The role of receptor tyrosine kinases in mediating glioblastoma resistance to radiotherapy and temozolomide Zammam Areeb ORCID: 0000-0003-1405-6139 Submitted to the University of Melbourne in total fulfilment of the requirements of the degree of Doctor of Philosophy January 2020 The Department of Surgery The Royal Melbourne Hospital The University of Melbourne ABSTRACT Glioblastoma is the most common and aggressive form of malignant glioma. Currently, despite treatment with surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ), mean patient survival time is approximately 12 months and the 5-year survival rate is close to 0%. A key factor for the dismal prognosis is tumour recurrence post- treatment which is largely due to: 1) the infiltrative nature of glioblastoma rendering complete resection impossible and 2) glioblastoma cell resistance to radio-chemotherapy. In this thesis we aimed to investigate the cellular mechanisms of receptor tyrosine kinases in conferring resistance to therapy. We first performed a literature search and found that almost all studies that advocated for the utility of targeting RTKs in overcoming treatment resistance did not employ both therapeutic agents comprising standard therapy – radiotherapy and TMZ. We next generated an in vitro glioblastoma resistant model via short-term treatment with radiotherapy and TMZ and found that these cells had down-regulated RTK activity in addition to down-regulated protein and gene expression of the commonly altered and studied epidermal growth factor receptor (EGFR) and MET receptor. After generating an in vitro glioblastoma recurrent model via long-term treatment we demonstrated that the surviving sub-population of cells also displayed down-regulated EGFR and MET expression compared to treatment naïve cells. Furthermore, we also showed that the resistant cell population already pre-exists within the parental population which suggests the possibility of pre-emptively targeting the inherently resistant population. Interestingly, we also observed differential microRNA expression in radiotherapy- and TMZ- treated cells and, specifically, found that miR-221 confers resistance to glioblastoma cells and is capable of down-regulating EGFR expression. We validated this relationship in a human cohort of 105 primary and 36 recurrent glioblastoma patients, showing a significant inverse relationship between miR-221 and EGFR. Consistently, we showed that high miR-221 and low-EGFR expression at recurrence is associated with a poorer prognosis. Lastly, we investigated the relevance of epithelial to mesenchymal transition markers after observing that migration rates were maintained in resistant cells despite low EGFR and MET. Both N-Cadherin and CD44 were found to be highly expressed in treatment-resistant cells [ii] and the down-regulation of AKT activity with wortmannin led to reduced levels of EMT markers, suggesting that AKT is a regulator of key EMT transcription factors that are specific to N-Cadherin and CD44. The thesis gains it significance by providing an explanation to the failure of RTK inhibitors in the glioblastoma clinic by suggesting that standard radio-chemotherapy down-regulates RTK activity and expression, thereby diminishing any theorised benefit of targeting RTKs. Furthermore, the thesis advocates for microRNAs to be crucial regulators of therapy resistance, potential biomarkers and targetable molecules for the clinic. [iii] DECLARATION This declaration is to certify that: i. The thesis comprises only their original work towards the PhD except where indicated in the preface ii. Due acknowledgement has been made in the text to all other material used iii. The thesis is fewer than 100,000 words in length, exclusive of table, maps, bibliographies and appendices Zammam Areeb The Department of Surgery The Royal Melbourne Hospital The University of Melbourne [iv] PREFACE The experimental data described in this thesis comprises only my work, except for the following, obtained in collaboration: The temozolomide resistant U87, U251 and U118 cell lines were generated by Dr Rodney Luwor and Dr Stanley Stylli [v] ACKNOWLEDGEMENTS It is impossible for me to thank the people who have aided me in my PhD journey adequately on a single page. I first would like to thank my primary supervisor, Dr Rodney Luwor, for not only being a great mentor during my PhD but also for the last six years. Your mentorship has helped me become an independent scientist and the characteristics that you have embedded in me serve as a strong platform to build on, not just for my scientific endeavours but also outside the laboratory. Always accessible, genuinely concerned for his students and not shy to let me know when I was slacking, I could not have asked for more. Hopefully you being my mentor does not end here but continues for decades to come. I would next like to thank my secondary supervisor, Dr Stanley Stylli. Your willingness to help whenever I required it, your ability to critique my approach and offer ways to improve my work and your overall demeanour were all truly appreciated. Thank you. To Lucy Paradiso, thank you for your assistance throughout my time here. I would have struggled to achieve any of this if it was not for you. I would also like to thank my committee, Dr Hong-Jian Zhu and Dr Jacqueline Donoghue for their advice. Thank you Fiona Tan and Ryan Atkins for being there at the very beginning for me and creating a jovial office culture, perhaps an often overlooked requirement for good science. Thank you Hong Nguyen for putting up with me constantly asking for tips and providing constructive criticism. I would like to thank all other past and current lab members. To Juliana Gomez, I really appreciate all that you have done for me; it is too difficult to appropriately thank you and list all the different ways you have helped me. Thank you to my parents for being the best parents they could possibly be – may God reward you both. And may God reward all those who have helped me. I thank God and acknowledge that all affairs, including my blessings and failings are in the Hands of God. Studying cell biology always reminds me how glorious Your creation is. [vi] Table of Contents ABSTRACT .................................................................................................................................... ii DECLARATION ............................................................................................................................. iv PREFACE ...................................................................................................................................... v ACKNOWLEDGEMENTS ............................................................................................................... vi LIST OF ABBREVIATIONS .............................................................................................................. xi LIST OF TABLES ......................................................................................................................... xiv LIST OF FIGURES ......................................................................................................................... xv CHAPTER 1: INTRODUCTION ......................................................................................................... 1 PART 1: Defining glioblastoma ............................................................................................................ 2 1.1 Epidemiology ............................................................................................................................. 2 1.2 The problem of classifying glioblastoma ................................................................................... 3 1.3 Primary and secondary glioblastoma ........................................................................................ 7 1.4 The four subtypes ................................................................................................................... 12 1.5 The cancer stem cell model .................................................................................................... 18 PART 2: The Stupp Protocol .............................................................................................................. 23 1.6 Current treatment of glioblastoma ......................................................................................... 23 1.7 Molecular mechanisms of TMZ ............................................................................................... 35 1.8 Mechanism of resistance to TMZ ............................................................................................ 39 PART 3: Overcoming chemo-radiotherapy resistance with targeted therapy ................................. 50 1.9 The oncogene addiction model .............................................................................................. 50 1.10 Introducing receptor tyrosine kinases .................................................................................. 51 1.11 Platelet-derived growth factor receptor ............................................................................... 56 1.12 Epidermal growth factor receptor ........................................................................................ 64 1.13 MET (also known as c-MET) .................................................................................................. 78 1.14 RTK-driven signalling pathways ...........................................................................................