconi’s anemia complementation group (FANCE), 73 Fas ligand (FasL)-dependent macrophages, 197 Fat globule membrane (FGM), 327 Fe3O4 nanoparticles (FeNPs), 97 Fermented milks, 330, 332 Fermented skim milk extracts, 333 Fibroblastic growth factor (FGF), 198 Fibulin (FBLN1), 10 First-line treatment, 107 Flavone, 252 Flavoring agents artificial sweeteners, 317 aspartame, 318 MSG, 318 saccharin, 317 taste enhancers, 317 5-Fluoro-2-deoxyuridine (5-FUDR), 126

449 5-Fluorouracil (5-FU), 47, 108–111, 113, 124, 125, 136, 230–232, 340, 387 Fluorodeoxyuridine monophosphate (FdUMP), 126 Fluoropyrimidines (FPs), 125, 134, 135, 389 FOLFOX, 155 Folinic acid (FOL), 155 Food additives beneficial effects, 310 carcinogenicity risk, 310, 311 categories (see Categories of food additives) chemical changes, 309 chemical substances, 308, 309 drinking beverages, 311 E numbers, 311 food-packaging materials, 309 legal limits, 310 mutagenic/carcinogenic, 308 preservatives, 311 purposes, 320 toxicity, 310 toxicological studies, 309 utilization, 309 Food coloring agents azo dyes, 315 benzidine, 317 food dyes, 315 groups, 315 visual appearance/acceptance improvement, 315 Food dyes, 315–317 Food ingredients, 308 Food preservatives antibrowning agents, 314 antimicrobial agents, 312 antioxidants, 314 chemical substances, 311 health risks, 313 negative health effects, 312 nitrates and nitrites, 313, 314 shelf life, 312 Free radical scavenging activity, 314 Fruits AKT activity, 280 anti-CRC activity, 280 apples, 282 banana, 284 Caco-2 cell lines, 280 CF, 281 grape, 283 pharmacological properties, 281 Punica granatum, 280 Fusion proteins, 24 Fusobacterium abundance, 265

Index

450 G G2/M phase, 405 Gas6 ligands, 202 Gastrointestinal (GI) tract, 176 Gastrointestinal (GI) tumors, 71 Gastrointestinal epithelium, 46 Gastrointestinal infections, 411 Gastrointestinal tract, 308, 408 Gastrointestinal tract (GIT) toxicity, 80 Gefitinib (Iressa), 148 Gelsolin (GSN), 10 Gemcitabine, 233 Gene transcription, 198 Gene-editing technologies, 354 Genetic engineering, 362, 377 Genetic mutation, 385, 386 Genetic susceptibility, 71, 83, 84 Genetically engineered bacteria, 363 Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO), 79 Genistein, 251 Genome, 27 Genome-wide association studies (GWAS), 27, 72 Genomic instability, 45 Genomic mutations, 45 Gingerol, 253 Global Cancer Observatory (GLOBOCAN), 3 Glucose transporters (GLUTs), 390 Glutathione S transferase (GST) activity, 280 Glycogen synthase kinase (GSK)-3β phosphorylation, 401 GO-Y030, 401 G-protein coupled estrogen receptor (GPER), 49 activation, 52 as GPR30, 43 cell surface receptors, 52 estrogenic action, 51 transmembrane protein, 51 Granulocyte-macrophage colony-stimulating factor (GM-CSF), 195 Grapes bioactive components, 283 cellular invasion, 283 Growth Arrest Unique gene, 202 Growth factor EGFR, 344 Growth factor signaling molecules, 145 Gut microbial profile, 409 Gut microbiome alterations/imbalance, 319 anaerobes, 319 carcinogenesis process, 320 development, 319

microorganisms community, 319 phyla, 319 symbiotic association, 319 Gut microbiota, 309 H Hammerhead ribozymes, 345 Hand-foot syndrome, 219 HCT116, 396 HDAC inhibitor, 207 Health food, 326 Healthcare infrastructure, 143 Heat shock proteins (HSPs), 406 Heat-inactivated bacteria, 363 Heavy-Ras (HARS), 147 Hedgehog (Hh) signaling pathway, 154 HEF1, 407 Helicobacter pylori, 363 Heme oxygenase (HO)-1 protein, 393 Hemiscorpius lepturus, 393 Hepatocyte growth factor (HGF), 132 HER2 overexpression, 112 Hereditary CRC, 308 Hereditary nonpolyposis colorectal cancer (HNPCC), 26, 43, 45, 308, 386 Hexahydrocurcumin analogue, 402 Hexamers, 370 HGF/c-MET pathway, 149, 150 Hh-signaling pathway inhibitor, 154 High-fat diet (HFD), 133 Highly upregulated in liver cancer (HULC), 27, 30 Hippo signaling pathway, 399 Histone deacetylases (HDACs), 405 Histone deacetylation, 350 Histone demethylase, 349 Histone modification, 26 Hollow gold nanoshell (HAuNS), 97 Homeobox protein CDX-2 (CDX2), 399 Homeostatic, 430 Hormone replacement therapy (HRT), 57, 60, 61 Hormone-associated cancers, 55 Host’s well-being, 408 HOTAIR, 351 HOX transcript antisense RNA (HOTAIR), 27, 29 HT-29 cell line, 332 HT-29 colon cancer, 405 Human gastrointestinal tract, 319 Hyaluronidase facilitated separation, 407 Hydrolysis, 326 Hypermethylation, 26, 405

