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Imperial College London Department of Medicine Imperial College London Department of Medicine A Genetic Screening for the Tumour Suppressive Anticancer Genes A thesis submitted for the degree of Doctor of Philosophy Qize Ding December 2016 Supervisor: Professor Eric Lam 1 Declaration of Originality I, Qize Ding, hereby declare that I am the sole author of this thesis, the whole of which contains my original research work conducted at the Department of Medicine, Imperial College London from 2012 to 2015. Contents such as figures, data and material from other sources are appropriately cited and acknowledged in the text and a full list of reference is shown at the end of my thesis. I declare that this is the true copy of my thesis and the content of this thesis has not been and will not be submitted in any form for a higher degree from any other university or institution. 2 Acknowledgements I would like to express my sincere gratitude to my current main supervisor Professor Eric Lam, as well as my former main supervisor Professor Stefan Grimm who supervised my project for the first two and half years. Their knowledge on the research areas and valuable inputs, directions and guidance have been of great value to me throughout the project. I would also like to say many thanks to my co-supervisor Dr Mona A El-Bahrawy, who advised me on the thesis write-up and corrections. Many thanks go to my colleagues, the PhD student Motasim Masood, who was helpful and supported the MTT, clonogenic assay and apoptosis assay. Another former PhD student, Ming Hwang who helped with some Western blotts. I also warmly thank the former MSc student Nazhif Zaini for efficient and hard work for 4 months under my supervision in the lab. My deep gratitude also goes to all other members of the lab, Dr Ana Gomes, Dr Stefania Zone, Dr Chun Gong, Upekha Karunarathna, Catherine Yao, as well as my former lab members Dr Ming Hwang, Dr Bevan Lin, Dr Wanwisa Chaisaklert, Dr Christoph Datler, Dr Evangelos Pazarentzos, for their enthusiasm to help and advice on technical expertise and knowledge. 3 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commerical 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 commerical purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers much make clear to others the licence terms of this work. 4 Abstract Here, using a gain of function genetic screen approach I investigated 377 human apoptosis inducer genes that were previously isolated in HEK293T cells, and performed several rounds of genetic screens to isolate 22 human anticancer genes that only induce cell death in transformed but not normal cells. Later, using the genetically well-characterised cell line HEK293T as well as establishing transformed variants overexpressing individual oncogene, mutated genes or by knocking down tumour suppressor genes, I performed a separate genetic „synthetic lethal screen‟ to identify a number of transformation scenarios in which these anticancer genes are targeted. Of these 22 anticancer genes, 16 showed a pronounced cell death inducing effect against c-Myc upregulation. c-Myc overexpressing cells were characterised to show phenotypic differences compared to their normal counterpart. On further validation, c-Myc specificity for these 16 genes was confirmed in a different cellular background in which some of these genes show inverse correlation to c- Myc expression. The last part of my project is to explore the molecular mechanism by which these 16 c-Myc specific anticancer genes induced cell death in c-Myc overexpressing cells. For the gene 1A3 (TMEFF2), my data indicated that it inhibited NF-B activity and CDK5 by inducing c-Myc specific cell death. In addition, 3 other genes 3B6, 4G3 and 4G4 also showed to induce c-Myc specific cell death through inhibition of FOXK2 in c-Myc overexpressing cells. In conclusion, I demonstrate that this „gain of function‟ genetic screening strategy is useful for the isolation of tumour suppressor genes, but the establishment of their potential „anticancer‟ functions would require further understanding of their molecular modes of action. 5 Publications and Conferences Publications Dominant Suppression of Inflammation via Targeted Mutation of the mRNA Destabilizing Protein Tristetraprolin. Ross EA, Smallie T, Ding Q, O'Neil JD, Cunliffe HE, Tang T, Rosner DR, Klevernic I, Morrice NA, Monaco C, Cunningham AF, Buckley CD, Saklatvala J, Dean JL, Clark AR. J Immunol. 2015 Jul 1;195(1):265-76. doi: 10.4049/jimmunol.1402826. The anticancer gene ORCTL3 targets stearoyl-Coa desaturase-1 for tumour-specific apoptosis. AbuAli G, Chaisaklert W, Stelloo E, Pazarentzos E, Hwang MS, Qize D, Harding SV, Al-Rubaish A, Alzahrani AJ, Al-Ali A, Sanders TA, Aboagye EO, Grimm S.Oncogene. 