AN ANALYSIS OF ESTROGEN METABOLISM AND BREAST CANCER RISK by Stacy Monique Lloyd BS, Prairie View A&M University, 2004 MPH, University of Pittsburgh, 2007 Submitted to the Graduate Faculty of Graduate School of Public Health in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2010 UNIVERSITY OF PITTSBURGH Graduate School of Public Health This dissertation was presented by Stacy Monique Lloyd It was defended on June 30, 2009 and approved by Committee Member: Robert Ferrell, PhD, Professor Department of Human Genetics Graduate School of Public Health University of Pittsburgh Committee Member: Susanne Gollin, PhD, Professor Department of Human Genetics Graduate School of Public Health University of Pittsburgh Committee Member: Seymour Garte, PhD, Professor, Department of Environmental and Occupational Health Graduate School of Public Health University of Pittsburgh Committee Co-Chair: Emanuela Taioli, MD, PhD, Professor Department of Epidemiology Graduate School of Public Health University of Pittsburgh Dissertation Advisor: Eleanor Feingold, PhD, Professor Department of Human Genetics Graduate School of Public Health University of Pittsburgh ii Copyright © by Stacy Monique Lloyd 2010 iii AN ANALYSIS OF ESTROGEN METABOLISM AND BREAST CANCER RISK Stacy Monique Lloyd, PhD University of Pittsburgh, 2010 Breast cancer is the most common noncutaneous form of cancer among women in the US. In recent years, the overall mortality rate has declined, yet there still exists a significant racial disparity in the incidence and mortality between African American and Caucasian women. While numerous hypotheses have been proposed to explain this difference, few offer a biological explanation. There is a well established association between estrogens and breast cancer risk, and the ratio of two estrogen metabolites, 2-hydroxyestrone (2OHE1) and 16α-hydroxyestrone (16OHE1), has been implicated as a marker of breast cancer risk. Many studies have also assessed the relationship between endogenous estrogens and mammographic density. Mammographic density is one of the strongest predictors of breast cancer risk, but the mechanism by which it influences this risk remains unknown. Nonetheless, few have examined mammographic density in relation to the 2OHE1:16OHE1 estrogen metabolite ratio (EMR). Research suggests that the Cytochrome P450 1B1 (CYP1B1) gene may also mediate breast cancer risk, as this gene is very active in estrogen metabolism. In fact, the Leu432Val polymorphism has reportedly been associated with urinary levels of the 2OHE1:16OHE1 EMR. The objective of this study was to investigate some of the relationships found among the 2OHE1: 16OHE1 EMR, CYP1B1 Leu432Val polymorphism, mammographic density, race, and iv breast cancer risk. The 2OHE1:16OHE1 EMR was associated with both breast cancer risk and the CYP1B1 Leu432Val polymorphism, yet, no association with breast cancer risk and this polymorphism was observed. This suggests that if the CYP1B1 Leu432Val polymorphism alters breast cancer risk, it does so through variations in the 2OHE1:16OHE1 EMR. When taking race into account, no association between mammographic density and the 2OHE1:16OHE1 EMR was observed. In culture, evidence was found to suggest that the 2OHE1:16OHE1 EMR is influenced by subcellular effects or other intrinsic factors (i.e. genetic variation), as passage number was the only significant contributor to the 2OHE1:16OHE1 EMR. The results of this study have great public health significance, as it provides a better understanding of the risk factors, including racial differences, and etiology of breast cancer, which will ultimately lead to better prevention and treatment for all women. v TABLE OF CONTENTS PREFACE .................................................................................................................................... XI 1.0 INTRODUCTION....................................................................................................... 1 1.1 BACKGROUND AND SIGNIFICANCE ...........................................................3 1.1.2 Breast Cancer ................................................................................................ 3 1.1.3 Incidence and Prevalence ............................................................................. 4 1.1.4 Etiology .......................................................................................................... 5 1.1.5 Risk Factors ................................................................................................... 6 1.1.6 Prevention and Screening........................................................................... 13 1.2 CLASSIC ENDOCRINOLOGY .......................................................................13 1.3 ESTROGENS AND BREAST CANCER .........................................................19 1.4 INTRACRINOLOGY ........................................................................................24 1.5 CYTOCHROME P450 1B1..................................................................................28 1.6 SUMMARY AND CONCLUSIONS .................................................................31 2.0 SPECIFIC AIMS……………. .......................................................................... 34 3.0 MANUSCRIPT ONE: THE EFFECT OF A CYP1B1 POLYMORPHISM ON THE 2OHE1:16OHE1 ESTROGEN METABOLITE RATIO AND BREAST CANCER RISK.....................................................................................................................37 3.1 ABSTRACT.........................................................................................................38 vi 3.2 INTRODUCTION ............................................................................................. 39 3.3 MATERIALS AND METHODS ...................................................................... 41 3.3.1 Study Population ......................................................................................... 41 3.3.2 DNA Extraction and Genotyping .............................................................. 42 3.3.3 2OHE1:16OHE1 Estrogen Metabolite Assay ............................................ 42 3.3.4 Statistical Analysis ...................................................................................... 43 3.4 RESULTS ........................................................................................................... 44 3.5 DISCUSSION ..................................................................................................... 47 4.0 MANUSCRIPT TWO: THE 2OHE1:16OHE1 ESTROGEN METABOLITE RATIO, MAMMOGAPHIC DENSITY, AND RACE: EXAMINING THE RELATIONSHIP BETWEEN COMPLEX TRAITS...............................................................53 4.1 ABSTRACT........................................................................................................ 54 4.2 INTRODUCTION ............................................................................................. 55 4.3 MATERIALS AND METHODS ...................................................................... 57 4.3.1 Study Population ......................................................................................... 57 4.3.2 Measures of Mammographic Density ....................................................... 58 TM 4.3.3 ESTRAMET 2OHE1 and 16αOHE1 Serum Assay .............................. 59 4.3.4 Statistical Analysis ...................................................................................... 60 4.4 RESULTS ........................................................................................................... 61 4.5 DISCUSSION ..................................................................................................... 65 5.0 MANUSCRIPT THREE: AN IN VITRO ANALYSIS OF THE 2OHE1:16OHE1 ESTROGEN METABOLITE RATIO……………. ................................................................. 71 5.1 ABSTRACT........................................................................................................ 72 vii 5.2 INTRODUCTION ............................................................................................. 73 5.3 MATERIALS AND METHODS ...................................................................... 75 5.3.1 Cell Lines and Chemicals ........................................................................... 75 5.3.2 Liquid Chromatography – Mass Spectrometry ....................................... 76 5.3.3 Statistical Analysis ...................................................................................... 77 5.4 RESULTS ........................................................................................................... 79 5.5 DISCUSSION ..................................................................................................... 83 6.0 AN ANALYSIS OF ESTROGEN METABOLISM AND BREAST CANCER RISK DISCUSSION.................................................................................................................... 89 APPENDIX A: TABLE 1: HIGHLY PENETRANT CANCER SYNDROMES AND BREAST CANCER RISK CITATIONS ................................................................................ 100 APPENDIX B: TABLE 2: LOW PENETRANCE BREAST CANCER SUSCEPTIBILITY VARIANTS CITATIONS ........................................................................................................ 102 BIBLIOGRAPHY ..................................................................................................................... 103 viii LIST OF TABLES Table 1. Highly Penetrant Breast Cancer Syndromes