Role of Signaling Pathways in Brain Tumorigenesis 9811584729, 9789811584725

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Mehdi Hayat Shahi

Role of Signaling Pathways in Brain Tumorigenesis

Role of Signaling Pathways in Brain Tumorigenesis

Mehdi Hayat Shahi

Role of Signaling Pathways in Brain Tumorigenesis

Mehdi Hayat Shahi Interdisciplinary Brain Research Centre, Faculty of Medicine Aligarh Muslim University Aligarh, Uttar Pradesh, India

ISBN 978-981-15-8472-5    ISBN 978-981-15-8473-2 (eBook) https://doi.org/10.1007/978-981-15-8473-2 © Springer Nature Singapore Pte Ltd. 2023 This work is subject to copyright. All rights are reserved 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 Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

This book is dedicated to Our beloved parents, wife, children, and family members for their love and constant support to accomplish this work.

Preface

This book is focused on the role of cell signaling pathways in brain tumor development. There are various pathways that contribute to brain tumorigenesis. However, comprehensive and detailed information of all these pathways in brain tumorigenesis is limited. Therefore, in this book the author has attempted to incorporate most of the major pathways contributing to brain tumor development. This book has been compiled with a comprehensive approach to defining the role of major cell signaling pathways in brain tumor origin, growth, and progression. Even many pathways which regulate early embryonic brain development have also shown significant contributions to brain tumor growth and are included. Moreover, in this book, the author has highlighted the involvement and regulatory approaches to inhibit or modulate these pathways in brain tumorigenesis. Henceforth, this approach is not only beneficial for molecular brain tumor researchers but also for neuro-oncologists, neuropathologists, postgraduate and graduate students who want to understand the role and regulation of cell signaling pathways in brain tumor growth and therapeutic approaches for the treatment of brain tumors.

Organization of the Book This book is divided into six parts. Part I: This introductory part has only one chapter in which the major types of cell signaling pathways and brief classification of brain tumors are mentioned including medulloblastoma and glioma. Part II: This part has four chapters, which include Chaps. 2–5. This part deals with major embryonic developmental cell signaling pathways, viz., Wnt, Notch, and Sonic hedgehog (Shh) in brain tumor development. Part III: This part also has four chapters, which include Chaps. 6–9. This part explains the role of major stem cell factors Nanog, Sox, and Oct4 in brain tumor development. Part IV: This part covers four chapters, namely, Chaps. 10–13. It defines the role of tumor suppressor gene p53, NFkβ, and neurotrophins contribution to brain tumor development.

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Preface

Part V: This part has three chapters, which include, Chaps. 14–16. It illustrates the role of epidermal growth factor receptor (EGFR) and MAP Kinase in brain tumor development. Part VI: This part is the last section with two chapters (Chaps. 17 and 18). This part explains the role of hypoxia inducing factor (HIF1) and Nmyc in brain tumorigenesis. Aligarh, Uttar Pradesh, India

Mehdi Hayat Shahi

Acknowledgments

First and foremost, I am thankful to Allah SWT, who blessed me to write this book titled Role of Signaling Pathways in Brain Tumorigenesis. I am grateful to the Springer Nature team, especially Mr. Ejaz Ahmad and Dr. Bhavik Sawhney, Project Coordinators, who gave me the golden opportunity to do this wonderful project to submit a proposal for a book on brain tumors and cell signaling pathways. After that, I prepared the proposal and submit ted it to Springer Nature, where it was reviewed and accepted. The book proposal acceptance was a great moment for me. Although it took a lot of time to finish this book because of other academic engagements, I am indebted to the Springer Nature team and management for their patience and cooperation. Fortunately, my parents always encouraged me, especially my father, who had motivated me for this book project. I am also thankful to my beloved wife, Sharia, for her constant support during the compilation of this book. She had supported me tirelessly even on weekends, and holidays. It is pertinent to mention that I am also thankful to my cute and lovely kids Hamzah, Ibrahim, and Zainul for minimum disturbance during the completion of this book. I want to express my special thanks to my supervisors and mentors, Professor Mohammed Afzal, Professor Subrata Sinha, and Professor Javier S. Castresana, for developing my research interest in cell signaling pathways and brain tumor biology. Nevertheless, I am also grateful to Professor Davis N. Louis, Harvard Medical School, USA, and Professor Arie Perry, University of California, San Francisco, USA, for providing a detailed classification of brain tumors. Mehdi Hayat Shahi

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Contents

Part I 1

 Introduction to Cell Signaling Pathways and Brain Tumors����������������   3 1.1 Cell-Signaling Pathway Classification������������������������������������������������   5 1.1.1 Endocrine Signaling����������������������������������������������������������������   5 1.1.2 Autocrine Signaling����������������������������������������������������������������   6 1.1.3 Paracrine Signaling ����������������������������������������������������������������   6 1.1.4 Intracrine Signaling����������������������������������������������������������������   6 1.1.5 Juxtacrine Signaling����������������������������������������������������������������   7 1.2 Introduction to Brain Tumors��������������������������������������������������������������   7 1.2.1 Primary Brain Tumors������������������������������������������������������������   8 1.2.2 Brain Tumor Metastases ��������������������������������������������������������   8 1.2.3 Benign Brain Tumors��������������������������������������������������������������   8 1.2.4 Malignant Brain Tumors ��������������������������������������������������������   8 1.2.5 Symptoms ������������������������������������������������������������������������������  10 References����������������������������������������������������������������������������������������������������  11

Part II 2

 Wnt Cell Signaling Pathway in Brain Tumor Development������������������  15 2.1 Canonical Pathway������������������������������������������������������������������������������  18 References����������������������������������������������������������������������������������������������������  24

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 Notch Cell Signaling Pathway and Brain Tumors����������������������������������  29 References����������������������������������������������������������������������������������������������������  35

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 Sonic Hedgehog Cell Signaling Pathway and Medulloblastoma ����������  39 References����������������������������������������������������������������������������������������������������  51

5

 Role of the Shh Signaling Pathway in the Most Common Brain Tumor “Glioma” role in Glioma Growth������������������������������������������������  57 References����������������������������������������������������������������������������������������������������  65

Part III 6

 NANOG Stem Cell Marker and Its Role in Brain Tumor Development ����������������������������������������������������������������������������������������������  71 References����������������������������������������������������������������������������������������������������  80 xi

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 Stem Cell Factor Sox2 and Brain Tumor Development ������������������������  83 References����������������������������������������������������������������������������������������������������  90

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 Role of Neurotrophins in Brain Tumor Development����������������������������  93 8.1 Role of Neurotrophins in Neuron Development ��������������������������������  95 8.2 NGF and Brain Tumors����������������������������������������������������������������������  96 8.3 BDNF and Brain Tumor����������������������������������������������������������������������  97 8.4 NT3 and Brain Tumors ����������������������������������������������������������������������  98 8.5 NT-4 and Brain Tumors���������������������������������������������������������������������� 100 8.6 Dehydroepiandrosterone (DHEA) and Brain Tumor�������������������������� 100 References���������������������������������������������������������������������������������������������������� 101

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 Role of NF-kB in Brain Tumor Development������������������������������������������ 105 References���������������������������������������������������������������������������������������������������� 110

Part IV 10 Role  of Myc in Brain Tumor Development���������������������������������������������� 115 References���������������������������������������������������������������������������������������������������� 124 11 Role  of p53 in Medulloblastoma Development���������������������������������������� 129 References���������������������������������������������������������������������������������������������������� 139 12 Role  of TP53 Gene in Glioma�������������������������������������������������������������������� 143 References���������������������������������������������������������������������������������������������������� 152 13 Role  of HIF-1 in Brain Tumor Development ������������������������������������������ 155 References���������������������������������������������������������������������������������������������������� 164 Part V 14 EGFR  and Its Role in Glioma Development ������������������������������������������ 169 References���������������������������������������������������������������������������������������������������� 179 15 Role  of EFGR in Medulloblastoma Development���������������������������������� 181 References���������������������������������������������������������������������������������������������������� 189 16 Role  of OCT4 in Glioblastoma����������������������������������������������������������������� 191 References���������������������������������������������������������������������������������������������������� 198 Part VI 17 Role  of OCT4 in Pediatric Brain Tumor “Medulloblastoma” Development ���������������������������������������������������������������������������������������������� 203 References���������������������������������������������������������������������������������������������������� 207 18 Mitogen-activated  protein (MAP) kinase Roles in Paediatric Brain Tumor “Medulloblastoma” ���������������������������������������������������������������������� 209 References���������������������������������������������������������������������������������������������������� 216

About the Author

Mehdi  Hayat  Shahi  is an Assistant Professor at the Interdisciplinary Brain Research Centre, Aligarh Muslim University, Aligarh. Before joining AMU, he was a postdoctoral fellow at the University of California, Davis, USA, from 2010 to 2014. He was also an instructor at the University of California Davis, besides his postdoctoral research. From 2009 to 2010, he worked as a postdoctoral research fellow at Uppsala University, Uppsala, Sweden. His current research interests are focused on molecular neuro-oncology. Towards this pursuit, he is exploring the early stem cell biomarkers that can be utilized for the early diagnosis of brain tumors. He has been honored with many prestigious awards, notably CAEN Award from the International Society of Neurochemistry, and Mrs. Farah Deeba Haq Award from the Indian Academy of Biomedical Sciences for being an outstanding researcher in Brain Tumour Biology. He has also served as a referee for several international journals, including Life Sciences, PLoS One and Tumor Biology. He was also appointed as Guest Editor of the Special issue of Frontier in Oncology. He has 18 years of research experience in molecular brain tumor biology and 11 years of teaching experience. He has published more than 30 research articles in peer-reviewed international journals and co-authored four book chapters. He has been as a Co-chair of the Postdoctoral Scholar Association, University of California, USA, from 2013 to 2014. He is a member of many international scientific societies and organizations, importantly International Society for Neurochemistry, Indian Academy of Neurosciences, Indian Association for Cancer Research, and Indian Academy of Biomedical Sciences.

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Part I

1

Introduction to Cell Signaling Pathways and Brain Tumors

Cell signaling is the process by which cells coordinate and communicate with other cells, allowing cells to act in the correct way in response to specific external stimuli. Moreover, cell signaling affects cell structure and function. Cell communication is critical for developmental processes, tissue repair, immunity, and normal cellular homeostasis (Fig. 1.1). Cell signaling pathway deregulation is one of the reason for the for the development of various diseases such as cancer, autoimmune disorders, incompetency, and diabetes (Fig. 1.2) (Vlahopoulos et al. 2015; Wang et al. 2013; Solinas et al. 2007). These diseases might be treated more effectively and efficiently by understanding the regulatory mechanisms of cell signaling (Smith et al. 2015). Normally, most cells usually communicate with each other with the help of extracellular messenger molecules. The extracellular messenger molecules move a short distance and then activate target cells, which are in the vicinity of the originator of the extracellular messenger. Generally, the cell-signaling pathway is activated by release of a messenger molecule from the cell that is responsible for transfer of the information to other cells inside the body. Interestingly, the extracellular surrounding of the cell contains hundreds of different informational molecules, which vary from small to large molecules, soluble proteins, hormones, and glycoproteins. These molecules bind to the surface of target cells. Moreover, a cell receives the particular extracellular messenger molecule if it has a receptor that specifically identifies the messenger (also known as the ligand) to bind it to the cell receptor. Most cells have different types of complement receptors that allow them to respond to different extracellular messenger molecules. Therefore, understanding cell signaling pathways is crucial to understanding the developmental process and disease pathogenesis. The role of a cell signaling pathway in the metabolic process is the activation of liver receptors by circulating adrenaline, which accomplice glycogen breakdown in liver cells and smooth muscle cell relaxation. Hence, interaction with the same initial stimulus can lead to different intracellular protein involvement in the response to two types of cells. Therefore,

© Springer Nature Singapore Pte Ltd. 2023 M. H. Shahi, Role of Signaling Pathways in Brain Tumorigenesis, https://doi.org/10.1007/978-981-15-8473-2_1

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1  Introduction to Cell Signaling Pathways and Brain Tumors Cell communication

Developmental process Cell communication

Normal cellular homeostasis

Tissue repair

Fig. 1.1  Role of cell communication. Cell communication plays a critical role in the developmental process, tissue repair, immunity, and normal cellular homeostasis of the body

Deregulation in cell signalling pathway

Many diseases

Cancer Autoimmune disease

Incompetency

Diabetes

Fig. 1.2  Deregulation of the cell signaling pathway interaction is responsible for various diseases such as cancer, autoimmune diseases, incompetency, and diabetes

the type of activity in which a cell is involved depends on both the stimuli it receives and the intracellular machinery that it possesses at a specific time. Generally, the extracellular messenger molecule binds to the cell membrane receptor of the responding cell. The binding initiates conformational changes in the receptor and consequently the signal transmits froms the membrane to the receptor’s cytoplasmic domain. When the signal reaches the inner layer of the plasma membrane, there are two major directions for it to be transmitted to the cell interior, in order to start any appropriate action. The specific route preference depends on the receptor type that is activated. Therefore, each cell-signaling pathway consists of a series of distinct proteins or transcription factors that act in a cascade process. Interestingly, most of the cell-signaling protein molecules have multiple domains that facilitate dynamic interaction with several different partners, either simultaneously or sequentially.

