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The Role of Brachyury in the Pathogenesis of Chordoma

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The Role of Brachyury in the Pathogenesis of Chordoma P a g e | 1 The role of brachyury in the pathogenesis of chordoma A thesis submitted to University College London (UCL) for the degree of Doctor of Philosophy Nischalan Pillay 2013 UCL Cancer Institute, London, UK P a g e | 2 I, Nischalan Pillay, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. P a g e | 3 Abstract Chordoma is a rare malignant tumour of bone, the molecular marker of which is the expression of the transcription factor T (also referred to as brachyury). Silencing of T induces growth arrest in chordoma cell lines; however its downstream genomic targets are unknown. In this thesis I have identified these targets with validation in human chordoma samples. This was achieved by using an integrated functional genomics approach involving shRNA-mediated brachyury knockdown, gene expression microarray, ChIP-seq experiments and bioinformatics analyses. The results show that T regulates a downstream network that involves key cell cycle related genes amongst other potential oncogenic programmes. This is the first documentation of genomic T targets in humans and provides a molecular insight into the pathogenesis of chordoma. In a separate but related piece of work, a genetic association study was conducted to determine the genetic susceptibility determinants in patients with sporadic chordomas. Whole-exome and Sanger sequencing of T exons revealed a strong risk association with the common non-synonymous SNP rs2305089 in chordoma. The degree of risk imparted by this variant is large and is an exceptional finding in cancer genetics. Overall the work presented in this thesis contributes to the evolving understanding of T’s role in the pathogenesis of this rare bone cancer. P a g e | 4 Acknowledgments I owe an enormous debt of gratitude to my supervisor, Prof Adrienne Flanagan for granting me the opportunity to pursue a doctoral research degree when conventional wisdom may have suggested otherwise. I continue to benefit immensely from her mentorship and tutelage. I am grateful to all members of Adrienne’s group, past and present in the UCL Cancer Institute and the Royal National Orthopaedic Hospital. There are too many to mention by name but there is no doubt that the questions that I sought to address in this thesis would not have been possible without the seminal and erudite discoveries made by both previous and current group members. Moreover the easy sharing of the varied expertise within the group has allowed me to broaden my knowledge- base on areas of molecular biology and pathology that would not have been possible otherwise. I am also grateful to my secondary supervisor Prof Claudio Stern whose all-round passion for designing “clean” experiments to help unravel fundamental biological mechanisms through the lens of a gene or a cell is infectious. Although he may not have known it, it was through valuable discussions with him at the infancy of my thesis that I was “bitten by the research bug”. I am also appreciative of the collaborative research environment provided at the UCL Cancer Institute by Prof Chris Boshoff. I have found sharing the laboratory space with his group both stimulating and beneficial. In particular I am grateful to Dr Leonid Nikitenko. Leonid is an oracle, disarmingly in multiple facets of science and life, through whom I have avoided many a rookie mistake by talking through my experiments for the day. He is meticulous and his scepticism, P a g e | 5 fastidiousness and perfectionism provided the perfect foil for my inexperience. Although I hope to have paid this back in part through providing some histopathology insights through collaborative projects, I fear the balance of favour weighs heavily on his side. I am grateful that our paths have crossed. He has become a close friend. It is said that “no man is an island” and I have learned that is no truer than in science. In that vein I am indebted to all the collaborators I have worked with on the various projects that make up this thesis. They are all named co-authors in the published papers. In particular, I would like to thank Drs Fiona Wardle and Andrew Nelson for their invaluable assistance and expertise in helping generate and analysing the data for the ChIP-seq experiment. This project was initiated when ChIP-seq was just about becoming a mainstream technique and much of the analytical and bio-informatic approaches were rapidly evolving over this period. That it took us more than 18 months from the time of