Index Hyperphosphorylated tau protein, 395 Hypoxia, 227, 230 Hypoxia-inducible factor (HIF) 1α, 390 Hypoxic factor-1 (HIF-1α), 198 I ICA [(1E,6E)-1,7-di(1H-indol-3-yl)hepta-1,6-­ diene-­3,5-dione], 403 IFN-γ-STAT-1 axis signalling, 207 IGF receptors, 397 IL-1–mediated inhibition, 405 Immune cell modulation, 409, 410 Immune cells, 11 Immune escape, 154 Immune system, 174 Immune system ailments, 390 Immune-regulatory activity, 392 Immune-related adverse events (irAEs), 184 Immunohistochemistry, 199 Immunohistological morphometric analysis, 199 Immunomodulatory effects, 409, 410 Immunostimulatory, 411 Immunotherapy, 95, 96, 125, 128–130, 135, 209, 436 Immunotoxins, 367, 372 Independent/combined modality treatment, 218 Indian saffron, 291 Indirect food additive, 309 Indocyanine green (ICG) liposome, 97 Indoleamine 2,3-dioxygenase (IDO), 198 Inducible nitric oxide synthase (iNOS), 249 Industrialization, 309 Inflammation and tumor progression, 186 Inflammatory bowel disease (IBD), 43, 44, 320 Inflammatory cytokines, 186 Inflammatory immune response, 174 Inflammatory leukocyte extravasation, 202 Inflammatory microenvironment, 185 Inflammatory treatments, 175 Inhibitor κB (IκB), 151 Insulin-like growth factor (IGF), 397 Interferon-β signaling, 183 Interleukin-2 (IL-2), 368 International Agency for Research on Cancer (IARC), 314 International numbering system (E numbers), 311 Intestinal dysbiosis, 319, 320 Intestinal flora, 116, 117 Intestinal homeostasis, 320

451 Intestinal inflammation, 174 Intestinal microbiome, 319 Intestinal microbiota, 320 Intestinal microflora, 319 Intestine’s epithelium, 174 Intraoperative radiation therapy (IORT), 220 Intrinsic apoptotic signalling pathway, 261 Ipilimumab, 158, 159 Irinotecan, 125–127, 134, 136, 232, 387, 389 Irreversible EGFR inhibitor, 148 Irreversible genomic modifications, 194 Irreversible tyrosine kinase inhibitor, 148 irritable bowel syndrome (IBS), 385 J JAK/STAT pathway, 264 Janus kinase and signal transducer and activator of transcription (JAK/ STAT) signaling pathway, 391 JNK signaling pathway, 395 Jumonji domain (JMJD) 2C, 401 K Kirsten Ras (KRAS), 147 Kirsten rat sarcoma viral oncogene homolog (KRAS), 5 KRAS-mutated CRC cell, 349 KRAS-mutated LARC, 231 L Lactate dehydrogenase (LDH), 390 Lactic acid bacteria (LAB), 332, 366 Lactobacillus, 12 Lactobacillus casei, 332 Lactobacillus rhamnosus, 332 Lactococcus, 12 Lactoferrin (Lf), 330 Lactose, 326 Lamiaceae family’s rosemary, 253 Lamina propria macrophages (lpMFs), 200 67 kDa laminin receptor (67LR), 263 Laparoscopic colectomy, 119 Laparoscopic surgery, 119, 144 Lapatinib, 148 Left-sided colon (LSC) tumors, 78 Left-sided colorectal cancer (LCRC), 176 Leuconostoc, 12 Leucovorin (LV), 232 Leucovorin calcium, 155 Lipopolysaccharide (LPS), 405