1.1  Cell-Signaling Pathway Classification

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1.1 Cell-Signaling Pathway Classification There are two types of cell-signaling pathways on the basis of signaling cascades, mechanical and biochemical. The mechanical signal is the force that acts upon a cell and the force produced by the cell. The biochemical signal corresponds to the involvement of molecules viz., proteins, lipids, ions, and gases. Moreover, these signals can be further classified based on the distance between signaling and responder cells (Miller and Davidson 2013). Cell signals are subdivided into the following: • • • • •

Endocrine signaling. Autocrine signaling. Paracrine signaling. Intracrine signaling. Juxtacrine signaling.

1.1.1 Endocrine Signaling In this signaling process hormone molecules are synthesized in specific endocrine cells and are exported by exocytosis to the extracellular regions. Thereafter, hormones are transmitted to target cells of the body with the help of the circulatory system. Only those cells or tissues with a specific receptor for the hormone initiate a hormonal response (Fig. 1.3) (Berridge 2014).

Fig. 1.3  Endocrine signaling. The signal as a hormone molecule is synthesized in specific endocrine cells and it is then it is transported by exocytosis process in the extracellular region via the circulatory system

Endocrine system

Endocrine cells

Signal

via Blood

Target cell

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1  Introduction to Cell Signaling Pathways and Brain Tumors

1.1.2 Autocrine Signaling In the autocrine signaling, similar cell types communicate with each other. If the autocrine hormone was secreted simultaneously by multiple cells, then a strong response is shown by the cells. Interestingly, autocrine signaling is critical for the immune response (Berridge 2014).

1.1.3 Paracrine Signaling The paracrine signaling is a characteristic of operating in the medium range. In this signaling pathway, passive diffusion helps the hormone-secreting cell to reach the target cells. The secreting cell is normally present near the receiving cells in the paracrine signaling. This is local and the contributing signaling molecules which are sometimes called local mediators or hormones. Interestingly, in the process of synaptic neurotransmission, a nerve cell communicates with either other nerve cell or a muscle cell, and it is included under a special type of paracrine signaling (Fig. 1.4) (Berridge 2014).

1.1.4 Intracrine Signaling In intracrine cell signaling cell stimulates itself by the factor which is produced within the cell. (Fig. 1.5) (Berridge 2014). Fig. 1.4  Paracrine signaling is a medium-range characteristic. In this system a signal (hormone) reaches the target cell by passive diffusion

Paracrine system

Source cell

Signal

Passive diffusion

Target cell

1.2  Introduction to Brain Tumors Fig. 1.5  The intracrine signal is generated within the target cell

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Intracrine system

Source cell

Signal

Target cell (inside the source cell)

Fig. 1.6  The juxtacrine system signal reaches the adjacent cells with the help of the cell membrane

Juxtacrine system

Source cell

Signal

Target cell (adjacent cell)

1.1.5 Juxtacrine Signaling The juxtacrine signal targets adjacent (touching) cells. This signal progresses along the cell membranes with the help of integral components of the cell membrane, including protein or lipid. It has the potential to affect either emitting cells or immediately adjacent cells (Fig. 1.6) (Berridge 2014).

1.2 Introduction to Brain Tumors Brain tumors are constituted by a mass-like structure inside the brain, containing cells developed under abnormal growth conditions. These tumors appear in adults and children and cause substantial mortality. Most familial gliomas are associated with hereditary syndrome. Ionizing radiation is one of the well-identified risk factors for the development of the brain tumors (McNeill 2016). Glioblastoma is the most important among all types of brain tumors due to both its incidence and its devastating effect on the patients (Aldape 2003). Brain tumors are also divided on the basis of cells of origin. There are two major divisions:

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1  Introduction to Cell Signaling Pathways and Brain Tumors

1 . Primary brain tumor. 2. Metastasis.

1.2.1 Primary Brain Tumors It originates inside the tissue of the brain or brain-adjacent cells: glial cells or nonglial cells (nerve, gland, and blood vessels, benign or malignant).

1.2.2 Brain Tumor Metastases Brain tumor metastases originate from breast or lung cancer cells which migrate to the brain via the circulatory system. Brain tumors are also divided based on cell proliferation rate: 1. Benign. 2. Malignant.

1.2.3 Benign Brain Tumors Meningioma originates from the meninges and the most common benign intracranial tumors which are membrane-like structures that cover the brain and the spinal cord.

1.2.4 Malignant Brain Tumors Gliomas are the most malignant brain tumor and they make up almost 78% of all malignant brain tumors. They originate from  glial cells  “supporting cells of the brain”. These cells are divided into the following: • • • •

Astrocytes. Ependymal cells. Oligodendroglial cells. Medulloblastoma.

1.2.4.1 Astrocytoma Astrocytoma is the most common type of glioma. It comprises almost 50% of all primary brain and spinal cord tumors. Astrocytomas originate from star-shaped glial cells, mainly in the cerebrum. They are prevalent in adults.

1.2  Introduction to Brain Tumors

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1.2.4.2 Ependymoma Ependymoma originates from a neoplastic transformation of the lining of ependymal cells in the ventricular system. It constitutes 2–3% of all brain tumors. 1.2.4.3 Glioblastoma Glioblastoma is a highly malignant glial tumor consisting of several different types of cells, mainly astrocytes and oligodendrocytes. It commonly occurs in people in the 50–70 years age range. Intriguingly, it is more prevalent in men than in women. 1.2.4.4 Oligodendroglioma Oligodendroglioma originates in the myelin sheath of the brain. The myelin sheath is the insulation for the wiring of the brain. 1.2.4.5 Medulloblastoma Medulloblastoma originates in the cerebellum of children. It is a high-grade tumor. WHO has also classified brain tumors based on histological features: • • • • •

Highly malignant. Rapid growth and Aggressiveness and rapid growth Infiltration in nature Recurrence Prone to necrosis.

World Health Organization (WHO) brain tumors classification is based on tumor types (Table 1.1) (Louis et al. 2016a, b).

Table 1.1  WHO Brain Tumors Classification 2007 Grade Low

Grade I

II High

III

IV

Characteristic Least malignant Non-infiltrative Long-term survival Relatively slow growing Somewhat infiltrative Malignant Infiltrative Most malignant Aggressive Widely infiltrative Rapid recurrences Necrosis prone

Tumor types Pilocytic astrocytoma

Diffuse astrocytoma Oligodendroglioma Anaplastic astrocytoma Anaplastic ependymoma Anaplastic oligodendroglioma Glioblastoma multiforme Medulloblastoma Ependymoblastoma

1  Introduction to Cell Signaling Pathways and Brain Tumors

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1.2.5 Symptoms • • • • • • • • •

Headaches: severe in the morning Seizures or convulsions. Difficulty thinking, speaking, or normal articulation of words. Weakness or paralysis in one part or one side of the body. Loss of balance or dizziness. Blur Vision. Loss of hearing ability Nausea or vomiting, swallowing difficulty. Confusion and disorientation.

Most brain tumor classifications are based on histology, i.e., hematoxylin and eosin-stained with the light microscope. In 2007, the WHO provided the classification of brain tumors that is mentioned in the Table 1.1 (Louis et al. 2007). Years later, there was a revision of the WHO 2007 brain tumor classification in a meeting that was held in Haarlem, the Netherlands, by the International Society of Neuropathology, in 2014 (Louis et al. 2014, Brain Pathology). In this meeting almost 117 contributors had participated in this meeting including 35 neuropathologists, neurological clinical advisors, and different scientists across 20 countries (Table 1.2) (Louis et al. 2014; Louis 2016).

Table 1.2  Central nervous system tumor classification by the WHO in 2016 IDH status 1. Diffuse astrocytic and oligodendroglial tumor Diffuse astrocytoma Gemistocytic astrocytoma Diffuse astrocytoma Diffuse astrocytoma 2. Anaplastic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma Anaplastic astrocytoma 3. Glioblastoma Glioblastoma Giant cell glioblastoma Gliosarcoma Epithelial glioblastoma Glioblastoma Glioblastoma 4. Anaplastic oligodendroglioma Anaplastic oligodendroglioma Oligoastrocytoma Anaplastic oligoastrocytoma

IDH mutant IDH mutant IDH wild type NOS IDH mutant IDH wild type NOS IDH wild type

IDH mutant NOS IDH mutant and 1p/19q co-deleted NOS NOS NOS

References

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Table 1.3  Embryonal tumors and their genetic changes S. no. 1. 2. 3. 4. 5. 6.

Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma

Table 1.4  Embryonal tumors and their histological changes

Genetically defined WNT-activated SHH-activated and TP53-mutant SHH-activated and TP53-wild type Non-Wnt/non-SHH Group 3 Group 4 S. no. 1. 2. 3. 4. 5. 6.

Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma Medulloblastoma

Histologically defined Classic Basic Desmoplastic/nodular With extensive nodularity Large cell/anaplastic NOS

Moreover, embryonal tumors such as medulloblastoma are also divided based on genetic and cell-signaling pathways, which are potentially involved in their development. They are also mentioned in Table 1.3 (Louis 2016). In addition, embryonal tumors are classified based on histological changes. They are mentioned in Table 1.4 (Louis 2016).

References Berridge MJ (2014) Module 2: cell signaling pathways. Cell Signal Biol 6:csb0001002 Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumors of the central nervous system. Acta Neuropathol 114(2):97–109. Epub 2007 Jul 6. Erratum in: Acta Neuropathol. 2007 Nov;114(5):547. PMID: 17618441; PMCID: PMC1929165. https://doi.org/10.1007/s00401-­007-­0243-­4 Louis DN, Perry A, Burger P, Ellison DW, Reifenberger G, von Deimling A, Aldape K, Brat D, Collins VP, Eberhart C, Figarella-Branger D, Fuller GN, Giangaspero F, Giannini C, Hawkins C, Kleihues P, Korshunov A, Kros JM, Beatriz Lopes M, Ng HK, Ohgaki H, Paulus W, Pietsch T, Rosenblum M, Rushing E, Soylemezoglu F, Wiestler O, Wesseling P, International Society of Neuropathology--Haarlem (2014) International Society of Neuropathology--Haarlem consensus guidelines for nervous system tumor classification and grading. Brain Pathol 24(5):429–435. Epub 2014 Sep 10. https://doi.org/10.1111/bpa.12171 Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW (2016a) The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 131(6):803–820. Epub 2016 May 9. https://doi.org/10.1007/s00401-­016-­1545-­1 Louis DN, Ohgaki H, Wiestler OD, Cavenee WK (2016b) World Health Organization histological classification of tumors of the central nervous system, vol 131. International Agency for Research on Cancer, Lyon, p 803 Miller CJ, Davidson LA (2013) The interplay between cell signaling and mechanics in developmental processes. Nat Rev Genet 14(10):733–744

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1  Introduction to Cell Signaling Pathways and Brain Tumors

Smith RJ, Koobatian MT, Shahini A, Swartz DD, Andreadis ST (2015) Capture of endothelial cells under flow using immobilized vascular endothelial growth factor. Biomaterials 51:303–312 Solinas G, Vilcu C, Neels JG (2007) JNK1 in hematopoietically derived cells contributes to diet-­ induced inflammation and insulin resistance without affecting obesity. Cell Metab 6(5):386–397 Vlahopoulos SA, Cen O, Hengen N, Agan J, Moschovi M, Critselis E, Adamaki M, Bacopoulou F, Copland JA, Boldogh I, Karin M, Chrousos GP (2015) Dynamic aberrant NF-κB spurs tumorigenesis: a new model encompassing the microenvironment. Cytokine Growth Factor Rev 26:389–403 Wang K, Grivennikov SI, Karin M (2013) Implications of anti-cytokine therapy in colorectal cancer and autoimmune diseases. Ann Rheum Dis 72(Suppl 2):ii100–ii103 (2003) Molecular Epidemiology of Glioblastoma The Cancer Journal 9(2) 99-106 ­10.1097/00130404-200303000-00005

