DISCERNING THE ROLE OF LMO4 AS A GLOBAL MODULATOR OF G2/M CELL CYCLE PROGRESSION AND CENTROSOME CYCLE IN BREAST CANCER CELLS by MARJORIE E. MONTAÑEZ-WISCOVICH Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Dr. Ruth A. Keri Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January, 2010 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____Marjorie E. Montanez-Wiscovich________ candidate for the _Doctor of Philosophy_ degree *. (signed) _____George R. Dubyak_______ (chair of the committee) _______Yu-Chung Yang_______ _________Julian Kim_________ _________Ruth Keri__________ (date) _October 21, 2009____________ * We also certify that written approval has been obtained for any proprietary material contained therein. DEDICATION To Lelo and Lela for one day we will have a cure. TABLE OF CONTENTS Dedication iii Table of Contents iv List of tables viii List of figures ix Acknowledgments xi List of Abbreviations xiii Chapter 1 Introduction 1 1.1 Background 1 a. Origin of Molecular Subtypes 3 b. Gene Expression Profiling and Patient Care 5 1.2 Chromosome Cycle and Centrosome Cycle 6 1.3 Luminal-like breast cancer subtype 15 1.4 ErbB2-amplified breast cancer subtype 21 1.5 Basal-like breast cancer subtype 26 1.6 LIM-only protein 4 (LMO4) 31 1.7 Statement of Purpose 42 Chapter 2 LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression 44 2.1 Introduction 44 2.2 Results and Discussion 47 a. LMO4 mRNA and protein are increased in ErbB2-induced tumors. 47 b. ErbB2 and PI3K activity are necessary for LMO4 expression. 49 iv c. LMO4 regulates proliferation of ErbB2-dependent breast cancer cells. 54 d. LMO4 is required for maintenance of Cyclin D1 mRNA and protein expression in cells with constitutive ErbB2 signaling as well as for heregulin-induced upregulation of Cyclin D1. 56 e. LMO4 regulates G2/M transition by inducing cullin-3 expression. 58 f. LMO4 expression oscillates throughout the cell cycle with peak expression occurring at G2/M phase. 62 2.3 Materials and Methods 66 Chapter 3 Aberrant expression of LMO4 induces centrosome amplification and mitotic spindle abnormalities in breast cancer cells 74 3.1 Introduction 74 3.2 Results and Discussion 77 a. LMO4 mRNA is highly expressed in BRCA1 mutant and ERα-negative breast cancers. 77 b. LMO4 expression is elevated in tumors with high mitotic indices. 80 c. LMO4 regulates G2/M progression in luminal and basal-like breast cancer cells. 84 d. Alterations in LMO4 levels induce centrosome amplification. 87 e. Altered LMO4 levels induce the formation of abnormal mitotic spindles. 90 f. LMO4 localizes to the centrosome. 92 3.3 Materials and Methods 95 Chapter 4 Summary and Future Directions 101 4.1 Summary 101 v a. Does LMO4 directly regulate cul3 transcription? Does cul3 mediate LMO4- dependent G2/M arrest and/or centrosome amplification? 104 b. Does LMO4 regulate the G2/M checkpoint and/or the centrosome cycle by modulating BRCA1 activity? 107 c. Does LMO4 regulate centrosome amplification and/or G2/M arrest via transcriptional mechanisms? 110 d. Do hyperplastic glands and tumors derived from mammary-specific overexpression of LMO4 exhibit amplification of centrosomes and aneuploidy? Do embryonic fibroblasts from LMO4-null mice have centrosome amplification and aneuploidy? 111 e. Does LMO4 regulate the cellular response to ionizing radiation? 112 f. Does LMO4 bind to partners in a cell cycle specific manner? 113 Appendix 1 Protocols 115 A1.1 DNA Extraction from Cell Lines 115 A1.2 Protein Isolation from Cells 116 A1.3 Protein Isolation from Tissue 116 A1.4 Trizol RNA extraction from cells 117 A1.5 Removing DNA contamination from extracted RNA 118 A1.6 RT with SuperScript II (Invitrogen) 119 A1.7 Taqman ABI Assay 120 A1.8 Promoter Transfection and Luciferase Assay 121 A1.9 siRNA transfections 122 A1.10 Western Blot Protean Xi System 123 vi A1.11 Propidium Iodide Protocol for FACS 124 A1.12 BrdU Immunofluorescense 125 A1.13 γ-tubulin and α-tubulin staining 126 Bibliography 127 vii LIST OF TABLES Table 2.1: LMO4 mRNA expression in murine models of breast cancer. 48 Table 2.2: LMO4 overexpression is associated with high-grade, poorly differentiated breast cancers. 65 Table 3.1: LMO4 overexpression is associated with basal-type, BRCA1 mutation-associated and ER-negative breast cancers 80 viii LIST OF FIGURES Figure 1.1: Breast cancer is a heterogeneous disease. 2 Figure 1.2: The origins of breast cancer subtypes. 4 Figure 1.3: Regulation of the cell cycle. 7 Figure 1.4: Estrogen Receptor signaling mechanisms. 17 Figure 1.5: ErbB2 signaling pathways. 23 Figure 1.6: LMO4 regulates transcription in a context-dependent manner. 34 Figure 2.1: LMO4 mRNA and protein are increased in ErbB2-induced tumors. 