INTERRELATIONSHIPS of the ESTROGEN-PRODUCING ENZYMES NETWORK in BREAST CANCER DISSERTATION Presented in Partial Fulfillment Of

Home , Atamestane, Estriol sulfamate, Niflumic acid, Toremifene

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

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 postmenopausal 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. Conclusions………………………………………………………………... 109 5.4. References…………………………………………………………………. 111

6. Effects of cyclooxygenase inhibitors on mRNA expression, STS enzyme activity and cellular proliferation……………………………………………………………….. 114 6.1. Introduction………………………………………………………………... 114 6.2. Results and discussion…………………………………………………….. 118 6.2.1. Gene expression……………………………………………………... 118 6.2.2. Steroid sulfatase enzyme activity……………………………………. 123 6.2.3. Breast cancer cell growth …………………………………………… 127 6.3. Conclusions………………………………………………………………... 129 6.4. References…………………………………………………………………. 131

7. Effects of estrogen receptor antagonists and aromatase inhibitors on mRNA expression, STS enzyme activity and cellular proliferation……………………….. 133 7.1. Introduction………………………………………………………………... 133 7.1.1. Aromatase…………………………………………………………… 133 7.1.2. Estrogen receptors…………………………………………………… 134 7.2. Results and discussion…………………………………………………….. 135 7.2.1. Gene expression……………………………………………………... 135 7.2.2. Steroid sulfatase enzyme activity……………………………………. 139 7.2.3. Breast cancer cell growth…………………………………………..... 141 7.3. Conclusions………………………………………………………………... 143 7.4. References…………………………………………………………………. 144

8. Effects of steroid sulfatase inhibitor combinations with aromatase and cyclooxygenase inhibitors………………………………………………………….. 145 8.1. Introduction………………………………………………………………... 145 8.2. Results and discussion…………………………………………………….. 146 8.2.1. COX inhibitor combinations………………………………………… 146 8.2.2. Aromatase/ER inhibitor combinations………………………………. 157 8.3. Conclusions………………………………………………………………... 167 8.4. References…………………………………………………………………. 170

9. Conclusions and future directions………………………………………………….. 171

Bibliography…………………………………………………………………………… 176

ix LIST OF TABLES

Table Page

1.1 Breast cancer risk factors……………………………………………………… 4

1.2 List of human sulfatases………………………………………………………...15

1.3 Structures of steroid sulfatase sulfamate-based inhibitors …………………….. 24

1.4 Structures of selective estrogen receptor modulator and aromatase inhibitors… 28

1.5 Structures of cyclooxygenase inhibitors……………………………….………. 36

3.1 Oligonucleotide primer and probe sequences for real time PCR……………… 53

3.2 Primer Mix ratios for use in RT-PCR experiments……………………………. 53

3.3 Mix ratios for use in RT-PCR experiments……………………………………. 53

5.1 Previous evidence of STS, aromatase, COX-2 regulation in breast cancer cells.89

5.2 Genomatrix Model Inspector search results…………………………………… 93

5.3 mRNA expression and STS activity ratios of LTED cells / normal MCF-7 cells……………………………………………………………………………. 107

6.1 Selectivity of various NSAIDs for COX-1 and COX-2 isoforms……………… 115

x LIST OF FIGURES

Figure Page

1.1 Side cross-section representation of the human breast………………………… 3

1.2 Steroidogenesis of androgens and estrogens from adrenal cortex products….... 11

1.3 Hormone-dependent breast cancer……………………………………….…….. 13

1.4 Representation of sulfamate-type STS inhibitors by SAR findings...... ……….. 19

1.5 Reaction mechanism of action of STS on natural substrate E1S and known STS sulfamate inhibitors…………………………………………………………….. 21

1.6 Actions of cyclooxygenase enzymes…………………………………………... 29

2.1 Tissue microarray analysis of biomarkers and molecular targets from fixed tissue specimens from breast cancer patients………...……………………………….. 45

4.1 STS sulfamate inhibitors designed by P.-K. Li and K. Selcer…………………. 57

4.2 Suppression of steroid sulfatase activity in MCF-7 and MDA-MB-231 breast cancer cells by DU-14 and DU-15……………………………………………... 62

4.3 DU-14 suppression of steroid sulfatase activity in MCF-7 and MDA-MB-231 breast cancer cells……………………………………………………………… 63

4.4 Effect of steroid sulfatase inhibitors with estrogens on STS mRNA expression..67

4.5 DU-15 suppression of STS mRNA expression in MCF-7 and MDA-MB-231 cells……………………………………………………………. 68

4.6 Effect of steroid sulfatase inhibitors with estrogens on CYP19 mRNA expression……………………………………………………………………… 70

xi 4.7 Effect of steroid sulfatase inhibitors with estrogens on COX-2 mRNA expression……………………………………………………………………… 72

4.8 Dose-response of DU-15 on COX-2 mRNA expression in MCF-7 cells……… 73

4.9 Proliferation assay of DU-14 in MCF-7 and MDA-MB-231 cells over 2, 4, and 7 days of treatment……………………………………………………………….. 75

4.10 Proliferation assay of DU-15 in MCF-7 and MDA-MB-231 cells over 2, 4, and 7 days of treatment……………………………………………………………….. 76

5.1 Schematic representation of STS exon structure………………………………. 84

5.2 NF-κB cellular activation by TNFα, IL-1, and phorbol esters………………… 88

5.3 STS mRNA expression in MCF-7 and MDA-MB-231 cells treated with potential growth effectors………………………………………………………………... 96

5.4 COX-2 mRNA expression in MCF-7 and MDA-MB-231 cells treated with potential growth effectors……………………………………………………… 98

5.5 CYP19 mRNA expression in MCF-7 and MDA-MB-231 cells treated with potential growth effectors……………………………………………………… 100

5.6 Effects of selective pathway effectors and key hormones on STS enzyme activity and STS mRNA expression……………………………………………………. 103

5.7 IC50 calculation of H-89 on STS enzyme activity…………………………….. 104

5.8 STS enzyme activity of Bay 11-7082 in MCF-7, MDA-MB-231, and MDA-MB- 231-ERα1 cells………………………………………………………………… 105

5.9 Basal mRNA expression in STS, COX-2 and CYP19 in normal MCF-7 cells and MCF-7-S cells………………………………………………………………….. 108

6.1 Effects of NSAIDs and COX inhibitors on aromatase activity in breast cancer cells…………………………………………………………………………….. 116

xii 6.2 Effects of COX inhibitors on STS mRNA expression in MCF-7 and MDA-MB-231 cells……………………………………………………………. 120