Part II

2

Wnt Cell Signaling Pathway in Brain Tumor Development

The Wnt cell signaling pathway originated from wingless and the name Int-1 (Nusse et  al. 1991) has both paracrine (nearby cell–cell communication) and autocrine (same-cell communication) cell signals. Interestingly, this pathway is highly conserved from Drosophila to humans (van Amerongen and Nusse 2009; Nusse and Varmus 1992). The pathway is activated by the Wnt-protein ligand binding to the frizzled receptor. The signaling pathway cascade progresses inside the cell with the help of the protein dishevelled. There are three types of Wnt pathway patterns: I. Canonical Wnt pathway: It is responsible for the regulation of gene transcription and negative control in part by the SPATS1 gene (Zhang et al. 2010). II. Noncanonical Wnt-planar cell polarity pathway: It is responsible for the cytoskeleton development. III. Noncanonical Wnt/calcium pathway: It  maintains the calcium  balance in the cell. The role of the Wnt cell signaling pathway was first reported in cancer progression and later its role was also confirmed in early embryonic development process comprising body axis, cell patterning, specification of cell fate, cell proliferation, and migration. A genetic mutation in Wnt gene causes abnormal Drosophila larvae and this investigation further revealed its significant contribution to  embryonic development (Goessling et al. 2009). Intriguingly, this pathway also regulates tissue regeneration in adult bone marrow, intestines, and skin (Goessling et  al. 2009). Moreover, mutations of Wnt cause metabolic disorder type II diabetes and various cancers including breast, prostate, and glioblastoma (Logan and Nusse 2004; Komiya and Habas 2008). The Wnt cell signaling pathway was revealed due to the discovery of retroviral oncogenes. In 1982, Harold Varmus and  Roel Nusse infected mice with mouse mammary tumor virus, which may potentially mutate mouse genes, to understand © Springer Nature Singapore Pte Ltd. 2023 M. H. Shahi, Role of Signaling Pathways in Brain Tumorigenesis, https://doi.org/10.1007/978-981-15-8473-2_2

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which of the mutated genes was responsible for the development of breast tumors. This experiment was successful and they were able to explore a novel mouse proto-­ oncogene named int1 (integration 1) (Nusse et al. 1984). Recently, inhibitors of the Wnt cell signaling pathway were used in the mouse models of disease to understand the role of this pathway in various diseases (Zimmerli et al. 2017). Wnt cell signaling promotes the secretion of various cellular cysteine-rich glycoproteins, which control cell-fate determination, cell behavior in early embryos and adults, and also tumor progression (Fig. 2.1) (Nusse and Varmus 1992). Notably, the role of Wnt-1 role is crucial for the neural development process (McMahon and Bradley 1990; Thomas and Capecchi 1990). Wnt1 knockout and mutation in mice are responsible for defective brain development, including cerebellum, midbrain, and posterior cerebellum (Fig. 2.2) (Nusse and Varmus 1992). The Wnt cell signaling pathway is activated when Wnt-ligand binds to frizzled receptor (Yang-Snyder et al. 1996), which further activates the intracellular protein, dishevelled (Sokol et al. 1995; Yang-Snyder et al. 1996). Now, activated dishevelled further down-regulates glycogen synthase kinase 3β (GSK3β) activity (Cook et al. 1996; Hedgepeth et al. 1997). Then, inactivated GSK3β increases the concentration of cytosolic β-catenin by augmentation of its half-life (Papkoff et  al. 1996; Yost et al. 1996). Thereafter, β-catenin interacts with the family of transcription factors TCF/LEF (T cells factor/lymphocyte enhance binding factor) (Molenaar et al. 1996; Behrens et al. 1996; Huber et al. 1996). Finally, both β-catenin and TCF/LEF bind to the consensus-specific sequence A/TA/TCAAG of the promoter region of target genes such as siamois, ultrabithorax, and nodal related 3 (Carnac et  al. 1996; Brannon et al. 1997; van de Wetering et al. 1997; Carnac et al. 1996; Brannon et al. 1997). There are many genes like the  homeobox gene cell adhesion,  E-cadherin and Engrailed-1 , which are also regulated by β-catenin and the TCF/LEF complex (Danielian and McMahon 1996; Huber et al. 1996). Wnt-1 up-regulates E-cadherin, plakoglobin and β-catenin, which further localize at cell-cell junctions (Hinck et al. 1994; Papkoff et al. 1996). It is noted that

Wnt cell signaling pathway

Secretion of various cellular cysteine rich glycoproteins

Cell fate determinant

Cell behavior in early embryonic And adult stage

Tumor growth

Fig. 2.1  Role of the Wnt cell signaling pathway in the development process. The Wnt pathway regulates the expression of cysteine-rich glycoproteins. These proteins are responsible for cell fate determination, cell behavior in early embryos, adult stage development, and tumor growth

2  Wnt Cell Signaling Pathway in Brain Tumor Development Fig. 2.2 Deregulation effect of the Wnt pathway on brain development. Either mutation or knockdown of Wnt gene is responsible for the abnormality of different parts of the brain, including the cerebellum, mid-brain, and posterior cerebellum

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WNT1

Mutation

Knock down

Defective different parts of the brain development

Cerebellum

Mid brain

Posterior cerebellum

Canonical WNT Pathway WNT Cell signaling WNT Cell signaling Off

LRP6

WNT

brane

a mem

CK1 GSK3

P

Fz DVL

APC

Plasm b-Catenin

CK1 APC

GSK3 Axin

ol Cytos

Fz

Axin

Cell brane a mem Plasm

LRP6

WNT

On

P b-Catenin b-Catenin

DVL

Degradation

Nucleus

Nucleus b-Catenin

TCF

OFF

WNT target genes

TCF

ON

WNT target genes

Fig. 2.3  Canonical Wnt cell signaling pathway. Inactivated Wnt cell signaling promotes β-catenin phosphorylation and its further degradation. In activated Wnt cell signaling pathway Wnt ligand binds to Frizzled (Fz) and LRP6 receptor. Thereafter, β-catenin enters the nucleus and binds to TCF/Lef-1 family of transcription factors and promotes Wnt cell signaling downstream target gene transcription. (Adapted from Angers and Moon 2009; Clevers and Nusse 2012)

Gap-junctions acquire selective permeability with the help of differentl connected family members that show diverse expression patterns in the process of development (Ruangvoravat and Lo 1992). The Wnt signaling pathway is involved in various cellular developmental processes like  proliferation, differentiation, and epithelial–mesenchymal transition (Fig. 2.3). It mainly operates in two ways.

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2  Wnt Cell Signaling Pathway in Brain Tumor Development

1. Canonical. 2. Noncanonical.

2.1 Canonical Pathway Wnt cell signaling activation is conferred via β-catenin and its deregulation is responsible for neoplastic transformation of the cells. The Wnt cell signaling pathway downstream target genes are Cyclin D1 and c-myc.(Fig. 2.3) (Smalley and Dale 1999). It is important to mention that β-catenin triggers the activation of the Wnt cell signaling pathway. Inactivated, Wnt signal promotes the proteasomal degradation of β-catenin. Activated, Wnt cell signaling pathway stabilizes β-catenin and stable β-catenin interacts with HMG box transcription factors (HMG) LEF/TCF family and forms a complex. This complex LEF/TCF/β-catenin further activates specific target genes of the Wnt cell signaling pathway. During the process of tumorigenesis the pathway is deregulated and β-catenin degradation is inhibited by either direct activating mutations or by inactivating mutations of the APC tumor suppressor gene product. Therefore, stable β-catenin forms the complex with LEF/ TCF to bind the promoter region of various target oncogenes such as c-Jun, c-myc, and Cyclin D1 (Behrens 2000) (Fig. 2.3). Interestingly, another cytoplasmic factor, conductin, regulates the Wnt cell signaling pathway in both normal and tumorigenesis. It controls Wnt signaling through the enhancement of the assembling process of major components that are responsible for the β-catenin degradations. Moreover, a significantly high concentration of β-catenin in the cytoplasm or nucleus indicates its potential involvement in tumorigenesis. In the absence of Wnt cell signaling cytoplasmic β-catenin phosphorylation occurs after binding with APC, glycogen synthase kinase 3β, and axin/conductin. Thereafter, β-catenin is rapidly degraded under the ubiquitin–proteasome pathway. Moreover, APC mutation typically gives a truncated protein that lacks regulatory ability and causes β-catenin to accumulate in the cytosol or nucleus (Huang et al. 2000). Additionally, the novel human gene BRCTNNB1A expression is down-regulated by β-catenin (Kawasoe et al. 2000). Medulloblastoma is the most common and aggressive brain tumor among children. It mostly appears sporadically; however, its chance of occurrence  incidence increased in two inherited tumor predisposition syndromes: Gorlin and Turcot syndromes. The genes causing these syndromes have been identified. Gorlin’s syndrome is due to germline mutations in the gene named “patched” (PTCH).’s Turcot’s syndrome with medulloblastoma carry germline mutations in the adenomatous polyposis coli (APC) gene. The APC gene product is the part of a multi-protein complex, which regulates β-catenin degradation. In this complex, axin acts as a major scaffold protein. However, APC mutations are rare in sporadic medulloblastoma. Moreover, β-catenin (CTNNB1)  hot sports region was identified  in a subset of medulloblastomas. In addition, deletion of axin in medulloblastoma samples and cell lines may suggest its potential role as a tumor suppressor gene  (TSG). It is

2.1  Canonical Pathway

19

noted that axin was initially identified as the gene product of the mouse fused locus, which inhibits Wnt cell signaling (Zeng et al. 1997). Axin is a scaffold protein of this multi-protein complex, and it can drag β-catenin and GSK-3β into proximity for β-catenin phosphorylation and further ubiquitin-mediated degradation by the proteasome system (Aberle et al. 1997). It has been revealed that axin forms dimers to act as an inhibitor of TCF/LEF transcription factors (Fagotto et al. 1999). Moreover, mutation of axin1 was detected in a subset of sporadic medulloblastoma samples and therefore, it suggests that such mutation can activate Wnt pathway in these tumors (Dahmen et al. 2001). The human DKK-1 gene expresses a Wnt cell signaling pathway antagonist, which responds to DNA damage and its overexpression further promotes brain tumor cells for apoptosis and  alkylation of damaged DNA  alkylation (Fig.  2.4) (Shou et al. 2002). The DKK-1 gene also known as Dickopf-1 (German for big head, stubborn), expresses glycoprotein to regulate cell fate and neural patterning during the development  process. (Glinka et  al. 1998; Kazanskaya et  al. 2000). There have been many studies that suggest that the Wnt cell signaling pathway is under tight regulation of DKK-1. The DKK-1 acts a Wnt cell signaling pathway inhibitor to regulate the correct positioning, embryonic brain development, and other parts of the body (Fig. 2.5) (Glinka et al. 1998). The Wnt cell signaling pathway comprises various highly conserved Wnt ligands that bind to frizzled receptors and propagate a cell signal transduction cascade. The cascade glycogen synthase kinase 3 (GSK3) and β-catenin play critical roles (Dale 1998; Wodarz and Nusse 1998). In the inactivated Wnt cell signaling pathway, β-catenin is associated with a cytoplasmic complex consisting of GSK-3, axin, and APC (Hayashi et  al. 1997; Sakanaka et  al. 1998). In this complex GSK3 phosphorylates serine and threonine residues of β-catenin protein. Thereafter, phosphorylated β-catenin  undergoes for  ubiquitination and degradation by the proteasome pathway (Hart et al. 1998). However, in the activated Wnt cell signaling pathway, Fig. 2.4  Effect of DKK-1 or Dickopf-1 gene on brain tumor cells. Owing to DNA damage the expression of DKK-1 or Dickopf-1 gene expression is enhanced. This high expression of DKK-1 further promotes apoptosis of brain tumor cells and alkylation of damaged DNA