48 Figure 2.2: LMO4 expression requires ErbB2 and PI3K activity. 50 Figure 2.3: ErbB2 and PI3K activity are necessary for LMO4 expression. 51 Figure 2.4: Overexpression of ErbB2 is sufficient to induce LMO4 expression. 53 Figure 2.5: LMO4 regulates proliferation of ErbB2-dependent breast cancer cells. 55 Figure 2.6: LMO4 is required for maintenance of Cyclin D1 mRNA and protein expression in cells with constitutive ErbB2 signaling as well as for heregulin-induced upregulation of Cyclin D1. 57 Figure 2.7: LMO4 regulates G2/M transition by inducing cullin-3 expression. 59 Figure 2.8: LMO4 expression oscillates throughout the cell cycle with peak expression occurring at G2/M phase. 63 Figure 2.9: LMO4 is an essential intermediate of ErbB2-induced proliferation. 64 Figure 3.1: LMO4 mRNA is highly expressed in BRCA1 mutant and ERα-negative breast cancers. 78 Figure 3.2: LMO4 expression is elevated in tumors with high mitotic indices. 82 Figure 3.3: Cytoplasmic LMO4 expression in human tumors. 83 ix Figure 3.4: LMO4 regulates G2/M progression in luminal and basal-like breast cancer cells. 85 Figure 3.5: Loss of LMO4 increases cell death in breast cancer cells representing luminal and basal subtypes. 86 Figure 3.6: Alterations in LMO4 levels induce centrosome amplification. 88 Figure 3.7: LMO4 overexpression in T47D breast cancer cells. 89 Figure 3.8: Altered LMO4 levels induce the formation of abnormal mitotic spindles. 91 Figure 3.9: LMO4 localizes to the centrosome. 93 Figure 3.10: LMO4 co-localizes to the centrosome in T47D cells. 93 Figure 4.1: Does LMO4 regulate the G2/M checkpoint and/or centrosome amplification by modulating BRCA1 activity? 107 x ACKNOWLEDGMENTS The best science is the work of many and I owe many special thanks. First, to Dr. Ruth Keri, thank you for taking me under your wings and teaching me all about transcription as well as tenacity and vision. Thank you also for all those yummy peanut butter blossom cookies! Second, I am especially grateful to my other thesis committee members – Yu-Chung Yang, George Dubyak, Julian Kim, David Schultz and Pamela Davis – for their helpful advice throughout the years and for keeping me focused. If I have learned one thing from my committee is to step back, analyze and question everything. Although this work has taken several years, I admit that it would not have been as full of fun experiences where I not in the Keri Lab. I am indebted to the Keri Lab members for throwing me into the pool of science and the labyrinth of life. Thank you also for helping me evaluate so many personal and scientific issues as well as for your guidance and patience. In particular, a belated apology to Kristen Lozada for suffering the brunt of (my few) obsessive- compulsive tendencies. Also, thanks Kris for lending me your magic centrosome yuyu! Thanks to Darcie Seachrist for her wonderful optimism and for providing a balance of sweetness for the Keri Lab’s sometimes way-too-frank attitude and many more thanks for your beautiful in situ and IHC expertise! I am also most obliged to Melissa Landis and Erin Milliken for their mentoring during my formative lab years. To Jonathan Mosley, thanks for all your statistical help and for sharing your perspective on life, “life is not a dress-rehearsal”, after all! I am also appreciative of Emhonta Johnson’s help with cloning and viral work but most of all for his attempts to keep me social and for improving my vocabulary with such terms as “jibby-jabba” and “excessive eye- xi ballage”. Many, many thanks to Gina for all our scientific ‘consultations’. I will never earn enough money to repay you! In addition, I am very grateful to Melissa for rejuvenating my interest in LMO4 and for introducing me to the magical world of Killer Bunnies and the sweet, sweet world of cake decorating. Muchas gracias también a mi nueva familia en Cleveland, mis hermanitas Dolly y Yeritza, y el desertor Carlitos as well as Candice and Jeremy for keeping my life merry. Finally, I appreciate my family’s constant support over the years. Moving to Cleveland was not an easy move and I am much obliged to my family (Mami, Papi and Ian) and my cousins, Yamil and Mairim, for taking time to visit and rescuing me from the every day routine. Furthermore, I am infinitely grateful to Matthew for being my pillar and my source of encouragement and laughter. Many more thanks for keeping me so well-fed and so much loved! xii LIST OF ABBREVIATIONS AIB1 Amplified In Breast Cancer AKT Murine thyme viral (v-AKT) oncogene homolog-1 APC/C Anaphase Promoting Complex ATM Ataxia Telangiectasia ATR Ataxia Telangiectasia and Rad3-related BARD BRCA1-Associated Ring Domain1 BMP Bone Morphogenetic Protein BRCA1/2 Breast Cancer