The Role of Frizzled-7 in Gastric Cancer Chloe Rachel Austin Thesis submitted for the award of Ph.D. July 2020 The European Cancer Stem Cell Research Institute School of Biosciences Cardiff University Preface Abstract Gastric cancer (GC) has an extremely low 5-year survival rate of only 30% and is the third-leading cause of cancer-related deaths worldwide. This is predominantly due to the highly metastatic nature of GC and the lack of available treatment strategies, highlighting the urgent and unmet need to identify novel therapeutic targets. The Wnt receptor Frizzled-7 (FZD7) regulates cell proliferation, epithelial-mesenchymal-transition (EMT), and invasiveness in many cancers. GC patients have mutations in genes that participate in or regulate Wnt signalling at the level of the Wnt receptor binding. Moreover, FZD7 is reported to be overexpressed in human gastric tumours suggesting that aberrant FZD7-mediated Wnt signalling drives GC growth and highlights FZD7 as a potential therapeutic target. However, the precise involvement of FZD7 in GC remains unclear and the specific Wnt receptor transmitting oncogenic Wnt signalling is unknown. Additionally, loss of function mutations to the negative regulator of the Wnt pathway, RNF43, has been implicated in the poor prognosis of GC. However, its functional significance in GC remains unknown. We have implicated FZD7 as the predominant Wnt receptor involved in the growth, EMT, migration and invasion of GC cells irrespective of APC mutation. Here we used inhibitors of Wnt/FZD (OMP-18R5/LGK-974) and shFZD7 to test the therapeutic potential of targeting Wnt signalling in GC. Pharmacological targeting of FZD inhibited the growth of GC in vitro and in vivo. Furthermore, we have confirmed the functional significance of RNF43 in GC. Conditional deletion of RNF43/ZNRF3 led to gastric tumours supporting the hypothesis of stratifying GC patients based on RNF43 mutations. OMP-18R5 and LGK-974 are currently in phase Ib clinical trials for multiple cancers. Our data expands the scope of patients that may benefit from these therapeutic approaches as we have demonstrated that these drugs are effective in treating GC patients regardless of APC mutation status. i Preface Acknowledgements Firstly, I would like to thank my supervisor Dr Toby Phesse for the constant support, guidance, and his confidence in me. Toby has been instrumental in shaping my scientific perspective, always challenging me to think of things from new angles. I want to say a special thank you to Dr Valerie Meniel, without whom I would not have been able to complete this PhD. She has been a constant support from my very first day and always had the answer to every one of my questions. Throughout my PhD, she has been there to reassure me and encourage me during the more difficult days and to laugh and celebrate with on the good ones. I would also like to give a big thank you to Dr Sarah Koushyar for her continuous encouragement, help and support, and most importantly for brightening up the lab, especially on the days when the science decided to stop working. Further I would like to thank all the staff and students of ECSCRI, who have made my 3 years at Cardiff unforgettable. There was always someone to pick me up and put a smile one my face, often over a slice of cake and a cup of coffee, during the roller-coaster that is a PhD. The members of ECSCRI are more like a family than co-workers and I was lucky to get to spend my PhD in their company. Finally, I would like to dedicate this thesis to my partner James Baker, who has supported me in every way possible and encouraged me through this journey. Thank you for keeping me grounded throughout the long days of writing, for the diligent proof-reading, endless patience and understanding, and for being my chief supplier of chocolate and coffee upon which this thesis was fueled. ii Preface Contents Abstract ...................................................................... i Acknowledgements ........................................................ ii Contents .................................................................... iii List of Figures ............................................................ viii List of Tables ............................................................... xi Abbreviations .............................................................. xii Chapter 1: Literature Review .......................................... 1 1. Introduction .............................................................. 2 1.1 Gastric Cancer ........................................................ 2 1.1.1 Risk Factors ................................................................... 2 1.1.2 Gastric Cancer Treatment Options .......................................... 3 1.2 Normal Gastric Architecture & Dynamics ......................... 6 1.2.1 Chief cells ....................................................................... 9 1.2.2 Parietal cells .................................................................... 9 1.2.3 Mucous-secreting cells ....................................................... 11 1.2.4 Enteroendocrine cells ....................................................... 12 1.2.5 Gastric epithelial renewal ................................................ 14 1.3 Gastric Cancer Initiation .......................................... 17 1.3.1 GC Classification ........................................................... 20 1.4 Gastric Cancer Metastasis ......................................... 23 1.4.1 Stages of Metastasis ....................................................... 23 1.4.2 EMT .......................................................................... 26 1.4.3 Tumour Microenvironment ............................................... 26 1.5 Wnt Signaling Pathways ........................................... 28 1.5.1 Canonical Wnt/β-catenin Pathway ........................................ 28 1.5.2 Wnt ligand synthesis, secretion and signalling ........................... 31 1.5.3 Frizzled receptors ............................................................ 32 1.5.4 LRP5/6 ......................................................................... 38 1.5.5 Ror and Ryk ................................................................... 39 iii Preface 1.5.6 The β-catenin destruction complex ....................................... 40 1.5.7 Nuclear Events ................................................................ 44 1.5.8 Wnt Antagonists & Agonists ................................................. 46 1.5.9 R-Spondin & E3 ligases (Rnf43 & Znrf3) ................................... 47 1.5.10 Wnt signalling in the homeostasis of the Gastric Epithelium ......... 48 1.6 Aberrant Wnt Signalling & Gastric Cancer ...................... 50 1.6.1 H.pylori-mediated Wnt signalling .......................................... 51 1.6.2 Intracellular Component Mutations ........................................ 53 1.6.3 Dysregulation of Wnt signalling at the Plasma Membrane ............. 56 1.6.4 The role of Frizzled Receptors in GC ...................................... 64 1.6.5 Oncogenic Wnt signalling & GC metastasis ............................... 68 1.7 Aims of thesis ....................................................... 73 1.7.1 Aim 1. .......................................................................... 73 1.7.2 Aim 2. .......................................................................... 73 1.7.3 Aim 3. .......................................................................... 73 Chapter 2: Materials & Methods ...................................... 74 2. Materials & Materials ................................................. 75 2.1 Cell Culture ......................................................... 75 2.1.1 Cell lines .................................................................... 75 2.1.2 Passaging cells ................................................................ 75 2.1.3 Long term storage ............................................................ 75 2.1.4 Raising cells from storage ................................................... 76 2.1.5 Cell seeding ................................................................... 76 2.2 Transfection of cell lines .......................................... 77 2.2.1 Plasmids ....................................................................... 77 2.2.2 Transient transfection ....................................................... 77 2.3 Cell Based Assays ................................................... 79 2.3.1 CellTiter-Glo Assay ........................................................... 79 2.3.2 Soft Agar Colony Forming Assay ............................................ 79 2.3.3 Migration Assays .............................................................. 79 2.3.4 Invasion Assays ................................................................ 80 iv Preface 2.3.5 EMT stimulation .............................................................. 81 2.3.6 Fluorescence immunocytochemistry ...................................... 81 2.4 In vivo experiments ................................................ 83 2.4.1 Gastric cancer xenografts ................................................... 83 2.4.2 In vivo peritoneal dissemination model ................................... 83 2.4.3 Experimental Animals........................................................ 83 2.4.4 Colony maintenance & breeding ........................................... 84 2.4.5 Genetic Mouse Model .......................................................