This Thesis Has Been Submitted in Fulfilment of the Requirements for a Postgraduate Degree (E.G
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This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. OMICs based identification of the mechanisms that underpin FAK’s regulation of gene expression Billie Georgina Cooper Griffith Doctor of Philosophy The University of Edinburgh 2020 Declaration I declare that this thesis has been composed by myself and details my own research unless stated otherwise in the text. No part of this thesis has been submitted elsewhere for any other degree or personal qualification. Billie Georgina Cooper Griffith January 2020 ii Abstract Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase, commonly over- expressed in cancer that is well known to be a key regulator of cancer cell proliferation, survival, migration and invasion. It has recently been shown to modulate the anti- tumour immune response via transcriptional regulation of chemokines and cytokines. These findings have fuelled interest in FAK as a plausible therapeutic target in cancer and FAK kinase inhibitors are currently in Phase I and II clinical trials. To date, much of the research on FAK has focused on its role at focal adhesions, detailing its major functions in cell motility and cancer cell invasion. However, 16 years ago, FAK was shown to translocate to the nucleus and it is now well established that this occurs under conditions of cellular stress such as oncogenic transformation. In the nucleus, FAK binds to a number of transcription factors such as P53, MDM2, IL33, RUNX1 and SP1. These nuclear interactions have been linked to proliferation, survival, differentiation and regulation of the anti-tumour immune response and cell cycle. However, despite these findings, the mechanism by which FAK regulates gene expression in the nucleus has yet to be defined. With FAK kinase inhibitors currently in clinical trials, it is important to understand the molecular mechanisms associated with FAK’s transcriptional regulation, the role of the kinase activity, and the potential therapeutic benefits of targeting FAK’s nuclear function. We therefore investigated the mechanism by which FAK regulates transcription by integration of a number of ‘omics’ approaches to define the nuclear FAK interactome and FAK-dependent gene programmes, as well as FAK’s role in regulating chromatin accessibility and transcription factor binding. Integration of these datasets predicted that FAK regulates the binding of AP-1 and ETS transcription factors to chromatin. Furthermore, our findings indicate that a subset of FAK-regulated genes display changes in chromatin accessibility. Chromatin ImmunoPrecipitation (ChIP) validation experiments on the enhancer region of one of these genes, IL33, show reduced binding of JUN upon loss of FAK’s kinase activity. This predicts that FAK is regulating chromatin accessibility at this region of the IL33 gene. iii The work presented in this thesis therefore identifies a novel mechanism by which FAK regulates transcription; further experiments will be required to define the epigenetic complexes responsible for FAK-dependent chromatin accessibility changes. This work adds to our understanding of how an integrin adhesion protein can control cancer cell behaviour via transcription in the nucleus. iv Lay abstract Focal adhesion kinase (FAK) is a protein that is used by cells when they grow and stick to their surroundings. When someone has cancer, one of the main risks is that the cancer may spread (or metastasise) causing cancers elsewhere in the body that are more difficult to treat. It has been shown in laboratory experiments, that when FAK is blocked, the risk of the cancer spreading can be reduced. More recently FAK has also been shown to move to the cell compartment where the regulation of genetic information (genes) occur – called the nucleus. Here FAK has been shown to interact with proteins called transcription factors that bind to regions next to genes (genomic regions) to turn genes on or off. By binding these transcription factors in the nucleus, FAK can regulate inflammation which has long been linked to the development of cancer. Therapies that target the body’s immune system to boost its own tumour killing activity offer a new form of cancer treatment with fewer negative side effects than chemotherapy. My work has investigated the mechanism by which FAK regulates the expression of genes that are important in cancer, in particular inflammatory genes. To do this, I have integrated a number of datasets defining 1) the proteins that FAK binds in the nucleus, 2) the genomic regions that FAK is regulating and 3) whether this turns on or off these genes that are FAK-regulated. My work has shown that FAK does indeed regulate a transcription factor to enhance the expression of a gene involved in cancer-inflammation. This work shows that FAK in the nucleus is an important regulator of the expression of genes in cancer by impacting a number of key transcription factors in the nucleus. v Acknowledgements Firstly, I would like to thank my supervisor Val Brunton – who has taken over overseeing the final year of my PhD and has helped me greatly with the challenging task of writing a thesis. I would also like to thank Margaret Frame who provided good advice on thesis writing and in lab meetings. Bryan Serrels for being my supervisor for most of my PhD, who provided immense support in experiments and contributed significantly to this work. Niamh McGivern for being a huge support during my PhD and for generating the FAK BirA* SCC cell lines. I would like to thank Rosanna Upstill-Goddard for performing the ATAC-seq bioinformatic analysis and therefore has contributed significantly to this work. Niall Quinn and Adam Bryon for running the mass spectrometry samples and for assistance with the data analysis, respectively. Graeme Grimes for the raw sequence alignment and differential expression analysis for the mRNA-seq data. I would like to thank Ben – I wouldn’t have been able to do this without you! Last but not least I would like to thank my mum, dad, stepmum and little Ava for being a tremendous emotional support in this difficult time. vi Contents Declaration………………………………………………………………………………………………………………………ii Abstract…………………………………………………………………………………………………………………………. iii Lay abstract……………………………………………………………………………………………………………………..v Acknowledgements ........................................................................................................vi Table of figures ...............................................................................................................x Table of abbreviations ................................................................................................... xv Nomenclature ............................................................................................................. xvii Chapter 1 Introduction ............................................................................................... 1 1.1 The hallmarks of cancer.................................................................................. 2 1.1.1 Sustaining proliferative advantage ................................................................. 2 1.1.2 Deregulated cell energetics ............................................................................ 3 1.1.3 Resisting cell death ......................................................................................... 3 1.1.4 Genome instability and mutation .................................................................. 4 1.1.5 Inducing angiogenesis .................................................................................... 4 1.1.6 Induction of invasion and metastasis ............................................................. 5 1.1.7 Tumour promoting inflammation .................................................................. 6 1.1.8 Enabling replicative immortality .................................................................... 7 1.1.9 Avoiding immune destruction ........................................................................ 7 1.1.10 Evading growth suppressors .......................................................................... 9 1.2 Targeting cancer .......................................................................................... 10 1.3 Introduction to FAK ...................................................................................... 14 1.3.1 FAK discovery and early studies ................................................................... 14 1.3.2 Proteomics identified that FAK is a key component of integrin adhesion complexes (IACs) ............................................................................................................ 15 1.3.3 FAK general structure ................................................................................... 17 1.3.4