INTERRELATIONSHIPS OF THE ESTROGEN-PRODUCING ENZYMES NETWORK IN BREAST CANCER DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Wendy L. Rich, M.S. ∗∗∗∗∗ The Ohio State University 2009 Dissertation Committee: Approved by Pui-Kai Li, Ph.D., Adviser Robert W. Brueggemeier, Ph.D. Karl A. Werbovetz, Ph.D. Adviser Graduate Program in Pharmacy Charles L. Shapiro M.D. ABSTRACT In the United States, breast cancer is the most common non-skin malignancy and the second leading cause of cancer-related death in women. However, earlier detection and new, more effective treatments may be responsible for the decrease in overall death rates. Approximately 60% of breast tumors are estrogen receptor (ER) positive and thus their cellular growth is hormone-dependent. Elevated levels of estrogens, even in post- menopausal women, have been implicated in the development and progression of hormone-dependent breast cancer. Hormone therapies seek to inhibit local estrogen action and biosynthesis, which can be produced by pathways utilizing the enzymes aromatase or steroid sulfatase (STS). Cyclooxygenase-2 (COX-2), typically involved in inflammation processes, is a major regulator of aromatase expression in breast cancer cells. STS, COX-2, and aromatase are critical for estrogen biosynthesis and have been shown to be over-expressed in breast cancer. While there continues to be extensive study and successful design of potent aromatase inhibitors, much remains unclear about the regulation of STS and the clinical applications for its selective inhibition. Further studies exploring the relationships of STS with COX-2 and aromatase enzymes will aid in the understanding of its role in cancer cell growth and in the development of future hormone- dependent breast cancer therapies. ii After confirming the high potency of two STS enzyme inhibitors DU-14 and DU- 15 in MCF-7 and MDA-MB-231 breast cancer cells, our initial studies investigated the effects of these compounds on STS, aromatase and COX-2 gene expression by Real- Time RT-PCR, on cancerous cellular growth using the Promega® MTS assay, and on STS enzyme activity using a tritium conversion radioassay. We then examined the individual effects, if any, of several COX isozyme inhibitors (celecoxib, NS-398, SC-560 and six non-steroidal anti-inflammatory drugs), aromatase inhibitors (letrozole and exemestane), and anti-estrogen 4-hydroxy tamoxifen. We found that these alternate target therapies can affect STS in vitro. To examine combinational effects, DU-14 and DU-15 were also combined with these same drugs. Furthermore, breast cancer cells were treated with potential regulatory factors, such as cytokines, steroids, and PKA and NK- κB pathway inhibitors, to better understand the regulation of STS and its interrelationships with aromatase and cyclooxygenase enzymes. iii Dedicated to my parents, Donal and Diana Rich, and their mothers, Beverly Rich and Betty Patterson. iv ACKNOWLEDGMENTS I wish to thank my co-advisors, Dr. B and Dr. Li, for all of their support and creativity in developing my thesis work. I am also grateful to my committee members for their precious time and patience. Thank you to my lab partners in crime, Drs. Yasuro Sugimoto, Danyetta Davis, Bin Su, Edgar Diaz-Cruz, Jennifer Whetstone, Michael Darby, and Ms. Serena Landini. I couldn’t have asked for a more entertaining lab group! Thank you to College of Pharmacy and CBIP staff members, especially Kathy Brooks, Ruth Luketic, Kelli Ballouz , Mary Kivel, Yolanda Hampton, Nancy Gilbert, Jonathan Gladden, and Casey Hoerig. A great big hug goes to my best friend and big sister, Lindsey Rich. Even when we’re both “doctors,” I’ll always be your Toots. I would also like to thank Tom, Maddie and Emma for the love and distractions along this tedious but interesting journey. And to Mr. Ralph (Rock) Sylvester Dunlap, III, I will never forget your loving Christian spirit and silent encouragement during your battle and loss to breast cancer. For funding, I greatly appreciate and am indebted to the NIH Chemistry-Biology Interface Program (Training Grant NIH/NIGMS T-32-GM008512), the College of Pharmacy Chih-Ming and Jane Chen Graduate Fellowship in Medicinal Chemistry, and the American Foundation for Pharmaceutical Education (APFE) Graduate Fellowship. v VITA April 30, 1981………………………Born – Wichita, Kansas, USA 2003…………………………………B.S. Chemistry, Georgia Institute of Technology 2006…………………………………M.S. Medicinal Chemistry and Pharmacognosy, The Ohio State University 2003 – present………………………NIH Trainee/Graduate Research Fellow, The Ohio State University PUBLICATIONS Research Publication 1. M.R. Hibbs; M. Vargas; J. Holtzclaw; W. Rich; D.M. Collard; D.A. Schiraldi. Synthesis and Characterization of PET-Based Liquid Crystalline Copolymers Containing 6-Oxynaphthalene-2-carboxylate and 6-Oxyanthracene-2-carboxylate Units. Macromolecules, 2003, 36, 7543 – 7551. 