Loss of DACH2 is a candidate early event in breast and ovarian carcinogenesis By Rania Chehade A thesis submitted in conformity with the requirements for Degree of Master of Science Medical Biophysics Department University of Toronto Copyright by Rania Chehade 2013 Loss of DACH2 is a candidate early event in breast and ovarian carcinogenesis Rania Chehade Master of Science Medical Biophysics Department University of Toronto 2013 Abstract Mechanistic insights into how enduring menstrual cycle hormonal signaling promotes tumorigenesis are emerging. We performed a genome-wide screen in primary epithelial cells to identify hormonally-regulated candidates that initiate pro-tumorigenic phenotypes in normal cells. One candidate, DACH2, has been described as part of a network that regulates organogenesis during development. In vitro, we find that DACH2 expression is regulated by estrogen and progesterone in a dosage dependent manner. Lentiviral-mediated shRNA silencing of DACH2 in hormonally responsive tissues promotes expansion of cells with progenitor characteristics. Within gene expression profiles of fallopian tubes, DACH2 is a member of a minimal gene classifier that distinguishes follicular versus luteal phases. Decreased DACH2 is characteristic of luteal-phase tubal cells and the majority of ovarian serous carcinomas. These studies suggest that DACH2 may orchestrate a physiological program that, when deregulated, locks cells in a progenitor-state, induces uncontrolled proliferation, and predisposes cells for breast and ovarian carcinogenesis. ii Acknowledgements Graduate school has been a bountiful journey of learning opportunities in academic, work and life relationships. I have developed various skills and I have matured both as a researcher and a human being. First and foremost, I would like to acknowledge my supervisor Dr. Mona Gauthier for her support throughout graduate school, our enriching discussions and her role in shaping my scientific way of thinking and writing. I officially started my graduate school in January 2010 and Mona was diagnosed with ovarian cancer a month after I had started. Thinking back to our meeting where Mona had asked me whether I would prefer to stay or look for another lab, I opted to stay. Since then, I have had a unique relationship with Mona as a researcher and cancer fighter. It added a sense of commitment and holiness to our daily work as scientists and as members of the society actively involved in raising awareness on cancer research. I would like to acknowledge my co-supervisor Dr. Hal Berman for his help on the project and in preparing presentations and writing the thesis. His door has always been open to interesting scientific discussions and advice on career choices. I would like to also acknowledge my committee members, Dr. Brad Wouters and Dr. Ben Neel for their helpful suggestions. The experience could not have been more rewarding without the interactions with lab members, graduate school colleagues and coworkers. I would like to thank in particular Jennifer Cruickshank who has been a role model with her meticulous lab experience, humbling work ethic and undying support. I would also like to thank my graduate colleague Noor Salman for her support in the lab, and for being there whenever I had a question or needed an advice. Previous summer students, Nymisha Chilukuri and Sara Halawi were exciting to work with and learn from. I would also like to acknowledge members of Dr. Shaw’s lab, Dr. Sophia George for iii sharing her lab experience, her passion for driving health outreach programs and fruitful discussions and Anca Milea for discussions and for being my fundraising partner in the Weekend to End Women Cancers. I would also like to acknowledge our visiting postdoctoral fellow Dr. Taeko Nagao who shared her experience in surgical oncology in Japan and was humbling to work with on research projects. I appreciate helpful discussions and reagents from members of the Okada lab in particular Rashi Gupta and Dr. Goto Kouichiro, members of the Reedjik lab and members of Mak lab. Last but not least, I am very grateful for my family’s support, for being the safe harbor and the inspirational anchor that motivates my every step. My love for science has me waking up at night thinking of the next experiment, the next project and the next exploration; this experience will be helpful in my future medical endeavors. iv TABLE OF CONTENTS CONTENT PAGE Abstract ii Acknowledgement iii-iv Table of Contents v-viii Statement of Contribution ix List of Figures x-xi List of Tables xii Abbreviations xiii- xvii CHAPTER 1: INTRODUCTION 1-21 1.1 The menstrual Cycle and risk for carcinogenesis 1-3 1.1.1 An overview of the menstrual cycle 4 1.1.2 Epithelial changes in the fallopian tube during the menstrual cycle 4-6 1.2 Cellular senescence: a barrier to tumorigenesis 6-7 1.2.1 Senescence characterization in vitro 7 1.2.2 Senescence occurs in vivo 8 1.2.3 Threshold levels and the balance between cell