6.3 Effects of COX inhibitors on COX-2 mRNA expression in MCF-7 and MDA-MB-231 cells……………………………………………………………. 121

6.4 Effects of COX inhibitors on CYP19 mRNA expression in MCF-7 and MDA-MB-231 cell……………………………………………………………...122

6.5 Effect of COX inhibitors and NSAIDs on STS activity in MCF-7 cells………. 125

6.6 Effect of COX inhibitors and NSAIDs on STS activity in MDA-MB-231 cells. 126

6.7 Proliferation assay of MCF-7 and MDA-MB0231 cells over 2, 4, and 7 days of treatment with COX inhibitors………………………………………………….128

7.1 mRNA expression in MCF-7 cells with ER and aromatase inhibitors of STS, COX-2, and CYP19…………………………………………………………….. 136

7.2 mRNA expression in MDA-MB-231 cells with ER and aromatase inhibitors of STS, COX-2, and CYP19……………………………………………………….. 137

7.3 STS enzyme activity in MCF-7 and MDA-MB-231 cells with ER and aromatase inhibitors……………………………………………………………………….. 139

7.4 Cellular growth proliferation in MCF-7 and MDA-MB-231 cells with ER and aromatase inhibitors…………………………………………………………… 141

8.1 STS mRNA expression in MCF-7 and MDA-MB-231 after 24 incubation with combinations of COX inhibitors with STS inhibitors……………………. 149

8.2 COX-2 mRNA expression in MCF-7 and MDA-MB-231 after 24 incubation with combinations of COX inhibitors with STS inhibitors………… 150

8.3 CYP19 mRNA expression in MCF-7 and MDA-MB-231 after 24 incubation with combinations of COX inhibitors with STS inhibitors………… 151

8.4 STS enzyme activity in MCF-7 cells after 24 incubation with combinations of COX inhibitors with STS inhibitors…………………………………………… 153

xiii 8.5 STS enzyme activity in MDA-MB-231 cells after 24 incubation with combinations of COX inhibitors with STS inhibitors…………………………. 154

8.6 Proliferation assay of MCF-7 and MDA-MB0231 cells over 2, 4, and 7 days of COX inhibitor treatment……………………………………………………….. 156

8.7 STS mRNA expression in MCF-7 and MDA-MB-231 after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors…………. 159

8.8 COX-2 mRNA expression in MCF-7 and MDA-MB-231 after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors…………. 160

8.9 CYP19 mRNA expression in MCF-7 and MDA-MB-231 after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors…………. 161

8.10 STS enzyme activity in MCF-7 cells after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors…………………………... 163

8.11 STS enzyme activity in MDA-MB-231 cells after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors…………. 164

8.12 Proliferation assay of MCF-7 and MDA-MB-231 cells over 2, 4, and 7 days of aromatase/ER inhibitor treatment……………………………………………… 166

xiv LIST OF ABBREVIATIONS

4-MUS – 4-methylumbelliferyl sulfate, 4-OHT – 4-hydroxytamoxifen 5-FU – 5-fluorouracil AG – aminoglutethimide ATCC – American Type Culture Collection DbcAMP – dibutyryl-cyclic adenosine monophosphate BRCA 1/2 – breast cancer genes 1 and 2 cAMP – cyclic adenosine monophosphate cDNA – complementary deoxyribonucleic acid COX 1/2 – cyclooxygenases 1 and 2 CYP19 – aromatase gene DASI – dual aromatase and sulfatase inhibitor DCIS – ductal carcinoma in situ DEX – dexamethasone DHEA(S) – dehydroepiandrosterone (sulfate) DMSO – dimethyl sulfoxide DNA – deoxyribonucleic acid E1 – estrone E1S – estrone sulfate E2 - estradiol ED50 – 50% effective dose EP – prostaglandin receptor ER – estrogen receptor (α or β) ERE – estrogen response element DMSO – dimethyl sulfoxide ER – estrogen receptor FBS – fetal bovine serum HER2/neu – epidermal growth factor receptor 2 (also known as ErbB2) HDBC – hormone dependent breast cancer HRT – hormone replacement therapy HSD – hydroxysteroid dehydrogenase IC50 – 50% inhibitory concentration IL-6 – interleukin 6 MAPK – mitogen activated protein kinase MCF-7 – ER-positive breast cancer cell line xv MDA-MB-231 – ER-negative breast cancer cell line mRNA – messenger ribonucleic acid NADPH - nicotinamide adenine dinucleotide phosphate NF-κB – nuclear factor κB NSAID – nonsteroidal anti-inflammatory drug OATP-B – organic anion transporter polypeptide B PBS – phosphate-buffered saline PGE2 – prostaglandin E2 PKA – protein kinase A PKC – protein kinase C RNA – ribonucleic acid rRNA – ribosomal RNA RT-PCR – reverse transcriptase polymerase chain reaction SAM – selective aromatase modulator SERM – selective estrogen receptor modulator ST – sulfotransferase STS – steroid sulfatase T-47D – ER-positive breast cancer cell line TAM – tamoxifen TGFβ – tumor growth factor β TNFα - tumor necrosis factor α

xvi CHAPTER 1

INTRODUCTION

1.1. Breast cancer: Overview

1.1.1. Breast cancer statistics

In the United States, breast cancer is the most common non-skin malignancy and

the second leading cause of cancer-related death, behind lung cancer, in women.

According to the National Cancer Institute, the average lifetime risk of an American woman developing breast cancer has increased from less than 10% in the 1970s to

12.28% (or 1 in 8 women) as of 2007 1. In Europe and Japan, the risk decreases to 1 in

12 and only 1 in 80 women, respectively 2. In 2008 there will be an estimated 184,450

new cases of invasive breast cancer diagnosed in the United States; of those, 1,990 are

expected in men. Almost 41,000 Americans will die from the invasive form of the

disease this year; only 1% of that value will be men. Another 67,770 new cases of breast

carcinoma in situ are also expected to occur, with about 85% as ductal carcinoma in situ

(DCIS), the predecessor of invasive ductal carcinoma (IDC). The remaining 15% of in situ cases originate in the lobules of the breast.

1 1.1.2. Breast cancer development

Breast cancer begins when cells start growing uncontrollably in the breasts’ milk

producing glands, called lobules, and the ducts that connect them to the nipple (see

Figure 1.1.). If the cancer is detected at this early stage, data has shown 98% of patients

survive for at least five years after diagnosis 1. This stage of the disease is called

carcinoma in situ because its cells exist and grow in their site of origin and have not yet

spread to other sites of the breast or other organs of the body. Invasive breast tumors

develop when the cancerous cells break through the walls of the lobules or ducts to

invade the surrounding fatty tissue; detection at this stage decreases the 5-year survival

rate to 81%. Metastatic breast cancer occurs when the cancer spreads beyond the breast

to other tissues of the body. If the cancer is not found until this late stage, only 26% of

these patients survive five years later.

Figure 1.1. Side cross-section representation of the human breast 3.

1.1.3. Breast cancer risk

Breast cancer risk is associated with both controllable and uncontrollable factors

(see Table 1.1.). Certain lifestyle choices have been statistically correlated to increased breast cancer risk, including those related to poor nutrition and fitness. Additional choices relating to pregnancy and breast-feeding may increase the risk of breast cancer.