2. B. Pandit; Y. Sun; P. Chen; D.L. Sackett; Z. Hu; W. Rich; C. Li; A. Lewis; K. Schaefer; P.-K. Li. Structure-Activity-Relationship Studies of Conformationally Restricted Analogs of Combretastatin A-4 Derived from SU5416. Bioorg. & Med. Chem. 2006, 14, 6492 – 6501. FIELDS OF STUDY Major Field: Pharmacy vi TABLE OF CONTENTS Page Abstract............................................................................................................................ ii Dedication....................................................................................................................... iv Acknowledgments .......................................................................................................... v Vita ................................................................................................................................. vi List of Tables................................................................................................................... x List of Figures ................................................................................................................ xi List of Abbreviations ...................................................................................................... xv Chapters: 1. Introduction 1.1. Breast cancer: Overview.…….………………………………..………….. 1 1.1.1. Breast cancer statistics….………………………......….……………. 1 1.1.2. Breast cancer development..………………...……...……….............. 2 1.1.3. Breast cancer risk…………..………………...………........………… 3 1.1.4. Molecular profiling in breast cancer………………………………… 5 1.1.5. Breast cancer in men………..…………………...…………………... 6 1.1.6. Current treatments…………..……………………...…………........... 6 1.1.7. Hormone dependence in breast cancer…..…………...…...………….9 1.1.8. Origins of estradiol precursors…………………….........…………… 10 1.2 Steroid Sulfatase…………………………………………………………… 15 1.2.1. Expression, function and regulation………………………….……... 14 1.2.2. Role in breast cancer………………………………………………… 17 1.2.3. STS inhibitors……………………………………………………….. 18 1.3 Aromatase………………………………………………………………….. 25 1.3.1. Expression, function and regulation………………………………… 25 1.3.2. Role in breast cancer………………………………………………… 26 1.3.3. Aromatase inhibitors………………………………………….…...... 26 1.4. Cyclooxygenases ………………………………………………………......29 1.4.1. Function and regulation…………………………………...….…....... 29 1.4.2. Role in breast cancer …………………………………………...…… 30 1.4.3. COX inhibitors and NSAIDs…………………………………...…… 32 1.5. Summary: Interrelationships between enzymes…………………………… 37 1.6. References…………………………………………………………………. 39 vii 2. Statement of the research problem and specific aims……………………………… 43 2.1. The research problem………………………………………….………….. 43 2.2. Specific aims…………………………………………………………..…... 46 3. Materials and methods……………………………………………………………... 47 3.1. Chemicals, biochemicals and reagents……………………………………. 47 3.2. Cell culture………………………………………………………………… 48 3.3. RNA extraction……………………………………………………………. 48 3.4. cDNA synthesis…………………………………………………………… 51 3.5. Real-time quantitative RT-PCR…………………………………………… 51 3.6. STS enzyme radioactivity assay………………………………………….. 54 3.7. DNA assay………………………………………………………………… 54 3.8. Cell proliferation assay (MTS) …………………………………………… 55 3.9. Statistical analysis…………………………………………………………. 55 4. Effects of steroid sulfatase inhibitors on mRNA expression, STS enzyme activity and cellular proliferation……………………………………………………………….. 56 4.1. Introduction………………………………………………………………... 56 4.2. Results and discussion…………………………………………………...... 61 4.2.1. Steroid sulfatase enzyme activity……................................................ 61 4.2.2. Gene expression……………………………………………………... 64 4.2.3. Breast cancer cell growth……………………………………………. 74 4.3. Conclusions………………………………………………………………... 77 4.4. References…………………………………………………………………. 79 5. Effects of potential regulatory factors on mRNA expression, STS enzyme activity and cellular proliferation……………………………………………………………….. 81 5.1. Introduction………………………………………………………………... 81 5.1.1. STS promoters………………………………………………………. 81 5.1.2. Previous examinations of potential regulatory factors………………. 85 5.1.3. Estrogen deprivation………………………………………………… 90 5.1.4. Conflicting reports with estrogen treatment………………………… 91 5.2. Results and discussion…………………………………………………….. 93 5.2.1. STS promoter search………………………………………………… 93 5.2.2. Steroid sulfatase expression…………………………………………. 94 5.2.3. Cyclooxygenase-2 expression……………………………………….. 97 5.2.4. Aromatase expression……………………………………………….. 99 5.2.5. NF-κB and cAMP pathway regulators……………………………… 101 5.2.6. Estrogen deprivation………………………………………………… 106 viii 5.3.