DISCERNING THE ROLE OF KRÜPPEL-LIKE FACTOR 4 IN BREAST CANCER by JENNIFER L. YORI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Ruth A. Keri Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2011 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION This work is dedicatied to Beverly Ann McMillan for all that she taught me and Fiona Campbell Yori for all that she teaches me everyday. iii TABLE OF CONTENTS List of Tables vii List of Figures viii Acknowledgements x Abbreviations xii Abstract 1 Chapter I INTRODUCTION, REVIEW OF THE LITERTURE, AND 3 STATEMENT OF PURPOSE 1.1 Molecular/Histopathological subtypes and cellular origin of 3 breast cancer 1.1.1 Luminal breast cancer 5 1.1.2 HER2/ERRB2 breast cancer 6 1.1.3 Basal-like breast cancer 8 1.1.4 Comparison of mouse models and human tumors generate a 10 new molecular subtype 1.2 Metastasis, epithelial-to mesenchymal transition (EMT) and 12 the cancer stem cell theory 1.2.1 The epithelial-to-mesenchymal transition 13 1.2.1.1 Transcriptional regulation of EMT 15 1.2.2 The cancer stem cell theory 19 1.2.2.1 Cancer stem cells and EMT 21 1.2.2.2 iPS cells 23 iv 1.3 Krüppel-like factor 4 (KLF4) 24 1.3.1 The Sp1/Krüppel-like factor family 24 1.3.2 Krüppel-like factor 4 (KLF4) 26 1.3.2.1 Structure and regulation 26 1.3.2.2 Mechanisms of activation and repression 28 1.3.3 Function of KLF4 in normal biological processes 30 1.3.3.1 KLF4 as a regulator of development and differentiation 30 1.3.3.2 KLF4 regulation of proliferation and apoptosis 31 1.3.4 The role of KLF4 during carcinogenesis 34 1.3.4.1 KLF4 as tumor suppressor 34 1.3.4.2 KLF4 as an oncogene 35 1.3.4.3 The role of KLF4 in breast cancer and metastasis 35 1.3.4.4 Krüppel-like factors, EMT and metastasis 37 1.4 Statement of Purpose 39 Chapter 2 KRÜPPEL-LIKE FACTOR 4 (KLF4) INHIBITS 52 EPITHELIAL-TO-MESENCHYMAL TRANSITION THROUGH REGULATION OF E-CADHERIN GENE EXPRESSION 2.1 Introduction 53 2.2 Materials and Methods 56 2.3 Results 61 2.4 Discussion 67 2.5 Acknowledgements 74 v Chapter 3 KRÜPPEL-LIKE FACTOR 4 INHIBITS TUMORIGENIC 88 PROGRESSION AND METASTASIS IN A MOUSE MODEL OF BREAST CANCER 3.1 Introduction 89 3.2 Materials and Methods 91 3.3 Results 95 3.4 Discussion 101 3.5 Acknowledgements 105 Chapter 4 SUMMARY AND FUTURE DIRECTIONS 120 4.1 Summary 120 4.2 Future directions 121 4.2.1 Pharmacological targeting of KLF4 121 4.2.2 Does TGF-β signaling regulate KLF4 expression during EMT? 130 4.2.3 Can forced expression of KLF4 block TGF-β-induced EMT? 131 4.2.4 Does the TGF-β “switch” during tumorigenesis alter its effect 132 on the regulation of KLF4? 4.2.5 What are the mechanisms by which KLF4 suppresses Snail? 133 4.2.6 Does HER2 signaling regulate KLF4 expression? 134 4.2.7 What is the role of KLF4 in the maintenance and self-renewal 135 of MaSCs and CSCs? References 139 vi LIST OF TABLES Table 1.1 Regulators and Targets of KLF4 48 Table 3.1 Incidence of lung and liver micrometastases in AdGFP-4T1 and 106 AdKLF4-4T1 tumor bearing mice at 21 days post injection vii LIST OF FIGURES Figure 1.1 The link between normal mammary epithelial hierarchy, 41 molecular subtypes, gene expression signatures and clinical markers of breast cancer Figure 1.2 Organization of the human KLF4 gene and protein 43 Figure 1.3 KLF4 is a central player in the regulatory networks modulating 45 EMT and MET during generation of iPSCs and CSCs Figure 2.1 KLF4 is required for the maintenance of mammary epithelial 75 cell morphology Figure 2.2 KLF4 silencing results in loss of acinus formation and 77 decreased proliferation of mammary epithelial cells Figure 2.3 KLF4 is required to sustain E-cadherin expression in non- 79 transformed mammary epithelial cells Figure 2.4 KLF4 binds and activates the E-cadherin promoter 81 Figure 2.5 KLF4 induces expression of E-cadherin protein and a 83 transition to epithelial morphology in the mesenchymal-like MDA-MB-231 breast cancer cells Figure 2.6 KLF4 transcriptional activation of E-cadherin results in 85 decreased migration and invasion of MDA-MB-231 cells Figure 3.1 Loss of KLF4 expression in Her2/Neu-induced mouse 107 mammary tumors Figure 3.2 KLF4 expression inversely correlates with metastatic 110 viii progression Figure 3.3 KLF4 inhibits growth of 4T1 cells 112 Figure 3.4 KLF4 inhibits primary tumor growth of 4T1 cells 114 Figure 3.5 KLF4 overexpression decreases lung and liver micrometastases 116 Figure 3.6 KLF4 inhibits Snail expression in