Wayne State University Wayne State University Dissertations 1-1-2011 Estrogen sulfotransferase (sult1e1) expression and function in mcf10a-series breast epithelial cells: role as a modifier of breast carcinogenesis and regulation by proliferation state Jiaqi Fu Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Pathology Commons, and the Toxicology Commons Recommended Citation Fu, Jiaqi, "Estrogen sulfotransferase (sult1e1) expression and function in mcf10a-series breast epithelial cells: role as a modifier of breast carcinogenesis and regulation by proliferation state" (2011). Wayne State University Dissertations. Paper 308. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. ESTROGEN SULFOTRANSFERASE (SULT1E1) EXPRESSION AND FUNCTION IN MCF10A-SERIES BREAST EPITHELIAL CELLS: ROLE AS A MODIFIER OF BREAST CARCINOGENESIS AND REGULATION BY PROLIFERATION STATE by JIAQI FU DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2011 MAJOR: MOLECULAR AND CELLULAR TOXICOLOGY Approved by: Advisor Date DEDICATION I dedicate this work to my parents for all the sacrifices they have made on my behalf. To Luan, for his love and support during the preparation of this work. ii ACKNOWLEDGEMENTS I owe my deepest gratitude to Dr. Melissa Runge-Morris and Dr. Thomas A. Kocarek, for the encouragement and support throughout all these years of study. Their guidance helped me in all the time of research and writing of this thesis. It is an honor for me to thank the members of my dissertation committee, Drs. Fred Miller, John J. Reiners Jr., Malathy Puthan Veedu Shekhar for all their time and insightful criticisms that made this thesis possible. I would like to show my gratitude to my fellow graduate students, the current and former lab members for all their help and encouragement over the years. In particular, thank you Mary Dorothy Gargano and Patricia A. Mathieu for all your support and willingness to help in any way possible. Thanks to the support of National Institutes of Health National Institute of Environmental Health Sciences [Grant ES05823] which made this work possible. Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the projects. iii TABLE OF CONTENTS DEDICATION...................................................................................................................ii ACKNOWLEDGEMENTS................................................................................................iii LIST OF ABBREBIATIONS..............................................................................................v LIST OF FIGURES.........................................................................................................vii CHAPTER 1: INTRODUCTION.......................................................................................1 Breast cancer…………………………………………...................................1 Estrogen and breast cancer……………………………………………….....3 MCF10A lineage cell lines…………………………………………….….....20 Statement of problem………………………………………………………..30 CHAPTER 2: Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression…………………………….………....32 CHAPTER 3: Regulation of SULT1E1 expression by confluency of MCF10A breast epithelial cells: Role of the AhR………..…………………...……....52 CHAPTER 4: Tobacco smoke condensate (TSC) down-regulates estrogen sulfotransferase (SULT1E1) expression in MCF10A human breast epithelial cells through an AhR-mediated mechanism……………………80 CHAPTER 5: Knockdown of estrogen inactivating enzyme SULT1E1 by RNAi accelerates tumorigenesis in vivo………………………………..………....90 REFERENCES.………………………………………………………………………………109 ABSTRACT…….……………………………………………………………………………..188 AUTOBIOGRAPHICAL STATEMENT……………………………………………………..192 iv LIST OF ABBREVIATIONS 2-OH 2-hydroxylation 2-OHE 2-hydroxylation catechol estrogen 2-OHE2 2-hydroxyestradiol 4-OH 4-hydroxylation 4-OHE 4-hydroxylation catechol estrogen 4-OHE2 4-hydroxyestradiol 17β-HSD 17β-Hydroxysteroid Dehydrogenases AhR Aryl Hydrocarbon Receptor ARNT AhR Nuclear Translocator BMI Body mass index BP benzo[a]pyrene BrU bromouridine cAMP Cyclic AMP CE catecholestrogen COMT Catechol-O-methyltransferase Ct cycle threshold CYP Cytochromes P450 CYP19 Aromatase CYP1A1 Cytochrome P450 1A1 CYP1B1 Cytochrome P450 1B1 DCIS Ductal Carcinoma in situ DHEA dehydroepiandrosterone DMEM/F12 Dulbecco‟s Modified Eagle Medium/Ham‟s F12 DMSO Dimethyl Sulfoxide DRE dioxin response element or xenobiotic response element or AhR•ARNT heterodimer binding site E1 estrone E1S Estrone Sulfate E2 17β-Estradiol E3 estriol EREs Estrogen response elements EGF Epidermal growth factor ER Estrogen