DNA Damage

DKK-1 gene or Dickopf-1

Apoptosis of Brain Tumor cells

Alkylation of damaged DNA

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2  Wnt Cell Signaling Pathway in Brain Tumor Development

DKK-1 gene or Dickopf-1

P53

Wnt Cell signaling

Secreted glycoprotein

Regulates

Cell fate

Neural patterning in the early embryonic stage

Fig. 2.5  Role of p53 in normal development and Wnt pathway regulation. Tumor suppressor gene p53 activates the expression of DKK-1 or Dickopf-1 gene and also inhibits the activation of Wnt cell signaling pathway with the help of the DKK-1 gene. The DKK-1 gene further regulates the secretion of glycoprotein that is responsible for cell fate and neural patterning in the early embryonic stage

GSK3 is inactivated and free unphosphorylated β-catenin accumulated in the cytoplasm. Thereafter, accumulated β-catenin transfers to the nucleus and interacts with  transcription factors lymphocyte enhancer factor (LEF)/TCF.  Finally, this complex binds the promoter regions of various genes that are responsible for the development and oncogenesis process such as Cyclin D1, engrailed-2, Cycloocyhenase-2, c-myc, and metalloproteases (McGrew et al. 1999; Polakis 2000). The Wnt antagonist DKK-1 (Hdkk-1) controls the Wnt cell signaling pathway by inhibiting the free β-catenin in the accumulation in the  cytoplasm and nucleus (Moon et al. 1997). DKK-1 inhibits the action of Wnt signaling at the upstream not by direct interaction with the frizzled receptor but by binding to an adjacent LDL-­ receptor-­related protein 6 (LRP6) (Mao et al. 2001; Nusse 2001). Moreover, it has also been reported that the Hdkk-1 gene is a direct target of the p53 tumor suppressor gene (Wang et al. 2000). Notably, E-cadherin is a protein that is associated with β-catenin and contributes a critical role in the cell–cell adhesion. The interaction and role of both Wnt and E-cadherin genes in brain tumorigenesis has been demonstrated (Howng et  al. 2002). The expression patterns of four Wnt genes, Wnt1 (41/45), Wnt5a (37/45), Wnt10b (35/45), and Wnt13 (6/45), was found in brain tumors. E-cadherin showed expression in  astrocytoma (2/16), meningioma (14/15), and adenoma (12/14) tumors. Although, there was no difference in β-catenin expression profile in brain tumors, two mutations of β-catenin on the speculative phosphorylation sites S73F and S23G were reported in astrocytomas.

2.1  Canonical Pathway

21

In addition, both Akt and Erk pathways are activated in medulloblastoma (Włodarski et al. 2006). It has been estimated that mutations of these proteins of the Wnt pathway may appear in almost 15% of sporadic medulloblastomas (Gilbertson 2004). Moreover, the reactivation of drivers of development cell signaling cascades such as (EGF)/EGFR, stem cell factor (SCF)/KIT, platelet-derived growth factor (PDGF)/PDGFR, Shh/Ptch1 (GLI TF), and Wnt/β-catenin seems to be associated with increased DNA repair mechanisms and ABC transporter-mediated multi-drug efflux in cancer progenitor cells, which could be responsible for their resistance to current clinical therapies (Fig. 2.6) (Mimeault et al. 2007). P53 inactivation or continuous activation of the Wnt/β-catenin pathway may also result in increased expression of downstream oncogenes that further contribute to developing medulloblastoma (Mimeault et al. 2007). It has been shown that conductin promotes the assembly of essential factors of the β-catenin degradation pathway  to control the Wnt cell signaling paway. Therefore, conductin functional defects can lead to tumorigenesis. It has been investigated that the multi-protein complex assembled by the cytoplasmic component conductin triggers the degradation of cytoplasmic β-catenin. This complex is composed of APC, the serine/threonine kinase, GSK3-β, and β-catenin, which bind to conductin at distance domains. Henceforth, this suggests that conductin negatively

Reactivation of various development cell signaling pathways

EGF/EGFR

Stem cell factor/KIT Platelet derived Shh-Gl1i/Patch1 PDGF/PDGFR

Wnt/β-catenin

DNA Repair mechanism

ABC transporter-mediated multidrug efflux in cancer progenitor cells

Resistant to current Clinical Therapies

Fig. 2.6  Role of the Wnt cell signaling pathway in the drug resistance of cancer cells. Wnt signaling, together with various developmental cell pathways, including EGF/EGFR, stem cell factor/ KIT, PDGF/PDGFR, and Shh-Gli1, increase the DNA repair mechanism of cancer progenitor cells. This leads to ABC transporter-mediated multidrug efflux in cancer progenitor cells and causes drug resistance to current therapy

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2  Wnt Cell Signaling Pathway in Brain Tumor Development

regulates Wnt signaling  pathway. Moreover, degradation of conductin promotes tumor growth (Behrens 2000). The expression of the novel human gene DRCTNNB1A is also downregulated by β-catenin (Kawasoe et al. 2000). β-catenin plays a significant role in cell adhesion and in the Wnt/Wg signal transduction pathway. Accumulation of β-catenin in the cytoplasm and nucleus was observed in a variety of tumors including brain tumors, owing to due to adenomatous polyposis coli (APC) gene mutation. Interaction of Accumulated β-catenin  interaction  with TCF/LEF TFs is known to deregulate expression of some downstream genes, but the exact mechanism is not cleared. The murine gene, Drctnnbla (downregulated by ctnnb1a) expression was experimentally downregulated in response to the activated form of β-catenin. This gene human homolog, DRCTNNB1A, shows 91% identity with murine protein. It is located on chromosome 7p15.3, which was confirmed by in situ hybridization. The expression of the DRCTBBB1A gene has been shown to be significantly increased in SW480 colon cancer cells in response to a reduction of intracellular β-catenin by adenovirus-­ mediated transfer of the β-catenin binding domain of the APC gene into the cells (Kawasoe et al. 2000). The cerebellar medulloblastoma (WHO grade IV) is a highly malignant embryonal tumor among the children. It has been observed that several molecular alterations appear to be involved in the development of the medulloblastoma, including isochromosome 17q and p53, mutation of  PTCH, and β-catenin gene. Forty-six sporadic medulloblastoma samples were screened for the presence of APC and β-catenin mutation. Three miscoding APC mutations were found in 2 (4.3%) of medulloblastomas. One sample showed GCAGTA mutation at codons 1296 (Ala-­ Val) and another case showed double mutation at codons 1472 (GTAATA, Val Ile) and 1495 (AGT GGT), Ser-Gly. Four tumors’ samples (8.7%) showed miscoding β-catenin mutation. Three of the miscoding were located at codon 33 (TCT TTT, Ser, Phe) and another one at codon 37 (TCT GCT, Ser Ala). The APC and β-catenin gene mutations were mutually exclusive and appeared in 13% of the total medulloblastoma samples. Although germline APC mutations are a well-established cause of familial colon and brain tumor (Turcot syndrome). Moreover, in a subset of sporadic medulloblastoma samples showed the APC gene mutation. Axin1 contributes as a scaffold protein in the β-catenin/TCF/Axin1 complex. The role of Axin1 in the pathogenesis of sporadic medulloblastomas has been investigated. Mutation of the Axin1 gene was checked in 86 medulloblastoma samples and 11 medulloblastoma cell lines with the help of single-strand conformation polymorphism analysis. There are seven large deletions (12%) and single somatic point mutation in exon 1(pro255ser) of the Axin1 gene. Therefore, it suggests that Axin1 may act as a tumor suppressor gene in medulloblastoma development (Dahmen et al. 2001). Notably, glioblastoma is usually treated with temozolomide (TMZ) but this treatment is not very effective owing to resistance developed by glioblastoma against TMZ. It has been reported that micro-RNA “miR-126-3P” was down-regulated and showed a tumor suppressor nature in various cancers, including glioblastoma. Interestingly, miR-126-3p was also able to induce the effectiveness of TMZ on

2.1  Canonical Pathway

23

glioblastoma. The sensitization of glioblastoma cells to TMZ executed by miR-­126-3p is due to SOX2 repression and blocking of Wnt/β-catenin cell signaling. This shows the therapeutic possibilities of miR-126-3p in TMZ resistance in glioblastoma (Luo et al. 2019). The spalt-like transcription factor1 (SALL1) gene is a member of the kruppel-­ associated box-containing zinc finger proteins (KRAB-ZFPs). SALL1 gene expression was reduced in human glioblastoma and glioma cells derived from brain gliomas. Interestingly, downregulation of SALL1 showed an association with decreased survival rate. Overexpression of SALL1 inhibited cell proliferation and induced cell-cycle arrest at the Go/G1phase. Moreover, epithelial–mesenchymal transition (EMT) and cell migration were inhibited owing to downregulation of stem cell marker expression by SALL1. Inhibition of β-catenin expression, downregulation of c-myc and cyclin D1 and upregulation of p21 and p27 expression were induced by high SALL1 expression. Therefore, SALL1 acts as a tumor suppressor in human glioblastoma cells and brain gliomas by inhibiting Wnt/β-catenin signaling (Chi et al. 2019). It has been reported that glioma tumor samples and cell lines showed  miR-­362-3p  downregulation and Pax3 upregulation.  It was shown that miRNA-362-3p ectopic expression on glioma cells; downregulates the expression of Pax3 and suppresses the epithelial mesenchymal transition due to inhibition of the Wnt/β-catenin pathway (Xu et al. 2019). Glioblastoma cells vampirize Wnt from neurons and initiate a JNK/MMP signaling cascade that increase glioblastoma progression and neurodegeneration. Wingless (wg)-related integration site (Wnt) pathway activation in glioblastoma is associated with a poor prognosis. Clinically, the disease is characterized by neurological defects. Glioblastoma cells show a tumor microtubule networks (TMs) that enwrap neurons, accumulate Wg receptor Frizzled1 (Fz1), and consequently, deplete Wg from the neuron, resulting in neurodegeneration. Therefore, this process is called vampirization. Moreover, glioblastoma cells showed  a positive feedback loop to promote their expansion in which the Wg pathway activates C-jun N-terminal kinase (JNK). Thereafter, JNK signaling leads to the post-transcription up-­regulation and accumulation of matrix metalloproteinase (MMPs), which further facilitate TM infiltration throughout the brain. Henceforth, glioblastoma cells proliferate with the help of the g Wg cell signaling activation target, and β-catenin and Wg cell signaling extinction  degenerate neurons. These findings have uncovered a molecular mechanism for TM production, infiltration, and maintenance that can explain both neuron-dependent tumor progress and also the neural death associated with glioblastoma (Marta Portela et al. 2019). Glioma stem cells (GSCs) can  decrease T cell cognition and escape systemic immunosurveillance with the help of down-regulation or a defect of the major histocompatibility complex Class I (MHC-1) molecule and antigen-processing machinery (APM) components. However, improvement of GSC tumor surface antigen can be an effective strategy to enhance an adaptive immune response and to activate cytotoxic T cells to the killing of glioma cells. It has been reported that down-regulation of MHC-1 and APM was associated with the activated Wnt cell signaling pathway in

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2  Wnt Cell Signaling Pathway in Brain Tumor Development

GSCs. Histone deacetylase inhibitors improved MHC-1 and expression  of APM components, which can be reverted by Wnt cell signaling pathway activation. It has been revealed that stemness pathway inhibition may be effective for GSC-based immunotherapy to counter immune-escape tumors (Yang et al. 2020).