Notably, the use of Hormone-Replacement Therapy (HRT) has received much attention in the media for its controversial, yet inconclusive risk.

Controllable Factors Uncontrollable Factors Smoking First degree family history of breast cancer Alcohol Consumption Inherited BRCA1 and/or BRCA2 gene abnormalities Dietary intake Early first menstruation or late menopause Post-menopausal obesity and Exposure to environmental toxins physical inactivity Delayed child-bearing or having Increased age no/few children; not breast-feeding Use of Hormone-Replacement Gender, race, height Therapy (HRT)

Table 1.1. Breast cancer risk factors 1;4.

The list of uncontrollable factors begins with those relating to family history,

including the mutations in the breast cancer genes 1 and 2 (BRCA1, BRCA2). Normal

BRCA genes are involved in DNA stability; therefore, mutated BRCA genes may have

defective DNA-repair or DNA-damage-sensing processes 5. Approximately 80% of those who carry these mutations develop breast cancer by the age of 70, as well as 20 to

40% who develop ovarian or prostate cancers. BRCA1/2-positive patients should keep aware of their breast health. Some people additionally elect to have preventative mastectomies and/or take anti-estrogen therapy in an attempt to avoid breast cancer development.

Age is important in assessing breast cancer risk. The highest occurrence of breast cancer cases are in women aged 75 – 79 years old, but a dramatic increase in risk is observed after the age of 40 1. Additionally, race is a distinguishing factor. While white

4 American women have a higher incidence rate of breast cancer, they are statistically less

likely to die from the disease than African American women. Other ethnicities studied,

including Asian American/Pacific Islander, Hispanic/Latina, and American

Indian/Alaskan Native women, all have lower incidence and mortality rates than white

and African American women.

1.1.4. Molecular profiling in breast cancer

Due to recent findings of incongruity between cultured breast cancer cell lines and primary tumor samples, one cannot expect any single cell line to fully represent the heterogeneity actually seen in breast cancers 6. It is now more accurate to examine a

panel of cell lines, with varying gene expression profiles and multiple genomic

alternations, to get a better picture of how tumor systems can behave. Upon analysis of

51 cell lines, Gray et al identified five subtypes of invasive ductal carcinoma by profiling

the molecular differences between cultured cell lines and primary tumors. The genetic

subtypes that comprise approximately 80% of all breast cancers are Luminal A (ER+ or

PR+/HER2-), luminal B (ER+ or PR+/HER2+), HER2+/ER- (ER-/PR-/HER2+), basal-

like, and triple-negative (ER-/PR-/HER2-). Invasive lobular carcinomas make up another

10 to 15% of breast cancers and also have distinct genetic profiles. Luminal A is the

most common genetic subtype in patients and has the most favorable projected outcome;

HER2+ and basal-like subtypes have the worst prognosis. The remaining 5 to 10% of

breast cancers are attributed to additional rare genetic types.

5 1.1.5. Breast cancer in men

Though breast cancer in men is rare, these cases are no less important and may in

fact be more difficult to diagnose and treat. Most cases of breast cancer in men are invasive, and thus the cancerous cells have already acquired the ability to spread

throughout the body. Since males have decreased estrogen production compared to females, the growth stimulus is more uncertain. Males with the highest risk have

Klinefelter’s syndrome, in which the patient is born with two X sex chromosomes (XXY) instead of the standard XY combination (genetic females are born XX) 4. As expected,

these patients have increased estrogen levels. However, men without Klinefelter’s

syndrome can also be at risk for breast cancer, especially if they are positive for a

mutation within the breast cancer gene BRCA2. The overall lifetime risk of male breast

cancer is 1 in 1000 men 1. Other risk factors, diagnosis, and treatment are very similar to

those for women. Like women, the majority of breast cancers in men are hormone-

dependent (also known as hormone receptor-positive) and they are likely to undergo

endocrine therapy.

1.1.6. Current treatments

There are four main routes for the treatment of breast cancer. These are different

procedures, but may be used in combination or sequence.

Surgery as a treatment is the physical removal of the cancerous region(s) from the

breast and sometimes the regional lymph nodes in the underarm. The removal can either

be a lumpectomy, which removes only the tumor itself and some surrounding tissue, or a

mastectomy, by which the entire breast is removed and possibly some of the axillary

6 lymph nodes. Radiation therapy is a method that kills cancerous cells by destroying their

DNA using high-energy X-rays. This process is tissue-targeted and intended to be a subsequent step to surgery, radiating the surrounding tissue from the excision site for any cancer cells remaining in the breast.

Chemotherapy is the use of anticancer medicines to kill or inhibit the growth of cancerous cells. Treatment can be given before surgery to attempt to shrink the tumor to be excised. It can also be given after surgery with or without radiation to either reduce

the likelihood of reoccurrence (adjuvant chemotherapy) or, in the case of metastatic breast cancer, to decrease the effects on organs to which the cancer cells may have

spread. The most commonly used chemotherapeutic agents, administered either alone or

in combinations, for in situ or locally invasive breast cancer are the following:

cyclophosphamide, doxorubicin, 5-fluorouracil (5-FU), methotrexate, and paclitaxel

(Taxol) or docetaxel 1. The mechanisms of action of the first four compounds disrupt

DNA synthesis and transcription via DNA cross-linking, intercalation, false nucleoside incorporation (antimetabolite), and inhibition of thymidine synthesis. The latter options

Taxol and its derivatives inhibit cellular division by stabilizing microtubules from depolymerization during mitosis. Taxols have additionally been found to further induce apoptosis in cancer cells. Metastatic breast cancer chemotherapy may use any of these compounds, in addition to other nucleoside analogs gemcitabine and capecitabine (pro- drug for 5-FU). All of these chemotherapeutics are intended to act on cancer cells; since

7 cancerous cells grow unchecked, they are more rapidly dividing and should thus be more affected by the drug’s action. However, since healthy cells grow and divide as well, they are also affected by the drugs and thus chemotherapy is commonly known to have large and non-specific side effects.

Endocrine (hormone) therapy takes advantage of some cancers’ use of the female hormones estrogen and/or progesterone. It has the added advantage over general chemotherapy in that it specifically targets those cells whose growth is dependent on hormones; therefore, endocrine therapy should have fewer side effects. This method of treatment counters that stimulatory effect by either inhibiting the hormone’s action or its synthesis. As will be described in Section 1.1.7., active estrogens act with the estrogen receptor (ER) to stimulate the growth of cells with those receptors. However, the compound tamoxifen competes with the substrate for the receptor, and upon binding blocks the action of estrogen’s downstream stimulatory effects. Tamoxifen is known as an “anti-estrogen” or selective estrogen receptor modulator (SERM) for this reason.