mammary epithelial cells and 118 tumor cells ix ACKNOWLEDGEMENTS It is very rare that one gets the opportunity to spend their time pursuing a career in something they are genuinely passionate about. I am ever grateful to the numerous people in my life who have made this possible. To Dr. John Nilson, who first brought me into the Pharmacology Department, imparting upon me his great words of wisdom, “It doesn‟t matter if you‟re not as smart as everyone else, it just means you have to work harder than everyone else”. His support and advice through my first year of graduate school made me realize this truly was an attainable goal. To Helai Mohammad, who taught me everything I needed to know in order to realize how much I really didn‟t know, I thank you for your persistence and attention to detail, as well as a solid understanding of cloning! I would especially like to thank Dr. Ruth Keri, who upon John‟s departure accepted me into her laboratory, allowing me to work in the area of breast cancer research. As a mentor, she has provided me with the opportunity to develop a research project from the ground up and call it my own. She has challenged me to critically evaluate my own experimental designs and data as well as the scientific literature. In addition, unlike most mentors, she has allowed her students to be an integral part of the grant writing process, contributing at all levels, from suggested experiments to critical review. She is truly a role model for young women in science. I‟m also grateful to my committee members, Dr. Amy Wilson-Delfosse, Dr. Mukesh Jain, and Dr. Noa Noy for their input and guidance. x During my time in the lab, many new members have come as old ones have moved on. Throughout the duration there has been comfort in consistency. To Darcie Seachrist and Kristen Lozada, who have been with me since the beginning and are still there today, I don‟t think I could have endured this journey without your friendships and support. Kris, thank you for teaching me all the ways to manipulate a mouse and for your many hours in the mouse room, especially when I was on maternity leaves. Darcie, your willingness to listen and offer advice, both scientific and personal, is so appreciated. I cannot thank you enough for all the hard work you put in towards the end to help me finish up. I‟d like to also thank several past lab member, including Jonathan Mosley, Erin Milliken, and Melissa Landis, for keeping me excited about science and showing me how it was done. To Monty Johnson, our first post-doc, I owe many thanks. His willingness to share his experience, knowledge and time was really a turning point for me in my graduate career, and I am forever grateful. Finally, I would like to thank my family and friends who have provided me with all the encouragement, love and support I could ever need. Rick and Louise Yori, the best in- laws anyone could ask for, thank you for your generosity and willingness to help, always! Dad, even though you‟ve teased me about being in school since I was five, you‟ve always found a way to let me know how proud you were of me, and that has meant so much.. To my husband Rich, who has helped me raise two wonderful children during these busy years, words alone could never convey my thanks and gratitude for all your sacrifice. Most of all, I would like to thank my mother, who provided me with a true appreciation for education, an understanding of true commitment and strength, and the realization that life is what you alone make of it – Thank you. xi ABBREVIATIONS AI Aromatase inhibitor AKT Protein kinase B APC Adenomatous polyposis coli bHLH Basic helix-loop-helixβ β-TrCP Beta-transducin repeat containing BRCA1 Breast cancer 1, early onset CBP CREB binding protein Cdk Cyclin-dependent kinase CK5/6 Cytokeratin 5/6 CRB3 Crumbs-3 CSC Cancer stem cell CYP1A1 Cytochrome P450 family 1, member A1 EGFR Epidermal growth factor receptor (HER1) EMT Epithelial-to-mesencymal transition ER Estrogen receptor ES Embryonic stem FOXC2 Forkhead box C2 GSK3β Glycogen synthase kinase 3 beta H3K4 Histone H3 lysine 4 HDAC1/2 Histone deacetylase 1/2 xii HDC Histidine decarboxylase HER2 Human epidermal growth factor receptor 2 (ERRB2, NEU) HER3 Human epidermal growth factor receptor 3 hNMSC Human normal mammary stem cell IHC Immunohistochemistry iPSC Induced pluripotent stem cell KLF4/GKLF/EZF Krüppel-like factor 4 / Gut-enriched krüppel-like factor 4 / Epithelial