2015 Mar 26;34(13):1718-28. doi: 10.1038/onc.2014.93. IκΒα inhibits apoptosis at the outer mitochondrial membrane independently of NF-κB retention. Pazarentzos E, Mahul-Mellier AL, Datler C, Chaisaklert W, Hwang MS, Kroon J, Qize D, Osborne F, Al-Rubaish A, Al-Ali A, Mazarakis ND, Aboagye EO, Grimm S. EMBO J. 2014 Dec 1;33(23):2814-28. doi: 10.15252 Selectin ligand sialyl-Lewis x antigen drives metastasis of hormone-dependent breast cancers. Julien S, Ivetic A, Grigoriadis A,QiZe D, Burford B, Sproviero D, Picco G, Gillett C, Papp SL, Schaffer L, Tutt A, Taylor-Papadimitriou J, Pinder SE, Burchell JM. Cancer Res. 2011 Dec 15;71(24):7683-93. doi: 10.1158/0008-5472.CAN-11-1139. Anti-inflammatory effects of selective glucocorticoid receptor modulators are partially dependent on up-regulation of dual specificity phosphatase 1. Joanny E, Ding Q, Gong L, Kong P, Saklatvala J, Clark AR. Br J Pharmacol. 2012 Feb;165(4b):1124-36. doi: 10.1111/j.1476-5381.2011.01574.x. Conference 21 March 2012 High Throughput/High Content Technology Symposium, Imperial College London 6 Table of Contents Declaration of Originality 2 Acknowledgement 3 Copyright Declaration 4 Abstract 5 Publications and Conference Attendence 6 Table of contents 7 Abbreviations 11 Chapter 1. Introduction 17 1.1 Cell death 17 1.1.1 Apoptosis 17 1.1.2 Necrosis 19 1.1.3 Autophagy 21 1.1.4 Apoptosis pathways 21 1.1.5 Other apoptosis pathways 24 1.1.6 Caspase cascade 25 1.1.7 Imbalance and disruption of apoptotis 26 1.1.8 Apoptosis and Cancer therapy 28 1.2 Anticancer genes 32 1.3 Synthetic lethality 40 1.3.1 Background information 40 1.3.2 BRCA and PARP inhibition mediated synthetic lethality 42 1.3.3 Mutated P53, Rb and Ras related synthetic lethality 43 1.3.4 Myc related synthetic lethality 45 7 1.4 Myc 50 1.4.1 History of Myc 50 1.4.2 My family proteins 51 1.4.3 Functions of Myc 52 1.4.4 Regulation of Myc 54 1.4.5 Roles of Myc in Cancers 55 1.4.6 Therapy against Myc-driven Cancers 57 1.5 Roles of Forhead box protein FOXM1, FOXK2 and FOXO3 in human cancers and apoptosis 58 1.6 Role of CDK5 in Cancers and apoptosis 64 1.7 Hypothesis and Objectives 66 Chapter 2. Methods and Materials 69 2.1 Reagents and Materials 69 2.2 Molecular Biology 72 2.2.1 Molecular Cloning 72 2.2.2 Plasmid DNAs Section 72 2.2.3 Bacterial culture 73 2.2.4 Transformation of Plasmids 73 2.2.5 Ultra-pure Silica oxide large scale plasmid DNA isolation 74 2.2.6 DNA isolation with commercial kits 75 2.2.7 Quantification of DNA concentration 75 2.2.8 Restriction enzyme reactions 76 2.2.9 DNA Gel electrophoresis 76 2.3 Cell culture and Transfection 77 2.3.1 Mammalian cell culture 77 8 2.3.2 Xfect transfection 77 2.3.3 Other commercial transfection kits 78 2.3.4 Production of stable transfected cell lines 78 2.4 Gene expression measurement 78 2.4.1 RNA extraction 79 2.4.2 Reverse transcription 79 2.4.3 Running of quantitative Polymer chain reaction 79 2.4.4 All Primers 80 2.5 Cell death measurement 82 2.5.1 Propidium iodide staining (PI) 82 2.5.2 DIOC6 staining 82 2.6 Functional assays 83 2.6.1 Cell cycle distribution assay 82 2.6.2 CPRG assay 82 2.6.3 MTT assay 85 2.6.4 Clonogenic cell surivial assay 85 2.6.5 Cellular transformation assay 85 2.6.6 Mitochondrial reactive oxygen species measurement (MitoSox assay) 85 2.7 Protein SDS-PAGE Gel electrophoresis 86 2.7.1 Preparation of cell lysates 86 2.7.2 Protein quantification 86 2.7.3 SDS-PAGE Gel electrophoresis 87 2.7.4 Western Blotting 87 2.7.5 Antibody list 88 9 2.8 Statistical Analysis 89 Chapter 3 Synthetic lethality screen-Results and Discussion 90 3.1 Background information 89 3.2 Transfection in CV1 cells 92 3.2.1 Commercial transfection reagents 92 3.2.2 Non-commercial transfection reagents 95 3.3 Optimization of in vitro cell death assay (CPRG assay) 97 3.4 Implementation of genetic screens 102 3.4.1 First round of screen (Primary screen) 105 3.4.2 Second round of screen (Primary screen) 107 3.4.3 Third round of screen (Primary screen) 107 3.4.4 Validation of candidate genes in CV1 normal cells 111 3.4.5 Validation of candidate genes in HEK293T cells 113 3.4.6 Identifying genetic changes linked to tumour specific effects (Secondary screen) 115 3.5 Discussion 123 Chapter 4 Functional validation of 22 putative anticancer genes -Results and Discussion 130 4.1 Background Information 129 4.2 Cell death validation in different cellular systems 129 4.3 Cell proliferation 131 4.4 Long term impact on cell growth 133 4.5 Characterisation of c-Myc overexpressing cells 134 10 4.5.1 Change of cell morphology 134 4.5.2 Loss of cell - cell contact inhibition 135 4.5.3 Cell cycle and cell proliferation
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