Studies have also shown tamoxifen to be a good prophylactic medication, in that women who are at high risk of breast cancer but have not shown any signs of disease can take it to reduce their chances of uncontrolled estrogen-stimulated growth 7. Other therapies to date are aromatase inhibitors, which inhibit the biosynthesis of estrogen. These are discussed in detail in Section 1.3.3. The downside of endocrine therapy for breast cancer is that it is not effective on those cancers that are hormone-independent.

Alternate options include targeted therapies, such as the engineered antibody trastuzumab (Herceptin®), for hormone-independent breast cancers 8. Specifically,

Herceptin is used for cancers testing positive for high levels of HER2/neu receptors on

8 the cancer cells’ surfaces. Unchecked, these receptors are used to promote cellular

growth; therefore, blocking their action on cancer cells with the antibody inhibits their

growth signaling cascades. Only about 20-30% of all breast cancers are HER2/neu-

positive and this treatment is only effective for those cancers.

1.1.7. Hormone dependence in breast cancer

As previously stated, more than 60% of all breast cancers require estrogen for

growth. Even more breast cancers, approximately 95%, are initially hormone-

2 dependent . The estrogen receptor (ER) first binds estradiol (E2) and then dimerizes

with another unit in the nucleus. There, the ER-E2 dimer complex binds DNA at estrogen

receptor response element (ERE) sequences to promote the production of specific

growth-stimulatory genes 9. Elevated levels of estrogens, even in post-menopausal

women (in which ovarian estrogen production has ceased), are believed to be responsible

for the development and progression of hormone-dependent breast cancer (HDBC).

Though most breast tumors are initially estrogen-dependent, a tumor can be or

become hormone-independent by a mechanism that is not yet fully understood. The

conversion may be explained by mutation of the ER, causing the ER to become non-

functional and thus unresponsive to estrogen binding 2. These cells do not have or need

estrogen receptor signaling for their growth. Interestingly, their growth is more rapid and

aggressive than HDBC. Growth factors and receptors, as seen in cancers positive for

HER2/neu receptor involvement, are other sources of stimulation 8. Mitogen-activated protein kinase (MAPK) signaling has also been implicated as a pathway for growth of hormone-independent tumors 10.

1.1.8. Origins of estradiol precursors

Endogenous estrogen is predominantly produced in the ovaries of pre- menopausal, non-pregnant women. However, after menopause, there are other internal

sources of estrogen. These include adipose tissue, brain, hair follicles, muscle, normal and malignant breast tissue 11. Hormone-dependent tumors can grow in locations with

little estrogen production because the estrogen is acting locally in an intracrine (on the

same cell) or paracrine (on a neighboring cell) fashion, and therefore acts where it is

produced. However, for these other tissues to synthesize estrogens, they must receive the

building blocks from the adrenal cortex.

Figure 1.2. Steroidogenesis of androgens and estrogens from adrenal cortex products. Androgens: DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; Adione, androstenedione; Testo, testosterone; and Adiol, androstenediol. Estrogens include: E1S, estrone sulfate; E1, estrone; and E2, estradiol, which acts on the ER, estrogen receptor.

The adrenal cortex, positioned on the adrenal gland, produces within its three distinct layers glucocorticoids (e.g. cortisol), mineralocorticoids (e.g. aldosterone), and androgens (e.g. dehydroepiandrosterone/DHEA) from the steroidogenesis of cholesterol.

In women, the adrenal cortex is the major source of androgens. In men, however, the testes dominate androgen production and thus the amount produced by the adrenal cortex is physiologically insignificant. Other androgenic products from the adrenal cortex include androstenedione (Adione) and DHEA sulfate (DHEAS). Adione, like 11 androstenediol (Adiol), can also be produced indirectly from DHEA by two different

hydroxysteroid dehydrogenases (HSD); they both can be converted into testosterone

(Testo). By means of the enzyme aromatase, testosterone’s A-ring is aromatized and

loses one carbon, converting the androgen (19 carbon structure) to estradiol (E2).

Estrogens have 18 carbons atoms, so the aromatase reaction is known as the conversion

of androgens to estrogens.

Estrogens are biosynthesized as described from androgens using the “aromatase

pathway” as well as from steroid sulfates using the “sulfatase pathway.” Sulfated

steroids such as estrone sulfate (E1S) and dehydroepiandrosterone sulfate (DHEAS) are hydrolyzed by steroid sulfatase (STS) to the unconjugated estrone (E1) and DHEA,

respectively. The precursor estrone is then converted to estradiol (E2) by 17β-

hydroxysteroid dehydrogenase type 1 (17β-HSD1). If not converted extracellularly, a

+ Na -dependent organic anion transporter polypeptide (OATP-B) can directly take E1S (or

DHEAS) into the cell to be converted. Though much E1 is produced from

androstenedione via the aromatase pathway, it can be converted into E1S by estrone

sulfotransferase (ST) and stored for later use 11. Therefore, it is believed that up to 10

times more E1 is produced by the sulfatase pathway. E1S plasma and tissue

concentrations have been shown to be greater than the other unconjugated steroids. E1S also has a greater half-life (10 – 12 hours) than either E1 or E2 (20 – 30 minutes).

Therefore E1S can act as a reservoir to store estrogens in their inactive sulfated forms.

Figure 1.3: Hormone-dependent breast cancer. E1S, estrone sulfate; E1, estrone; E2, estradiol; T, testosterone; PGE2, prostaglandin E2; ER, estrogen receptor; PTK, protein tyrosine kinase. Figure adapted from Brueggemeier, et al 12.

The aromatase pathway, which converts testosterone into estradiol, is present in

both breast stromal cells and breast epithelial cancer cells. Though not directly involved,

cyclooxygenases enzymes 1 and 2 (COX-1, COX-2) are also part of the extended

aromatase pathway. They produce thromboxane and prostaglandins D, E, and F within

epithelial cells of the breast. One particular product, prostaglandin E2 (PGE2), is released

from the epithelial cells and to bind G-protein-coupled-prostaglandin receptors (EPs) on

neighboring stromal cells. There, PGE2 performs its hormone regulatory action by

stimulating aromatase transcription and furthering the production of estradiol. 13 Upon entering the cell, two estradiol molecules bind with two nuclear estrogen

receptors (ERs) to form a dimer that can modulate transcription of mRNAs of growth-

related proteins. By this outline, estradiol is the most active endogenous estrogen and is

therefore viewed as the end product of both pathways.

In the post-menopausal state, there are low levels of circulating estrogens.

However, these concentrations are elevated in breast cancer tissue, indicating tumor-

specific hormone biosynthesis and accumulation. The enzymes indicated here in the

biosynthesis of estradiol (aromatase, STS, and COX-2) are all present in human breast

cancer tissue 2. Additionally, they have all been shown to be overexpressed in malignant breast tissue and are therefore the focus of the following research. Much previous research within and outside of this laboratory has focused on the gene and enzyme of aromatase. However, the current project seeks to more fully understand STS and its relationships to the other estrogen-producing enzymes. To note, significant discussion of the enzymes sulfotransferase (ST) and 17β-HSD1 will not be included in this particular

dissertation. For further information, please see listed references in Section 1.6. 2;13.

