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TRANSCRIPTIONAL REGULATION OF ESTROGEN RECEPTOR ALPHA TARGET GENES BY HEXAMETHYLENE BISACETAMIDE-INDUCIBLE GENE 1 (HEXIM1) AND ITS ROLE IN MAMMARY GLAND DEVELOPMENT AND BREAST CANCER by NDIYA OGBA Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Monica M. Montano Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January, 2010 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. TABLE OF CONTENTS Title page i Signature sheet ii Table of contents 1 List of Tables 4 List of Figures 5 Acknowledgements 8 List of abbreviations 9 Abstract 15 CHAPTER I: Introduction, review of literature and statement of purpose Introduction 17 Review of literature 18 Statement of purpose 47 CHAPTER II: HEXIM1 regulates 17β-estradiol/Estrogen Receptor-α- mediated expression of cyclin D1 is modulated by P-TEFb in mammary cells Abstract 50 1 Introduction 51 Materials and methods 54 Results 60 Discussion 70 Acknowledgments 74 Figures and Tables 76 CHAPTER III: HEXIM1 modulates vascular endothelial growth factor expression and function in breast cancer cells Abstract 97 Introduction 98 Materials and methods 100 Results 106 Discussion 115 Acknowledgments 119 Figures 120 CHAPTER IV: Summary and future directions Summary 133 Future directions 136 2 Concluding remarks 154 BIBLIOGRAPHY 155 3 LIST OF TABLES Table II-1. Primer sequences used for transgenic mice genotyping 93 Table II-2. Primers used for reverse transcriptase PCR (RT-PCR) reactions 94 Table II-3. miRNA oligonucleotide sequences used for HEXIM1 knockdown experiments 95 Table II-4. Primers used in chromatin immunoprecipitation (ChIP) experiments 96 4 LIST OF FIGURES Figure I-1. Structures of Estrogen Receptors alpha and beta with identified activation function regions 21 Figure I-2. Structure of HEXIM1 44 Figure II-1. Increased HEXIM1 expression inhibits estrogen- regulated mammary gland morphogenesis due to changes in proliferation and apoptosis 76 Figure II-2. Increased HEXIM1 expression inhibits E2-induced cyclin D1 expression and serine 2 phosphorylation of RNAP II in mouse mammary gland 78 Figure II-3. Increased HEXIM1 expression does not inhibit c-Myc expression in mouse mammary gland 80 Figure II-4. HEXIM1 regulates E2-induced cyclin D1 expression in breast epithelial cells 81 Figure II-5. Increased HEXIM1 expression inhibits E2-induced pS2 expression in breast epithelial cells 83 Figure II-6. Increased HEXIM1 expression leads to increase in HEXIM1 occupancy on DNA of ER target genes 85 Figure II-7. Effect of increased HEXIM1 expression on E2- dependent recruitment of ERα and P-TEFb (cyclin T1) to ER-responsive genes 87 Figure II-8. Increased HEXIM1 expression inhibits E2-induced P-TEFb activity and recruitment of serine 2 5 (hyperphosphorylated) RNA polymerase II to the coding region of ER-responsive genes 89 Figure II-9. Increased HEXIM1 expression inhibits E2-induced P-TEFb activity 91 Figure II-10. Proposed model for HEXIM1 action on ERα and P-TEFb at ER-responsive genes, pS2 and CCND1, in mammary cells 92 Figure III-1. Increased HEXIM1 expression inhibits E2-induced transcription of VEGF via ERα in a P-TEFb- independent manner in breast cancer cells 120 Figure III-2. Increased HEXIM1 expression inhibits E2-induced VEGF expression in ERα-expressing MDA-MB-231 cells 122 Figure III-3. Increased HEXIM1 expression inhibits E2-induced VEGF mRNA expression under hypoxia that correlates with a decrease in E2-induced HIF-1α expression 123 Figure III-4. Increased HEXIM1 expression inhibits E2-induced recruitment of HIF-1α to VEGF Hypoxia Response Element 125 Figure III-5. HEXIM1 modulates VEGF and HIF-1 expression and vascularization in mouse mammary gland 127 6 Figure III-6. Expression of HEXIM1 C-terminus mutant enhances carcinogen-induced mammary tumorigenesis and correlates with increased vascularization of tumors 129 Figure III-7. HEXIM1 C-terminus mutant inhibits P-TEFb activity and does not affect proliferation in mammary gland 131 Figure III-8. Model: HEXIM1 regulates VEGF expression via ER HIF-1 to modulate angiogenesis and tumorigenesis 132 Figure IV-1. Summary model of HEXIM1 mechanism of action in breast cancer 137 7 ACKNOWLEDGEMENTS I would like to take this opportunity to thank so many people who have guided me during this dissertation process. I was very privileged to work under the supervision and guidance of Dr. Monica Montano. Thank you very much, Monica, for looking out for my interests and giving me