The Role of Inhibitors of Differentiation Proteins ID1 and ID3 in Breast Cancer Metastasis
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The role of Inhibitors of Differentiation proteins ID1 and ID3 in breast cancer metastasis Wee Siang Teo A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy St Vincent’s Clinical School, Faculty of Medicine The University of New South Wales Cancer Research Program The Garvan Institute of Medical Research Sydney, Australia March, 2014 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Teo First name: Wee Siang Abbreviation for degree as given in the University calendar: PhD (Medicine) School: St Vincent’s Clinical School Faculty: Faculty of Medicine Title: The role of Inhibitors of Differentiation proteins ID1 and ID3 in breast cancer metastasis Abstract 350 words maximum: (PLEASE TYPE) Breast cancer is a leading cause of cancer death in women. While locally-confined breast cancer is generally curable, the survival of patients with metastatic breast cancer is very poor. Treatment for metastatic breast cancer is palliative not curative due to the lack of targeted therapies. Metastasis is a complex process that still remains poorly understood, thus a detailed understanding of the biological complexity that underlies breast cancer metastasis is essential in reducing the lethality of this disease. The Inhibitor of Differentiation proteins 1 and 3 (ID1/3) are transcriptional regulators that control many cell fate and developmental processes and are often deregulated in cancer. ID1/3 are required and sufficient for the metastasis of breast cancer in experimental models. However, the mechanisms by which ID1/3 mediate metastasis in breast cancer remain to be determined. Little is known about pathways regulated by ID1/3 in breast cancer as well as their functional role in the multiple steps of metastatic progression. The current body of work was aimed at exploring the role of ID1/3 and their transcriptional targets that mediate their function in breast cancer. This was achieved through the use of a range of in vitro and in vivo techniques to; firstly, examine ID1 expression in a cohort of breast cancer patients. Secondly, a model of inducible, stable Id1/3 knockdown system was employed to establish the function of Id1/3 in the 4T1 breast cancer cell line and to identify Id1/3 target genes. Thirdly, the requirement of Id1/3 in controlling mammary tumour growth and metastasis was determined by conditional knockdown of Id1/3 expression during tumour progression. The data gathered is the first study to identify the global transcriptional targets of Id1/3 in breast cancer as well as to investigate the role of Id1/3 in the multi steps of metastatic cascade. The results presented here showed that ID1 expression is associated with the triple-negative and HER2-enriched subtypes of breast cancer. ID1 expression is enriched in brain metastases compared to patient matched primary breast cancers. Silencing of Id1/3 reduces primary tumour growth and significantly impairs spontaneous lung metastasis. These data strongly suggest Id1/3 as central controllers of the metastatic phenotype in breast cancer. Transcript profiling experiments revealed a multitude of genes that are regulated by Id1/3. Subsequent validation identified several novel Id1/3 target genes and suggested that the function of Id1/3 in 4T1 cells is possibly mediated by Bmi1/Mel18 and TGF-β signalling pathway. Further functional validation is required to confirm this finding. 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Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: I Copyright and Authenticity Statements COPYRIGHT STATEMENT 'I hereb;• grant t1e Universil;' of Ne'l•' South Wales or ~s agents the right to (ifChi•J@an d t<J make <t'lail~ble rn~· th~si s or d1sseri(ltion in whole or r.>art in the University libraries in all forms of media. no•!l or here affer known. subject to the provisions of the Copyrigh1 Act 1968. I retain all proprietary righls, such as patant rights. I \-ilso ret~in the right ~o use in fulvre ....'Q rks (such as ;;ut icles or b!Xlfts) <ill or pari of this thesis or dissertation. I (llso authonse Unr;erslty M1crofllms to vse the 350 •!iOrd (ltstract of my thesis in Disserta~ion Abstract lnternatiooal o; this is o:iPP~icable to doC:oral (heses onl;•}. I hav& either used no substantial ~ rtions of cop~'ligh t material in m~' th$Sis or I have obta;ned permission to use copyright m.<1terial; where pennission has not b&ecl granted I haV& ap~ ied/will apply fc( a pattial f&slnctc n o f th& .aigilal COPt of my tl"'~sis or dissertaOCo.' Signed 30 March 2014 Oat(,! ALITHENTICITY STATEMENT 'I carlify 1h(lt the Library de;::os t digitdl oo;;y is a direct equiva1Etnt of the fin(tl ott:cially appro,·ed versicn of my thesis. No emendation oi content has occurred ~nd if thsfe are any minor -.rari~tions in ionn~n:ng , they are t1e rGsu:t of ihe con\•er5ion to digital forrrot: Signed 30 March 2014 Dal<! II Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of materials which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgment is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that the assistance from others in project’s design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed: Date: III Acknowledgements No man is an island, but rather it is through the help and support of many people that made this research possible. First and foremost, I would like to express my deepest appreciation and gratitude to my supervisor, Alex Swarbrick. Thank you for taking me under your tutelage and for your patience, invaluable guidance and constant encouragement throughout my PhD candidature. Secondly, I would like to extend my gratitude to my co-supervisor, Radhika Nair. I am deeply indebted to you for everything you have done to help and support me over the past five years. I would also like to thank the many people who have contributed to my PhD project. I am very grateful for having labmates who are there all the time to help and support me. Without them, I would have never overcome all the hurdles that I had encountered in this journey. I express my sincere thanks to Andrea McFarland, Jessica Yang and Nicola Foreman for their assistance in the animal work. I would also like to acknowledge Dr Daniel Roden for his expertise in bioinformatics, Dr Natasha Sharma for her input in the TFG-β experiments and Kate Harvey for her assistance on IHC. I would also like to thank Yuwei Phua, Drs Akira Nguyen and Simon Junankar for their very helpful discussion and help along the way. Not forgetting all the other wonderful people in the CTP group, Laura, Mun, Aurelie, Iva, Sunny, Jaynish, Dorothy, Astrid and Stephanie. Thank you for making the lab such a positive and enjoyable place to work in! And finally to my parents, I would not be able to make it without your love and support. I am forever indebted to you. IV Publications, Conferences and financial support Publications: 1. Nair R, Roden DL, Teo WS, McFarland A, Junankar S, Ye S, Nguyen A, Yang J, Nikolic I, Hui M, Morey A, Shah J, Pfefferle AD, Usary J, Selinger C, Baker LA, Armstrong N, Cowley MJ, Naylor MJ, Ormandy CJ, Lakhani SR, Herschkowitz JI, Perou CM, Kaplan W, O'Toole SA, Swarbrick A. c-Myc and Her2 cooperate to drive a stem-like phenotype with poor prognosis in breast cancer. Oncogene. 2013 Sep 23. doi: 10.1038/onc.2013.368. [Epub ahead of print]. 2. Teo WS, Nair R, Swarbrick A. New insights into the role of ID proteins in breast cancer metastasis: a MET affair. Breast Cancer Research. 2014, 16:305 doi:10.1186/bcr3654. 3. Nair R*, Teo WS*, Mittal V, Swarbrick A. ID proteins regulate diverse aspects of cancer progression and provide novel therapeutic opportunities. Molecular Therapy. 2014 May 14. doi: 10.1038/mt.2014.83. (*These authors contributed equally) 4. Nair R*, Teo WS*, Ye S, McFarland A, Harvey K, Roden D, Junankar S, Baker L, Yang J, Fluke N, Millar E, Mellick A, Naylor M, Ormandy C, Lakhani S, O’Toole S, Swarbrick A.