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The Role of Inhibitor of DNA Binding 4 (Id4) in Mammary Gland Development and Breast Cancer

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The Role of Inhibitor of DNA Binding 4 (Id4) in Mammary Gland Development and Breast Cancer The role of Inhibitor of DNA binding 4 (Id4) in mammary gland development and breast cancer Simon Junankar A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy UNSW Garvan Institute of Medical Research St. Vincent’s Hospital Clinical School Faculty of Medicine November 2012 Copyright and Authenticity statement I 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 material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement 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: 14/11/12 II Acknowledgements Firstly I would like to thank Alex Swarbrick for being a great supervisor, friend and mentor. You gave me the freedom to follow my own ideas and also gave direction when I needed it. I would also like to thank the other members of the Swarbrick group for their help, advice and friendship. In particular I would like to thank Radhika for conceptualising the Id4 project and for so much advice and help along the way. I would like to thank my family and friends for their support and forbearance during my PhD studies. In particular my wife Marcel Batten for all of her love, help and support over the last four years and also our son Perry who has made the last 20 months all the more exciting and enjoyable. I would also like to acknowledge the support of both my parents and Marcel’s parents over the last four years. I hope now to be able to spend some more time with my friends and family. III Conferences, Publications and Awards Conferences Simon Junankar, Sandra O’Toole, Ewan Millar, Radhika Nair, Andrea McFarland, Kyuson Yun, Robert L. Sutherland, Alexander Swarbrick. 2012, A critical role for Id4 in breast cancer and mammary gland development. Poster presentation. 24th Lorne Cancer Conference, Lorne, Victoria, Australia. Simon Junankar, Sandra O’Toole, Ewan Miller, Radhika Nair, Andrea McFarland, Kyuson Yun, Robert L. Sutherland, Alexander Swarbrick. 2011. Inhibitor of DNA binding 4 plays a crucial role in breast cancer and mammary gland development. Poster presentation. 19th NSW Scientific Meeting, ASMR, The University of Sydney, NSW, Australia. Simon Junankar, Sandra O’Toole, Ewan Miller, Radhika Nair, Andrea McFarland, Robert L. Sutherland, Alexander Swarbrick. 2011. A crucial role for Id4 in breast cancer and mammary gland development. Poster presentation. Stem Cells, Cancer and Metastasis Keystone Symposia, Keystone, Colorado, USA. Simon Junankar, Sandra O’Toole, Ewan Miller, Radhika Nair, Andrea McFarland, Robert L. Sutherland, Alexander Swarbrick. 2011. A crucial role for Id4 in breast cancer and mammary gland development. Poster presentation. 23rd Lorne Cancer Conference, Lorne, Victoria, Australia. Simon Junankar, Emie Roy, Alex Swarbrick. 2009. Id1 overexpression protects mammary tumours from senescence and immune system recognition. Poster presentation. 39th Annual Scientific Meeting Australasian Society for Immunology, Gold Coast, Queensland, Australia. Simon Junankar, Emie Roy, Alex Swarbrick. 2009. Id1 overexpression plays a role in protecting from senescence and immune cell recognition in Ras overexpressing mammary tumours. Poster presentation. 21st Lorne Cancer Conference, Lorne, Victoria, Australia Publications Junankar S, Baker L, Yang J, O’Toole S, McFarland A, Phua Y, Yun K, Nair R, Swarbrick A. Id4 regulates stem cell maintenance and luminal differentiation during mammary gland development, (manuscript in preparation). Nair R, Junankar S, O’Toole S, Shah J, Borowsky AD, Bishop JM, Swarbrick A. Redefining the expression and function of the inhibitor of differentiation 1 in mammary gland development. PLoS One. 2010; 5 (8):e11947 Awards 2012 – 24th Lorne Cancer Conference – Poster award. 