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Expression, Regulation and Roles of Semaphorins and Their Receptors in Prostate and Prostate Cancer

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Expression, Regulation and Roles of Semaphorins and Their Receptors in Prostate and Prostate Cancer Thesis for the Doctor of Philosophy Award of a Research Degree From the University of London Veronlque Maxence BLANC Expression, regulation and roles of semaphorins and their receptors in prostate and prostate cancer Supervisors Professor John Masters, University College London Doctor Magali Williamson1, University College London Examiners Professor Paul Abel, Imperial College London Professor Fouad Habib, University of Edinburgh Prostate Cancer Research Centre (PCRC) Institute of Urology and Nephrology University College London (UCD 63-67 Riding House Street London W1W7EJ +44(0) 207 679 95971 1 UMI Number: U592524 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U592524 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 To Nicole and Yves, To Helene, Lucie and Marcel, To Erwann, 2 ACKNOWLEDGEMENTS I thank the Pilkington Trust for funding the following research project. I thank Professor John Masters and Doctor Magali Williamson for allowing and supervising my research work at the Prostate Cancer Research Centre (Institute of Urology and Nephrology, University College London), as well as for their support and supervision during completion. I thank all my colleagues for their encouragements and support, particularly Dr. Simon Bott, Dr. Charlotte Foley, Dr. Manit Araya, Dr. Aamir Ahmed, Dr. Andy Symes, Dr. Tharani Nitkunan, Dr. Joseph Nariculam, Dr. Jason Constantinou... I deeply thank all my friends for their support in this hard and long labour, to which they have largely contributed in one way or another, particularly Dr. Isabelle Bisson, Dr. Roger Tatoud, Dr. Rena Meimaridou, Dr. Tahirah Alam, Marthin Louis Vorster, Patrice Renault, Jennifer McNair Wilson... I dedicate this work to my parents, my grand parents, and especially to Erwann Thomassain, who has seen the worst of me during completion, and without whom, I would not have succeeded. Thank you for your continuous support and your confidence in my success. 3 Declaration that the work presented in the following thesis is my own I, V6ronique Maxence BLANC, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis (e.g. site-directed mutagenesis and subsequent stable transfection, SSCP, some figures in the introduction annotated with a reference). 4 ABSTRACT Prostate cancer (PCa) has become a major public health issue; it is the second most common cause of cancer-related death in the UK. PCa preferentially metastasises to the bone. Semaphorins are an important family of membrane bound or secreted signalling proteins, which control cell movement (or chemotaxis). They have a role in the nervous, immune and vascular systems, but have not previously been studied in prostate. To determine if semaphorins and their receptors are expressed in normal and pathological prostate, profiling experiments were performed by (q)RT-PCR in prostate cell lines and tissues. Results show that semaphorins and receptors are ubiquitously expressed, suggesting autocrine and paracrine regulation of semaphorin signalling in prostate; particularly an autocrine signalling of Semaphorin3A in bone metastasis derived cells. Results also show that expression of secreted semaphorins is regulated by hypoxia; which strongly downregulates semaphorin 3e in primary tumour derived cells, whereas semaphorin 3c is strongly upregulated. Particular focus was brought on semaphorin 3e, whose expression is strongly increased in epithelial cells derived from benign prostate hyperplasia, non-neoplastic tissue, and bone metastasis derived cells. A Semaphorin3E expression construct was made and the protein produced in mammalian cells to study its effects on cell morphology and adhesion by im mu nocytochemistry and adhesion assays. Results show that Semaphorin3E inhibits integrin-mediated cell adhesion while it initiates vinculin-mediated focal adhesion formation, which suggest that Semaphorin3E may play important regulatory roles in prostate cancer metastasis. The effects on cell movement of frequent missense mutations of PlexinBl receptor, associated with prostate primary tumours and metastases, was studied by migration assays. Results show that the three mutations of PlexinBl increase cell migration in absence of ligand Semaphorin4D, and further increase migration in its presence. Together these results suggest that semaphorin signalling has a key role in prostate cancer progression. 5 INTRODUCTION The following thesis is divided into four chapters: (1 ) literature review, (2 ) materials and methods, (3 ) results and discussions, (4 ) conclusion and future work. (1 ) The 'literature review' chapter comprises three sections, (a ) The prostate and its pathologies, with emphasis on prostate adenocarcinoma, the second most common cause of death due to cancer in the UK. (b ) The theories of cancer progression, summarising the molecular-based theory of cancer, which describes nine cancer gene pathways, eventually all leading to activation of the hypoxia/HIFl pathway, which promotes angiogenesis and survival of the cancer cells, (c) The sema domain containing superfamily describes three family members with common and specific features, details the HGF and MET, and Semaphorin4D and PlexinBl signalling pathways, and summarise their roles in cell guidance and migration, invasive growth, and tumour angiogenesis. (2 ) The 'materials and methods' chapter is divided into two sections, (a ) materials and (b ) methods. (3 ) The 'results and discussions' chapter contains three sections, (a ) The first section resumes the results of expression profiling experiments under normoxic and hypoxic conditions in 10 prostate cell lines. Results show that semaphorins and their receptors are largely expressed in prostate cell lines, and that the expression of secreted semaphorins is strongly regulated by hypoxia, (b ) The second section summarises the study of three mutations of PlexinBl receptor, associated with prostate tumours and metastases. The mutations modulate cell migration in presence or absence of Semaphorin4D ligand, (c ) The last section describes the production of Semaphorin3E protein and its effects on cell phenotype and behaviour. Semaphorin3E inhibits pl-integrin-mediated cell adhesion and initiates vinculin-mediated cell motility, spreading. (4 ) Finally, the 'conclusions and future work' chapter summarises all findings and suggests further studies. 6 TABLE OF CONTENTS ABSTRACT................................................................................................................................5 INTRODUCTION....................................................................................................... 6 TABLE OF CONTENT.................................................................................................................7 LIST OF FIGURES.................................................................................................................. 10 LIST OF TABLES........................................................................ 12 LIST OF ABBREVIATIONS..................................................................................................... 13 GtiAPTElUi-LITERATURE-REYIEWt ...................... 1$ Section 1: The Prostate and Its pathologies .................................. 1. Role and structure of the prostate ................ 17 2. Pathologies of the prostate .........................................................................................................................17 3. Prostate cancer: risk factors, treatments, staging and grading .......................................................... 18 4. Molecular biology and genetics of prostate cancer ................................................................................19 Section 2: Theories of cancer progress/on...,., . .,.,20 1. Genetic alterations ......................................................................................................................... 21 a. Oncogenes, tumour suppressor genes and stability genes ................. ........................21 b. The nine cancer gene pathways ..........................................................................................................22 2. The hallmarks of cancer cells .................................................................................................................... 24 a. Acquiring self sufficiency in growth signals .......................................................................................24 b. Acquiring insensitivity to growth-inhibitory signals .........................................................................24 c. Acquiring resistance to programmed cell death (apoptosis) ...........................................................25 d. Acquiring unlimited proliferative potential (resistance to senescence) ........................................26 e. Acquiring sustained angiogenesis (VEGF secretion) .........................................................................27
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