Epstein-Barr Virus Induction of the Hedgehog Signalling Pathway Imposes a Stem Cell-Like Phenotype on Human Epithelial Cells – Implications for the Pathogenesis of Nasopharyngeal Carcinoma by REBECCA J. PORT A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY School of Cancer Sciences College of Medical and Dental Sciences University of Birmingham September 2013 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Nasopharyngeal carcinoma (NPC) is endemic in Southern China and South East Asia, causally linked to Epstein-Barr virus (EBV) infection, and frequently shows dysregulation in a number of stem cell maintenance signalling pathways. This thesis has endeavoured to investigate the status of one of these pathways; the Hedgehog (HH) signalling pathway, in NPC tumours, and reveals the novel finding that EBV is able to active the HH signalling pathway through autocrine induction of the SHH ligand in the C666.1 authentic EBV-positive NPC-derived cell line and latently infected epithelial carcinoma cell lines. This study demonstrates that constitutive engagement of the HH pathway in EBV-infected epithelial cells in vitro induces the expression of a number of stemness-associated genes and imposes stem-like characteristics. Using epithelial cells expressing individual EBV latent genes, this study also investigates the viral protein responsible for HH dysregulation demonstrating that EBNA1, LMP1 and LMP2A are all capable of inducing SHH ligand and activating the HH pathway, but only LMP1 and LMP2A are able to induce expression of stemness-associated marker genes. These findings not only identify a role for dysregulated HH signalling in NPC oncogenesis but also provide a novel rationale for therapeutic intervention. ACKNOLEDGMENTS Firstly I would like to thank my supervisor, Chris Dawson, without whom none of this work would have been possible, I truly could not have hoped for better supervisor and guidance. Chris is one of the most intelligent people I have ever met and, over the course of the last four years, has become a great friend. Thank you for wanting this to be the best thesis ever and for driving me to try and achieve that. I would also like to thank Prof Lawrence Young who made this PhD project possible and for all the advice he has given. Thanking the remainder of the NPC group will not take long but fortunately what the group lacked in size was made up for by the skill of such a dedicated technician – thank you Sonia for your help, patience and words of wisdom. I will also put out a thanks to past NPC members; Kathryn, John, Mhairi and, particularly, Khil who showed me the ropes. Your help and support at the start of my journey is remembered with great fondness and without you I may have never found my love for public engagement. There are so many other staff and students at the University of Birmingham who deserve a mention, I can only hope that you know who you are and know how grateful I am. It is the people and not the place that makes a University, and the University of Birmingham is such as wonderful place to have spent the last four years! I would like to thank my parents for all their encouragement and support. I am very touch by my dad who, despite not being able to say ‘Nasopharyngeal Carcinoma’, carries my project written down on a piece of paper so he can point to it and say “this is what my daughter is studying”. Finally I would like to thank some very special people; my boyfriend Jonny, for his understanding and assistance, and Sarah for her encouragement. Contents List of tables INDEX LIST OF FIGURES .................................................................................................... VI LIST OF ABBREVIATIONS ......................................................................................... I CHAPTER ONE: INTRODUCTION ............................................................................. 1 1.1 Introduction to cancer .................................................................................... 2 1.2 The biology of cancer ..................................................................................... 2 1.3 The role of infectious agents in cancer ........................................................ 4 1.4 Viruses and cancer ......................................................................................... 5 1.4.1 Viruses implicated in cancer ...................................................................... 8 1.4.2 DNA and RNA viruses ............................................................................. 10 1.4.3 RNA tumour viruses ................................................................................ 10 1.4.4 DNA tumour viruses ................................................................................ 11 1.4.5 Herpesviruses family ............................................................................... 12 1.5 Epstein-Barr virus (EBV) .............................................................................. 12 1.5.1 The EBV genome .................................................................................... 13 1.5.2 EBV biology ............................................................................................. 16 1.6 EBV infection of B-cells and epithelial cells .............................................. 18 1.6.1 EBV infection of B-cells in vitro ................................................................ 18 1.6.2 EBV infection of epithelial cells in vitro .................................................... 18 1.7 Latency programs in EBV-associated malignancies ................................. 23 1.7.1 EBV associated lymphoma malignancies ................................................ 24 1.7.2 EBV associated epithelial malignancies .................................................. 26 1.8 EBV gene expression and the pathogenesis of NPC and EBV-aGC ........ 34 1.9 EBV latent proteins ....................................................................................... 36 1.9.1 Latent membrane protein 1 (LMP1) ......................................................... 36 1.9.2 Latent membrane protein 2 (LMP2) ......................................................... 39 1.9.3 Epstein-Barr virus nuclear antigen 1 (EBNA1) ........................................ 42 1.9.4 EBV-encoded RNAs (EBERs) ................................................................. 43 1.9.5 The EBV-encoded micro-RNAs (miRNAs) .............................................. 44 1.9.6 BamH1-A reading frame-1 (BARF1) ........................................................ 46 1.10 The cancer stem cell hypothesis ................................................................ 46 1.10.1 Stem cells ................................................................................................ 46 1.10.2 Cancer stem cells (CSCs) ....................................................................... 49 1.10.3 Isolation of CSCs ..................................................................................... 51 1.10.4 Evidence for CSCs in NPC ...................................................................... 54 1.11 Deregulation of signalling pathways associated with “stemness” and cancer ....................................................................................................................... 55 Contents List of tables 1.11.1 The Hedgehog (HH) Signalling Pathway ................................................. 57 1.11.2 Regulators of the HH Signalling Pathway ................................................ 62 1.11.3 Targets of activated GLI transcription factors .......................................... 63 1.11.4 Interaction of HH signalling pathway with other stem cell maintenance pathways ................................................................................................................ 64 1.12 The HH signalling pathway in cancer ......................................................... 67 1.12.1 Mechanisms of HH signalling pathway activation .................................... 67 1.12.2 Targeting the HH signalling pathway in tumour therapy .......................... 70 CHAPTER TWO: MATERIALS AND METHODS ..................................................... 72 2.1 Cell culture .................................................................................................... 73 2.1.1 Basic media ............................................................................................. 73 2.1.2 Supplements and sterile solutions ........................................................... 73 2.2 Cell lines ........................................................................................................ 74 2.2.1 Maintenance of cell lines ......................................................................... 76 2.2.2 Cryopreservation of cell lines .................................................................. 76 2.3 Generation of EBV positive cell lines ........................................................