ROLES OF THE ESTROGEN RECEPTORS AND THE NUCLEAR MATRIX IN BREAST CANCER DEVELOPMENT AND TAMOXIFEN RESISTANCE by Miranda Jean Sarachine B.S. Biology, Allegheny College, 2004 Submitted to the Graduate Faculty of The University of Pittsburgh School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2009 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Miranda Jean Sarachine It was defended on September 10th, 2009 and approved by Bruce Freeman, Ph.D., Pharmacology and Chemical Biology Thomas Conrads, Ph.D., Pharmacology and Chemical Biology Jean Latimer, Ph.D., Obstetrics, Gynecology, and Women’s Health Guillermo Romero, Ph.D., Pharmacology and Chemical Biology Billy W. Day, Ph.D., Major Thesis Advisor, Pharmaceutical Sciences ii Copyright © by Miranda Jean Sarachine 2009 iii ROLES OF THE ESTROGEN RECEPTORS AND THE NUCLEAR MATRIX IN BREAST CANCER DEVELOPMENT AND TAMOXIFEN RESISTANCE Miranda Jean Sarachine, PhD University of Pittsburgh, 2009 In the United States in 2009, 192,370 women are expected to be diagnosed with invasive breast cancer, and 62,280 with in situ disease. About 70% of these cases are estrogen receptor positive (ER+). There are two isoforms of the ER, α and β, that differ somewhat in structure and action. ERβ expression plays a protective role in breast cancer, and selective targeting of this isoform would have many beneficial effects. Tamoxifen has long been the standard of care for patients with ER+ breast cancer. A major problem with tamoxifen is the development of drug resistance. One of the mechanisms proposed for the development of tamoxifen resistance involves the loss of ERβ expression. The first objective of this study was to screen a library of biphenyl C- cyclopropylalkylamides for their ability to function as ERβ-selective ligands. Two compounds were identified with modest selectivity for ERβ and anti-proliferative effects in breast cancer cells where they inhibited expression of c-Myc. The nuclear matrix (NM), the structural scaffolding of the nucleus, plays a major role in many fundamental processes of the cell. Using the ER+ breast cancer cell line MCF-7 and an antiestrogen resistant derivative, along with subtype selective ER ligands, alterations in the abundance of specific proteins present in the NM were identified using a mass spectrometry (MS)-based relative quantitative methodology. Some of the most interesting proteins with altered abundance are NuMA, serpin H1, hnRNP R, and dynein heavy chain 5. These proteins iv may represent putative biomarkers to customize treatment. The alterations also provide a mechanistic understanding of tamoxifen resistance. The NM was also investigated by MS in the earliest stage of breast cancer, ductal carcinoma in situ (DCIS), utilizing novel cell lines derived from normal (breast reduction), DCIS, and non-diseased contralateral breast surgical specimens. Two of the interesting proteins found to be altered in DCIS were HSP90 and EEF1D. These studies may provide biomarkers to aid in the diagnosis and treatment of breast cancer. In addition by understanding the mechanism behind the development of breast cancer, prevention becomes a possibility. v TABLE OF CONTENTS ACKNOWLEDGEMENTS ......................................................................................................XV ABBREVIATIONS..................................................................................................................XIX 1.0 INTRODUCTION ............................................................................................................... 1 1.1 THE ESTROGEN RECEPTORS.............................................................................. 1 1.1.1 Overview ........................................................................................................... 1 1.1.2 Structure ........................................................................................................... 3 1.1.3 Mechanism of Action ....................................................................................... 4 1.1.3.1 Genomic Signaling................................................................................ 5 1.1.3.2 Non-genomic Signaling......................................................................... 6 1.1.4 Ligands.............................................................................................................. 7 1.2 BREAST CANCER................................................................................................... 10 1.2.1 Overview ......................................................................................................... 10 1.2.2 The Estrogen Receptors in Breast Cancer................................................... 11 1.2.3 Therapy Strategies ......................................................................................... 13 1.2.3.1 Surgery................................................................................................. 14 1.2.3.2 Radiation Therapy.............................................................................. 14 1.2.3.3 Systemic Therapy................................................................................ 15 1.2.4 Tamoxifen Resistance .................................................................................... 18 vi 1.2.5 Biomarkers ..................................................................................................... 21 1.2.5.1 Overview.............................................................................................. 21 1.2.5.2 Breast Cancer Biomarkers in Clinical Use....................................... 22 1.2.5.3 Isobaric Tags for Relative and Absolute Quantitation ................... 24 1.3 THE NUCLEAR MATRIX ...................................................................................... 26 1.3.1 Overview ......................................................................................................... 26 1.3.2 Alterations to the Nuclear Matrix in Cancer............................................... 28 1.3.3 Alterations to the Nuclear Matrix in Breast Cancer................................... 31 1.4 HYPOTHESIS AND SPECIFIC AIMS .................................................................. 36 1.4.1 Hypothesis....................................................................................................... 36 1.4.2 Specific Aims .................................................................................................. 36 2.0 AN ER BETA SELECTIVE BIPHENYL C-CYCLOPROPYLALKYLAMIDE THAT INHIBITS THE GROWTH OF BREAST CANCER CELLS IN VITRO ............................ 38 2.1 ABSTRACT ............................................................................................................... 38 2.2 INTRODUCTION ..................................................................................................... 39 2.3 RESULTS................................................................................................................... 44 2.3.1 Binding to the Estrogen Receptors ............................................................... 44 2.3.2 Inhibition of the Growth of Breast Cancer Cells ........................................ 46 2.3.3 Effects on the Protein Levels of c-Myc, p21, and p27................................. 49 2.4 DISCUSSION............................................................................................................. 51 2.5 MATERIALS AND METHODS.............................................................................. 54 2.5.1 Fluorescence Polarization Assay................................................................... 54 2.5.2 Cell Proliferation Assay................................................................................. 55 vii 2.5.3 Western Blots.................................................................................................. 56 3.0 SPECIFIC ALTERATIONS TO THE NUCLEAR MATRIX IN A TAMOXIFEN RESISTANT CELL LINE ......................................................................................................... 58 3.1 ABSTRACT ............................................................................................................... 58 3.2 INTRODUCTION ..................................................................................................... 59 3.3 RESULTS................................................................................................................... 63 3.3.1 Estrogen Receptor Expression in MCF-7/LY2 Breast Cancer Cells ........ 63 3.3.2 Nuclear Matrix Proteins Identified in MCF-7 Cells ................................... 64 3.3.3 Nuclear Matrix Protein Abundance Levels Altered in MCF-7/LY2 Cells 68 3.3.4 Nuclear Matrix Protein Abundance Levels Altered in MCF-7 and MCF- 7/LY2 Cells in Response to PPT ............................................................................... 70 3.3.5 Nuclear Matrix Protein Abundance Levels Altered in MCF-7 and MCF- 7/LY2 Cells in Response to MPP .............................................................................. 71 3.3.6 Nuclear Matrix Protein Abundance Levels Altered in MCF-7 and MCF- 7/LY2 Cells in Response to DPN............................................................................... 73 3.3.7 Western Blot Validation ................................................................................ 75 3.3.8 Comparison of the Different Workflows ..................................................... 77 3.4 DISCUSSION............................................................................................................