generation of data to finalising the analysis and publishing bears testament to this. I am eternally grateful to my maternal aunt Sundree and my parents for their encouragement and all their sacrifices on my behalf. I am grateful also to my siblings for tolerating me pursuing my dream away from home and entrusting many domestic responsibilities upon them. Lastly, I would not have been able to undertake this PhD without the moral and financial support of my wife, Ula. She has provided me with the space and time to fully commit myself to satisfying this rather self-indulgent exercise. Moreover, P a g e | 6 her unwavering love and faith carried me through my many moments of self- doubt. She is the greatest person that I know.1 1 “Fate gives us all three teachers, three friends, three enemies and three great loves in our lives. But these twelve are always disguised and we can never know which one is which until we’ve loved them, left them or fought them.” Gregory David Roberts, Shantaram, 2007 P a g e | 7 Publications My thesis is partly based on the following journal articles, which are appropriately referenced in the main body of the thesis and reference list. Permission for use of the figures and content from these articles has been obtained from the publishers where necessary. 1. Presneau N, Shalaby A, Ye H, Pillay N, Halai D, Idowu B, Tirabosco R, Whitwell D, Jacques TS, Kindblom LG, Bruderlein S, Moller P, Leithner A, Liegl B, Amary FM, Athanasou NN, Hogendoorn PC, Mertens F, Szuhai K, Flanagan AM: Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study, J Pathol 2011, 223:327-335 2. Pillay N, Amary FM, Berisha F, Tirabosco R, Flanagan AM: P63 does not regulate brachyury expression in human chordomas and osteosarcomas, Histopathology 2011, 59:1025-1027 3. Nelson AC and Pillay N*, Henderson S, Presneau N, Tirabosco R, Halai D, Berisha F, Flicek P, Stemple DL, Stern C, Wardle FC, Flanagan AM: An integrated functional genomics approach identifies the regulatory network directed by brachyury (T) in chordoma. J Pathol. 2012 Nov;228(3):274-85. *Joint first authors P a g e | 8 4. Pillay N, Plagnol V, Tarpey PS, Lobo SB, Presneau N, Szuhai K, Halai D, Berisha F, Cannon SR, Mead S, Kasperaviciute D, Palmen J, Talmud P, Kindblom LG, Amary FM, Tirabosco R, Flanagan AM: A common single nucleotide variant in T is strongly associated with chordoma. Nat Genet. 2012 Nov;44(11):1185-7 P a g e | 9 Contents Page Table of Figures 17 List of Tables 19 Abbreviations 20 Chapter 1: General Introduction 23 1.1. Background 24 1.1.1. Epidemiology and natural history 24 1.1.2. Therapeutic options 26 1.1.3. Pathology 28 1.1.3.1. Macroscopy 28 1.1.3.2. Histology 30 1.1.3.3. Immunohistochemistry 31 1.1.4 Genetics of chordoma 32 1.1.4.1. Gene expression studies 32 1.1.4.2. Somatic mutations in chordoma 33 1.1.4.3. Somatic copy number alterations in chordomas 35 1.1.4.4. Chromothripsis and chordoma 38 1.1.5 Mouse models of chordoma 39 1.2. T and T-box genes 40 1.2.1. Background 40 1.2.2. Structure of T 42 1.2.3. T-box domain 42 1.2.4. Genes bound by the T protein during development 44 1.2.5. T-Box genes and disease 45 1.3. The relationship between T and chordomas 46 1.3.1. The origin of chordoma 46 P a g e | 10 1.3.2. Benign notochordal cell tumours or Giant notochordal rests? 49 1.4. T and other malignancies 51 1.5. T, chordoma and stem cells 52 1.5.1. Cancer stem cells 53 1.5.2. T and stem cells 53 1.5.3. T and cancer stem cells 54 1.6. Overall scope of my thesis 55 Chapter 2: Materials and Methods 56 2.1 Clinical samples 57 2.2 Cell culture 58 2.2.1 Chordoma cell lines 58 2.2.2 Media and supplements 58 2.2.3 Other cell lines 58 2.3 Stable shRNA knockdown of T in U-CH1 and U-CH2 59 2.3.1 Virus production 59 2.3.2 Stable lentiviral transduction 59 2.4 Cell proliferation and cell cycle assay 60 2.5 Migration assay 60 2.6 Adhesion assay 61 2.7 Molecular biology: RNA 62 2.7.1 RNA extraction 62 2.7.2 cDNA synthesis 62 2.7.3 qRT-PCR 63 2.7.4 RT-PCR 65 P a g e | 11 2.7.5 Primers 65 2.7.6 Gene expression microarray 66 2.8 Molecular biology: DNA 68 2.8.1 Genomic DNA isolation 68 2.8.2 PCR amplification 69 2.8.3 Taqman genotyping 70 2.8.4 Whole exome sequencing (in collaboration with ICGC-Wellcome Trust Sanger Institute) 70 2.8.5 ArrayCGH 72 2.8.6 Chromatin immunoprecipitation and next generation sequencing (ChIP-seq) (in collaboration with Dr Fiona Wardle, Kings College, London) 73 2.9 Molecular biology: Protein 74 2.9.1 Western blotting 74 2.9.2 Immunohistochemistry 75 2.10 Bioinformatics 76 2.11 Statistical analysis 80 Chapter 3: The gene expression profile of chordoma cells depleted of T.
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