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Nusse R, van Ooyen A, Cox D, Fung YK, Varmus H (1984) Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307(5947):131–136. https://doi.org/10.1038/307131a0 Nusse R, Brown A, Papkoff J et al (1991) A new nomenclature for int-1 and related genes: the Wnt gene family. Cell 64(2):231. https://doi.org/10.1016/0092-­8674(91)90633-­a Papkoff J, Rubinfeld B, Schryver B, Polakis P (1996) Wnt-1 regulates free pools of catenins and stabilizes APC-catenin complexes. Mol Cell Biol 16(5):2128–2134. https://doi.org/10.1128/ mcb.16.5.2128 Polakis P (2000) Wnt signaling and cancer. Genes Dev 14(15):1837–1851 Portela M, Venkataramani V, Fahey-Lozano N et  al (2019) Glioblastoma cells vampirize WNT from neurons and trigger a JNK/MMP signaling loop that enhances glioblastoma progression and neurodegeneration. PLoS Biol 17(12):e3000545. Published 2019 Dec 17. https://doi. org/10.1371/journal.pbio.3000545 Ruangvoravat CP, Lo CW (1992) Connexin 43 expression in the mouse embryo: localization of transcripts within developmentally significant domains. Dev Dyn 194(4):261–281. https://doi. org/10.1002/aja.1001940403 Sakanaka C, Weiss JB, Williams LT (1998) Bridging of beta-catenin and glycogen synthase kinase-3beta by axin and inhibition of beta-catenin-mediated transcription Proc Natl Acad Sci USA. 95(6):3020–3023. https://doi.org/10.1073/pnas.95.6.3020 Shou J, Ali-Osman F, Multani AS, Pathak S, Fedi P, Srivenugopal KS (2002) Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA. Oncogene 21(6):878–889. https://doi.org/10.1038/sj.onc.1205138 Smalley MJ, Dale TC (1999) Wnt signaling in mammalian development and cancer. Cancer Metastasis Rev 18(2):215–230. https://doi.org/10.1023/a:1006369223282 Sokol SY, Klingensmith J, Perrimon N, Itoh K (1995) Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled. Development 121(10):3487 Thomas KR, Capecchi MR (1990) Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature 346(6287):847–850. https://doi.org/10.1038/346847a0 van Amerongen R, Nusse R (2009) Towards an integrated view of Wnt signaling in development. Development 136(19):3205–3214. https://doi.org/10.1242/dev.033910 van de Wetering M, Cavallo R, Dooijes D et al (1997) Armadillo coactivates transcription driven by the product of the drosophila segment polarity gene dTCF. Cell 88(6):789–799. https://doi. org/10.1016/s0092-­8674(00)81925-­x Wang J, Shou J, Chen X (2000) Dickkopf-1, an inhibitor of the Wnt signaling pathway, is induced by p53. Oncogene 19(14):1843–1848. https://doi.org/10.1038/sj.onc.1203503 Włodarski P, Grajkowska W, Łojek M, Rainko K, Jóźwiak J (2006) Activation of Akt and Erk pathways in medulloblastoma. Folia Neuropathol 44(3):214–220 Wodarz A, Nusse R (1998) Mechanisms of Wnt signaling in development. Annu Rev Cell Dev Biol 14:59–88. https://doi.org/10.1146/annurev.cellbio.14.1.59 Xu G, Fang P, Chen K, Xu Q, Song Z, Ouyang Z (2019) MicroRNA-362-3p targets PAX3 to inhibit the development of glioma through mediating Wnt/β-catenin pathway. Neuroimmunomodulation 26(3):119–128. https://doi.org/10.1159/000499766 Yang W, Li Y, Gao R, Xiu Z, Sun T (2020) MHC class I dysfunction of glioma stem cells escapes from CTL-mediated immune response via activation of Wnt/β-catenin signaling pathway. Oncogene 39(5):1098–1111. https://doi.org/10.1038/s41388-­019-­1045-­6 Yang-Snyder J, Miller JR, Brown JD, Lai CJ, Moon RT (1996) A frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr Biol 6(10):1302–1306. https://doi.org/10.1016/ s0960-­9822(02)70716-­1 Yost C, Torres M, Miller JR, Huang E, Kimelman D, Moon RT (1996) The axis-inducing activity, stability, and subcellular distribution of beta-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 10(12):1443–1454. https://doi.org/10.1101/gad.10.12.1443

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3

Notch Cell Signaling Pathway and Brain Tumors

Most of the organisms show conserved Notch signaling pathway (Artavanis-­ Tsakonas et  al. 1999). There are four different types of Notch receptors (Kumar et al. 2016): 1. 2. 3. 4.

Notch1 Notch2 Notch3 Notch4

The Notch receptor is one of the single transmembrane receptors composed of hetero-oligomers with a large extracellular part. This extracellular part is calcium-­ dependent, and showed non-covalent interaction with a smaller piece of notch protein composed of a short extracellular region, a single transmembrane pass, and a small intracellular region (Brou et al. 2000) (Fig. 3.1). John S. Dexter reported a Notch for the first time in the wings of the fruit fly Drosophila melanogaster. Thomas Hunt Morgan identified this gene allele in 1917 (Morgan 1917). Spyros-Artavanis-Tsakones and Michael W. Yong performed the molecular analysis and sequenced this gene in the 1980s. Moreover, two alleles of the Notch gene were identified in C. elegans based on development phenotypes lin-12 (Greenwald et al. 1983) and glp-1 (Austin and Kimble 1987). The Notch protein receptor spans the cell membrane, with one part of it inside and the other part outside. Notch ligand-protein binds to the extracellular domain, which further enters the nucleus and regulate expression of various genes (Oswald et  al. 2001). The Notch/Lin-12/Glp-1 receptor family was reported due to their involvement in the specification of cell fates during development in Drosophila melanogaster and C. elegans (Artavanis-Tsakonas et al. 1995; Singson et al. 1998). The intracellular domain of Notch forms a complex with CBF1 to activate transcription of target genes (Singson et al. 1998; Wilson and Kovall 2006). The Notch signaling pathway is crucial for cell–cell communication, which involves a gene

© Springer Nature Singapore Pte Ltd. 2023 M. H. Shahi, Role of Signaling Pathways in Brain Tumorigenesis, https://doi.org/10.1007/978-981-15-8473-2_3

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3  Notch Cell Signaling Pathway and Brain Tumors

Fig. 3.1  Notch cell signaling pathway: Notch signaling pathway is responsible for the proliferation in the neurogenesis process, and Numb inhibits it promotes neural differentiation. Therefore, the Notch cell signaling pathway shows significant contribution in embryonic development (Fig. 3.2)

Notch Cell Signalling Pathway

Proliferation in Neurogenesis

Numb

Promote Neural differentiation

Embryonic Development

Fig. 3.2  Role of Notch cell signaling pathway in early embryonic development: Notch cell signaling pathway promotes neurogenesis and helps in early embryonic development. Proliferation in neurogenesis is inhibited by the Numb protein, which further promotes neural differentiation

3  Notch Cell Signaling Pathway and Brain Tumors

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Notch signalling Pathway

Crucial for cell-cell communication

Gene Regulation

Multiple Cell differentiation process during embryonic/adult life

Neural Function and Development

Expanasion of the hemogenin endothelial cells with Hedgehog and SCI

Deregulation of this pathway causes many cancers T-ALL,CADASIL. Leukoencephalopathy, multiple sclerosis

Fig. 3.3  Role of Notch cell signaling pathway in developmental processes: the Notch cell signaling pathway is important for cell–cell communication and this further causes gene regulation for the multiple cell differentiation processes during embryonic and adult life. Deregulation of this pathway causes various cancers including T-ALL, CADASIL, leukoencephalopathy, and multiple sclerosis

regulation mechanism that controls multiple cell differentiation processes during embryonic and adult life. This pathway contributes to the following developmental process (Fig. 3.3): 1. Neural function and development (Hitoshi et al. 2002). 2. Expansion of the hemogenin endothelial cells with signaling axis including Hedgehog and SCI (Kim et al. 2013). 3. Notch cell signaling pathway is dysregulated in many cancers and also in many diseases including T-ALL (T-cell acute lymphoblastic leukemia (Sharma et al. 2007), CADASIL (Cerebral autosomal-Dominant arteriopathy) with sub-­cortical infarcts, leukoencephalopathy, and multiple sclerosis. Notch pathway inhibition showed anti-proliferative effects on T-cell acute lymphoblastic leukemia in vitro and in vivo models (Moellering et al. 2009; Arora and Ansari 2009). Rex1 inhibits the expression of Notch in mesenchymal stem cells and prevents differentiation (Bhandari et al. 2010). Notch and most of its ligands are transmembrane proteins, and therefore cells expressing the Notch ligands are very close to the target Notch cells for the activation cell signaling cascade. The Notch ligands are also single transmembrane proteins and a member of the DSL (Delta/Serrate/LAG-2) family of proteins. Drosophila melanogaster has two

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ligands, namely delta and serrate, and mammals have the corresponding ligands that are delta-like and jagged. There are various delta-like jagged ligands as well as other ligands like F3/Contactin (Lai 2004). The composition of Notch extracellular domain is a cysteine-rich motif called EGF-like repeats (Ma et al. 2006). Notch1 has 36 of these repeats. Each repeat is composed of 40AAs and its structure is defined largely by six conserved cysteine residues that form three conserved disulfide bonds. Modification of EGF-like repeat by o-linked glycans at specific sites (Shao et al. 2002). A embryonic lethal phenotype in Drosophila melanogaster due to Notch dysfunction (Poulson 1937). Notch mutation can lead to failure of the neural and epidermal cell segregation processes in early Drosophila embryos. The Notch pathway is also able to contribute to the maintenance and self-renewal ability of neural progenitor cells (NPC). There are also other important functions of Notch cell signaling: 1. Glial cell specification (Furukawa et al. 2000; Scheer et al. 2001) 2. Neurites development (Redmond et al. 2000) 3. Learning and memory (Costa et al. 2003) Notch pathway activation is sufficient to sustain NPCs in a proliferative stage, although loss of function mutation in the major components of the pathway causes precocious neuronal differentiation and NPC deletion (Bolós et  al. 2007). Numb protein inhibits the Notch cell signaling pathway and then consequently causes cell cycle arrest and differentiation of NPCs (Zhong et al. 1997; Li et al. 2003). However, the fibroblast growth factor promotes Notch signaling ability to maintain cerebral cortex stem cells in a proliferative stage. Therefore, gyrification occurs due to growth of this cortical surface area (Rash et al. 2013). In vitro studies showed that neurite development is also contributed by the Notch cell signaling pathway (Redmond et al. 2000). Deletion of Numb, which is the modulator of the Notch cell signaling pathway, disturbed neuronal maturation in the developing cerebellum (Klein et al. 2004). Moreover, Numb deletion disturbed axonal arborization in sensory ganglia (Huang et al. 2005). Glial cells and their subtypes differentiation is regulated by the Notch cell signaling pathway (Furukawa et  al. 2000; Scheer et  al. 2001). Moreover, Notch cell signaling also regulates neural apoptosis, neurite retraction, and neurodegeneration of ischemia stroke in the brain (Woo et al. 2009). Adult neural cells also expressed Notch protein and ligands, which indicates that this pathway has a significant role in CNS plasticity. Almost 60% of T-cell acute lymphoblastic leukemia (T-ALL) cases showed mutation in Notch cell signaling pathway (Weng et  al. 2004). A Notch inhibitor (gamma secretase) is a potential therapeutic approach for cancer treatment and approximately seven Notch inhibitors were under clinical trials (Espinoza Ingrid 2013 Pharmacology and Therapeutics). Another inhibitor, MK-0752, showed promising results in an early clinical trial for breast cancer. Presenilin-dependent gamma secretase activity regulates neurite outgrowth (Figueroa et  al. 2002). Notch dysregulation may contribute to the neuritic

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33

dystrophy, and it was reported in Alzheimer’s disease. Inhibition of gamma secretase/PS1 may have a beneficial effect on the neuritic pathology of Alzheimer’s disease. Specific inhibition of Presenilin (PS1)/gamma-secretase modulates PS1 activity in human CNS neurons and also Notch cell signaling pathway activity. Notch1 receptor intracellular domain ubiquitination and degradation are regulated by Numb protein (McGill and McGlade 2003). Numb recruits ubiquitination system components to the Notch receptor and inhibit Notch1 ubiquitination at the membrane. Thereafter proceed degradation of the intracellular domain to circumventing its nuclear translocation and inhibition of downstream Notch1 target genes activation. The Numb also influences development fate by antagonizing the Notch cell signalling pathway. Neural stem-like cells are present in human cortical glial tumors which showed expression of astroglial and neuronal markers in vitro (Ignatova et al. 2002). The author suggested that the latent critical stem cell characteristics can be epigenetically induced by growth conditions in cells from the neurogenesis region of normal CNS also in cells from the cortical glial tumors. Moreover, tumor stem-like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the development of pathological heterogeneity of glial tumors. Notch1 and Notch2 have opposite effects on embryonal brain tumor growth (Xing Fan et al. 2004). The role of Notch cell signaling in tumorigenesis showed how Notch1 acts as an oncogene in some neoplasms and as a tumor suppressor in others. It was reported that different Notch receptors showed opposite effects in a single tumor type. Expression of truncated, constitutively active Notch1 or Notch2 in embryonal brain tumor cell lines had antagonist effects on tumor growth. Cell proliferation, soft agar colony formation, and xenograft growth showed that Notch2 is oncogenic while Notch1 acts as a tumor suppressor gene (TSG). Progenitor cells