Though DHEAS is less discussed in the following research, it is also a valuable

substrate of STS. DHEAS, which is secreted by the adrenal cortex, is a precursor to

androstenediol, which is mainly converted into testosterone and later estradiol. Adiol is

also mildly estrogenic itself and can weakly bind the estrogen receptor and elicit its own

growth stimulatory response 14. Though its ER affinity is not near that of estradiol’s,

Adiol can exist in increasing concentrations in breast cancer tissue and, due to over active

STS, may make its effects significant in post-menopausal women. For more information

on DHEAS in relation to breast cancer, see references at the end of this chapter 14.

14 1.2. Steroid Sulfatase

1.2.1. Expression, function and regulation

Steroid sulfatase is one enzyme of a 12-member protein family of human

sulfatases 15. Seven lysosomal sulfatases function in acidic conditions to degrade

glycosaminogycans and sulfolipids 16. The remaining five sulfatases (arylsulfatases C –

G) function in pH neutral conditions and are associated with intracellular membranes.

Arylsulfatase C is also known as steroid sulfatase (STS, E.C. 3.1.6.2) or previously

“estrone sulfatase” for one of its specific substrates.

Table 1.2. List of human sulfatases 16. 4-MUS = 4-methylumbelliferyl sulfate, a substrate for a fluorimetric STS activity assay 15.

15 In protein form, human STS is a 63 – 73 kDa (583 amino acids) glycosylated

monomer located in cellular membranes, particularly those of the endoplasmic reticulum

and cis-Golgi complex 16;17. Studies have shown STS to be located in almost all

mammalian tissues. However, research further suggests that there are tissue-specific STS

isoforms, and that they additionally have differing enzyme kinetics for DHEAS and E1S

substrates. STS is present in the liver, endometrium, ovaries, bone, brain, prostate, white

blood cells, adipocytes, placenta, and breast carcinoma tissue 2. The STS crystal structure was isolated in 2001 by X-ray crystallography 18. It contains two anti-parallel,

hydrophobic α−helices that likely traverse membranes to anchor the catalytic domain

near the membrane surface. The active site contains a formylglycine residue that

complexes with a bivalent cation, such as Ca2+.

In general terms, the function of the enzyme is hydrolysis of 3-O-sulfate

monoester groups from several biologically relevant 3-hydroxy phenolic steroids,

including sulfates of cholesterol, estrone, pregnenolone, and dehydroepiandrosterone, via

a covalent intermediate 11;16. The most important substrates in breast cancer for

hydrolysis are DHEAS and E1S since they are the precursors for the synthesis of active

estrogens and androgens.

Besides breast cancer, STS and its inhibition may be relevant to other diseases 15.

First, since STS also converts inactive DHEAS to DHEA, a precursor to androgens

dihydrotestosterone and testosterone, STS inhibition may be important to the treatment of

endometrial and prostate cancers, other hormone-dependent cancers 16. Likewise, STS

may be important in several androgen-dependent skin disorders, such as alopecia and acne. Finally, STS may play a role in cognitive dysfunction. Since STS controls the

16 amount of DHEA present, it therefore controls the amount of that neurohormone in the

brain. In several studies, rats treated with STS inhibitors showed enhanced learning

ability and spatial memory. The regulation of STS is still greatly unknown, but studies

have been and are being performed to understand the roles regulatory elements play on

STS expression and activity. More detailed discussion of this topic can be found in

Chapter 5 of this dissertation.

1.2.2. Role in breast cancer

Despite the low levels of circulating estrogens in post-menopausal women,

estrogen concentrations, including E1, E2, E1S, ad E2S, are elevated in breast tumors

compared to levels in the plasma and normal tissues of the breast 2;19. This indicates the

local formation of estrogens in the breast, and therefore the importance of estrogen-

producing enzymes. Several studies have shown that estrone sulfate may be the more

likely precursor to estradiol than those substrates from the aromatase pathway 20.

Estrogen sulfates are present in high concentrations in circulation even in healthy tissues.

In postmenopausal women, E1S plasma levels are 5 – 10 times greater than those of the

21 unconjugated estrogens (E1, E2, and estriol E3) . Levels of E1 and E2 in plasma are

generally unchanged between normal and breast cancer patients both before and after

menopause. However, concentrations of E2 are approximately 23 times greater and E1S

nine times greater in breast tumors than in plasma of postmenopausal women 20. These elevated levels of STS substrate and downstream product are evidence of the importance of the sulfatase pathway in breast cancer initiation.

17 In further evidence of this point, STS itself has increased effects in cancerous

tissue over that of normal tissue. In a recent study, 87% of patients tested had elevated

levels of STS mRNA in malignant tissue over that found in normal breast tissue 11.

Furthermore, evidence has shown the STS enzyme to be highly active in breast tumors, beyond that of the aromatase enzyme; in fact, STS levels are 40 to 500 times higher than aromatase in patient breast tumor tissue.

1.2.3. STS inhibitors

Multiple types of compounds not designed for such have shown inhibitory affects on STS activity and/or expression. Several progestin derivatives, such as medrogestone and Promegestone (R-5020), reduce E2 formation when co-incubated with E1S in ER-

positive breast cancer cell line T-47D 2. Promegestone additionally inhibited STS mRNA

expression 50% in T-47D cells, which positively correlated to reduced enzymatic activity. Tibolone, a synthetic steroid mainly used in hormone replacement therapy, and its metabolites have shown potent inhibition of STS and stimulation of sulfotransferase

(ST) activities in HDBC cells and total breast cancer tissues. Estradiol also inhibits its own synthesis via STS in low and sub-nanomolar ranges, though this is likely by means of a negative feedback loop 22.

Nevertheless, the most potently developed STS inhibitors are steroidal and non-

steroidal sulfamates designed to mimic the natural substrate E1S and compete for the

active site residues. As determined in structure-activity relationship tests, potent and

irreversible STS inhibitors require N-unsubstituted arylsulfamate groups, a linker

segment if on a non-steroidal compound, and a long or bulky side chain 16.

18 Alkyl-substituted sulfamates are typically inactive or act as reversible inhibitors. The

sulfamate group should be attached to the A-ring in a β-position and must be

unsubstituted (or can be easily degraded to that status) to retain inhibitory activity. The

linker has higher activity when it contains a heteroatom or is cyclic to create a B-ring to

the aryl A-ring. The side chain should be located para (opposite) of the sulfamate

substituent and contain a long linear chain or a bulky aliphatic group of 13 – 16 atoms.

The optional space beyond the side chain is only relevant for reversible inhibitors and

does not seem to be needed for the most potent irreversible inhibitors. Based upon the 3-

D structure of the enzyme, the optional space appears to allow for hydrophobic interactions with the transmembrane region.

