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RUNX1 Control of Mammary Epithelial and Breast Cancer Cell Phenotypes University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2017-12-12 RUNX1 Control of Mammary Epithelial and Breast Cancer Cell Phenotypes Deli Hong University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Cancer Biology Commons, and the Cell Biology Commons Repository Citation Hong D. (2017). RUNX1 Control of Mammary Epithelial and Breast Cancer Cell Phenotypes. GSBS Dissertations and Theses. https://doi.org/10.13028/M21Q2F. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/949 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. RUNX1 CONTROL OF MAMMARY EPITHELIAL AND BREAST CANCER CELL PHENOTYPES A Dissertation Presented By Deli Hong Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 12, 2017 Program of Cell Biology RUNX1 CONTROL OF MAMMARY EPITHELIAL AND BREAST CANCER CELL PHENOTYPES A Dissertation Presented By Deli Hong This work was undertaken in the Graduate School of Biomedical Sciences Program of Cell Biology The signature of the Thesis Advisor signifies validation of Dissertation content Gary S. Stein, Ph. D., Thesis Advisor The signatures of the Dissertation Defense Committee signify completion and approval as to style and content of the Dissertation Leslie Shaw, Ph. D., Member of Committee Kendall Knight, Ph. D., Member of Committee Jeffery Nickerson, Ph. D., Member of Committee Robert Weinberg, Ph. D., Member of Committee The signature of the Chair of the Committee signifies that the written dissertation meets the requirements of the Dissertation Committee Hong Zhang, Ph.D., Chair of Committee The signature of the Dean of the Graduate School of Biomedical Sciences signifies that the student has met all graduation requirements of the School. Anthony Carruthers, Ph.D., Dean of the Graduate School of Biomedical Sciences December 12, 2017 Acknowledgements Foremost, I would like to express my deepest gratitude to my thesis advisor Prof. Gary Stein, Dr. Jane Lian and Dr. Janet Stein for their continuous guidance and generous support throughout my doctoral study, without which this experience would not have been the positive experience it was. They always support me to pursue my own scientific interests in the lab. This great experience working in their lab helped me to mature into a well-rounded scientist. I would like to give thanks to Jason Dobson for his guidance during the initial stage of my doctoral study doctoral study. Specific thanks go to Dr. Andrew Fritz, who is my primary collaborator in the lab. Additional thanks to Dr. Coralee Tye and Natali Page for their help in RNA-seq analysis, Joseph Boyd for his assistance with bioinformatics analysis, and Kristiaan Finstad and Morgan Czaja for their help in animal studies. Thanks to Terri Messier for her kind help in the lab. Additional thanks to all current and previous members of the Stein-Lian laboratory, Nick, Gillian, Prachi, Kaleem, Jonathan, Kirsten, Mingu, Areg, Josh, Helena, Gileade, Phil, Hai, Xuhui, Rodrigo, Jennifer, Mark, Cesear, Ryan, Alex for their help with my projects and scientific discussions. I would like to thank my thesis research advisory committee members Dr. Leslie Shaw, Dr. Kendall Knight, Dr. Jeffrey Nickerson, Dr. Stephen Jones and my external committee Dr. Robert Weinberg and my committee chair Dr. Hong Zhang for their time, valuable input and support for my graduate studies and career development. I would like to extend my gratitude to my beloved parents Dr. Xiuping Hong and Dr. Yiping Sun for their unconditional love and support throughout my life. I also want to thank all of my friends for their support and company. iii Abstract Breast cancer remains the most common malignant disease in women worldwide. Despite the advantages of early detection and improved treatments, studies into the mechanisms that initiate and drive breast cancer progression are still required. Recent studies have identified RUNX1, which is an essential transcription factor for hematopoiesis, is one of the most frequently mutated genes in breast cancer patients. However, the role of RUNX1 in the mammary gland is understudied. In this dissertation, we examined the role of RUNX1 in both normal mammary epithelial and breast cancer cells. Our in vitro studies demonstrated that RUNX1 inhibits epithelial to mesenchymal transition (EMT), migration, and invasion, reflecting its tumor suppressor activity, which was confirmed in vivo. Moreover, RUNX1 also contributes significantly to inhibition of the phenotypes of breast cancer stem cells (CSC), which is responsible for metastasis and tumor relapse. We showed that Runx1 overexpression reduces the tumorsphere formation and cancer stem cell population. Overall, our studies provide mechanistic evidence for RUNX1 repression of EMT in mammary cells, anti-tumor activity in vivo and regulation of CSC-like properties in breast cancer. Our results highlight crucial roles for RUNX1 in preventing epithelial to mesenchymal transition and tumor progression in breast cancer. This RUNX1 mediated mechanism points to novel intervention strategies for early stage breast cancer. iv Table of Contents Acknowledgements……………………………………….