so many opportunities to advance as a graduate student and scientist and helping me grow as a person. I also want to give many thanks to all the members of my thesis committee, Dr. Yu-Chung Yang, Dr. George Dubyak, Dr. Koh Fujinaga and Dr. Noa Noy. I am thoroughly grateful for giving me your time, guidance and advice throughout the dissertation process. I am also indebted to the department of Pharmacology for giving me the opportunity to gain an education here and will always be grateful for all I have learned through my interactions with the faculty, students and staff during my time here at Case. The support from my lab mates, friends and family cannot really be put into words, so all I will say is thank you. There are so many of you, so I will save the trees and not name everyone. Just know that I thank God for putting all of you in my way so that we could get to know, learn from and support each other. I wish you all Godspeed on your journey and keep up a good attitude when you are able. Lastly, to my dear parents, thank you for being my heroes and supporting my dreams. 8 LIST OF ABBREVIATIONS 7SK snRNA Ubiquitous, non-coding small nuclear RNA AF Activation Function AI Aromatase inhibitor AR Androgen receptor AP1 Activator protein 1 AhR Aryl hydrocarbon receptor AIB Amplified In Breast cancer AKT V-Akt murine thymoma viral oncogene homolog 1 AML Acute myeloid leukemia BPA Bisphenol A BRCA1 Breast cancer-1 early onset CD Cluster of differentiation molecule CK Cytokeratin CR Coiled-coil region CBP Cyclic AMP response element binding protein CDK Cyclin dependent kinase 9 CLP-1 Cardiac lineage protein-1 CRE Cyclic AMP response element CSC Cancer stem cell CTD Carboxy terminal domain CYP2D6 Cytochrome P450 2D6 CARM1 Co-activator-associated arginine methyltransferase-1 ChIP Chromatin immunoprecipitation assay COBRA1 Cofactor of BRCA1 DBD DNA binding domain DPN Diarylpropionitrile DRB 5,6-dichloro-1-b-D-ribofuranosylbenzimidazole DMBA 7,12-dimethylbenz[a]anthracene DRIP Vitamin D-interacting protein DSIF DRB-sensitivity inducing factor E1 Estrone E2 17-beta estradiol E3 Estriol 10 EP300/p300 E1A-binding protein 300 ERα Estrogen Receptor alpha ERβ Estrogen Receptor beta ECM Extracellular matrix EGF Epidermal growth factor ERE Estrogen response element ERK Extracellular signal-regulated kinase EDG1 Estrogen down-regulated gene 1 EGFR Epidermal growth factor receptor FDA Food and drug administration FCP1 TFIIF-interacting CTD phosphatase GR Glucocorticoid receptor GTF General transcription factor GPR30 G-protein couple receptor 30 HAT Histone acetyltransferase HDM2 Human double minute-2 HIF Hypoxia inducible factor 11 HRT Hormone Replacement Therapy HSP Heat shock protein HAND1 Heart and neural crest derivatives expressed 1 HDAC Histone deacetylase HMBA Hexamethylene bisacetamide HEXIM1 Hexamethylene bisacetamide-inducible protein 1 HIV-LTR Human Immunodeficiency Virus long terminal repeat IGF-1 Insulin-like growth factor 1 IGF-1R Insulin-like growth factor 1 receptor Kd Dissociation constant MEC Mammary epithelial cell MAPK Mitogen-associated protein kinase MMTV-LTR Mouse mammary tumor virus long terminal repeat nM nanomolar NF-κB Nuclear factor kappa B NLS Nuclear localization signal NPM Nucleophosmin 12 NCoR Nuclear receptor corepressor NELF Negative elongation factor PR Progesterone receptor PARP-1 Poly(ADP)-ribose polymerase 1 PCAF p300/CBP-associated factor PI3K Phosphatidylinositol-3-kinase PLGA Poly(lactic-co-glycolic acid) PPAR-γ Peroxisome proliferation activated receptor gamma P-TEFb Positive transcription elongation factor b REA Repressor of ERα activity RTK Receptor tyrosine kinase RNAP II RNA polymerase II SP1 Specificity protein 1 SNP Single nucleotide polymorphism SRC Steroid receptor complex SERM Selective Estrogen Receptor Modulator SMRT Silencing mediator for retinoid and thyroid hormone 13 receptor STAT Signal Transducer and Activator of Transcription TFF1 Trefoil factor 1 TRAP Thyroid receptor-associated protein VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor 14 Transcriptional Regulation Of Estrogen Receptor Alpha Target Genes By Hexamethylene Bisacetamide-Inducible Gene 1 (HEXIM1) And Its Role In Mammary Gland Development And Breast Cancer Abstract by NDIYA OGBA Breast cancer is the second leading cause of death in women in the United States, making the search for more therapeutic targets in breast cancer etiology very significant.
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