THE ROLE OF TETRASPANIN 6 IN COLORECTAL CANCER by Regina Andrijes A thesis submitted to the University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Institute of Cancer and Genomics College of Medical and Dental Sciences University of Birmingham October 2018 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties.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 Colorectal cancer is one of the most common cancer types and the second leading cause of cancer-related deaths worldwide. Poor survival of patients highlights the importance of identification of novel prognostic markers. The work in this thesis presents Tspan6 as a potential candidate. Tspan6 is a poorly studied member of the tetraspanin family of proteins that has been implicated in cancer initiation, progression and metastasis. The expression of Tspan6 in a cohort of genetically profiled colorectal adenocarcinomas in this study demonstrated that Tspan6 expression is significantly reduced in tumours vs. adjacent non-cancerous tissues. To illustrate the role of Tspan6 in colorectal cancer (CRC) Tspan6 KO mice carrying APCmin/+ allele were generated. It was found that loss of Tspan6 gene accentuates APC-driven tumorigenesis in vivo. Specifically, these animals developed larger numbers of intestinal and colonic polyps. Interestingly, these polyps were significantly bigger in size and presented with a more severe neoplastic phenotype. The RNAseq analysis of polyps derived from APCmin/+ and APCmin/+Tspan6 KO showed substantial enrichment of differentially expressed genes within the MAPK signalling pathway. Additionally, our experiments with intestinal organoids derived from Tspan6 KO mice and a Caco-2 CRC cell model confirmed the important role of Tspan6 in EGFR-dependent signalling in colonic epithelium via a pathway involving autocrine production of TGF-α. Furthermore, it was demonstrated that Tspan6 regulates the production of EVs through the involvement of an adapter protein syntenin-1, an established partner of Tspan6 which is known to play a critical role in biogenesis of multivesicular bodies (MVBs) and exosomal production. Therefore, it is hypothesised that the Tspan6-syntenin-1 complex plays a critical role in suppressing of colorectal tumorigenesis by controlling autocrine secretion of EGFR ligands via i extracellular vesicles. The better understanding of the Tspan6-dependent mechanisms of EGFR regulation can underpin further development of EGFR- targeting therapy and improve survival of CRC patients. ii I dedicate this thesis to my mother, Luiza, and my siblings, Viorika and Artjom, who always love, understand and support me. ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS This PhD is undoubtedly one of the biggest achievements in my life. The whole journey has been a mixture of failure and success, excitement and frustration, discouragement and motivation, but overall very rewarding in my life. I would like to thank everyone who has helped me on the way. Firstly, I would like to express my gratitude to my PhD supervisors, Dr Fedor Berditchevski, Dr Andrew Beggs and Dr Chris Tselepis for the advice, guidance, constructive criticism, your help, support and encouragement. A special thank you to Dr Rahul Hejmadi and my friend and colleague Dr Maha Ibrahim for helping me with pathology examination and scoring of samples. I am grateful to Celina Whalley for helping me with RNA sequencing and all the members of Beggs lab for being helpful, kind and friendly. A special thank you to Richard Horniblow for consulting me, willingness to proof read countless pages of my reports and thesis and becoming a good friend in the process. I would like to thank all the staff, colleagues and fellow students of the Institute of Cancer and Genomics, you all have made this place a second home to me. I am also thankful to all the members of our laboratory, Maryam, Jing, and especially Isaac. Thank you for the stimulating discussions, for all the long hours we shared in the lab and the fun we had outside the lab, I could not ask for a better team to work with. I wish to express my eternal gratitude to Vera Novitskaia for always being there for me, for teaching, advising and listening. You have become my dearest friend, a shoulder to cry on, you have provided me with strength and motivation and taught me invaluable life lessons. I would like to thank all my friends in the UK and in Estonia for your unconditional friendship. Especially to my best friend Kristina, who iv ACKNOWLEDGEMENTS has shared with me the moments of happiness, when I was accepted to the program, the moments of frustration, when I was writing my thesis, and been there for me for over 18 years of my life. I would also like to express my gratitude, appreciation and love to Andrea for believing in me, supporting me and making me happy. And finally, I would like to thank my whole family: my mother, sister and brother for supporting me not only during my PhD but throughout my life. I would not be here if it was not for you. v TABLE OF CONTENTS TABLE OF CONTENTS ABSTRACT ................................................................................................................................. I ACKNOWLEDGEMENTS ......................................................................................................... IV TABLE OF CONTENTS ............................................................................................................ VI LIST OF FIGURES .................................................................................................................... XI LIST OF TABLES .................................................................................................................... XV ABBREVIATIONS ................................................................................................................... XVI 1 INTRODUCTION ............................................................................................................... 1 1.1 COLORECTAL CANCER ..................................................................................................... 1 1.1.1 Definition ............................................................................................................... 1 1.1.2 Types of cancers in the colon and rectum ............................................................ 1 1.1.3 Colorectal cancer incidence ................................................................................. 2 1.1.4 Colorectal cancer mortality and survival ............................................................... 2 1.1.5 The worldwide distribution of colorectal cancer .................................................... 3 1.1.6 Colorectal cancer risk factors ............................................................................... 5 1.1.7 Treatment of colorectal cancer ............................................................................. 9 1.2 MOLECULAR BASIS OF CRC ........................................................................................... 11 1.2.1 The anatomical structure of the colon ................................................................ 11 1.2.2 Molecular pathways of colorectal carcinogenesis .............................................. 14 1.2.3 Adenoma-Carcinoma sequence ......................................................................... 15 1.2.4 Serrated Pathway ............................................................................................... 17 1.3 SIGNAL TRANSDUCTION PATHWAYS IN COLORECTAL CANCER ........................................... 20 1.3.1 Wnt signalling ..................................................................................................... 20 1.3.2 EGFR signalling in colorectal cancer.................................................................. 27 1.4 MOUSE MODELS OF CRC ............................................................................................... 33 1.5 ORGANOIDS AS A MODEL OF COLORECTAL CANCER AND NORMAL INTESTINAL EPITHELIUM .. 41 1.6 TETRASPANINS .............................................................................................................. 45 vi TABLE OF CONTENTS 1.6.1 Overview ............................................................................................................. 45 1.6.2 Tetraspanin structure .......................................................................................... 45 1.6.3 Tetraspanin function ........................................................................................... 48 1.6.4 Tetraspanins and colorectal cancer.................................................................... 54 1.6.5 Tetraspanin 6 ...................................................................................................... 54 1.6.6 Tetraspanins, extracellular vesicles and exosomes ........................................... 59 1.7 RESEARCH OBJECTIVES ................................................................................................. 65 1.7.1 Aims ...................................................................................................................