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Regulation of ABCG2 Expression in the Lactating Mammary Gland by Alex Man Lai Wu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Pharmacology and Toxicology University of Toronto © Copyright by Alex Man Lai Wu (2014) ABSTRACT Regulation of ABCG2 Expression in the Lactating Mammary Gland Alex Man Lai Wu, Doctor of Philosophy (2014) Department of Pharmacology and Toxicology University of Toronto The Breast Cancer Resistance Protein (ABCG2) is a multidrug efflux transporter that is upregulated in certain drug-resistant cancer cells and in the mammary gland during lactation. It is unclear how ABCG2 is regulated in the lactating mammary gland. This thesis sought to understand the role of prolactin (PRL) and its downstream signalling cascades, and epigenetic mechanisms, in the regulation of ABCG2 expression in the mammary gland during lactation. Using T-47D human breast cancer cells, I showed that PRL upregulated ABCG2, in part, by inducing the recruitment of Signal Transducer and Activator of Transcription-5 (STAT5) to an interferon-γ activation sequence (GAS) motif in the human ABCG2 gene. Pharmacological inhibition of phosphoinositide 3-kinase and mitogen-activated protein kinase signalling further demonstrated the involvement of these pathways in the induction of ABCG2 by prolactin. To further investigate whether STAT5 regulates ABCG2 in vivo, I performed a series of experiments using mammary glands from non-lactating (virgin and forced involution) and lactating mice. These experiments revealed that the E1b alternative mouse Abcg2 mRNA isoform was predominantly expressed and induced in the mouse mammary gland during lactation. Using published STAT5 ChIP-seq datasets and ChIP-qPCR, I showed that STAT5 was bound to multiple regions along the mouse Abcg2 gene during lactation. In particular, one of these STAT5 binding regions, which contains a GAS motif, showed functional activity in luciferase reporter assays. I further investigated epigenetic mechanisms that may regulate Abcg2 in the mouse mammary gland. Despite the presence of a CpG island at the E1b ii promoter, which when hypermethylated in vitro dramatically reduced promoter activity, the E1b promoter was already hypomethylated in the virgin mammary gland and continued to be hypomethylated during lactation. Analysis of published ChIP-seq data revealed that the E1b promoter region in virgin mouse mammary epithelial cells was already enriched with the open chromatin histone mark H3K4me2 but that there was further accumulation of H3K4me2 during lactation. Collectively, these results suggest that ABCG2 is already poised for expression in the virgin mammary gland and that lactation-associated activation of STAT5, perhaps by prolactin, induces its expression. iii ACKNOWLEDGEMENTS I must thank a long list of people who have made it possible for me to endure both the challenges and triumphs of graduate studies: I must first thank my mentor and supervisor Dr. Shinya Ito for providing me with the opportunitity to work with him. Dr. Ito was always open to new ideas, new experiments, some of which may have failed whereas others succeeded, but it was this openness that has afforded me the opportunity to develop my scientific curiosity – to ‘think outside the box’. I must also thank members of my PhD supervisory committee (Drs. Patricia A Harper, David S. Riddick, and Jason Matthews) for having the foresight at the first year and most criticial time of my PhD training that my thesis was a viable PhD project. I must thank each of them individually: Dr. Harper for going beyond the role of the committee member, often acting as a teacher, mentor, and above all, a listener of all the difficulties of graduate school and life-in-general; Dr. Riddick for being the one to introduce me to Professors at international conferences and for acting as ‘thesis reader’; and Dr. Matthews for generously allowing me to work in his lab for part of my PhD work. I would also like to thank past and present members of the Ito Lab: most notably Hendrick Tan, Mingdong Yang, Pooja Dalvi, Liana Dedina, Reo Tanoshima, Tohru Kobayashi, Andrew Chuang, Marie-Caroline Delebecque, Hisaki Fujii, and John Leon-Cheon. Some of them have contributed scientifically to this thesis but all of them have made it fun to work in the Ito Lab. Other people who I must sincerely acknowledge for contributing to this thesis are: Dr. Andrei Turinsky (Centre for Computational