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Heterogeneity in Melanoma and the Microenvironment Cerys Sian

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Heterogeneity in Melanoma and the Microenvironment Cerys Sian Heterogeneity in melanoma and the microenvironment Cerys Sian Manning University College London and Cancer Research UK London Research Institute PhD Supervisor: Dr Erik Sahai A thesis submitted for the degree of Doctor of Philosophy University College London January 2013 Declaration I Cerys Manning 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. 2 Abstract This thesis addresses the signalling pathways and gene networks associated with melanoma cell motility in vivo and heterogeneity in tumour-associated vasculature. Melanoma metastasis begins with the acquisition of motility in the primary tumour, however, there is little characterisation of signalling pathways or microenvironments associated with melanoma motility in vivo. Intra-vital imaging of clonal signalling reporter cell-lines showed heterogeneous cell behaviour in vivo and increased Notch, TGF-β and SRF dependent transcription in motile cells. Further investigations into Notch signalling in melanoma metastasis showed that Notch promotes metastasis through cell-autonomous and non-cell autonomous mechanisms. The reporter cell-lines were used to enrich for the motile melanoma cell population and genes up-regulated in this population were identified. Many cell-cycle regulators, including regulators of cytokinesis, were up-regulated in the B16 F2 melanoma population enriched for motile cells. Bioinformatic analysis indicated that EZH2, a histone methyltransferase in the Polycomb repressor 2 complex, can regulate the genes associated with motile cells and EZH2 depletion decreased melanoma cell motility in vitro and metastasis in vivo. EZH2 was also shown to regulate cell morphology and the actin cytoskeleton through regulating activation of a subset of ERM proteins. Due to association between melanoma metastasis and differentiation, the role of EZH2 in melanoma differentiation state was investigated. EZH2 depletion did not significantly affect expression of melanoma differentiation markers but increased Oca2 mRNA expression leading to increased pigment. This thesis also investigates heterogeneity in the tumour microenvironment. Morphologically and dynamically distinct regions of the tumour vasculature were identified by intra-vital imaging and quantitative image analysis, and these regions could be followed over time using imaging windows. The tumour vasculature showed a heterogeneous response to the anti-angiogenic drug, Sunitinib, possibly due to microenvironmental factors. Aspects of vascular morphology and dynamics correlated with melanoma motility and may be used to describe microenvironments favourable for motility. 3 Acknowledgement I would like to thank the many people who have helped me during my PhD. Foremost, I would like to sincerely thank Erik for the continuous support, patience, and motivation. His immense knowledge, guidance and teaching have helped me throughout the whole PhD. Secondly, thanks go to Steve who has answered every question I could throw at him, with a friendly face and without complaint. Thanks also to Rob for helping with data analysis in the angiogenesis project. The Tumour Cell Biology Lab has been a great place to work and that is down to the people in it, past and present. Thank you for being fun and friendly but also great role models and teaching me a lot (not just about science!). The work has been greatly aided by members of the Biological Resources Unit, Experimental Histopathology Lab, FACS Lab and Bioinformatics department, so thank you to everyone who has helped. Finally I would like to thank Fay, my long-suffering partner (now wife) and my family for the encouragement and distraction at times when it all seemed too much. 4 Table of Contents Abstract ............................................................................................................... 3 Acknowledgement .............................................................................................. 4 Table of Contents ................................................................................................ 5 Abbreviations .................................................................................................... 10 Table of figures ................................................................................................. 11 List of tables ...................................................................................................... 16 Supplementary movies and files ..................................................................... 17 Chapter 1. Introduction ................................................................................ 20 1.1 Chapter overview ............................................................................................... 20 1.2 Melanocyte and melanoma biology .................................................................. 20 1.2.1 Melanocytes .................................................................................................. 20 1.2.2 Melanin production ....................................................................................... 21 1.2.3 Melanocyte natural history and differentiation ............................................. 23 1.2.4 Melanoma and metastasis ............................................................................. 23 1.2.5 Melanoma differentiation and metastasis...................................................... 26 1.3 Metastasis and the microenvironment ............................................................. 28 1.3.1 Exiting the primary tumour ........................................................................... 28 1.3.2 Survival in the circulation ............................................................................. 29 1.3.3 Arrival and survival at secondary sites ......................................................... 29 1.3.4 Growth at secondary sites ............................................................................. 31 1.4 Cell motility......................................................................................................... 33 1.4.1 The actin cytoskeleton................................................................................... 33 1.4.2 The microtubule cytoskeleton ....................................................................... 35 1.4.3 Cell protrusions ............................................................................................. 36 1.4.4 Adhesions ...................................................................................................... 38 1.4.5 Contractile force ............................................................................................ 39 1.4.6 Cell polarity and directional cell motility ..................................................... 40 1.4.7 ERM proteins ................................................................................................ 42 1.4.8 Different modes of single cancer cell motility in 3D .................................... 43 1.5 Cancer cell motility in vivo ................................................................................ 47 1.5.1 Imaging approaches to study cancer cell motility in vivo ............................ 47 1.5.2 Different modes of cancer cell motility in vivo ............................................ 49 1.6 Control of gene expression ................................................................................ 54 1.6.1 TGF-β signalling ........................................................................................... 55 1.6.2 SRF signalling ............................................................................................... 59 1.6.3 Notch signalling ............................................................................................ 64 1.6.4 Interactions between signalling pathways ..................................................... 68 1.6.5 Chromatin control of gene transcription ....................................................... 69 1.6.6 PRC2 complex during development and differentiation ............................... 73 1.6.7 EZH2 in cancer ............................................................................................. 73 1.7 The tumour microenvironment and angiogenesis ........................................... 74 1.7.1 The tumour vasculature ................................................................................. 75 1.7.2 Induction of tumour angiogenesis ................................................................. 76 1.7.3 Sprouting angiogenesis ................................................................................. 77 1.7.4 Targeting tumour angiogenesis ..................................................................... 79 5 Chapter 2. Materials and Methods .............................................................. 82 2.1 Reagents and chemicals ..................................................................................... 82 2.1.1 Enzymes ........................................................................................................ 82 2.1.2 Buffers and solutions .................................................................................... 82 2.1.3 Cell-culture reagents ..................................................................................... 83 2.2 Cell culture manipulations ................................................................................ 84 2.2.1 Cell-lines ......................................................................................................
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