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The RNA-Binding Protein LARP1 As Potential Biomarker and Therapeutic Target in Ovarian Cancer

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The RNA-Binding Protein LARP1 As Potential Biomarker and Therapeutic Target in Ovarian Cancer The RNA-binding protein LARP1 as potential biomarker and therapeutic target in ovarian cancer A thesis submitted for the Degree of Doctor of Philosophy Imperial College London Department of Surgery and Cancer Thomas Glass Hopkins December1 2014 DECLARATIONS This thesis is submitted to Imperial College in support of my application for the degree of Doctor of Philosophy. It has been composed by myself and has not been submitted in any previous application for other degrees. The work presented (including data generated and analysis) represents original work carried out by the author in the laboratory of Dr Sarah Blagden in the Department of Surgery and Cancer, Imperial College London, except where clearly stated. The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 1 ACKNOWLEDGEMENTS Firstly, I would like to take this opportunity to thank my thesis supervisor, Dr Sarah Blagden, for all her help and advice throughout this project. Her patience and enthusiasm has kept me going, and she has been extremely supportive of everything I have done. I would also like to thank my co- supervisors, Professor Martin Wilkins, Dr Richard Wooster and particularly Professor Eric Aboagye for all their helpful input throughout the last three years. This work would not have been possible without the financial support provided by the Imperial College Wellcome Trust-GSK Translational Medicine and Therapeutics PhD Fellowship, and I would like to thank Professor Martin Wilkins and Professor Paul Matthews for selecting me for this prestigious scheme. Dr Manuela Mura in Dr Blagden’s lab has been a fantastic person to work with and I have learnt a huge amount from her over my PhD. I should also like to acknowledge Dr Normala Abd-Latip, who, together with Manuela, laid much of the groundwork for my research, and Dr Katrina Sweeney, who got the lentiviral work up and running. Everyone on the 4th floor of the Institute of Reproductive and Developmental Biology has been extremely helpful, but I would particularly like to thank Dr Jayantha Chatterjee for his support with all things ELISA-related, Haonan Lu for introducing me to ‘R’ and helping with data analysis, Camila Henrique De Sousa for keeping me sane, and Drs Erick Loomis, Elaina Maginn, Paula Cunnea, Rajpal Burmi and Jamie Studd for helpful discussions. Naina Patel, Jenny Steel and Nona Rama have gone above and beyond the call of duty in helping to retrieve and process patient samples. I have abandoned my girlfriend, Lizzie Monks, on numerous weekends and evenings when the cells have demanded my attention, but she has been tireless in her support. I could not have done it without her. Finally, and most importantly, I would like to dedicate this work to my parents, Professor Robert Glass and Linda Hopkins, without whom I would, literally and figuratively, not be here today! They have been unfailing in their support and unstinting in their time, and have selflessly read pretty much everything I have ever written. I hope I’ve done them proud! 2 ABSTRACT Ovarian cancer is the most lethal gynaecological malignancy, responsible for over 4,000 deaths each year in the UK. There is growing evidence that mRNA-binding proteins (RBPs) can be post-transcriptional drivers of cancer progression. Here, I investigated the expression of the RBP LARP1 in ovarian malignancies and role of the protein in ovarian cancer cell biology. LARP1 is highly expressed at both an mRNA and protein level in ovarian cancers compared with benign tumours and normal ovarian tissue. I show that higher levels of LARP1 in tumour tissue are predictive of poor patient survival. Consistent with this clinical finding, in xenograft studies knockdown of LARP1 expression causes a dramatic reduction in tumour growth. In vitro, LARP1 knockdown is associated with increased apoptosis, and is sufficient to restore platinum sensitivity in chemotherapy-resistant cell lines. Furthermore, LARP1 is required to maintain cancer stem cell marker-positive populations, and knockdown decreases tumour-initiating potential, as demonstrated by in vivo limiting dilution assays. Transcriptome deep-sequencing following LARP1 knockdown revealed altered expression of multiple genes linked to survival and evasion of apoptosis, including BCL2 and BIK. Transcripts of both genes are in complex with LARP1 protein, and LARP1 maintains the stability of BCL2 mRNA, whilst actively destabilising BIK transcripts. This effect is mediated at the level of the 3’ untranslated region. I therefore conclude that by differentially regulating mRNA stability, LARP1 is a key post-transcriptional driver of tumourigenicity and cell survival in ovarian cancer. 3 PUBLICATIONS ARISING FROM THIS THESIS Mura M, Hopkins, TG et al. LARP1 post-transcriptionally regulates mTOR and promotes cancer progression. 2014. Oncogene. Accepted and in press. Hopkins TG, et al. LARP1 promotes cancer cell survival and chemoresistance in ovarian cancer. Under submission. 4 TABLE OF CONTENTS Declarations ...................................................................................................................... 1 Acknowledgements ........................................................................................................... 2 Abstract ............................................................................................................................. 3 Publications arising from this thesis ................................................................................... 4 Table of Contents .............................................................................................................. 5 List of Figures .................................................................................................................... 8 List of Tables .................................................................................................................... 10 Abbreviations .................................................................................................................. 11 1 Chapter I – Introduction ............................................................................................. 13 1.1 Chapter one abstract ....................................................................................................... 13 1.2 Epithelial ovarian cancer ................................................................................................. 14 1.2.1 Ovarian tumour classification ......................................................................................... 15 1.2.2 Diagnosis ........................................................................................................................ 17 1.2.3 Treatment ....................................................................................................................... 20 1.2.4 Cell of origin ................................................................................................................... 23 1.2.5 The molecular basis of high-grade serous ovarian cancer ............................................. 24 1.2.6 Ovarian cancer stem cells ............................................................................................... 26 1.2.7 Summary ........................................................................................................................ 28 1.3 mRNA-binding proteins ................................................................................................... 29 1.3.1 mRNA ribonucleoproteins (mRNPs) ............................................................................... 30 1.3.2 mRNA-binding Proteins .................................................................................................. 31 1.3.3 The mRNA journey ......................................................................................................... 32 1.3.4 RNA-binding proteins in cancer ...................................................................................... 48 1.3.5 Summary ........................................................................................................................ 50 1.4 The LARP family ............................................................................................................... 51 1.4.1 LARP3/La/SSB ................................................................................................................. 52 1.4.2 LARP7 .............................................................................................................................. 59 1.4.3 LARP 4 and 4B ................................................................................................................. 62 1.4.4 LARP6 .............................................................................................................................. 63 1.4.5 LARP1 and 1B .................................................................................................................. 65 1.4.6 The LARP family in summary .......................................................................................... 73 1.5 Study Hypothesis ............................................................................................................
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