University of Cincinnati

UNIVERSITY OF CINCINNATI Date: 5/8/06 I, Emily E. Bosco Hereby submit this work as part of the requirements for the degree of: Doctor of Philosophy in: Cell and Molecular Biology It is entitled: RB Modifies the Therapeutic Response of Breast Cancer This work and its defense approved by: Chair: Erik Knudsen Sue Heffelfinger Kathy Hepner-Goss Sohaib Khan Yolanda Sanchez The Retinoblastoma Tumor Suppressor Modifies the Therapeutic Response of Breast Cancer A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctorate of Philosophy (Ph.D.) in the Department of Cell Biology, Neurobiology, and Anatomy of the College of Medicine 5/8/06 by Emily Elizabeth Bosco B.S. University of Notre Dame, 2001 Committee Chairman: Erik S. Knudsen Ph.D. Abstract: The retinoblastoma tumor suppressor (RB) is functionally inactivated in the majority of human cancers, and nearly half of all breast cancers. Here, we investigate the consequence of RB loss on the response to DNA damage and anti-estrogenic therapies used in the treatment of breast cancer. Initially, we demonstrate that downstream RB targets are severely mis-regulated following acute deletion in adult primary cells causing abrogation of the DNA damage checkpoint and consequently, accumulation of secondary DNA lesions upon treatment with chemotherapeutics. Additionally, we found that RB modifies the DNA repair response in adult primary fibroblasts, such that RB-deficient cells are able to repair UV-induced lesions at an accelerated rate. These initial studies reveal that RB loss in primary cells modifies the response to DNA damage by promoting aberrant replication and inappropriately accelerating repair, both of which may ultimately sensitize cells to DNA damaging therapies. To specifically recapitulate RB loss in breast cancer, we directed shRNA against RB in MCF7 cells. RB-deficiency resulted in RB/E2F target gene deregulation and accelerated tumorigenic proliferation, thereby demonstrating that even in the context of a complex tumor cell genome, RB status exerts significant control over proliferation. Furthermore, the loss of RB compromised the short-term cell cycle inhibition following anti-estrogen, cisplatin, and ionizing radiation therapies. In the context of DNA damaging agents this bypass resulted in increased sensitivity to these agents in cell culture and xenograft models. In contrast, the bypass of anti- estrogen signaling resulted in continued proliferation and xenograft tumor growth in the presence of tamoxifen. These effects of RB loss were reiterated by ectopic E2F expression, indicating that control of downstream target genes was important for the observed responses. Specific analyses of the RB/E2F gene expression signature in 60 human patients indicated that deregulation of this pathway was associated with early recurrence following tamoxifen monotherapy. Thus, because the RB-pathway is a determinant of tumorigenic proliferation and differential therapeutic response, it may represent a critical basis for informing therapy in the treatment of breast cancer. Acknowledgements: I am extremely grateful to my advisor, Dr. Erik Knudsen, whose guidance, creativity, and enthusiasm made four years feel much more like four days. Both Erik and Karen Knudsen have been tremendous role models and I am thankful for all of their advice and support. All members of the Knudsen laboratories, past and present, have made my graduate work possible and my time in the lab more enjoyable. I am especially appreciative of all of the time and assistance given to me by the members of my thesis committee; Dr. Sue Heffelfinger, Dr. Yolanda Sanchez, Dr. Kathy Hepner-Goss, and Dr. Sohaib Khan. Additionally, this work could not have been accomplished without the generosity and efforts of many collaborators and co-authors: Dr. Tyler Jacks, Dr. Julien Sage, Dr. Bob Hennigan, Dr. Scott Lowe, Dr. Jack Zilfou, Dr. Christopher Mayhew, Sejal Fox, Dr. Bruce Aronow, Huan Xu, Sandy Schwemberger, and Dr. George Babcock. I would like to thank my family for their constant encouragement and for giving me something to aspire to, both in life and in my career. Lastly, I must thank my husband, Chris, not only for bringing me to Cincinnati, but for his patience and love, neither of which I could be here without. TABLE OF CONTENTS Page List of Tables and Figures……………………………………………………………..2 Chapter I: Introduction…………………………………………………………………4 A. The Retinoblastoma Tumor Suppressor and Cancer……………………...4 B. RB and Cell Cycle Control…………………………………………………….5 C. RB and Breast Cancer Therapeutic Response…………………………….10 D. References……………………………………………………………………..13 Chapter II-IV: Results……………………………………………………………18-139 Chapter II: RB Signaling Prevents Replication-Dependent DNA Double-Strand