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Identification of KSR1 As a Novel Target and Decoding Tyrosine Kinase Proteome in Breast Cancer

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Identification of KSR1 as a novel target and decoding tyrosine kinase proteome in breast cancer By Hua Zhang A thesis submitted for the degree of Doctor of Philosophy at Imperial College London Faculty of Medicine, Department of Surgery and Cancer, 1st floor ICTEM Imperial College London Hammersmith Hospital, Du Cane Road London, W12 0NN Supervisor: Prof Justin Stebbing Co-supervisor: Dr Georgios Giamas Dedicated to my family and everyone who loves me to the 1,278 days to the stars in the night and the beautiful eyes in my heart 1 Abstract Kinase suppressor of Ras-1 (KSR1), originally identified as a novel protein kinase in the Ras- Raf cascade, plays a role in activation of mitogen-activated protein kinases (MAPKs). Although efforts have been devoted to study the role of KSR1 in certain tumour types, its biological functions in breast cancer have remained largely undefined. A SILAC-based proteomic analysis was conducted to identify the KSR1-regulated phospho- proteins in breast cancer. Our results revealed that KSR1 overexpression decreases deleted in breast cancer 1 (DBC1) phosphorylation. We then demonstrated that KSR1 decreases transcriptional activity of p53 by reducing phosphorylation of DBC1, which leads to a reduced interaction of DBC1 with sirtuin 1 (SIRT1); this in turn enables SIRT1 to deacetylate p53. We further examined the correlation between KSR1 expression and clinical outcome in breast cancer. Our results showed that patients with breast cancer with high KSR1 in our cohort (n>1000) had better disease free- and overall survival. Moreover, in KSR1-transfected stable cells, fewer and smaller size colonies were formed in comparison to parental cells, while an in vivo study demonstrated that the growth of xenograft tumours overexpressing KSR1 was inhibited. Mechanistically, the tumour suppressive action of KSR1 is BRCA1 dependent, which was shown by in vitro 3D matrigel and soft agar assays. KSR1 regulates BRCA1 ubiquitination through elevated BRCA1-associated RING domain 1 (BARD1) expression and increased BRCA1-BARD1 interaction. Deregulation of tyrosine kinases (TKs) signalling can contribute to tumourigenesis. A combined approach of RNAi and SILAC-based quantitative proteomics was employed to decode the TKs-regulated proteomes upon silencing individually each 65 validated TKs in MCF7 breast cancer cells. Bioinformatics analysis identified 10 new distinctive clusters based on similarity in the TKs-regulated proteomes. The biological relevance of our proteomic study in interpreting the TKs-regulated proteomes supports the essential role of TKs in regulating all aspects of cellular activities. 2 Statement of originality All the experimentation presented in this thesis has been conducted by myself, aside from the following: In result sections, SILAC proteomics analysis was performed by Dundee Cell Products Ltd. Immunohistochemistry staining of tumour tissues from Nottingham Cohort was carried out by Dr Pritesh Trivedi at Histopathology Department, Hammersmith Hospital. Statistical analysis of the significance of KSR1 expression with clinical outcome in breast cancer patients was performed by Dr Andrew Green at Department of Pathology, Nottingham City Hospital and Dr Filipovic, Aleksandra. In vivo animal studies were conducted by Dr Manikandan Periyasamy, Dr Ylenia Lombardo and myself. Immunohistochemistry and histopathological analysis of tissue samples isolated from mouse mammary primary tumour was performed by Dr Pritesh Trivedi and Dr Aleksandra Filipovic. SNP analysis of samples from Nottingham cohort was undertaken by Prof Heinz-Josef Lenz‟s lab at University of Southern California. LOH analysis was performed by Dr Kevin Blighe and Prof Jacqui Shaw at University of Leicester. cDNA samples from 24 different breast cancer cell lines were kindly provided by Prof Simak Ali. Soft agar and 3D-matrigel assays were carried out by myself with the help from Mrs Arnhild Grothey and Dr Ylenia Lombardo. RNAi screen in TKs was performed by Dr Georgios Giamas, Mrs Arnhild Grothey, Mr Yichen Xu and myself. Bioinformatics analyses of TKs clustering, GO classification and STRING network were conducted by Dr Nicos Angelopoulos. I am appreciative of the help from the aforementioned. Hua Zhang 3 In the introduction chapter, some paragraphs of sections 1.2 and 1.3 have been published on two separate articles with myself as the first author. These two papers are entitled “The role of pseudokinases in cancer” on Cellular Signalling, (2012) 24, 1173-1184 and “The