452 Lipopolysaccharide-coated CuS nanoparticles, 97 Lipoprotein receptor-related protein 1 (LRP1), 369 Liposomes, 406 Liquid chromatography–mass spectrometry (LC-MS), 398 Listeria monocytogenes, 208 Listeria monocytogenes LADD strain, 364 Liver ailments, 390 Liver cancer, 400 L-OHP, 389 Long non-coding RNAs (lncRNAs), 350, 351 Long-chain noncoding RNA (lncRNA) HOTAIR, 108 Low anterior resection (LAR), 387 Lung infections, 390 Lung metastases, 106 Luteolin, 254 Lymph system, 387 Lysine-specific demethylase 1 (LSD1), 349, 351 M M2 macrophage heterogeneity, 197 Macrophage infiltration, 197 Macrophage invasion, 199 Macrophages defenders, 205 innate immunity players, 194 localized propagation, 195 locations, 195 mononuclear phagocytic system, 194 myeloid-based antigen-presenting cells, 195 progenitors division, 195 Macrophages antitumour activation, 208 Magnetic resonance imaging (MRI), 388 Magnetococcus marinus, 364 Maintain cell cycle progression, 396 Maintenance therapy bevacizumab, 110, 111 cetuximab and panitumumab, 111 Major histocompatibility complex (MHC), 180 Malignancies, 390, 411 Malignancy (cancer), 308 Malignancy risk factors, 296 Malignant cells, 400 Malignant disorders, 363 Malignant tumor, 194, 308 MAPK pathway, 147 MARCO, 206

Index Matrix metalloproteases (MMPs), 200, 370 Matrix metalloproteinase (MMP)-9, 390 Matuzumab (EMD72000), 148 MDR phenotype formation, 397 Mechanistic target of rapamycin (mTOR), 395 Medicinal plant, 389 Medullary carcinoma, 385 MEK inhibitor, 149 MEK1/2 inhibitor, 149 Membrane-associated ERs, 51 Memory damage, 395 Mesenchymal stem cells (MSCs), 432 Messenger RNA (mRNA), 393 Metabolic sicknesses, 390 Metal-based drugs gold-based drugs, 13 platinum, 13 Metastasis, 269, 431 blood circulation/lymph vessels, 200 destined organs, 201 myeloid lineage cells, 201 pre-metastatic niches, 200 therapeutic options, 200 tumour development, 200 Metastasis-associated in colon cancer-1 (MACC1) gene, 350 Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), 27, 29 Metastasis-initiating cancer cells, 186 Metastatic colon cancer, 109, 110 therapy after progression, 113–114 Metastatic colorectal cancer (mCRC), 147 old and new targets, 129 Metastatic diseases, 3 Metastatic patterns, 106 Methylenetetrahydrofolate reductase (MTHFR), 79, 80, 126, 127 Micelles, 406 Microbial environment, 408, 409 Microbiology, 5 Microbiome profile, 408 Microbiota, 308, 408 Microorganisms, 308 MicroRNA (miRNA), 24, 45, 349, 350, 402 Microsatellite instability (MSI), 5, 26, 45, 177 Microsatellite instability (MSI) disruption, 405 Microsatellite stable (MSS), 79 Migration process, 431 Milk and dairy products butyric acid, 296 calcium, 295 conjugated linoleic acid (CLA), 296 vitamin D3, 295

Index Milk and dairy products, CRC anticancerous effect, 333 bioactive peptides, 330 calcium, 328, 329 cancer-modulating activity, 327 casein, 330 CLA, 329 cohort studies, 328 constituents, 326, 327, 330, 333 cytotoxicity, 328 epidemiological data, 327, 328 fermented milks, 330, 332 genotoxicity, 328 lactoferrin, 330 LfcinB, 330 meta-analysis studies, 328 prebiotic compounds, 333 probiotic consumption, 333 sarcopenia, 330 supplemental vitamin D3, 329 vitamin D, 329 whey proteins, 330 Milk fat globule-epidermal growth factor (EGF) 8 (MFG-E8), 198 Milk lipids, 327 miR-204-5p, 350 miRNA, 82, 83 miRNA therapy, 350 miRNA-143, 350 miRNA-based therapies, 108 Mismatch repair (MMR) genes, 78 Misonidazole, 230 Mitochondrial-dependent pathway, 396 Mitogen-activated protein kinase kinase 1 (MAP2K1), 80 Mitomycin C plus chemoradiation, 233 MLH1-silenced HCT116, 405 Molecular biology, 5 Molecular docking, 394 Molecular factors influencing CRC BRAF V600E, 179 environmental factors, 177 genomic instability, 177 MSI, 178 p53, 179 Ras proteins, 178 VEGF, 178 Molecular mechanism, drug resistance, 135, 136 Molecular pathways, 3 Molecular targeted therapy classification, 145 Monocarboxylate transporters (MCTs), 390