Breaks Following Genotoxic Insult…………………………………………………..18 A. Abstract………………………………………………………………………...19 B. Introduction…………………………………………………………………….20 C. Materials and Methods……………………………………………………….23 D. Results…………………………………………………………………………27 E. Discussion……………………………………………………………………..34 F. References…………………………………………………………………….38 Chapter III: Differential Role of RB in Response to UV and IR Damage……….56 A. Abstract………………………………………………………………………..57 B. Introduction……………………………………………………………………58 C. Materials and Methods………………………………………………………61 D. Results………………………………………………………………………...65 E. Discussion…………………………………………………………………….76 F. References……………………………………………………………………81 Chapter IV: RB Modifies the Therapeutic Response of Breast Cancer……….103 A. Abstract……………………………………………………………………….104 B. Introduction…………………………………………………………………...105 C. Materials and Methods………………………………………………………108 D. Results………………………………………………………………………...112 E. Discussion…………………………………………………………………….119 F. References……………………………………………………………………123 1 Chapter V: Summary and Conclusions…………………………………………….140 A. RB-mediated transcriptional repression and maintenance of cell cycle control………………………………………………………………………….140 B. Role of RB in Therapeutic Response……………………………………...143 C. Can RB Status Inform Therapy?.............................................................145 D. Interesting Questions and Future Investigations………………………….146 E. References…………………………………………………………………....149 LIST OF TABLES AND FIGURES Figure Page I-1 Two-Hit Hypothesis of Retinoblastoma Heritability……………………….....5 I-2 RB is Targeted in Cancer………………………………………………………6 I-3 Regulation of Cell Cycle by RB…………………………………………….….8 I-4 RB and Breast Cancer Therapy………………………………………………11 II-1 Infection of Primary RbloxP/loxP MAFs with Adenoviral Cre Yields Acute Downregulation of RB Proteinand Deregulation of RB Repression Targets……46 II-2 RB Loss in Primary Adult Fibroblasts Impairs Replication in Response to DNA Damage……………………………………………………………………….48 II-3 Acute RB Loss Influences the Accumulation of DNA DSBs……………….50 II-4 Replication-Dependent DSB Accumulation………………………………….52 II-5 BrdU and γH2AX Foci Co-localization………………………………………..54 III-1 Acute Downregulation of RB Protein Abrogates the DNA Damage Checkpoint Response to UV and IR………………………………………………...90 III-2 Acute RB Loss Compromises the Rapid Checkpoint Response to UV and IR…………………………………………………………………………………..92 III-3 Kinetics of RB Loss is Concomitant with Target Gene Deregulation to promote Abrogation of the DNA Damage Checkpoint Response…………….94 III-4 Differential Effects of Acute RB Loss on UV and IR Induced DNA Lesion Removal……………………………………………………………………….96 III-5 Acute RB Loss Accelerates UV Induced DNA Damage Repair…………..99 III-6 RB Modifies Repair Factor Dynamics Following DNA Damage………….101 IV-1 Efficient RB Knockdown in MCF7 Cells Causes Deregulation of 2 RB/E2F Target Genes and Increased Proliferation Kinetics……………………129 IV-2 Tumor Growth in Nude Mouse Xenografts is Accelerated in RB Knockdown Cells…………………………………………………………………….130 IV-3 RB-deficiency Enables Bypass of the DNA Damage Checkpoint Resulting in Increased Sensitivity………………………………………………….132 IV-4 RB is Necessary for Sensitivity to Anti-Estrogen Therapy and Long Term Growth Arrest…………………………………………………………..134 IV-5 E2F3 Overexpression in MCF7 Cells Allows Bypass of Anti- Mitogenic Checkpoints……………………………………………………………...135 IV-6 E2F Downstream Target Deregulation Correlates with Poor Prognosis in Human Breast Cancers Treated with Tamoxifen Monotherapy…………………136 IV- Supplemental 1. Stable RB Knockdown in Several MCF7 Clones………...138 IV- Supplemental 2. RB-Deficiency Enables Accelerated Cell Cycle Progression……….............................................................................................138 IV- Supplemental 3. Bypass of Anti-Estrogen Checkpoint is Evident in Several RB-Deficient MCF7 Clones……………………………………………….138 3 Chapter I: Introduction The Retinoblastoma Tumor Suppressor and Cancer Cancer is a disease that develops in multiple steps, and each of these steps is a genetic alteration that fuels the transformation of a normal cell into a malignant one. Most genetic mutations that drive tumorigenesis disrupt proteins involved in signaling networks controlling cell division. These genetic mutations allow for uncontrolled cell division via a variety of mechanisms, for example, cells may become able to evade apoptosis, become self-sufficient for growth, or become insensitive to anti-growth signals (1). In 1971, the “two-hit hypothesis” was proposed to explain a mechanism by which cells are able to achieve such genetic changes during tumorigenesis. Through a detailed statistical analysis of two forms of a pediatric eye tumor, retinoblastoma,

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