INTERFERON REGULATORY FACTOR-1 (IRF1) SIGNALING REGULATES APOPTOSIS AND AUTOPHAGY TO DETERMINE ENDOCRINE RESPONSIVENESS AND CELL FATE IN HUMAN BREAST CANCER A Dissertation Submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physiology & Biophysics By Jessica L. Roberts, B.S. Washington, DC September 27, 2013 Copyright 2013 by Jessica L. Roberts All Rights Reserved ii INTERFERON REGULATORY FACTOR-1 (IRF1) SIGNALING REGULATES APOPTOSIS AND AUTOPHAGY TO DETERMINE ENDOCRINE RESPONSIVENESS AND CELL FATE IN HUMAN BREAST CANCER Jessica L. Roberts, B.S. Thesis Advisor: Robert Clarke, Ph.D. ABSTRACT Interferon regulatory factor-1 (IRF1) is a nuclear transcription factor and pivotal regulator of cell fate in cancer cells. While IRF1 is known to possess tumor suppressive activities, the role of IRF1 in mediating apoptosis and autophagy in breast cancer is largely unknown. Here, we show that IRF1 inhibits antiapoptotic B-cell lymphoma 2 (BCL2) protein expression, whose overexpression often contributes to antiestrogen resistance. We proposed that directly targeting the antiapoptotic BCL2 members with GX15-070 (GX; obatoclax), a BH3-mimetic currently in clinical development, would be an attractive strategy to overcome antiestrogen resistance in some breast cancers. Inhibition of BCL2 activity, through treatment with GX, was more effective in reducing the cell density of antiestrogen resistant breast cancer cells versus sensitive cells, and this increased sensitivity correlated with an accumulation of autophagic vacuoles. While GX treatment promoted autophagic vacuole and autolysosome formation, p62/SQSTM1, a marker for autophagic degradation, levels accumulated. Moreover, GX exposure resulted in a reduction of cathepsin D and L protein expression that would otherwise digest autolysosome cargo. Taken together, these data highlight a new mechanism of GX- induced cell death that could be used to design novel therapeutic interventions for antiestrogen resistant breast cancer. iii In addition to establishing a functional role for BCL2 members downstream of IRF1, we also provided evidence that this IRF1 mediates antiestrogen sensitivity in breast cancer through autophagy signaling. We demonstrated that IRF1 and autophagy-related protein 7 (ATG7) expression were inversely correlated in vivo and in human breast cancer tissues. We then identified both ATG7 and the functionally related protein, beclin-1 (BECN1), as negative regulators of IRF1. Conversely, loss of IRF1 promoted antiestrogen resistance by increasing prosurvival autophagy, whereas combined ATG7 and IRF1 knockdown restored sensitivity to the antiestrogen fulvestrant (Faslodex, ICI 182780, ICI). Thus, inhibition of autophagy proteins, ATG7 and BECN1, enables IRF1- dependent and -independent signaling pathways and provides a novel therapeutic approach for inhibiting breast cancer cell proliferation. iv ACKNOWLEDGEMENTS I would first like to express my deepest appreciation to my mentor, Dr. Robert Clarke, for his continuous patience and support of my Ph.D. work, academic progress, and professional development. His positive attitude, creativity, and exceptional intellect continually inspired me with new ideas and motivated me to succeed as a cancer researcher. Without his guidance and persistent help this dissertation would not have been possible. I would also like to thank the members of his laboratory, especially Drs. Ayesha Shajahan-Haq and Katherine Cook for guiding my research and providing constant advice and encouragement. Assistance from the Hilakivi-Clarke and Riggins laboratories also proved invaluable and helped to make this dissertation possible. To the members of my committee, Drs. Ayesha Shajahan-Haq, Tom Sherman, Priscilla Furth, and Minetta Liu, thank you for all of your encouragement, insightful comments, and hard questions. I also thank my academic advisor, Dr. Kathryn Sandberg, for her guidance and support during my professional development. Lastly, this doctoral effort could not have been achieved without the support of my family and friends. Thanks to my parents, Kathy and Jerry Schwartz, for their persistent love and support. They built the foundation that allowed me to get this far and always encouraged me to work hard to achieve my goals. Without you, I would not be the person I am today. v Above all, I would like to thank my husband, Matt, for his unwavering love and support. Thank you for being my inspiration, editor, proofreader, and comic relief. But most of all, thank you for being my best friend. I could not have done this without you. Finally, this work would not have been possible without the financial support of a NRSA predoctoral fellowship provided by the National Cancer Institute