453 Monoclonal antibodies (mAb) bevacizumab, 156, 157 cetuximab, 157, 158 ipilimumab, 158, 159 nivolumab, 158, 159 panitumumab, 158 pembrolizumab, 159 ramucirumab, 157 Monocyte chemoattractant protein 1 (MCP1), 197 Monocyte chemotactic protein 1 (MCP-1), 432 Monocytes (F4/80low), 197 Monosodium glutamate (MSG), 318 mRNA expression, 406 MSG-induced diabetic mice, 318 Mucinous, 106 Mucinous adenocarcinoma, 385 Multidrug resistance (MDR), 133–135, 340, 387 Multikinase inhibitor, 148 Murine model, 397 Mutation, 72, 74–77, 83 MutL homolog (MLH1) gene, 78, 79 Mutual ontogenicity, 200 Mycobacterium bovis BCG, 364 Mycobacterium tuberculosis, 363 Myeloid-derived suppressor cells (MDSC), 95 Myeloid lineage cells, 201 Myeloid-originated innate immune cells myeloid cells, 181 neutrophils, 183 normal tissue function, 181 TAMs, 182, 183 TLRs, 181 TME, 181 Myeloid prerequisites, 195 Myh-associated polyposis (MAP), 25, 26 N Naked cuticle 2 (Nkd2) inhibition, 398 Nano-carriers, 353 Nanoformulation, 98 Nano-immunotherapy amplification nanoparticles, 96 anti-FAT mAb198.3, 97 antitumor response, 97 cancer immunotherapeutic approaches, 95 cancer immunotherapeutics, 94 cancer immunotherapy, 98 cellular models, 98 chemo-immunotherapy, 96 chemotherapy, 94

Index

454 Nano-immunotherapy (cont.) chitosan-TPP/nanoparticles, 99 conventional immunotherapies’ efficacy, 96 CRC, 93 gold nanoparticles, 97 immune checkpoint inhibitors, 96 immune system, 94 immune-related adverse effects, 96 immunity activation, 96 immunology, 95 immunotherapy, 95 immunotherapy-based nanoparticles, 96 lipid calcium phosphate nanoparticle, 98 lipid nanoparticles, 98 lipid-protamine-DNA nanoparticle system, 98 liposomal nanohybrid centrosomes, 99 MC-38 colon adenocarcinoma models, 97 mechanism, 94 nanoformulation, 98 nanoparticles, 96 polydopamine nanoparticles, 98 radiotherapy, 94 surgery, 94 T cells, 97 treatment, 97 Nanoparticles, 96, 353, 406 Nanoparticles (NP)-based cancer immunotherapy approach, 94 Nanotechnology, 95–99, 355 Napabucasin, 161 National Comprehensive Cancer Network (NCCN), 108, 111, 113 Natural antisense transcripts (NATs), 27 Natural food preservatives, 311 Natural killer (NK) cells, 180 Natural ribozymes, 345 Nature-derived components, 362 NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines), 106, 109, 114 Neoadjuvant chemoradiotherapy, 221–224 Neoadjuvant chemotherapy, 388 Neoadjuvant preoperative strategy, 232 Neovascularization, 186, 198 Neuroblastoma Ras (NRAS), 147 Neurodegenerative disorders, 395 Neurofibromatosis type-1 (NF1), 149 Neurological illnesses, 390 Neuropathy, 219 Neuroprotection, 394, 395 Neutralization, 204 Neutralizing pro-inflammatory cytokines, 186 Neutrophils, 183

NF-κB pathway, 264 NF-κB signaling, 186, 282 Nicotinamide adenine dinucleotide phosphate (NADPH), 393 Niemann-Pick C1-like 1 (NPC1L1), 393 Nigella sativa L., 294 Nintedanib, 161 Nisin A, 372 Nitrates and nitrites, 313, 314 Nitric oxide synthase, 390 Nivolumab, 109, 158, 159, 389 NK cell therapy, 180 Noncancerous growth, 384 Noncoding RNAs (lncRNA), 26 Nonribosomal bacterial peptides, 373, 374 Nonribosomal peptide synthetases (NRPSs), 373 Nonsteroidal anti-inflammatory drugs (NSAIDs), 11, 12, 14, 185, 411 Nontoxic drugs, 411 Non-vector methods, 353 Notch signaling pathway, 151 Novel therapeutics, 186 Nuclear estrogen receptors, 42 Nuclear factor (NF)-кB, 390 Nuclear receptors, 48–51, 59 Nuclease-resistant RNA oligonucleotides, 347 Nucleolin (NCL), 10 Nutrition, 327 Nutritional additives, 319 O Oat, 290 Obesity, 385 Oncogene miRNAs (oncomiRs), 350 Ornithine decarboxylase (ODC), 398 Orphan nuclear receptors, 49 Oxalato-platinum or 1-OHP (OHP), 127 Oxaliplatin, 125, 127, 135, 136, 231, 232 Oxaliplatin (L-OHP), 387 Oxidation stress, 260 Oxygen, 230 P p38 mitogen-activated protein kinase (MAPK), 393 p53 mutation, 179, 399 Palliative chemoradiotherapy, 221 Panitumumab, 8, 111–114, 148, 158, 387, 389 in the non-first-line setting, 114 with FOLFIRI, 113 with FOLFOX, 113