Medicine, Hospital for Sick Children) for running the ChIP-Seq data pipeline/analysis; Kelvin Wang and Dr. Sean Egan (Developmental and Stem Cell Biology Program, Hospital for Sick Children) for helping with the virgin mammary epithelial cell isolation; Youliang Lou and Drs. Rosanna Weksberg, Darci Butcher, Sanaa Choufani (Genetics and Genome Biology Program, Hospital for Sick Children) for pyrosequencing services and discussing about epigenetics and data interpretation; Dr. Charles V Clevenger (Department of Pathology, Northwestern University) for acting as a resource to learn about PRL signalling and treatment protocols. iv Most importantly, I must thank my parents Henry and Lilian, siblings Cecilia and Brian, for being great listeners and providing often well-needed encouragement thoughout my studies. I must thank all my friends who have stayed by me, even when I’m too tired or not in the right mood to socialize. Lastly, I must thank the taxpayers and others who still believe in the importance of training new PhD scientists. It is a constant reminder that graduate school is a privilege. v Table of Contents ABSTRACT ii ACKNOWLEDGEMENTS iv List of Figures x List of Tables xi List of Appendices xiii List of Abbreviations xiv 1. INTRODUCTION 1 1.1 Statement of Research Problem 1 1.2 ABCG2 expression and function 2 1.2.1 Discovery (BCRP/MXR/ABCP/ABCG2) 2 1.2.2 Expression, localization, and function 3 1.2.2.1 Placenta 4 1.2.2.2 Gastrointestinal tract 5 1.2.2.3 Liver 6 1.2.2.4 Kidney 7 1.2.2.5 Brain 8 1.2.2.6 Testis 9 1.2.2.7 Mammary Gland 10 1.2.2.8 Stem Cells 11 1.2.2.9 Cancer 13 1.2.2.10 ABCG2 Substrates and Inhibitors 14 1.3 Promoter Organization of the Human and Mouse ABCG2 gene 18 1.3.1 Human ABCG2 gene promoter 18 1.3.2 Mouse Abcg2 gene promoter 19 1.4 Regulatory control of ABCG2 expression 20 1.4.1 Hormones and inflammatory cytokines 20 1.4.1.1 Estrogen 20 1.4.1.2 Progesterone 21 1.4.1.3 Testosterone 21 1.4.1.4 Glucocorticoids 22 1.4.1.5 Proinflammatory Cytokines 23 vi 1.4.1.6 Other Growth factors 24 1.4.2 Stress and xenobiotics 27 1.4.2.1 Hypoxia 27 1.4.2.2 Aryl hydrocarbon receptor 27 1.4.2.3 Peroxisome-proliferator activated receptor 28 1.4.2.4 Pregnane X Receptor 28 1.4.2.5 Constitutive Androstane Receptor 29 1.4.2.6 Nuclear factor-erythroid 2 p45-related factor 2 29 1.4.2.7 Retinoic acid receptor/retinoid X receptor 29 1.4.2.8 Cyclooxygenase 2 30 1.4.2.9 Extracellular milieu: folate status 30 1.4.3 miRNA and Epigenetics 31 1.4.3.1 Micro-RNA (miRNA) 31 1.4.3.2 Promoter Methylation 32 1.4.3.3 Histone Modification 33 1.4.4 Cell signalling 34 1.4.4.1 PI3K/AKT signalling 34 1.4.4.2 MAPK signalling 36 1.4.5 Species 37 1.5 Mammary Gland Biology 39 1.5.1 General Anatomy 39 1.5.2 Development 39 1.5.2.1 Embryonic 39 1.5.2.2 Prepubertal 40 1.5.2.3 Puberty 40 1.5.2.4 Pregnancy 40 1.5.2.5 Lactation 41 1.5.2.6 Involution 42 1.6 Prolactin and Prolactin Receptors 44 1.6.1 The ligand 44 1.6.2 Prolactin receptor 44 1.6.3 Prolactin receptor signalling 45 vii 1.6.3.1 JAK2/STAT5 pathway 46 1.6.3.2 Mitogen Activated Kinase Pathway 47 1.6.3.3 PI3K/AKT pathway 48 1.6.3.4 Other pathways 48 1.7 Epigenetics 50 1.7.1 CpG Islands 50 1.7.2 Histone Modifications 51 1.8 Research Rationale 52 2. METHODS 54 2.1 Reagents 54 2.2 Animals 54 2.3 Cell Culture and serum starvation 55 2.4 Treatment with hormones or TCDD 56 2.5 Small molecule inhibitors of STAT5, MAPK and PI3K signalling 56 2.6 Isolation of mouse mammary epithelial cells (MEC) 56 2.7 RNA isolation from cells and tissues 58 2.8 cDNA synthesis - reverse transcription 58 2.9 Real-time RT-PCR to assess gene expression 59 2.10 Crude membrane fraction and whole cell lysate from human-derived cells 59 2.11 Preparation of mouse mammary gland tissue lysate 60 2.12 Immunodetection of protein by gel electrophoresis/western blot 60 2.13 Immunohistochemistry 61 2.14 Short-interfering RNA 62 2.15 Plasmid constructs 63 2.15.1 Activity of the human ABCG2 gene promoter and GAS element 63 2.15.2 Activity of the mouse E1b promoter and GAS elements 64 2.16 In vitro methylation of plasmid DNA 65 2.17 Transient transfection and luciferase assay 65 2.17.1 Human ABCG2 gene promoter and GAS element activity 65 2.17.2 Mouse Abcg2 gene promoter and GAS element(s) activity 66 2.18 Genomic DNA isolation, bisulfite treatment, and pyrosequencing 67 2.19 Chromatin Immunoprecipitation (ChIP) 67 viii 2.19.1 T-47D cells 67 2.19.2 Mouse mammary gland tissue 68 2.20 ChIP-seq data analysis 69 2.21 Statistical Analysis 70 3.