receptor ERDs Estrogen receptor down-regulators ERK Extracellular signal-regulated kinases GAPDH Glyceraldehydes-3-phophate dehydrogenase GPR30 G protein-coupled receptor 30 GPER G protein-coupled estrogen receptor HDACs Histone deacetylases HB-EGF heparin-binding EGF HPRT1 Hypoxanthine phosphoribosyltransferase 1 HRT hormone replacement therapy v ICI ICI 182,780 IHC Immunohistochemistry MAPK Mitogen-activated protein kinase MEs methoxyestrogen MC 3-methylcholanthrene MNF 3‟-methoxy-4‟-nitroflavone OVX Ovariectomized PAHs Polycyclic aromatic hydrocarbons PAPS 3‟-phopphoadenosine-5‟-phosphosulfate PI3k Phosphoinositide 3-kinase PBS Phosphate-buffered saline PR progesterone receptor RSK Ribosomal S6 kinase SERMs Selective estrogen receptor modulators STS Steroid sulfatase SULTs cytosolic sulfotransferases SULT1E1 Sulfotransferase family 1E; Estrogen sulfotransfrase SULT1A1 Sulfotransferase family 1A SULT2A1 Sulfotransferase family 2A SULT2B1 Sulfotransferase family 2B TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin TDLU Terminal duct lobular unit TNBC Triple negative breast cancer TSA Trichostatin A TSC Tobacco smoke condensate UGTs UDP-glucuronosyltransferases vi LIST OF FIGURES CHAPTER 1 Fig. 1.1. Pathways of oxidative estrogen metabolism....................................................16 Fig. 1.2. The process of human breast cancer progression….......................................21 Fig. 1.3. Histological features of hyperplasia.................................................................22 Fig. 1.4. Atypical hyperplasia.........................................................................................23 Fig. 1.5.Ductal carcinoma in situ ...................................................................................24 Fig.1.6. Comedo, solid, cribriform, and micropapillary ductal carcinoma in situ.............24 Fig. 1.7. Nuclear grading................................................................................................25 Fig. 1.8. The diagnostic distinction between atypical ductal hyperplasia and ductal carcinoma in situ...............................................................................................27 Fig. 1.9. The production of MCF10A lineage human breast cancer cell lines................29 CHAPTER 2 Fig. 2.1. Pathways of estrogen metabolism, bioactivation, and action...........................33 Fig. 2.2. Expression of SULT1E1 mRNA and protein in the MCF10A-derived lineage cell culture model for breast cancer progression and in MCF7 cells...............39 Fig. 2.3. Expression of estrogen metabolism enzymes in MCF10A series and MCF7 cell lines..........................................................................................................41 Fig. 2.4. ERα and ERβ mRNA expression in MCF10A series and MCF7 cell lines.......42 Fig. 2.5. Effects of TSA treatment on ERα and ERβ mRNA expression in MCF10A vii series and MCF7 cell lines and on E2-mediated activation of an estrogen- responsive reporter gene................................................................................43 Fig. 2.6. TSA treatment effects on estrogen metabolism enzyme expression in MCF10A series and MCF7cell lines................................................................46 CHAPTER 3 Fig. 3.1. SULT1E1 expression in pre-confluent and confluent MCF10A cells...............63 Fig. 3.2. Relative rates of SULT1E1 mRNA synthesis and degradation in pre-confluent and confluent MCF10A cells......................................................64 Fig. 3.3. Confluency-related decrease in miR-221 and miR-100*..................................65 Fig. 3.4. Overexpression of miR-100* or miR-221 did not suppress SULT1E1 expression in confluent MCF10A cells.............................................................66 Fig. 3.5. Indices of Aryl hydrocarbon receptor (AhR) activity in pre-confluent and confluent MCF10A cells..................................................................................67 Fig. 3.6. Expression of AhR and ARNT in pre-confluent and confluent MCF10A cells..68 Fig.3.7. Effects of AhR agonist and antagonist treatments on CYP1A1 and SULT1E1 expression in MCF10A cells............................................................69 Fig. 3.8. Effect of AhR knockdown on CYP1A1 and SULT1E1 expression in pre-confluent MCF10A cells............................................................................70 Fig. 3.9. Effects of MNF treatment, cell confluency, or AhR knockdown on luciferase expression from a reporter plasmid containing 7073nt