Index Parkinson’s disease (PD), 395 Pathophysiology, 3 Patient’s hospitalization, 119 PE38-based immunotoxins, 370 Pediocin, 372 Pediococcus acidilactici, 372 Pelvic radiation therapy, 232 Pembrolizumab, 116, 159, 389 Pentagamavunon (PGV)-1, 403 Peroxiredoxin-1 (PRDX1), 10 Peroxisome proliferator–activated receptor (PPAR) γ protein, 395 Pertuzumab (Perjeta), 148 Pexidartinib, 205 P-glycoprotein (P-gp) expression, 397 Phenolic compounds, 260 4-(2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo(1,5-a)-pyrimidin-3-yl) phenol (PHTPP), 55 Phosphatase and tensin homolog (PTEN) levels, 402 Phosphatidylethanolamine-binding protein (PEBP1), 10 Phosphoinositide 3-kinase (PI3K) signaling pathway, 151, 152 Phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway, 393 Phosphorylated Akt, 262 Photodynamic therapy (PDT), 31 Physical inactivity, 385 Phytochemicals, 25, 33 Phytoestrogen-based anticancer hormonal effect, 289 PI3 K signalling pathway, 255 PI3K pharmacological inhibition, 206 PI3K/Akt signalling, 262 Pigment epithelium-derived factor (PEDF), 10 Placebo-treated patients, 186 Plantaricin A, 373 Plasmacytoma variant translocation 1 (PVT1), 32 Platinum, 13 Plexiform neurofibromas (PN), 149 PLGA [poly(lactic-co-glycolic acid)], 407 PLGA–lecithin–PEG nanoparticles, 408 Pluripotent colon stem cells, 46 Poly(ethylene glycol methyl ether methacrylate) (PEGMEMA)–based micelles, 407 Polyamine synthesis, 398 Polycomb repressive complex 2 (PRC2), 351 Polydopamine nanoparticles, 98 Polymeric nanocarrier curcumin (PNCC), 407 Polypectomy, 387

455 Polyphenols, 12, 247, 248, 255, 260–263, 265, 267, 270, 434 availability, 243 binding affinity, 244 classification, 244–245 cooking methods, 243 dietary phlorizine, 243 factors, 246 function, 244 health benefits, 242 hereditary changes and epigenetic variations, 246 metabolism, 242 mixtures, 242 optional metabolites, 242 organic compounds, 242 pi-electron complexes, 244 semi-engineered natural synthetic substances, 242 sources, 243 Polyps, 94, 384, 386, 428 Pore-forming immunotoxins, 370 Post-marketing perception, 309 Postoperative chemoradiotherapy, 221 Postoperative intestinal obstruction (POI), 119 Potential biomarker, 436 Prebiotic compounds, 333 Preoperative chemoradiotherapy, 221, 232 Primary tumor sidedness, 111 Probiotic bacteria, 366 Probiotics, 12, 332 Processed foods, 320 Progenitor cells, 46 Progression-free survival (PFS), 107, 110, 113, 115, 116 Progression-free survival (PFS) analysis, 73 Pro-inflammatory “hot” environment, 185 Pro-inflammatory arbitrators, 199 Pro-inflammatory cytokines, 186 Pro-inflammatory therapy, 175 Propionibacterium freudenreichii, 333 Prosaposin (PSAP), 10 Prostaglandin D2 (PGD2), 119 Prostaglandin-E2 (PGE2), 197 Prostate cancer, 400 Protracted venous infusion (PVI), 231 Pro-tumoral tumor-induced monocytes (TIMCs), 181 Pro-tumour phenotype, 198 Pro-tumour polarized macrophages, 208 Pro-tumour TAM function, 197 Pseudomonas aeruginosa, 369 Pseudomonas Exotoxin A (PE), 369, 370 Psychoneuroimmunomodulation, 398