Notch1

Tumour Suppressor Gene

Notch 2

Oncogene

Oncogene

Expressed in the post mitotic differentiating cells Brain Tumour Medulloblastoma

Expressed in Proliferative Progenitor cell

Cerebellar Development

Fig. 3.4  Differentiated roles of Notch1 and Notch2: Notch1 and Notch 2 show differentiated roles during developmental and oncogenic processes. Notch1 acts as oncogene and also as a tumor suppressor gene in medulloblastoma. Notch1 is also expressed in post-mitotic differentiating cells to help in cerebellar development. Notch 2 acts as oncogene and is expressed in proliferating progenitor cells

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3  Notch Cell Signaling Pathway and Brain Tumors

derived from the most common pediatric brain tumor medulloblastoma showed high transcripts expression of Notch2, although Notch1 was undetectable. Notch2 is predominantly expressed in proliferation progenitor and Notch1 in post-mitotic differentiating cells in the process of cerebellar development (Fig.  3.4) (Fan et al. 2004). Notch cell signaling is crucial for the growth and survival of Sonic hedgehoginduced medulloblastoma in SmoA1 mouse model (Hallahan et al. 2004). Transgenic mouse model ND2:SmoA1 was developed by activating Shh pathway constitutive active form Smoothened in mouse cerebellar granule neuron precursors. This transgenic mouse model showed 48% incidence of medulloblastoma formation. The developed medulloblastoma showed high expression of Gli1 and Nmyc, and increasing hyperplasia. It was assumed that these factors are responsible for medulloblastoma tumor formation. Moreover, Notch2 and the Notch cell signaling target gene, HES5, was also highly expressed in Smoothened-induced tumors. Interestingly, Shh activation in this tumor was sufficient to induce the Notch pathway. Moreover, inhibition of the Notch pathway with soluble delta ligand or y-secretase showed marked reduction of numbers of viable cells in medulloblastoma cell lines and primary tumor medulloblastoma (Hallahan et al. 2004). Meningioma is the second most common occurence central nervous system tumor, although however, its molecular pathway mechanism is little known that are important for its pathogenesis. Induction of the Notch cell signaling pathway is carried out through three components in meningioma: transcription factor, hairy and enhancer of the spilit1 (Hes1), and two members of the Groucho/transducin-like enhancer of the split family of co-repressors, TLE2 and TLE3. Meningioma showed development. It was reported that meningioma tumor showed high expression of Hes1 transcript and protein, and Notch1 and Notch2 receptors were induced. The Jagged1 ligand was also induced in all grades of meningioma. However, induced expression of TLE2 and TLE3 appeared in higher-grade meningioma. Moreover, meningioma cell lines expressed Notch cell signaling receptor and this pathway suppressed meningioma cell growth (Cuevas et al. 2005). Musashi1 modulates cell proliferation genes in the Daoy, a medulloblastoma cell line Musashi1 (MsiI) is one of the RNA binding proteins which plays a significant role in nervous system development and stem cell maintenance. High expression of Musashi1 was reported in various tumors including a brain tumor. Daoy cells showed high expression of Msi1 when these cells were grown as neurospheres compared to monolayer cultures. This result suggested the potential role of Musashi1 in the proliferation of brain tumor stem cells. It was also reported that when MsiI was knockdown in Daoy cells with the help of MsiI shRNA, soft agar colonies formation and neurospheres of Daoy cells were reduced. Moreover, ShRNA-mediated MsiI knockdown in Daoy cells showed differential expression of Notch, Hedgehog, and Wnt pathway-related genes including MYCN, FOS, Notch2, Smo, CDKNIA, CCND2, CCND1, and DKK1 (Sanchez-Diaz et al. 2008). Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors. The Notch pathway plays an important role in both non-­ neoplastic neural stem cells and embryonal brain tumors like medulloblastoma. The

References

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Notch inhibitor suppresses the Notch pathway downstream target transcription factor Hes1 expression. This further leads to cell cycle exit, differentiation, and apoptosis in medulloblastoma cell lines. However, viable cells continue to grow but are unable to form soft agar colonies after the application of the Notch cell signaling inhibitor. These results indicate that Notch cell signaling inhibition depleted the ability of medulloblastoma cells to grow. It was also reported that the Notch pathway inhibitor reduced the expression of CD133+ and decreased the ability to form stem-like cells. It also enhanced the apoptosis rate approximately tenfold in primitive nestin-positive cells compared to nestin-negative cells. Therefore, stem-like cells in brain tumor are inhibited by Notch pathway inhibitors (Fan et al. 2006) Notch signaling enhances nestin expression in glioma. It was reported that brain tumor and stem cells showed active Notch cell signaling, and these stem cells promote brain tumor formation. Moreover, these stem cells expressed various markers including nestin, which is an intermediate filament protein. Interestingly, high-grade glioma tissue showed high expression of Notch receptors and ligands, which was also confirmed in vitro. Notch activates the nestin promoter. The mouse model of glioblastoma also showed activation of the Notch cell signaling pathway that was induced by K-ras. Moreover, activation of both Notch and K-ras in wild-type nestin-­ expressing cells leads to their expansion within the sub-ventriculation zone and retention of proliferation and nestin expression. Hence, Notch cell signaling activation alone was unable to induce this cellular expansion (Shih and Holland 2006) Notch1 regulates transcription of the epidermal growth factor through p53. Knockdown of Notch1 downregulated the expression of epidermal growth factor receptor (EGFR) in glioma, which is supposed to be highly expressed in glioma as well as other cancers. Moreover, Notch1 inhibitor decreased the EGFR mRNA and protein expression in glioma and other cell lines. There was significant correlation between EGFR and Notch1 mRNA expression in primary high-grade glioma. p53 also influenced the expression pattern of Notch1 on EGFR expression in glioma (Purow et  al. 2008). In addition, Notch cell signaling pathway activation was reported in a xenograft model of brain metastasis (Nam et al. 2008).

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Moellering RE, Cornejo M, Davis TN et al (2009) Direct inhibition of the NOTCH transcription factor complex [published correction appears in Nature. 2010 Jan 21;463(7279):384]. Nature 462(7270):182–188. https://doi.org/10.1038/nature08543 Morgan TH (1917) Goodale’s experiments on gonadectomy of fowls. Science 45(1168):483–484. https://doi.org/10.1126/science.45.1168.483 Nam DH, Jeon HM, Kim S et  al (2008) Activation of notch signaling in a xenograft model of brain metastasis. Clin Cancer Res 14(13):4059–4066. https://doi.org/10.1158/1078-­0432. CCR-­07-­4039 Oswald F, Täuber B, Dobner T et  al (2001) p300 acts as a transcriptional coactivator for mammalian Notch-1. Mol Cell Biol 21(22):7761–7774. https://doi.org/10.1128/ MCB.21.22.7761-­7774.2001 Poulson DF (1937) Chromosomal deficiencies and the embryonic development of drosophila melanogaster. Proc Natl Acad Sci U S A 23(3):133–137. https://doi.org/10.1073/pnas.23.3.133 Purow BW, Sundaresan TK, Burdick MJ et  al (2008) Notch-1 regulates transcription of the epidermal growth factor receptor through p53. Carcinogenesis 29(5):918–925. https://doi. org/10.1093/carcin/bgn079 Rash BG, Tomasi S, Lim HD, Suh CY, Vaccarino FM (2013) Cortical gyrification induced by fibroblast growth factor 2  in the mouse brain. J Neurosci 33(26):10802–10814. https://doi. org/10.1523/JNEUROSCI.3621-­12.2013 Redmond L, Oh SR, Hicks C, Weinmaster G, Ghosh A (2000) Nuclear Notch1 signaling and the regulation of dendritic development. Nat Neurosci 3(1):30–40. https://doi.org/10.1038/71104 Sanchez-Diaz PC, Burton TL, Burns SC, Hung JY, Penalva LO (2008) Musashi1 modulates cell proliferation genes in the medulloblastoma cell line Daoy. BMC Cancer 8:280. https://doi. org/10.1186/1471-­2407-­8-­280 Scheer N, Groth A, Hans S, Campos-Ortega JA (2001) An instructive function for Notch in promoting gliogenesis in the zebrafish retina. Development 128(7):1099–1107 Shao L, Luo Y, Moloney DJ, Haltiwanger R (2002) O-glycosylation of EGF repeats: identification and initial characterization of a UDP-glucose: protein O-glucosyltransferase. Glycobiology 12(11):763–770. https://doi.org/10.1093/glycob/cwf085 Sharma VM, Draheim KM, Kelliher MA (2007) The Notch1/c-Myc pathway in T cell leukemia. Cell Cycle 6(8):927–930. https://doi.org/10.4161/cc.6.8.4134 Shih AH, Holland EC (2006) Notch signaling enhances nestin expression in gliomas. Neoplasia 8(12):1072–1082. https://doi.org/10.1593/neo.06526 Singson A, Mercer KB, L’Hernault SW (1998) The C. elegans spe-9 gene encodes a sperm transmembrane protein that contains EGF-like repeats and is required for fertilization. Cell 93(1):71–79. https://doi.org/10.1016/s0092-­8674(00)81147-­2 Weng AP, Ferrando AA, Lee W et  al (2004) Activating mutations of NOTCH1  in human T cell acute lymphoblastic leukemia. Science 306(5694):269–271. https://doi.org/10.1126/ science.1102160 Wilson JJ, Kovall RA (2006) Crystal structure of the CSL-Notch-mastermind ternary complex bound to DNA. Cell 124(5):985–996. https://doi.org/10.1016/j.cell.2006.01.035 Woo HN, Park JS, Gwon AR, Arumugam TV, Jo DG (2009) Alzheimer’s disease and Notch signaling. Biochem Biophys Res Commun 390(4):1093–1097. https://doi.org/10.1016/j. bbrc.2009.10.093 Zhong W, Jiang MM, Weinmaster G, Jan LY, Jan YN (1997) Differential expression of mammalian Numb, Numblike and Notch1 suggests distinct roles during mouse cortical neurogenesis. Development 124(10):1887–1897

4

Sonic Hedgehog Cell Signaling Pathway and Medulloblastoma

The Sonic hedgehog cell signaling pathway is responsible for the cellular differentiation of embryonic cells. Interestingly, different parts of the embryo show variable concentrations of Hedgehog signaling protein. This cell signaling pathway is one of the crucial regulators in the  developmental process. Moreover, it shows its  presence in all bilaterians (Ingham et al. 2011). There are three types of HH homologous: 1. Desert Hedgehog (DHH) 2. Indian Hedgehog (IHH) 3. Sonic Hedgehog (Shh) Christiane Nusslein-Volhard and Eric Wieschaus identified a mutations in genes that regulate  segmented anterior-posterior body axis  development of Drosophila melangastor in 1970 (Nüsslein-Volhard and Wieschaus 1980). They discovered groups of genes that are involved in the development of the body segment with the help of the saturation mutagenesis technique. They shared the Nobel Prize in 1995 with Edward B. Lewis for exploring genetic mutations in Drosophila embryogenesis. Shh is the best studied ligand among the three in vertebrates. It is translated as a 45 kDa precursor and undergoes autocatalytic processing and produces a 20 kDa N-terminal protein with signaling nature (SHH-N) and a 25 kDa C-terminal domain protein without signaling nature (SHH-C). There is cholesterol modification to the carboxyl end of the N-terminal domain (Banavali 2020). Shh can process signaling in an autocrine pattern affecting the cells in which it is produced. It also acts as paracrine with the help of Dispatched (DISP). The Shh-N ligand hits its target cells; and binds to the Patched-1 (PTCH1) receptor. However, in the absence of Shh-N ligand, PTCH1 inhibits another transmembrane receptor, Smoothened (SMO), a downstream activator of the Shh cell signaling pathway (Fig. 4.1). The Patched gene expresses a 12-transmembrane receptor of the Sonic hedgehog cell signaling pathway. It is considered a potential tumor suppressor gene (TSG). The Patched gene is defective in basal cell nevus syndrome (BCNS). Homozygous

© Springer Nature Singapore Pte Ltd. 2023 M. H. Shahi, Role of Signaling Pathways in Brain Tumorigenesis, https://doi.org/10.1007/978-981-15-8473-2_4