Figure 1.4. Representation of sulfamate-type STS inhibitors by structure-activity- relationship (SAR) findings 16.

19 The reaction mechanisms of action of STS are similar for the natural substrate

11 E1S and sulfamate-based inhibitors (see Figure 1.5.) . The sulfatase active site residue

Cα-formylglycine is present where, based upon its cDNA, a cysteine should occur. It was discovered that newly synthesized STS polypeptides carry the cysteine residue, and upon oxidation of the thiol group to an aldehyde in the endoplasmic reticulum, the protein is modified to the FGly prior to folding to its native structure 23. The surrounding

peptide sequence of the cysteine residue directs the oxidation procedure. The aldehyde group of formylglycine then takes its reactive gem-diol form to attack the sulfur group of either substrate as a nucleophile. In the case of E1S, electrons of the sulfur-oxygen bond

connected to the C3 position of the steroid A-ring then push to the oxygen and the

resulting estrone (E1) product is released. The sulfated FGly residue then in two steps

- loses HSO4 and gains H2O to regenerate its gem-diol form and become ready to convert

another substrate.

In the inhibitor reaction, the nucleophilic gem-diol FGly also attacks the sulfur-

containing sulfamate group (-SO2NH2) to release the desulfated form of the inhibitor.

However, this time, the enzyme’s residue is left inactivated in an irreversible, covalent

sulfamoyl bond, terminating the ability of the active site FGly to reform.

A. loses HSO4 and gains H2O to regenerate gem-diol

O OH OH S O O O Estrone (E1) O OSO3 Estrone 3-sulfate H (natural substrate) Sulfated gem-diol form of FGly; gem-diol form loses HSO4 and gains H2O to regenerate gem-diol of FGly of enzyme B.

O OH OH S H N O 2 Desulfated form O OSO NH O 2 2 of inhibitor STS sulfamate inhibitor H Sulfamoylated (inactivated) gem-diol form enzyme residue of FGly of enzyme

Figure 1.5. Reaction mechanism of action of STS on (A) natural substrate E1S and (B) known STS sulfamate inhibitors 11. Both reactions involve the transfer of a sulfur- containing group to the active site residue formylglycine and the release of the desulfated substrate. In the inhibitor reaction, STS is inactivated by an irreversible, covalent sulfamoyl bond, terminating the ability of the active site FGly to react again.

21 The first generation of potent sulfamate-based inhibitors include steroidal

compound EMATE, estrone-3-O-sulfamate. EMATE is a time- and concentration-

dependent inhibitor with an IC50 of 18 nM in human placental microsomes (enzyme affinity Ki = 670 nM) and 65 pM in MCF-7 cells 11;16. These characteristics indicated to

researchers that the compound acts irreversibly on the enzyme active site. However,

EMATE has no future therapeutic use because it is strongly estrogenic, releasing estrone

upon hydrolysis. Several D-ring modified analogs have been synthesized with reduced

estrogenicity, but focus moved on to a second generation of compounds. Many of the

first nonsteroidal STS inhibitors, in a desire to mimic the potency of EMATE but reduce

its estrogenity, were based on 4-methylcoumarin-7-O-sulfamate. COUMATE, as it came

11;16 to be known, is also an STS irreversible inhibitor with an IC50 of 380 nM . Because

of this reduced potency, COUMATE analogs were developed and one tricyclic

derivative, 667-COUMATE, stood out with an IC50 = 8 nM in human placental

microsomes and a higher affinity for the enzyme active site (Ki = 40 nM) than EMATE.

It is believed that the phenolic coumarin is a better leaving group than E1 of EMATE.

667-COUMATE was the first ever STS inhibitor to undergo clinical trials in postmenopausal women. It is currently in trials under the name STX64, and is well-tolerated and achieving greater than 90% inhibition of STS in both peripheral blood lymphocytes and in tumor tissue 24;25. Interestingly, STX64 also reduces serum androstenedione, typically an aromatase substrate, by 86% in addition to estradiol and androstenediol (see

Figure 1.2. for clarification). This finding may indicate that more androstenedione is coming from the conversion of DHEAS to DHEA (a sulfatase pathway reaction) and not from DHEA directly secreted from the adrenal cortex 25.

22 Also in development are dual aromatase and sulfatase inhibitors (DASIs), which

target both enzymes with a single agent. They are based on templates of aromatase

inhibitors letrozole and anastrozole with an attached arylsulfamate ester to include anti- sulfatase activity 26;27. The best usable R groups consist of halides (Cl, Br) and/or small

substituent groups (H, OMe, CN, CF3).

The novelly designed STS inhibitors used in the following studies (DU-14 and

DU-15) are thoroughly discussed in Chapter 4.

Steroidal H

H H

H2NO2SO EMATE, estrone 3-O-sulfamate

Non-steroidal

H2NO2SO O O

STX64 (667-COUMATE)

N N R Dual aromatase OSO2NH2 and sulfatase N inhibitors N N R N N NC

NC O

OSO2NH2

Table 1.3. Structures of steroid sulfatase sulfamate-based inhibitors.

24 1.3. Aromatase

1.3.1. Expression, function and regulation

The aromatase enzyme is actually a complex made up of two proteins 11. The first

is a heme-containing estrogen synthetase (which uses an energy cofactor source) that is

cytochrome P450 number 19, also known as CYP19. The second protein is an NADPH- cytochrome P450 reductase, which transfers reducing equivalents to P450arom. The hemeprotein converts carbon-19 androgens to carbon-18 estrogens via an aromatization of the steroidal A-ring and eliminating the C19 methyl group. These carbon-19 androgens include androstenedione and testosterone. The carbon-18 estrogens produced are estrone (from androstenedione) and estradiol (from testosterone). The catalyzation of this reaction uses three moles of oxygen and NADPH each per mole substrate. The aromatase protein is glycosylated and is approximately 58 kDa. Aromatase is located in both normal and malignant tissue, though it is mainly located in the breast stromal/adipose cells; it is lowly expressed in the cancer cells themselves, but is nevertheless an increase from normal tissue. Therefore, stromal cells produce more estradiol to act in a paracrine fashion on the cancer cells. The tumor cells additionally produce and secrete stimulatory factors that act on the stromal cells to promote aromatase production in the adipose tissue and thus help progress cancerous growth.

The full length CYP19 cDNA contains 10 exons (3.4 kilobases) that encode a 503 residue polypeptide 11. However, the gene’s regulation is very complex; the regulatory

region has at least 10 tissue- and signaling pathway-specific promoters. Among the

multiple promoters is I.4, for normal breast and adipose tissue. Upon cancer

development, there typically are promoter switches from I.4 to I.3 and PII in breast

25 cancer tissues. While normal breast promoter I.4 is stimulated by glucocorticoids and

class I cytokines, PII is stimulated by PKA-cAMP pathways and I.3 by tumorigenic

phorbol esters. Aromatase’s promoter-specific regulation opens doors for specific

inhibitors of aromatase transcription to target only expression in the cancerous tissue.

