THE ROLES OF ATF3, AN ADAPTIVE-RESPONSE GENE, IN BREAST CANCER DEVELOPMENT DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xin Yin, B. M. ***** The Ohio State University 2008 Dissertation Committee: Approved by Dr. Tsonwin Hai, Advisor Dr. James DeWille Advisor Dr. Anthony Young The Ohio State Biochemistry Program Dr. Mike Xi Zhu ABSTRACT During cancer progression, cells encounter many stress signals and all along they have built-in mechanisms to eliminate themselves. The successful cancer cells managed to foil this hardwired stress response. Emerging evidence indicates that some of the genes that normally function to eliminate the cells are co-opted to become oncogenes. How the cellular (normal vs. cancerous) context determines that some genes undergo this “Jekyll-and-Hyde” conversion is an intriguing but largely unresolved issue in cancer biology. Breast cancer is the most common malignancy among women in the United States and is the second leading cause of cancer death in women, after lung cancer. The initiation and development of breast cancer rely on both cell- autonomous (genetic and epigenetic) alterations as well as stroma-cancer interactions. In this thesis work, I found ATF3, an ATF/CREB family transcription factor encoded by an adaptive-response gene, is a new regulatory molecule with a dichotomous role. It enhances apoptosis in untransformed cells, but protects the cells from stress-induced death and promotes cell motility in malignant cancer cells. In an in vivo xenograft mouse model, in addition to promoting primary tumor growth, ATF3 also increases lung metastasis. ii To explore the potential mechanisms by which ATF3 promotes metastasis, I set out to address three major questions. First, as a transcription factor, will ATF3 regulate the expression of some target genes involved in cell motility? Second, as an adaptive response gene, will ATF3 be induced in cancer cells in response to stromal signals and whether the induction of ATF3 mediates cancer cells’ response to stromal signals? Third, will the induction of ATF3 in cancer cells feed back on stromal cells to affect stroma-cancer interaction? By examining potential ATF3 target genes, I found ATF3 regulates a set of genes involved in cell motility, some of which were proved to be direct targets of ATF3. As an adaptive-response gene, ATF3 was induced by multiple stromal signals, such as TGFβ, TNFα and IL-1β. When focusing on one multifunctional cytokine, TGFβ, I found that ATF3 can mediate TGFβ effects on target gene expression and cell motility regulation. The interaction between ATF3 and Smad2/3 offers a partial mechanistic understanding. Besides mediating the intracellular signaling of stromal factors, ATF3 is also involved in stroma-cancer interaction, as demonstrated by the effect of ATF3 expression in cancer cells on macrophage recruitment and angiogenesis. Finally, by examining ATF3 status in human breast cancer, I found that wild- type ATF3 gene is frequently amplified and its expression elevated in human breast tumors, suggesting a pathophysiological relevance of ATF3 to human cancer. Through the database analysis of microarray data generated by two other groups, I found that the higher expression of ATF3 correlates with worse outcome of breast cancer patients. iii The significance of this work is several-fold. First, it identified a novel mediator that allows the cells to interpret the exogenous signals and initiate dichotomous cellular processes, in a manner dependent on the malignancy of the cells. Second, as a downstream mediator for TGFβ, another well-demonstrated stromal factor with paradoxical functions during cancer development, ATF3 constitutes a link in the stromal signaling and transcriptional networks during stroma-cancer interactions. Third, the dichotomous effect of ATF3 was demonstrated in isogenic breast cancer cells representing different stages of cancer development. Since ATF3 can regulate some genes in an opposite direction, this cell system may provide a handle to elucidate the “cellular contexts” (such as interacting proteins or co-factors) that allow ATF3 to regulate the same promoters in an opposite manner. This would help to unravel the mysteries behind the dichotomy in cancer development. Fourth, about 50% human breast tumors show up- regulation of wild-type ATF3. In combination with the functional consequences of ATF3 in both cell system and mouse model, we have discovered a new oncogene in malignant breast cancer cells that is likely to have pathophysiological relevance to human cancer. iv DEDICATION To Mom, Dad, Wei and my little girl, Cynthia v ACKNOWLEDGMENTS I would like to give my sincere thanks my advisor, Dr. Tsonwin Hai, for the tireless dedication