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4  Sonic Hedgehog Cell Signaling Pathway and Medulloblastoma

SHH-N

Cytoplasm

SMO

Patch1 Gli1

Nucleus

Gli1-A

Cyclin D1 Nmyc

Promoter of the target genes

Fig. 4.1 Canonical Shh cell signaling pathway: Active ligand of Shh (Shh-N) binds to 12-­transmembrane receptor Patch1 and this binding relieves the inhibition of Patch1 on seven-­ transmembrane receptor Smoothened (SMO). Now activated Smoothened further activates Gli1 (Gli1-A), enters the nucleus, and binds to the promoter regions of downstream target genes (e.g., Cyclin D1 and N-myc), enhancing their expression and increasing cell proliferation

mutation of the Patched gene produces an open and overgrown neural tube and death in mice at early embryonic stages (Fig. 4.2). The Shh gene is transcribed ventrally in normal conditions and abnormally in lateral and dorsal neural tube cells. Moreover, Patch1 also inhibits local activation of Shh cell signaling. Interestingly, mice with heterozygous Patch1 are larger than normal and showed defective hind limb or cerebellar medulloblastoma which showed  similar  abnormalities as BCNS patients (Goodrich et  al. 1997). Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder manifested with various developmental disorders and cancers including basal cell carcinomas (BCCS). Medulloblastoma and primitive neuroectodermal tumors (PNETs) are mainly pediatric tumors and almost 3–5% of NBCCS patients manifested with these tumors (Fig. 4.3). Moreover, the Patch1 gene locus (9q22-q23) showed allelic imbalances in PNETs patients. Approximately 23 of 26 PNETs patients showed LOH at 9q22-q23 Chromosome locus (Vorechovský et al. 1997). Patch1 mutation is causing almost 1/3rd of skin’s sporadic basal cell carcinomas (BCCs) and approximately 10–15% of primitive neuroectodermal tumors (PNETs). When Shh cell signaling is activated, Shh-ligands bind the 12-transmembrane receptor Patch1. Patch1 receptor also interacts with the seven-transmembrane receptor Smoothened (SMO) and

4  Sonic Hedgehog Cell Signaling Pathway and Medulloblastoma

41

Patch1 Gene

Acts as TSG

Defective in Basal Cell Nevus Syndrome(BCNS)

Inhibits local activation of Shh signaling

Heterozygous Patch

Homozygous Mutation

Defective hind limb and develop cerebellar medulloblastoma

Open and overgrown neural tube in mice

Mice dies at early embryonic stage

Fig. 4.2  Role of Patch1 gene: The Patch1 gene acts as tumor suppressor gene (TSG) of the Shh cell signaling pathway. It also inhibits the local activation of Shh cell signaling. It shows defective expression in basal cell nevus syndrome. It also shows defective limbs and develops medulloblastoma in heterozygous conditions. Homozygous mutation of Patch1 leads to open and overgrown neural tubes in mice, with high mortality at the early embryonic stage

Nevoid Basal Cell Carcinoma Syndrome(NBCCs)

Autosomal dominant disorder

Manifest various development abnormalities including Basal Cell Carcinoma

3-5% of NBCCs patients manifest

Medulloblastoma and Neuroectodermal Tumours (PNETs)

Fig. 4.3  Role of nevoid basal cell carcinoma syndrome (NBCCs): NBCC is an autosomal dominant disorder and shows various developmental abnormalities including basal cell carcinoma, primitive neuroectodermal tumors (PNETs) and medulloblastoma

inhibits Smoothened in the absence of Shh cell signaling activation. SMO gene somatic missense mutation was identified in codon 535 (TGG-TTG; Trp-Leu) and also another codon 533 (AGC-AAC; Ser-Asn) in PNETs samples. PNETs samples also showed high expression of Patch1, SMO, and GLI1 as compared to normal skin and non-neoplastic brain tissue. However, none of these tumor samples showed a

42 Fig. 4.4 Genes responsible for BCCs and PNETs: Both Patched1 and Smoothened are the target for genetic modification/ alteration to cause BCCs and PNETs (Reifenberger et al. 1998)

4  Sonic Hedgehog Cell Signaling Pathway and Medulloblastoma

Patched1

Smoothened

Genetic alteration target

BCCs and PNETs

mutation in the Shh gene. These findings suggested that Patch1 and SMO are genetic targets for the development of sporadic BCCs and PNETs (Fig. 4.4) (Reifenberger et al. 1998). Germline mutation in the Patch1 gene causes basal cell nevus syndrome (BCNS) or Gorlin Syndrome, which later manifests as the predisposition for skin and nervous system cancer. Patch1 gene is critical  for normal developmental processes because deletion of both alleles is lethal for the survival of the embryo. However, Patch1+/− mice survived and developed spontaneous cerebellar brain tumors. This character of the Patch1 gene shows its tumor suppressive nature. Hence, Patch1+/− mice are considered as models of human medulloblastoma. It was reported that almost 14% of Patch1+/− mice developed tumors at the age of 10 months, and between 16 and 24 weeks was the peak time to develop tumors. There were also several polymorphisms in the Patch1 gene sequence from normal mice 57BL/6 and 129SV. Moreover, Patch1 haploinsufficiency is sufficient to progress oncogenesis in the CNS (Wetmore et al. 2000). There is a specific zone that shows expression patterns of Shh and Gli in developing mice. Shh expresses mainly in the neocortex and tectum regions, and Gli expresses in the proliferative zones. Shh acts as a mitogen for neocortical and tectal precursors. Therefore, it is responsible for cell proliferation in the dorsal brain. This investigation was confirmed by in vitro and in vivo studies. Moreover, Shh also controlled all three major dorsal brain structures. Many primary brain tumors and cell lines expressed the Gli1 gene. Cyclopamine is a Smoothened antagonist that inhibits the proliferation of these tumor cells. Gli1 missexpression is responsible for CNS hyperproliferation and depends upon the endogenous Gli1 activation. Hence, Shh-Gli cell signaling controls the normal dorsal brain development by regulating the proliferation of precursor cells. Therefore, this pathway dysregulation may cause brain tumor development (Dahmane et al. 2001). Medulloblastoma is  the most common  pediatric malignant brain tumor  originates in the cerebellum. Shh cell signaling is responsible for developmental processes for cell fate and growth and is expressed by Purkinje cells in the growing cerebellum. Shh promotes granule cell precursors mitosis that may be the cells that are later responsible for medulloblastoma origin. Patch1 inhibits the Shh cell

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signaling effect and mutation in the Patch1  gene may cause persistent growth of granule cell precursor, and additional accumulation of genetic or environmental factors may promote medulloblastoma growth. Patch heterozygous mutations like heterozygous PTCH-human, showed a high rate of medulloblastoma and the growth of other tumors (Corcoran and Scott 2001). Three main CNS tumors viz. retinoblastoma, glioblastoma, and medulloblastoma, define the relation between tumorigenesis and normal development. In medulloblastoma, the Patch gene controls the cell fate of the cerebellum and the reduced function of the Patch gene promote medulloblastoma (Wechsler-Reya and Scott 2001). Gli3 is another transcription factor as a negative regulator of the Shh cell signaling pathway. It was identified a new nonsense germline mutation in Gli3 gene. This germline mutation in Gli3 promoted the development of Greig syndrome and medulloblastoma. This mutation  produces a stop codon in position 809 of Gli3. However, Gli3 is very rarely mutated in medulloblastoma (Erez et al. 2002). Direct high expression of Shh in the cerebellum is sufficient to induce medulloblastoma growth. The high expression of Shh was induced with the help of ultrasound biomicroscopy-guided injection of a Shh-expressing retrovirus into the cerebellum of 13.5-day-old mice. However, it was also reported that even Gli1 null mutant mice developed cerebellum tumors. Henceforth, an efficient mouse model of medulloblastoma was developed without Gli1 whenever Shh cell signal was activated upstream of the pathway (Fig. 4.5) (Weiner et al. 2002). It was reported that when 99 brain tumor samples were gone for DNA microarray gene expression profile for their  analysis revealed that medulloblastoma is molecularly distinct from, atypical teratoid/rhabdoid tumors (AT/RTs), primitive neuroectodermal tumors (PNETs) and malignant gliomas. They also further confirmed that medulloblastoma is derived from the cerebellar granule cells by the Shh cell signaling pathway activation (Pomeroy et al. 2002).

Shh-expressing retrovirus introduced in the cerebellum with the help of utero injection

Shh

in Cerebellum

Gli1 null mutant mice

Developed Medulloblastoma

Fig. 4.5  Development of medulloblastoma in a mouse model: With the help of a retrovirus vector, Shh is introduced in the mouse cerebellum with the help of utero injection. Then mice’s cerebellum shows high expression of Shh and developed medulloblastoma in the cerebellum. Even Gli1 null mutant mice developed medulloblastoma in the presence of high expression of Shh

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The Sufu (encoding the human suppressor of fused), which is another regulator of Shh cell signaling, showed both somatic and germline mutations, and loss of heterozygosity (LOH) of its wild-type allele leading to the predisposition of medulloblastoma. Mutation in Sufu produces a truncated form of Sufu which is unable to transport activated Gli1 from the nucleus to the cytosol, and Shh cell signaling remains activated and develop medulloblastoma. Henceforth, Sufu is identified as another tumor suppressor gene of this pathway (Fig. 4.6) (Taylor et al. 2002). Patch heterozygosity promotes tumor formation by the predisposition of a subset of granule cell precursor cells to the formation of proliferation sets and further developmental genes' downregulation. Mouse medulloblastoma model like human medulloblastoma showed low expression of the neurotrophin-3 receptor, trkc/Ntrk3, which were further  showed decreased apoptosis in  vivo, demonstrating the TrkC roles in regulating tumor cell survival (Kim et al. 2003). Proto-oncogene and transcription factor Nmyc are upregulated by Shh cell signaling in cultured cerebellar granule neuron precursors (GNPs) in the absence of new protein synthesis. Interestingly, exclusively Nmyc from the Myc family gene showed temporal-spatial precisely coincides with Shh proliferating activity regions in the developing cerebellum. This pattern of expression was observed in the Patch heterozygous mouse model of medulloblastoma (Kenney et al. 2003). The type I IFNs (IFN-α and IFN-β) are responsible for antiviral defenses and immune regulation via the Janus Kinase and activation of the transcription (JAK/ STAT) pathway with activation of STAT1 and STAT2. The role of STAT2 was reported in transgenic mice (GIFN/STAT2−/−) for medulloblastoma development. GIFN/STAT2−/− but not GIFN/STAT1-null transgenic mice with CNS production of IFN-a died prematurely of medulloblastoma. Moreover, it was reported that IFN-­ y, but not IFN-a, induced STAT-1-dependent Shh expression in cultured cerebellar granule neurons (Wang et al. 2003).