1.3.2. Role in breast cancer

Since the final reaction and rate-limiting step in estrogen biosynthesis is

aromatization (via the aromatase complex), inhibition of the aromatase enzyme should

only decrease the total amount of estrogen without affecting the concentrations of other

endogenous steroids (i.e. testosterone, cholesterol, DHEA, etc). Therefore, inhibition of

aromatase as a method of endocrine therapy for breast cancer is ideal to remove any non-

specific side effects.

Aromatase activity in human breast cancer cells and tissues is actually quite

low 22. Many times, to study aromatase in vitro, ER-positive cells lines are stably

transfected with aromatase cDNA to overexpress the gene. Nevertheless, aromatase is

present in both normal and malignant tissue 28. Like STS, estradiol itself can inhibit

aromatase activity in breast cancer cells 22.

1.3.3. Aromatase inhibitors

There are both steroidal and nonsteroidal aromatase inhibitors (see Table 1.4.).

The steroidal compounds typically are noncompetitive and mechanism-based enzyme inhibitors. Nonsteroidal compounds, on the other hand, are competitive inhibitors that contain a heteroatom to bind up the heme iron of P450arom, keeping it from acting on the

26 natural substrates. The first compound tested in patients was the “first generation”

nonsteroidal inhibitor aminoglutethimide (AG), but due to serious non-selective side effects on other cytochrome P450 enzymes, it was removed from trials. Second generation compounds 4-hydroxy androstenedione (also known as formestane), which is

a steroidal enzyme inactivator, and fadrozole (nonsteroidal) were more selective and

potent than AG. However, poor oral bioavailability limited their development as first

line breast cancer therapies. Third generation inhibitors letrozole (Femara®) and

anastrozole (Arimidex ®) are both nonsteroidal triazole ring compounds that have low

9 nanomolar IC50 values in human placental microsomes (11.5 and 15 nM, respectively) .

Exemestane (Aromasin ®), a steroidal third generation compound, is also a potent aromatase inactivator, with a Ki of 26 nM. For these reasons, we used letrozole and

exemestane in the relevant experiments to study the effects of aromatase inhibitors.

Current clinical studies are investigating these aromatase inhibitors as adjuvant therapies before, instead of, and in combination with tamoxifen. So far, the aromatase inhibitors have shown positive results, being more effective than tamoxifen alone in postmenopausal breast cancer patients. Nevertheless, the aromatase inhibitors have not yet become the first line therapy for the hormone-dependent disease. However, with studies showing their success in tamoxifen-resistant patients, it is likely this method of treatment will soon be the foundation of HDBC therapy.

Selective Estrogen Receptor Modulator

HO O

4-Hydroxy Tamoxifen

O CH Steroidal Aromatase 3 Inhibitor CH3

CH2

Exemestane

N Nonsteroidal Aromatase N Inhibitor N

NC CN Letrozole

Table 1.4. Structures of selective estrogen receptor modulator (4-hydroxy tamoxifen) (4-OHT) and aromatase inhibitors (exemestane and letrozole) used in the subsequent experiments.

28 1.4. Cyclooxygenases

1.4.1. Function and regulation

Cyclooxygenase enzymes are actually prostaglandin G/H endoperoxide synthases

(PGHS) 1 and 2. They are members of the eicosanoid biosynthetic scheme, converting arachidonic acid to prostaglandins, prostacyclin, and thromboxane (TXA2).

Figure 1.6. Actions of cyclooxygenase enzymes 29.

The two isoforms of the enzyme are similar in structure and catalytic activity, but

they have different substrate specificity due to the difference in sizes of their active sites.

COX-1 is a constitutively-produced enzyme and is typically involved in gastrointestinal

functions 30. COX-2, however, is inducible and works in response to stimuli for the

29 purposes of inflammation and cell proliferation, including cytokines (particularly IL-6 and TNFα), growth factors, and oncogenes 29. COX-2 activity can also be reduced by corticosteroids. Finally, COX-1 and COX-2 different by their intracellular locations;

COX-1 is found attached to endoplasmic reticulum membranes, whereas COX-2 is additionally found on nuclear membranes.

Prostaglandins, the major products of the COX enzymes, have a wide range of physiological functions, including contraction and relaxation of smooth muscle in the heart, intestines, stomach, uterus, and bronchial tissue. Because of these various activities, COX enzymes have been implicated in multiple other diseases beyond cancer.

1.4.2. Role in breast cancer

Inhibition of COX enzymes decreases cellular production of prostaglandins, which have been shown to stimulate aromatase gene expression and therefore downstream estrogen biosynthesis. To explain, increased COX-2 expression leads to increased production of PGE2. PGE2 then binds its target cells, acting on prostaglandin receptors EP1 and EP2 to stimulate intracellular cyclic adenosine monophosphate (cAMP) accumulation, signaling through increased adenylate cyclase activity 29;31. Finally, aromatase expression via cAMP-responsive promoter PII is activated downstream of

9 cAMP-PKA and PKC signaling pathways . PGE2 binding EP1 stimulates the PKC pathway, whereas EP2 binding stimulates cAMP formation and PKA pathway involvement. DbcAMP (dibutyryl cAMP) is a cAMP mimic which also has been shown to stimulate aromatase activity.

30 Studies have displayed elevated levels of PGE2 in breast cancer tissues compared

to normal breast tissue 32. Additionally, COX-2 gene expression has been found to be up-

regulated in human breast cancer 29;33. COX-2 mRNA is undetectable in normal breast tissues, but has been found to be overexpressed in 40% of invasive breast cancers, and in even more cases of preinvasive DCIS. High levels of COX-2 expression have also been correlated to large tumor size, hormone-receptor negative status, and HER2/neu

overexpression. Interestingly, it is believed that HER2/neu induces COX-2

transcription 29.

Some studies have found several non-steroidal anti-inflammatory drugs

(NSAIDs), including aspirin, indomethacin, and ibuprofen, to have small

chemopreventive effects against breast cancer, reducing its relative risk 30. It was shown

over 20 years ago that indomethacin suppresses induced-mammary tumor formation. In

more recent analysis, Rodrigeuz et al. reported that breast cancer risk was reduced (odds

ratio = 0.77) in women taking aspirin for one year or more 34. In another study, Ready et

al. showed that low-dose use of aspirin correlated to reduced risk (OR = 0.65), whereas

high-dose use of aspirin alone or other NSAIDs had marked risks of breast cancer (OR =

1.43, 1.26 respectively) 35. In a large study from the Women’s Health Initiative, is was

generally determined that regular, low-dose use of NSAIDs over 5 – 9 years correlated

with a 21% reduction in breast cancer incidence, and use extending beyond 10 years

produced a 28% reduction, with the highest risk reducers being ibuprofen and aspirin 30.