dual function of KSR1: a pseudokinase and beyond” on Biochemical Society Transactions, (2013) 41, 1078-1082. I would like to thank Dr Andrew Photiou and Dr Chuay Yeng Koo for their contribution to these publications. In the result chapter, some parts of section 3.1 have been published with myself as the first author on a research article entitled “SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1” on British Journal of Cancer, (2013) 109, 2675-2684. Hua Zhang Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial 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 commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 4 Acknowledgements NO words can really describe the greatest thanks and appreciation from my heart to my supervisor, Prof Justin Stebbing for this unique journey and unforgettable experience in my life. I am extremely grateful for everything he has done to take me on board as a member of his family, he made my study at Imperial a lifelong memory. Without his vision, wisdom and ambition, I wouldn‟t have been able to accomplish my PhD study. He is always by my side whenever I need help. I have to admit Justin has made a monumental impact on my life in a good way. I will never forget the email he sent to me in one afternoon in 2011. He advised me to concentrate on work and helped me to build courage and confidence. All the educational/inspiring/fighting/flirtatious emails between Justin, George and I have apparently become a culture at work, which I will pass on to the next generation. Due to space limit, I have to delete 1,000 words that I would like to say to Justin, but the future will tell our story. I would also like to express my sincerest thanks and appreciation to my co-supervisor, Dr Georgios Giamas for his guidance, his inspiration and his encouragement during my study. I especially appreciate his great efforts to help me think like a scientist and work like a medical doctor. He is always like a brother who has taken care of me since the very first day. He has kindest heart I have ever encountered. I still remember that on the first day of my work, he th gave me a ticket to watch the NBA game at O2 Arena on the 28 September 2010. As time flies, only true friends always stick with you through ups and downs of your life. George is that person for me and I will cherish our friendship forever. I would like to give my special thank you to Dr Andrew Photiou (the heavyset Mr Hardy) for being the most genuine friend there is. He was the best teacher for my research techniques and for my English. In certain ways, he has changed my way of living life for good. I would like to thank Dr Aleksandra Filipovic for her enormous help during my PhD study and her encouragement from the beginning. I would like to thank Yichen Xu for being a good mate at work and otherwise. Before he joined the group, I was the “hardest” working one, but he has raised the bar of our research ever since and has always inspired me with his ideas. A huge thank you to Arnhild Grothey who has been helping me from the very first day she joined the group. Without her excellent work, I wouldn‟t be able to finish my PhD in such a short time. 5 I would like to thank Alex de Giorgio for his kindness, his stylish humour and continuous help. It wouldn‟t be the same if he was not around. Life is nothing without good company. I would like to thank my colleagues for their exceptional help with my PhD study and for making every day full of happiness and sunshine, Dr Chun Fui Lai, Dr Darren Patten, Dr Jimmy Jacob, Dr Loredana Pellegrino, Dr Laura Roca- Alonso, Lei Cheng Lit, Dr Joao Farinha Garcao Nunes, Dr Jonathan Krell, Dr Adam Frampton, Dr Victoria Harding, Dr Christoph Richard Schlegel, Dr Nair Bonito, Dr Meng- Lay Lin, Dr Sebastain Trousil, Dr Chuay Yeng Koo, Dr Leandro Castellano, Dr Manikandan Periyasamy, Dr Hetal Dattani, Dr Holly Kramer, Dr Claire Fletcher, Dr Sue Powell, Dr Ylenia Lombardo and Dr Monica Faronato; my “invisible friend” Dr Rosa Williams. Special thanks are given to Dr Andrew Photiou, Dr Darren Patten, Alex de Giorgio and Dr Victoria Harding for the critical reading of my thesis; and to Dr Nicos Angelopoulos for his enormous help with data analysis and his extraordinary patience. I would also like to thank Dr Adam Frampton and Dr Laura Roca-Alonso for being good writing buddies in those unforgettable days during Christmas and New Year Holidays. I would also like to express my appreciation toward my friends from every corner of the world for their support, Dr Chen Lu, Dr Ming Liu, Dr Helena Lo, Chelsey Chu, Dr Jiaying Tang, Dr Yaozu Xiang, Dr Yiping Ge, Bo Han, Chao Gao, Tianhui He and Shuang Gao.
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