…………….……..……………iii Abstract………………………………………………………………….……...……….…...iv Table of Contents...…………………………………………………………………............v List of Figures……………………………………………………………...…………......….x List of Tables…………………………………………………..…………………….……..xiii List of Symbols and Abbreviations………..……..…………………..……………….….xiv Preface……………………………………………….……………………………………..xvi CHAPTER I. Introduction…………………………………...…………………………...1-52 1.1 The Introduction of Breast Cancer …………………………………………………1 1.1.1 Breast cancer overview………………………………………...……………1 1.1.2 Breast cancer molecular subtypes………………………………………....1 1.1.3 The origin of breast cancer and breast cancer subtypes………..………7 1.1.4 Cell line models used in breast cancer study………….........…………..11 1.2 The Runx Family………………………………………………..……………....….12 1.2.1 Runx family overview……………………………..………...…………......12 1.2.2 Structure of Runx………………………………..………………………....14 1.2.3 Evolutionary role of Runx………………………..………………………...17 1.3 The Runx Family and Development in Mammals…….........…………………..20 1.3.1 Overview……………………………………….……………………………20 1.3.2 Runx1………………………………………………..……………………….21 1.3.3 Runx2……………………………………………….……………………….24 1.3.4 Runx3………………………………………………….…………………….26 v 1.4 The Runx Family in Disease and Cancer…………………….………………….27 1.4.1 Overview………………………………………….…………………………27 1.4.2 Runx1………………………………………….…………………………….28 1.4.3 Runx2 and Runx3…………………………………………………....……..31 1.5 Runx1 and Mammary Gland Development and Breast Cancer……………....32 1.5.1 Mammary gland development and hierarchy……………………………32 1.5.2 Runx1 and mammary gland development…………………...…………..33 1.5.3 Runx1 and breast cancer………….…………………………...………….34 1.6 Epithelial Mesenchymal Transition in Breast Cancer…………..………………38 1.6.1 Overview of epithelial mesenchymal transition …………..…………….38 1.6.2 Epithelial mesenchymal transition in development…………..…………39 1.6.3 Epithelial mesenchymal transition in cancer………………….…………41 1.6.4 Runx and EMT………………………………………………….…………..43 1.7 Cancer Stem Cell and Breast Cancer……………..………………..……………45 1.7.1 Intra-tumor heterogeneity………………………………….………………45 1.7.2 Cancer stem cells………………………………………….……………….47 1.7.3 EMT and plasticity and cancer stem cells……………………....……….49 1.8 Rationale for the Dissertation………………………………………..…………….…50 CHAPTER II. Runx1 stabilizes the mammary epithelial cell phenotype and prevents epithelial to mesenchymal transition……………………………...………53-101 2.1 Abstract……………………………………………………….………….……….…….54 2.2 Introduction…………………………………………….….……………….…………...55 2.3 Materials and Methods……………………………….……….……….….………..…58 vi 2.4 Results……………………………………………………………..…………....…...…66 2.4.1 Runx1 expression is decreased in breast cancer …………..……...….….…66 2.4.2 TGFβ induced EMT decreases Runx1 expression in MCF10A cells …..….69 2.4.3 Runx1 reverses the TGFβ-induced EMT phenotype……………..….….……72 2.4.4 Decreased expression of Runx1 during TGFβ independent EMT in MCF10A cells…………………………………………..………………..................…..……..74 2.4.5 Gene expression profiling of growth factor-depleted MCF10A cells reveals the spectrum of EMT markers………………………………………..…………..……77 2.4.6 Directly Depleting Runx1 in MCF10A cells results in loss of epithelial morphology and activation of EMT.………………….……………………………….……....…82 2.4.7 Depleting Runx1 in MCF7 breast cancer cells promotes EMT.…...….….…88 2.4.8 Overexpressing Runx1 in mesenchymal like breast cancer cells drives mesenchymal to epithelial transition (MET).…….……………………...…….…89 2.4.9 Runx1 expression in breast tumors correlates with metastasis, tumor subtype and survival…………………………………...…….…………………...……….…92 2.5 Discussion for Chapter II…………………………………………….….……...……..96 Chapter III RUNX1 Genome-wide Regulation of Normal Mammary Epithelial Cells: Novel Functions for Mitosis and Genome Stability……………………………………....102-146 3.1 Introduction……………………………………………...……………….……………106 3.2 Materials and Methods……………………………………………..…….....……….106 3.3 Results…………………………………………………………….………………......111 3.3.1. RUNX1 knockdown
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