2009 – Cancer Institute NSW – Research Scholars award. IV Abstract The inhibitor of DNA binding (Id) proteins Id1-4 are transcriptional regulators that control many cell fate and developmental processes and are often deregulated in cancer. In this dissertation I examine the role of Id proteins in mammary development and neoplasia. Initially the role of Id1 in regulating the immune response to senescent tumour cells was examined, before a more thorough investigation into the role of Id4 during mammary gland development and breast cancer. Prior to the studies described in this thesis, the role for Id4 during mammary gland development had not been investigated and its role in breast cancer was controversial. Id4 expression patterns in the mammary gland were analysed throughout development by immunohistochemistry and the phenotype of Id4 loss was determined using the Id4 knockout mouse. The function of Id4 was further analysed using the normal mouse mammary epithelial cell line Comma-D . Id4 expression was examined in breast cancer cell lines and a number of cohorts of breast cancer patients. The results presented here show that Id4 is a critical regulator of mammary gland development through its control of differentiation, proliferation and extracellular matrix (ECM) remodelling pathways. Id4 expression is restricted to the myoepithelial cells of the mammary gland and its loss leads to impaired mammary gland development. Overexpression and knockdown studies utilising the Comma-D cells demonstrated that Id4 inhibited luminal differentiation and was required for cell proliferation. Furthermore Id4 overexpression promoted neoplastic transformation of the normal Comma- D cell line in vivo. Transcript profiling experiments further demonstrated Id4 regulated a number of ECM remodelling genes. Our results from examining patient cohorts demonstrate that Id4 expression associates with the Her2 and basal-like subtypes of breast cancer and that high expression significantly correlates with improved patient survival. Despite high Id4 expression associating with improved survival, our combined results demonstrate that Id4 nonetheless has the capacity to promote tumourigenesis. V Frequently used abbreviations ABC transporter ATP binding cassette transporter ABCTB Australian breast cancer tissue bank SMA alpha smooth muscle actin AREG Amphiregulin ATCC American type tissue collection BaA Basal A BaB Basal B bHLH basic helix-loop-helix BMP Bone morphogenic protein BRCA1 Breast cancer associated 1 C3TAg C3(1)/SV40 T-antigen CD Cluster of differentiation CFC Colony forming capacity CK14 Cytokeratin 14 (also known as Keratin 14/K14) CK5 Cytokeratin 5 (also known as Keratin 5/K5) CK6 Cytokeratin 6 (also known as Keratin 6/K6) CK8 Cytokeratin 8 (also known as Keratin 8/K8) CpG Cystein-phosphate-guanine CSC Cancer stem cell d.n.s. Data not shown D18 Day 18 DAPI 4',6-diamidino-2-phenylindole DCIS Ductal carcinoma in situ DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid Dox Doxycycline E16 Embryonic day 16 E2 Oestrogen ECM Extracellular matrix EcoR Ecotropic receptor EGF Epidermal growth factor EMT Epithelial to mesenchymal transition ER Oestrogen receptor ES Embryonic stem FACS Fluorescence activated cell sorter FBS Foetal bovine serum FCS Foetal calf serum FFPE Formalin fixed paraffin embedded FGF Fibroblast growth factor FISH Fluorescent in situ hybridisation FSC Forward scatter VI GFP Green fluorescent protein GSEA Gene set enrichment analysis H&E Haematoxylin and eosin H2B Histone-2B HA Haemagglutanin Her2 Human epidermal growth factor receptor 2 Het Heterozygous HR Hazard ratio IC Immunocytochemistry Id1 Inhibitor of differentiation/DNA binding 1 Id2 Inhibitor of differentiation/DNA binding 2 Id3 Inhibitor of differentiation/DNA binding 3 Id4 Inhibitor of differentiation/DNA binding 4 IF Immunofluorescence IHC Immunohistochemistry IL2R Interleukin 2 receptor gamma IP Intra-peritoneal IPA Ingenuity pathway analysis K14 Keratin 14 K8 Keratin 8 KD Knockdown KO Knockout LB Luria broth MHC Major Histocompatability complex mIR MicroRNA mMEC Mouse mammary epithelial cell MMP Matrix metalloproteinase MMTV Mouse mammary tumour virus mRNA Messenger RNA MRU Mammary repopulating unit MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium) n Number NA Not applicable ND Not done NK cell Natural killer cell NOD Non-obese diabetic NOG NOD/SCID/IL2R -/- NS Not significant OE Over expression OIS Oncogene induced senescence OS Overall survival OVX Ovariectomized p value Probability value P4 Progesterone VII PBS Phosphate buffered saline PCR Polymerase chain reaction PR Progesterone receptor Prl Prolactin PyMT Polyoma middle T-antigen RAG Recombination activating gene Rb Retinoblastoma protein RFS Recurrence free survival RNA Ribonucleic acid RT-PCR Reverse transcription polymerase chain reaction SASP Senescence associated secretion phenotype SA- -gal Senescence associated -galactosidase SCID Severe combined immunodeficiency SD Standard deviation SEM Standard error of the mean shRNA Short hairpin RNA siRNA Small interfering RNA SOC Super optimal broth SSC Side scatter TBS Tris buffered saline TDLU Terminal ductal lobular unit TEB Terminal end bud Tet Tetracycline TetR/TRE Tetracycline
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