of the National Institutes of Health (Award Number F31CA165514), for which I am extremely grateful. vi TABLE OF CONTENTS Chapter 1: Introduction 1.1 Breast Cancer Incidence and Risk Factors………………………………………….1 1.2 Endocrine Therapies for Breast Cancer…………………………………………….5 1.3 Antiestrogens……………………………………………………………………….7 1.4 Mechanisms of Antiestrogen Action……………………………………………….10 1.5 Cell Death and Antiestrogens………………………………………………………13 1.6 Antiestrogen Resistance……………………………………………………………15 1.7 Interferon Regulatory Factor-1 Mediates Antiestrogen Sensitivity………………..19 1.8 Hypothesis and Aims……………………………………………………………….20 Chapter 2: Effect of the BCL2 Inhibitor, GX15-070, on Apoptosis and Autophagy in Breast Cancer Cell Lines 2.1 Introduction…………………………………………………………………………22 2.1.1. Antiestrogen Resistance………………………………………………22 2.1.2. BCL2 Family Members……………………………………………….23 2.1.3. BH3 Mimetics…………………………………………………………24 2.1.4. Autophagy and Lysosomal Function………………………………….25 2.1.5. Normal Function of Lysosomes………………………………………26 2.1.6. Lysosomal Hydrolases………………………………………………..27 2.1.7. Cathepsin D…………………………………………………………...28 2.1.8. Cathepsins B and L……………………………………………………29 2.2 Materials and Methods……………………………………………………………...31 2.2.1. Cell Culture and Reagents…………………………………………….31 vii 2.2.2. Cell Proliferation……………………………………………………..31 2.2.3. RNA Interference…………………………………………………….32 2.2.4. Western Blot Analysis………………………………………………..32 2.2.5. Reverse Transcription PCR…………………………………………..32 2.2.6. Apoptosis and autophagosome formation assays…………………….33 2.2.7. Autophagosome Maturation………………………………………….33 2.2.8. Orthotopic xenografts in athymic nude mice………………………...34 2.2.9. Immunohistochemistry……………………………………………….34 2.2.10. Electron Microscopy…………………………………………………34 2.2.11. Cathepsin Activity…………………………………………………...35 2.2.12. Isolation of Cytosolic Enriched Fraction…………………………….35 2.2.13. Statistical Analysis…………………………………………………...35 2.3 Results……………………………………………………………………………...36 2.3.1. Breast cancer models for antiestrogen resistance……………………36 2.3.2. Endogenous BCL2 expression in breast cancer cell lines can be targeted with the BH3 mimetic, GX…………………………………38 2.3.3. GX alone and in combination with an antiestrogen inhibits breast cancer cell density through apoptosis………………………………..38 2.3.4. GX induces autophagosome formation………………………………42 2.3.5. GX induces BECN1-dependent autophagosome formation, but autophagy is not required for GX toxicity…………………………...42 2.3.6. GX induces autophagic vacuole and lysosome formation in antiestrogen resistant LCC9 breast cancer cells……………………..47 2.3.7. GX treatment results in the accumulation of LC3-II and p62 proteins………………………………………………………………47 viii 2.3.8. GX blocks autophagic degradation through attenuation of cathepsin activity…………………………………………………….50 2.3.9. Inhibition of cathepsin L and D results in breast cancer cell death by blocking autophagosomal degradation…………………………...58 2.4. Discussion………………………………………………………………………….61 2.4.1. GX inhibits breast cancer cell proliferation through caspase- and PARP-independent mechanisms……………………………………..61 2.4.2. GX-mediated autophagosome formation is dependent on BECN1 but independent of ATG7……………………………………………62 2.4.3. GX inhibits autophagic cargo degradation…………………………..64 2.4.4. GX blocks autophagic cargo degradation by inhibiting cathepsin D and L protein expression…………………………………………..64 Chapter 3: IRF1 Signaling Regulates the Switch between Apoptosis and Autophagy to Determine Endocrine Responsiveness and Breast Cancer Cell Fate 3.1 Introduction………………………………………………………………………...66 3.1.1. Structure of the IRF1 gene/protein…………………………………..66 3.1.2. Role of IRF1 in IFN signaling……………………………………….68 3.1.3. Regulation of IRF1 expression………………………………………69 3.1.4. Biological functions of IRF1………………………………………...70 3.1.5. Tumor suppressive activities of IRF1………………………………..71 3.1.6. IRF1 in breast cancer………………………………………………...72 3.1.7. IRF1 and antiestrogen sensitivity……………………………………73 3.1.8. IRF1 in glucose metabolism…………………………………………74 3.1.9. Interplay between various forms of cell death: apoptosis and autophagy……………………………………………………………75 3.1.10. Autophagy in immunity and inflammation………………………….75 ix 3.2 Materials and Methods…………………………………………………………….77 3.2.1. Cell culture, reagents, and small interfering RNA (siRNA) treatments…………………………………………………………….77 3.2.2. Lentiviral plasmid creation and infection; creation of stable cell lines…………………………………………………………………..78 3.2.3. Cell proliferation….………………………………………………….79 3.2.4. Microarray analysis…………………………………………………..79 3.2.5. Western blot analysis………………………………………………...79 3.2.6. Isolation of mitochondrial enriched fraction………………………....80 3.2.7. MMP, autophagosome formation, ROS, and glucose uptake assays...80 3.2.8. Autophagosome