456 PTEN/AKT/PI3K pathway, 350 Punica granatum, 280 Punica granatum (Pomegranate) anticancer activities, 280 tannins and anthocyanins, 280 Pyocin S2, 373 Pyruvate dehydrogenase (PDH), 251 Pyruvate dehydrogenase kinase 4 (PDK4), 399 Pyruvate kinase isoenzyme (PK) M2, 390 Q Quantitative trait loci (QTL), 71 Quercetin, 251, 394 R Radiation damage intensification, 228 Radiation therapy, 124, 388 brachytherapy, 220 CRC treatment, 219 EBRT, 219 endocavitary, 220 interstitial brachytherapy, 220 IORT, 220 side effects, 220 X-rays, 219 Radiation-induced cell death, 230 Radiation-sensitive phase, 229 Radioactive seeds, 220 Radiosensitizers, 228 Radiotherapy, 107, 375, 377, 386, 388, 389 Ramucirumab, 114, 115, 135, 157, 389 Randomized clinical investigation, 293 Randomized phase III trial, 130 RAS inhibitors, 147 Ras protein, 178, 251 RAS/MAPK pathway, 75 Ras-Raf-MEK extracellular signal-regulated kinase signaling pathway, 179 Rat sarcoma (RAS), 144 Reactive nitrogen species (RNS), 11 Reactive oxygen species (ROS), 11, 54, 393 Receptor binding domain, 368 Receptor tyrosine kinases (RTKs), 146, 161, 262 Rectal bleeding, 176 Rectal cancer, 385 Regional/local chemotherapy, 218 Regorafenib, 115, 135, 159, 387, 389 Regulatory T cells (Tregs), 197

Index Reprogrammed antitumour macrophages, 206 Resveratrol, 34, 251, 270 CamKKb/AMPK pathway, 252 COX-2 transcription, 252 metabolic enzymes and signalling pathways, 252 stresses, 251 Resveratrol functions, 270 Reversible addition−fragmentation chain-­ transfer (RAFT) polymerization technique, 407 Rhodamine 123 (Rh123), 397 Rhotekin (RTKN), 30 Ribonucleic acid (RNA)-based therapeutics advantages, 354 ASOs, 341, 344 CRC treatment, 341–343 delivery system efficacy, 353 disadvantages, 354 mechanisms, 341 molecules, 341 nanocarriers development, 354 nanoparticle, 353 non-vector methods, 353 ribozymes, 345, 346 RNA aptamers, 346, 347 RNA chemical structure modification, 352, 353 RNAi (see RNA interference (RNAi)) survivin siRNA, 353 target disease-causing genes, 341 viral vectors, 353 Ribozyme RPI.4610 (Angiozyme), 346 Ribozymes, 345, 346 Right-sided colon (RSC) tumors, 73 Right-sided colon cancer (RCC), 176 RKO cell lines, 405 RNA aptamers, 346, 347 RNA chemical structure modification, 352, 353 RNA delivery system, 353 RNA interference (RNAi) action mechanism, 348 Caenorhabditis elegans, 347 lncRNAs, 350, 351 miRNA, 349, 350 saRNAs, 352 siRNAs, 348, 349 RTKs inhibitor, 148 Ruminococcus-like species, 409 Runt-related transcription factor 3 (RUNX3), 398

Index S Saccharin, 317 Salmonella typhi, 363 Salmonella typhimurium VNP20009, 364 Sarcopenia, 330 Secreted protein, acidic and rich in cysteine (SPARC), 10 Selective estrogen receptor degrader (SERD), 55 Selective estrogen receptor modulators (SERMs), 55, 62 Self-immune cell populations, 396 Selumetinib, 149 Semaxanib, 161 Sensitizer enhancement ratios (SERs), 228 Sequential chemoradiotherapy, 221 Serratia marcescens, 363 Sex hormone, 44, 47, 56 androgens, 42 estrogen, 43, 61 Short hairpin RNAs (shRNAs), 348 Short-chain fatty acids (SCFA), 265, 333 SH-SY5Y human neuroblastoma cell line, 394 Signal transducer and activator of transcription (STAT) 3, 390 Signal transduction pathways, 144, 145, 147–149, 153, 156, 162, 163 Signalling pathways gut microbiota, 265 JAK/STAT pathway, 264 MAPKs signalling, 262 NF-μB pathway, 264 Nrf2 pathway, 264 p38 MAPK pathway, 262 phosphorylated Akt, 262 PI3K/Akt signalling, 262 Wnt pathway, 263 Signet cell carcinoma, 385 Signet ring adenocarcinomas, 106 Silibinin, 253 Single nucleotide polymorphisms (SNPs), 71 Single-chain protein diphtheria toxin, 367 Single-fraction in vitro assays, 228 Sirtuin-1 (SIRT1), 398 Skin cancer, 400 SMAD4, 433 Small activating RNAs (saRNAs), 352 Small interfering RNAs (siRNAs), 348, 349 Small molecules, 108 Small-molecule inhibitors, 146, 149, 156, 163, 384 Small-molecule therapy regorafenib, 159