and endless support at all levels throughout these years. I appreciate the trainings and challenges she offered to help me understand the logic, thought process and experimental design for biological sciences. She never lets me down when I need guidance and encouragement, in science or in daily life. I wouldn’t be even close to where I am now without her. I am also grateful for my committee members, Dr. James DeWille, Dr. Anthony Young and Dr. Mike Xi Zhu, for their encouragement and instructive comments on my research, as well as their generous help on my future career. I would like to thank the current and past members of the Hai lab: Dr. Matthew G. Hartman, Dr. Dan Lu, Milyang Kim, Dan Li and Shawn Behan, with special thanks to Erik Zmuda and Christopher Wolford. They help me on experimental details, project discussions and English communications. vi VITA April 28, 1977…………………………….….Born – Jing Dezhen, P. R. China 1994 – 2000 ………………………………..B.S. Clinical Medicine, China Medical University, Shenyang, P.R.China 2000 – 2002……………………………….…Assistant Editor, Chinese Medical Sciences Journal , Beijing, P.R.China 2002 – 2003……………………………….…Program Fellowship, The Ohio State University 2003 – present………………………………Graduate Research Associate The Ohio State University PUBLICATIONS 1. X. Yin , JW. DeWille and T. Hai. (2008) A potential dichotomous role of ATF3, an adaptive-response gene, in cancer development. Oncogene. 27(15): 2118-27 . 2. D. Li, X. Yin , EJ. Zmuda, CC. Wolford, X. Dong, MF. White and T. Hai. (2008) The repression of IRS2 gene by ATF3, a stress-inducible gene, contributes to pancreatic-cell apoptosis. Diabetes. 57(3): 635-44. FIELD OF STUDY Major Field: Ohio State Biochemistry Program vii TABLE OF CONTENTS Page ABSTRACT ...........................................................................................................ii DEDICATION ........................................................................................................v ACKNOWLEDGMENTS .......................................................................................vi VITA…................................................................................................................. vii PUBLICATIONS .................................................................................................. vii FIELD OF STUDY ............................................................................................... vii LIST OF TABLES .................................................................................................xi LIST OF FIGURES .............................................................................................. xii LIST OF FIGURES .............................................................................................. xii ABBREVIATIONS............................................................................................... xiv INTRODUCTION ...................................................................................................1 1. INTRODUCTION .......................................................................................1 1.1. An overview of the ATF/CREB family of transcription factors and ATF3...........................................................................................................3 1.1.A. The ATF/CREB family of transcription factors............................3 1.1.B. Cloning and Characterization of ATF3 .......................................5 1.1.C. ATF3 as an adaptive-response gene.........................................8 1.1.D. Biological consequences of ATF3 expression .........................11 1.1.E. Summary..................................................................................15 1.2. Overview of mammary development and tumorigenesis ...................16 1.2.A. Mammary gland anatomy and development ............................16 1.2.B. Epidemiology of breast cancer.................................................19 1.2.C. Causes, evolution and heterogeneity of breast cancer ............19 1.2.D. Classification of breast cancer .................................................22 1.2.E. The multi-stage development of breast cancer and cancer metastasis ..........................................................................................23 1.2.E. Stromal-cancer interaction .......................................................24 1.2.F. Breast cancer, adaptive response and “Jekyll-and-Hyde” conversion..........................................................................................26 1.2.G. Conclusion...............................................................................28 2. A POTENTIAL DICHOTOMOUS ROLE OF ATF3, AN ADAPTIVE- RESPONSE GENE, IN BREAST CANCER DEVELOPMENT