Human Suppressor of Fused(SUFU)

Loss of Heterozygosity of wild type allele

Somatic and Germline mutation

Another TSG of Shh pathway

Predisposition of Medulloblastoma

Fig. 4.6  Role of Sufu in medulloblastoma: Sufu also acts as tumor suppressor gene of the Shh cell signaling pathway. Loss of heterozygosity (LOH) of its wild-type allele, germline, and somatic mutations of Sufu gene are responsible for the predisposition to medulloblastoma

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The c-myc and Shh are well-established mitogens for neural progenitors. Nmyc acts as a proto-oncogene and Shh plays a critical role in determining the embryonic pattern of CNS. The role of c-myc and Shh was investigated in medulloblastoma development by specific cells in vitro. Replication-competent ALV splice receptor (RCAS) vector was used for the Shh and c-myc to nestin-expressing neural progenitor cells injected in the newborn mice cerebella. Thereafter, the RCAS-Shh vector injected in mice developed 9% (3/32) medulloblastoma. However, mice with RCAS-Shh plus RCAS-Myc developed 23% (9/32) medulloblastoma. Therefore, it was suggested that nestin-expressing neural progenitors are present in the cerebellum at birth and may act as the original cells for medulloblastoma, c-myc further coordinating Shh for the promotion of tumorigenesis (Rao et al. 2003). The most abundant neurons are Cerebellar granule cells in the brain. Their precursor cells are responsible for the origin of the most common pediatric brain tumor, medulloblastoma. The growth regulation of these cells is mainly controlled by the Shh cell signaling pathway. Based on DNA microarray profile analysis, two genes, Cyclin D1 and Nmyc, were identified as target genes that are induced by Shh. Cyclin D1 and Nmyc genes are responsible for cell cycle entry and DNA replication, respectively. It was revealed that Nmyc is a direct target of Shh because even in the presence of the protein synthesis inhibitor Nmyc transcription was induced. Moreover, induced expression of Nmyc into GCPs influences the expression of E2F1, E2F2 and Cyclin D1, which further leads to proliferation. Interestingly, Shh actions also required Nmyc because dominant-negative N-myc is sufficient to reduce Shh-induced proliferation. Therefore, it was suggested that both Nmyc and Cyclin D1 are target genes for Shh-mediated proliferation and tumorigenesis (Fig. 4.7) (Oliver et al. 2003). The small inhibitor HhAntag downregulates the Shh cell signaling pathway by inhibiting the activation of Smoothened and resulting in complete eradication of medulloblastoma growth in Ptch1+/− and p53−/− mice at the high doses of HhAntag (Romer et  al. 2004). Shh cell signaling, which is responsible for pediatric tumor medulloblastoma can be targeted with Smoothened antagonist cyclopamine. Moreover, Cyclopamine is under evaluation in preclinical studies (Newton 2004). Patch heterozygous mice are a model for medulloblastoma. It shows, the pre-­ neoplastic condition and consequently developed medulloblastoma. This model would provide insight to detect early biomarkers for the developmental process of medulloblastoma and its  effective and efficient therapy. However,  these  pro-­ neoplastic cells behave like tumor cells, show an active Shh cell signaling pathway, and continuously proliferate. Thus, there was no wild-type Patch alleles expression, henceforth, suggesting early patch loss in medulloblastoma genesis. Moreover, pre-­ neoplastic cells are like tumor cells and GCPs in various aspects but show distinct molecular signatures. In terms of molecular signatures, various genes are categorized as regulators of migration, apoptosis, and differentiation, these are still unexplored in medulloblastoma (Oliver et al. 2005).

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Shh Cell signalling

Direct Targets of Shh signalling

Cyclin D1

Nmyc

Cell Cycle entry

DNA replication

Regulates cerebellar Granule Cells

Shh-mediated proliferation and Tumoriogenesis

Fig. 4.7  Shh cell signaling pathway role in proliferation and tumorigenesis: Shh cell signaling pathway regulates cerebral granule cells. The activated Shh cell signaling pathway has many downstream target genes and Cyclin D1 and Nmyc are crucial which are responsible for the entry to the cell cycle and DNA replication, respectively. The high expression of these downstream target genes further promotes Shh-mediated cell proliferation and tumorigenesis

When the Shh cell signaling is activated the Shh ligand binds to the Patch receptor and this further activates another receptor, Smoothened. Then Smoothened further activates canonical target genes including Gli1 and Patch1. Gli1 is one of the members of zinc finger protein that binds to DNA including Gli2 and Gli3 and regulates transcription of many downstream target genes of the Shh cell signaling pathway. Interestingly, Gli1 alleles were inactivated in Patch1+/− mice and therefore the chance of spontaneous medulloblastoma was reduced. Therefore, Gli1 is a crucial transcription factor for medulloblastoma development. Moreover, Gli2 also showed expression in medulloblastoma cells and acts to support Gli1 in medulloblastoma genesis. However, Gli1 is more potent in promoting cell proliferation and inducing medulloblastoma development than Gli2 (Kimura et al. 2005). Another factor Insulin-like growth factor-2 (Igf2) is also essential for Patch1+/− mice to form medulloblastoma. Interestingly, Igf2 expression is not present in pre-­ neoplastic lesions but its expression was very high in medulloblastoma. Medulloblastoma precursor cells (GNPs) and a medulloblastoma cell line PZp53MED showed a high proliferative activity after exogenous treatment of Igf2. Cell line PZp53MED inhibition with Igf2 cell signaling led to fewer proliferation cells. Therefore, it was suggested that Igf2 may be a potential clinical target for medulloblastoma development (Corcoran et al. 2008). Another study reported high expression of Shh, Smoothened, Gli1, and Gli2 in medulloblastoma primary human samples and reduced expression of Gli3 and Patch1 (Shahi et al. 2008).

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MicroRNA (miRNA) is a small 20–21 sequence of nucleotides that significantly contributes to early embryonic developmental processes. Therefore, it would be expected that microRNA also contributes to medulloblastoma growth. To prove the hypothesis, two spontaneous mouse medulloblastoma models with specific initiating mutations, Ink4c−/−; Patch1+/− and Ink4c−/−; p53−/−. In this selection 26 miRNA showed high expression and 24 miRNA showed low expression in proliferating mouse GNPs and medulloblastoma compared to mature mouse cerebellum. Interestingly, among the 26 high-expressing miRNAs, 9 miRNAs were from the cluster of miR-17-92. This cluster miR-17-92 showed an oncogenic nature and is also expressed in other cancers. Moreover, human medulloblastoma also showed high expression of 3miR-17-91 cluster miRNAs (miR92, miR-19a, and miR-20) with a highly active Shh cell signaling pathway without any other disease. Moreover, it was further injected miRNAs in GNPs isolated from postnatal (P) cerebella day P6 from Ink4C−/−; Patch1+/− mice and also in Ink4C−/− p53−/− mice. Interestingly, both models of mice formed medulloblastoma orthotypically with complete penetrance ability. Finally, it was suggested that miR-17-92 cluster collaborates with Shh signaling for medulloblastoma development in both humans and mice (Uziel et al. 2009). High expression of miR-17/92 polycistron in medulloblastoma was identified with the help of a high-resolution single nucleotide polymorphism genotyping assay and by interphase fluorescence in situ hybridization (FISH) on a human medulloblastoma microarray. Molecular profiling of 90 human medulloblastoma samples showed high expression of miR-17/92 cluster among the 427 mature miRNAs. Moreover, miR-17/92 cluster high expression was mainly in the Shh cell signaling-­mediated medulloblastoma. These medulloblastoma samples showed high expression of miR-17/92 and Myc/Nmyc. Moreover, it was revealed that Nmyc and not Gli1 is the main effector of the Shh cell signaling pathway in primary cerebellar granule neuron precursors (CGNPs) to induce the expression of miRNA-17/92. Interestingly, ectopic miR-17/92 expression in CGNPs synergized with exogenous Shh to enhance the proliferation and give potential to these cells to proliferate even in the absence of Shh. Therefore, in the Shh-mediated medulloblastoma miR-17/92 acts as a proliferative enhancer of medulloblastoma development (Northcott et al. 2009). Another study showed the correlation between the Sonic hedgehog cell signaling and novel tumor suppressive nature hippo pathway in brain and the genesis of medulloblastoma development. It was reported that co-activator Yes-associated protein1 (Yap1) expression was high in the Shh-mediated medulloblastoma. Moreover, Yap1 is downregulated by the hippo pathway. It was revealed that Shh cell signaling also upregulated Yap1 expression in CGNPs and medulloblastoma. Even  Yap1 presence was reported in the post-irradiated recurrence of tumor cells. Henceforth, Yap1 may be another novel target to treat Shh-mediated medulloblastoma (Fernandez-L et al. 2009). It was reported that during the process of cerebellar development, two cell cycle regulators Cyclin-dependent-kinase inhibitors, p18Ink4c and p27kip1 play an important role. However, loss of any of these inhibitors switches the cerebellar cell more proliferative, and even  this proliferation was more intense when kip1 was inactivated. Therefore, both inhibitors act as tumor suppressors in cerebellar

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development. It was tested that mice with kip1 heterozygous or nullizygous kip1 developed medulloblastoma and showed  high penetrance ability. Interestingly, tumors developed from Patch+/−; kip1+/− or Patch1+/−; Kip1−/− mice were unable to express wild-type allele and this is considered a canonical “two hit” tumor suppressor inactivation theory. It was reported that Patch+/− mice medulloblastoma showed a heterogeneous nature of cells some were highly proliferating, and some were differentiating. Similarly, tumor cells that lacked kip1 also showed the same pattern of cells. Hence, it was suggested that both Patch 1 and p27kip1 tumor suppressors can suppress medulloblastoma formation (Ayrault et al. 2009). Suppressor of fused (Sufu), which is another transcription regulator of Shh cell signaling behaves as a tumor suppressor and inhibits the Shh cell signaling downstream target gene Gli1. Mutations in both Ptch1 and Sufu increase the chance of medulloblastoma development in both humans and mice. Sufu mainly binds to Shh cell signaling downstream effector Gli1. It was reported that Sufu was degraded rapidly in tumor and this was facilitated by Shh cell signaling. Shh cell signaling activates the ubiquitination process for Sufu to degrade it by the proteosome degradation pathway. It was also identified ubiquitin-binding site of Sufu at K257 and the mutation of this site K257R is more resistant to ubiquitination suppresses the transcription and inhibits the cell proliferation. Therefore, Shh cell signaling regulates the Sufu turnover by the ubiquitination process (Yue et al. 2009). Current tumor therapy is not very efficient due to functional heterogeneity within the tumor mass. It was reported that therapy resistance Sox2+ cells were responsible for the recurrence of Shh-mediated medulloblastoma. Several Sox2+ cells produced rapidly cycling double  cortin+ progenitor cells that, together with the post-mitotic progeny which expresses NeuN, formed the tumor bulk. After the treatment of Smoothened inhibitor Cyclopamine and the anti-mitotic drug, number of Sox2+ cells increased and the recurrence of medulloblastoma. The anti-neoplastic drug mithramycin was very effective to control the recurrence of medulloblastoma. Therefore, targeting functional heterogeneity and Sox2+ cells would be one of the novel therapeutic approaches to control relapses of Shh-mediated medulloblastoma (Vanner et al. 2014). Another tumor suppressor gene, GNAS, has been reported. It expresses a G-protein, GSα,  regulating the  Shh-mediated human medulloblastoma  development. Deletion of the GNAS gene is sufficient to develop in vivo Shh-mediated medulloblastoma in distinct progenitor cells, which are very similar to human medulloblastoma. GSα suppresses Shh signaling with the help of the cAMP-­ dependent pathway and ciliary trafficking of Shh cell signaling factors. Granule neuron precursor cells cilium is enriched with GSα. When GSα effector cAMP was increased it was able to suppress the tumor cell proliferation even in the GSα mutant cells. Henceforth, G-protein GSα was identified as a TSG in Shh-mediated medulloblastoma and provided one of the potential therapy opportunities for medulloblastoma (He et al. 2014). Molecular classification of medulloblastomas divided medulloblastoma into four categories, and among these Shh-mediated medulloblastoma is a major one. Various clinical data suggested that Smoothened inhibitors would be a specific target for

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medulloblastoma under Shh group. However, due to mutation downstream of Smo, these inhibitors are not effective for all Shh group medulloblastomas. Therefore, a more efficient and effective approach is needed to control them (Kieran 2014). MET kinase was identified as one of the novel biomarkers from a variety of large cohorts of Shh-mediated medulloblastoma patients. Active MET kinase in phosphorylated was identified by immunohistochemistry in various Shh-driven medulloblastoma which showed tumor relapses and poor survival. Henceforth, it was suggested that MET kinase-based therapy may efficiently target Shh-driven medulloblastoma. Foretinib was approved as a MET kinase inhibitor and was shown to inhibit MET activation and tumor cell proliferation, and also induced apoptosis in Shh-driven medulloblastoma. Moreover, Foretinib inhibitor activity was confirmed in vitro and in vivo studies. Interestingly, Foretinib was very effective for both primary and metastatic medulloblastoma because it can cross the blood–brain barrier. Moreover, Foretinib injection into a transgenic mouse model of medulloblastoma was effective to reduce the growth of the primary tumor, inhibit metastases, and enhance host survival. Therefore, altogether, suggested that Foretinib is  needed to evaluate clinically for the treatment for Shh-regulated medulloblastoma patients (Faria et al. 2015). Tumor microenvironment is also important for tumor growth and knowledge about this may help to elucidate better therapeutic approaches to medulloblastoma. It was assumed that genes related to tumor-associated macrophages (TAM) showed a strong correlation with a specific group of medulloblastomas. For this, gene profiling by human exon assay was performed in 168 medulloblastoma samples and tumors were divided into subgroups based on inflammation-related genes expression. Shh-driven medulloblastoma showed increased expression of the inflammation-­ related genes and higher infiltration of TAMs as compared to group 3 and group 4 (P