457 Ziv-aflibercept, 159, 160 Smoking, 385 Solid tumors, 372 Solute carrier (SLC) transporters, 24 Sorafenib, 231 Sorafenib (Nexavar), 148 Spatial assistance, 227 Spatial learning, 395 Spices antioxidants, 291 ginger, 293 turmeric, 291 Src family kinases (SFKs), 153 ssRNA induces transcriptional gene activation, 352 Staphylococcus aureus, 313 STAT3 inhibitors, 186 Staufen homolog 1 (STAU1)-mediated degradation, 351 Sterol regulatory element-binding protein-2 (SREBP-2), 393 Stomach cancer, 400 Streptococcus, 12 Streptococcus pyogenes OK-432, 364 Streptomyces caespitosus, 233 Stress-activated’ kinase pathway, 262 Sulfites, 315 Sulfotransferase family 2B member 1 (SULT2B1), 81 Surgical intervention, 46 Survival after relapse (SAR), 76 Survivin siRNA, 353 Synergistic anticancer effect, 349 Synthetic antioxidants BHA and BHT, 314 Synthetic food dyes, 315 Synthetic HDL (sHDL), 97 Systemic CCL2 level, 204 Systemic chemotherapy, 218 T T cells adaptive immunity, 179 CAR T-cell therapy, 180 NK cell therapy, 180 TCR therapy, 180 TIL, 180, 181 Tailor-made ribozymes, 345 TAM anti-tumorigenic activity, 197 TAM in anticancer therapy, 203 TAM polarization, 198, 205 TAM receptor-binding ligands, 202 TAM recruitment, 204

458 TAM reprogramming antitumour phenotype, 208 CD40L, 207 CD47 expression, 206 CD47-SIRPα axis, 206 clinical trials, 208 epigenetic regulation, 207 immunosuppressive articles, 206 IMO-2055, 208 M1 antitumour phenotype, 208 MHC-II expression, 206 monoclonal antibodies, 206 pattern recognition receptor, 206 pro-inflammatory behaviour, 207 pro-tumour activities, 206 receptors/signalling, 207 signalling mediators, 206 SIRPα, 206 strategies, 206 targets, 206 TLRs, 207 V-ATPase inhibition, 208 TAMs advantageous plasticity, 182 TAMs therapeutic targeting anticancer interventions, 202 cancer biology, 202 macrophages limiting, 204–205 reprogramming, 202, 205–208 strategies, 202 Tangeretin, 254 Target biomarkers, 372 Target RNAs, 345 Targeted chemotherapeutic drugs apatinib, 162 brivanib, 162 cediranib, 162 cellular toxicity, 155 CRC patients in clinic, 156 monoclonal antibodies (mAb), 156–159 napabucasin, 161 nintedanib, 161 semaxanib, 161 small-molecule therapy, 159, 160 vatalanib, 161 Targeted molecules, 145, 146, 155, 160, 163 Targeted oxaliplatin, 134 Targeted therapy, 47, 106, 144, 145, 340 cancer immunotherapy, 154 EGFR, 146, 147 EGFR, RAS/RAF/MEK/ERK signaling, 147–149 HGF/c-MET pathway, 149, 150 Hh signaling pathway, 154

Index immune escape, 154 notch signaling pathway, 151 PI3K signaling pathway, 151, 152 SFKs, 153 TGF-β signaling pathway, 152 VEGF/VEGFR pathway, 150, 151 Wnt signaling pathway, 153 TAS-102, 136 Temporal modulation, 227 Tetrahydrocurcumin, 401 Texturing agents, 318, 319 Texturizing agents emulsifiers, 318 stabilizers, 318 TGF-β pathway, 145 TGF-β secretions, 197 TGFβ-induced proteins, 200 The Cancer Genome Atlas (TCGA), 72, 144 Therapeutic approaches, 129 Therapeutic index enhancement approaches biologic cooperation, 227 normal tissues protection, 227 spatial assistance, 227 temporal modulation, 227, 228 toxicity independence, 227 tumor response improvement, 227 Therapeutics, 134–135 Thoracic metastases, 106 Thymidylate synthase (TS), 126 Thymoquinone, 294 Tie2+ TAM, 199 Time to recurrence (TTR), 76 Tipiracil, 389 Tirapazamine, 227, 230 Tissue inhibitor of metalloproteinase 2 (TIMP2), 10 Tissue-resident macrophage trait, 197 TME-driven epigenetic alteration, 207 TNF-Related Apoptosis-Inducing Ligand (TRAIL), 333 Toll-like receptor (TLR) 4, 409 Toll-like receptors (TLRs), 181, 207 Topoisomerase I (Topo I), 126, 127 Toxicity independence, 227 Trabectedin, 205 Traditional chemotherapeutic agents, 234 Traditional Chinese medicine, 106, 118 Traditional topical therapy, 201 TRAIL/caspase-8 targeting trabectedin, 205 Transanal endoscopic microsurgery (TEM), 24 Transcriptome, 27