cycle proliferation and arrest 9 1.3 Primary cell models 9-10 1.3.1 HMEC system: well explored and cells have limited lifespan 10-11 1.3.2 FTE system: recently explored and cells have limited lifespan 12 1.3.3 Advancements in primary cell models 13 v 1.4 Studying bypass of senescence in HMEC 13-14 1.5 The Dachshund gene family 14 1.5.1 Dachshund during Drosophila development 14-15 1.5.2 Dachshund during murine development 16-17 1.5.3 Retinal Determination Gene Network (RDGN) in breast and ovarian carcinogenesis 17 Sine Oculis 17-19 Eyes Absent 19 Dachshund 19-20 1.7 HYPOTHESES 21 CHAPTER 2: METHODS 22-37 2.1 Cells and cell culture 22 2.1.1 Human Mammary Epithelial Cells 22 2.1.2 Fallopian Tube Epithelial Cells 22 2.1.3 Human Cancer Cell lines 23 2.1.4 Estrogen and Progesterone treatment 23-24 2.2 Growth curve kinetics 24 2.3 Engineering of stable cell lines 24 2.3.1 Lentiviral vectors 25 2.3.2 Lentivirus production 25 2.3.3 Generation of E7 Retrovirus 26 vi 2.3.4 DACH2 expression construct 26 2.3.5 Generation of stable HMEC and FTE cell lines 27 2.4 Senescence associated β-galactosidase activity 27 2.5 QRT-PCR/RT-PCR 28 2.5.1 RNA Extraction 28 2.5.2 Reverse Transcription Polymerase Chain Reaction (RT-PCR) 28-29 2.5.3 QRT-PCR 29-30 2.6 Protein Extraction, Western Blot 31-32 2.7 Flow Cytometry 32-33 2.8 Colony Forming Cell (CFC) Assay 33 2.9 Mammosphere Assay 33-34 2.10 Immunofluorescence 34 2.11 Soft Agar Assay 34 2.12 Patient samples and immunohistochemistry 35 2.13 Statistical analysis 35 2.14 Microarray data analysis 36-37 CHAPTER 3: RESULTS 38-83 3.1 A loss of function screen for bypass of replicative senescence in HMECs 38-39 3.1.1 Identification of the dachshund homolog 2 as a candidate gene 40-41 3.1.2 Loss of DACH2 is sufficient for bypass of replicative senescence 41-47 3.2 Loss of DACH2 is sufficient for bypass of premature senescence 48-51 vii 3.3 DACH2 is sufficient to induce senescence 51-56 3.4 DACH2 regulates anchorage independent growth 56-57 3.5 Evidence to support a role of DACH2 in regulating differentiation 57-67 3.6 DACH2 is differentially expressed in the fallopian tube epithelium during the menstrual cycle 68- 74 3.7 Differential regulation of DACH2 by estrogen and progesterone 74-80 3.8 DACH2 expression in normal and malignant breast tissue 80-83 CHAPTER 4: DISCUSSION 84-97 4.1 Model of hormonal regulation of DACH2 during the menstrual cycle 84-88 4.2 DACH2 and Checkpoint Regulation 89-92 4.3 DACH2 and Cell Fate 92-94 4.4 DACH2 and Differentiation 94-95 4.5 DACH2 expression in breast and ovarian carcinogenesis 95-97 SUMMARY 98 REFERENCES 99-107 viii Statement of Contribution: 1- Growth curves in Figure 1A have been done by Bernie Martin. 2- Experiment and data analysis in Figure 1B and 1C has been done by Dr. Mona Gauthier, Bernie martin and Noor Salman. 3- Experiment and data analysis in Figure 13A has been done by Jennifer Cruickshank 4- Microarray Data analysis in Figures 17, 22 and 23 have been done by Dr. Hal Berman 5- Immunohistochemistry staining of DACH2 in Figures 18, and 24 has been done by Bernie Martin, and staining scoring by Dr. Mona Gauthier 6- The fallopian tube epithelial lines have been generated and propagated by Dr. Sophia George. 7- Association studies have been statistically analyzed by Dr. Mona Gauthier ix LIST OF FIGURES PAGE 1- A loss of function screen for bypass of replicative senescence in HMECs 42 2- Loss of DACH2 is sufficient for bypass of replicative senescence in HMECs 45 3- Loss of DACH2 is sufficient for bypass of replicative senescence in FTEs 47 4- Loss of BRCA1 induces premature senescence in HMECs 49 5- Loss of DACH2 is sufficient for bypass of premature senescence 50 6- DACH2 mRNA and protein levels increase with senescence 52 7- Generation of DACH2 overexpression construct 53 8- Overexpression of DACH2 induces senescence in HMECs 54 9- Overexpression of DACH2 induces senescence in FTEs 55 10- Loss of DACH2 induces anchorage independent growth 59 11- Overexpression of DACH2 decreases anchorage independent growth in breast cancer cell line, MDA231. 60 12- Overexpression of DACH2 decreases anchorage independent growth in select ovarian cancer cell lines 61 13- Human Mammary Epithelial cells (HMEC) show progenitor cell characteristics 62 14- Loss of DACH2 promotes luminal progenitor phenotypes 64 15- Loss of DACH2 does not affect mammosphere formation capacity in HMECs 66 16- Loss of DACH2 increases progenitor capacity of FTEs 67 17- Differentially expressed genes between follicular and luteal phases of the menstrual cycle in primary fallopian tube samples 69 18- Minimal gene classifier that distinguishes follicular phase from luteal x phase of the menstrual cycle in the fallopian tube 72 19- DACH2 protein expression is decreased in the luteal phase of the menstrual cycle 73 20- Estrogen regulates DACH2 expression and cellular localization 76 21- Loss of DACH2 impairs Estrogen Receptor Signaling 77 22- Differential regulation of DACH2 by estrogen and progesterone.