Index Transformed cells’ physiology, 209 Transforming growth factor B (TGF-β), 132, 183 Transforming growth factor beta (TGF-β) signaling pathway, 152 Transmembrane domain, 368 Traumatic pain, 176 Treg-cell-depleted tumor, 435 Trifluridine, 389 Trifluridine/tipiracil, 116 Tripartite motif 47 (TRIM47), 433 Tumor-associated neutrophils (TANs), 183 Tumor cells, 376 Tumor degradation, 183 Tumor-elicited inflammation, 409 Tumor endothelial marker 8 (TEM8), 371 Tumor-infiltrating immune cells, 187 Tumor-infiltrating lymphocytes (TIL), 180, 181 Tumor microenvironment (TME), 95, 108, 124, 131–132, 397 Tumor necrosis factor (TNF), 284, 390 Tumor necrosis factor receptor 2 (TNFR2), 97 Tumor-node-metastasis (TNM), 76 Tumor–Node–Metastasis (TNM) cancer staging system, 388 Tumour microenvironment (TME) abundance regulation, 204 CCL2 antagonists, 204 components, 194 M2 phenotypes, 204 macrophage migration, 205 precancerous niche, 204 role, 194 siltuximab, 204 TAM recruitment, 204 VEGF, 204 Tumour-associated macrophages (TAMs), 131, 132, 182, 183 angiogenesis, 198–199 chemoresistance, 201–202 ECM modelling, 199–200 functional properties, 196 human ovarian carcinoma, 195 key constituent, 195 manipulation, 208, 209 metastasis, 200–201 murine fibrosarcoma tumours, 195 phenotypic behaviour, 208 pro-tumorigenic impact, 196 regulatory effects, 195

459 therapeutic targeting (see TAMs therapeutic targeting) TME, 197, 198 tumor progression role, 196–197 tumour-induced immunosuppression, 197, 198 Tumour-derived cytokines, 197 Tumour-educated M2-like macrophages, 197 Tumour-induced immunosuppression, 207 Tumour-induced M2 polarization, 205 Tumour-secreted proteins, 201 TYMS gene expression, 126 Type 2 diabetes mellitus, 385 Tyrosine kinase inhibitor, 148 U Ulcerative colitis (UC), 44, 194, 385 United States for advanced non-small-cell lung cancer (NSCLC), 148 United States Preventive Services Task Force (USPSTF), 11 3′ Untranslated region (UTR), 349 Urothelial carcinoma-associated 1 (UCA1), 27 V Vascular density, 199 Vascular endothelial growth factor (VEGF), 109, 110, 115, 178, 198, 233 Vascular endothelial growth factor (VEGF)/ vascular endothelial growth factor receptor (VEGFR) pathway, 150, 151 Vascular endothelial growth factor receptor (VEGFR), 125 Vatalanib, 161 V-ATPase inhibition, 208 V-ATPase inhibitor, 208 Vegetables Allium, 285 balanced diet, 284 cruciferous, 284 melanoidins, 286 potato, 286 tomato, 286 VEGF and JAK/STAT3 pathways, 408 VEGF receptor-1 (VEGFR-1), 201 VEGFR1+ cell recruitment, 201 Viral vectors, 353 Vitamin D, 119, 329 Vitamin D receptor (VDR) gene, 81

Index

460 W Whey protein isolate (WPI), 330 Whey proteins, 330–332 WHO GLOBOCAN database, 384 WiDr colon cancer cell line, 407 Wild-type K-Ras mutation, 178 Wnt signaling, 25, 26 Wnt signaling pathway, 144, 153, 399 Wnt/β-catenin signaling pathway, 32, 246–247, 350, 398

Y Yes-associated protein (YAP), 399 Z Ziv-aflibercept, 109, 114, 115, 159, 160, 389 Zoledronic acid, 205