Due to greater specificity for the COX-2 isoform, COX-2 selective inhibitors

“COXibs” have been evaluated in both ER-positive and ER-negative models. Since most breast cancers are estrogen-dependent, COXibs’ abilities to inhibit ER-positive tumors

31 were expected due to the PGE2-aromatase connection. It was also determined that ER-

negative tumor formation was significantly delayed upon celecoxib treatment, giving

further evidence for a HER2/neu and COX connection 29. Though acting by different

mechanisms, it is suggested that COX-2 inhibition should have a positive effect in the

inhibition of breast cancer initiation and/or growth.

As seen over multiple studies, the relationship between NSAID use and breast

cancer risk is inconsistent 30. Nevertheless, these studies give reason to investigate the

effect of COX inhibitors on the estrogen biosynthetic pathways in breast cancer. Another

reason to further study NSAIDs pharmacology in breast cancer is their effect, though

variable, on tumors based upon estrogen receptor status. Both NSAIDs and selective

COXibs have been shown to suppress mammary tumor formation in rodent models of

breast cancer. Additionally, COX-2 knockout studies correlate the lack of COX-2 to

reduced tumorigenesis and angiogenesis.

1.4.3. COX Inhibitors and NSAIDs

COX inhibitors have been discovered and designed as selective and non-selective

inhibitors, distinguished by their selectivity for one COX isoform over another. Even

within each category, there still is variability in their favor between the isozymes.

NSAIDs are typically used as over-the-counter analgesics and are generally

deemed nonselective COX inhibitors. The most common NSAIDs are known by their

brand names. They are: indomethacin (Indocin®), ibuprofen (Advil®, Motrin ®),

naproxen sodium (Aleve®), acetylsalicylic acid (Aspirin), piroxicam (Feldene®), and niflumic acid (Nifuril, Forenol). Amongst others, indomethacin, ibuprofen, aspirin, and

32 piroxicam are relatively selective for and have greater activity against COX-1 36. Aspirin irreversibly acetylates COX-1, whereas the others can act reversibly or irreversibly by competing with the natural substrate, arachidonic acid 36. Naproxen sodium is

nonselective, being equally efficacious against both isozymes. Niflumic acid is slightly

more selective for COX-2. To note, acetaminophen (Tylenol®), though an analgesic, is

not considered an NSAID because it has relatively little anti-inflammatory activity.

COX-2 specific inhibitors, including celecoxib and NS-398 used here in these

studies, are able to differentiate between the COX isozymes due to the sizes of their

active sites and thus can selectively inhibit COX-2 without or minimally affecting the

COX-1 enzyme. This specificity should be able to remove the potential gastrointestinal

side effects of inhibiting COX-1 processes. Celecoxib (Celebrex ®) is a highly potent

COX-2 inhibitor, nearly 400 times more selective for COX-2 than COX-1 36. Celecoxib

produced marked reductions in mammary cancer incidence as well as tumor burden and

volume in a carcinogen-induced rat mammary tumor model 37. Celecoxib also has

antiangiogenic and proapoptotic activities.

The drug has found a place in clinical trials both in combination and as adjuvant

treatment with the aromatase inhibitor exemestane in advanced breast cancer patients.

These trials showed responses to celecoxib alone and in combination, but several had

toxicity issues 30. However, in one particular 2008 study, the effect of exemestane plus

celecoxib was no different than with exemestane alone, indicating that COX-2

suppression may not be as important in patients as initially shown in a preclinical rodent

model 38;39. Nevertheless, in this study the combination group had longer clinical benefit

and the addition of celecoxib did not change the tolerability of exemestane alone.

33 Interestingly, celecoxib has also been shown to delay the onset of HER2/neu-induced

tumors. In a Phase II trial, celecoxib has been combined with Herceptin® in metastatic

breast cancer patients 39.

Nimesulide cyclohexyl analog NS-398 (N-[2-(Cyclohexyloxy)-4- nitrophenyl]methanesulfonamide) is a COX-2 selective inhibitor (approximately 150-

36 fold) . Its IC50 value against human recombinant COX-2 is 1.77 μM, whereas that for

COX-1 is only 75 μM 40. Both were developed out of the N-arylmethanesulfonamide

series of NSAIDs. Nimesulide, also a COX-2 selective inhibitor, has shown reduced

tumor size and occurrence in carcinogen-induced tumors. Both have found most notable

use in the laboratory setting and not for clinical use due to potential risk of

hepatotoxicity. Both nimesulide and NS-398 have anti-proliferative and pro-apoptotic

effects in cells and in animal models, including head and neck squamous cell carcinoma,

colorectal cancer, human prostate cancer, human esophageal adenocarcinoma, and human

ovarian cancer 41. Independent of COX-2 inhibition, they appear to downregulate Akt

and/or correspond to the release of cytochrome c and activation of caspases 9 and 3.

Further evidence suggests NS-398 is capable of inducing G0/G1 cell-cycle arrest.

Regardless, NS-398 is actually a weak inhibitor against its COX target. Nimesulide and

NS-398 analogs have had more recent success in taking advantage of their activity

against the aromatase enzyme, while chemically removing their COX inhibitory

properties 42.

SC-560 (5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole) is

700-fold selective for COX-1 over COX-2. In human recombinant COX enzymes, the

42;43 drug has an IC50 of 9 nM for COX-1 and 6.3 μM for COX-2 . Additionally, SC-560 34 treatment in murine breast cancer model resulted in significant inhibition of tumor size 37.

In ovarian cancer model, SC-560’s inhibitory effects resulted from inhibited cell proliferation as well as accelerated apoptosis 44. In this particular study, it was

determined that the combination SC-560 with other agents (non-selective NSAIDs such as ibuprofen) proved to be most effective at promoting tumor responsiveness to all agents

involved. Furthermore, it was shown that SC-560, in addition to being a COX-1 selective inhibitor, also has the ability to indirectly limit tumor angiogenesis in ovarian cell cells.

Due to problems in the trials and use of rofecoxib (Vioxx ®), in which significant cardiovascular effects presented themselves, Vioxx was removed from the U.S. market.

Though celecoxib still remains on the market as a colorectal polyp chemopreventive agent, negative public feedback about COX inhibitors has limited it further acceptance as a cancer treatment.

“Nonselective” NSAIDs

CH3 O

N CH3 HO2C O Na

O O Naproxen Sodium Cl H3CO Indomethacin H3C CO2H CF Ibuprofen 3

CO2H O O

S CH3 O CH3 N N NH H O N

CO2H Acetylsalicylic Acid OH O N (Aspirin) Niflumic Acid Piroxicam

COX-selective inhibitors

CF3 CF3

N N O2N O N N

NH H3C Cl

SO2CH3

SO2NH2 NS-398 OCH3

Celecoxib SC-560

Table 1.5. Structures of cyclooxygenase inhibitors. “Nonselective” NSAIDs: indomethacin, ibuprofen, naproxen sodium, acetylsalicylic acid, piroxicam, niflumic acid. COX-2 specific inhibitors: celecoxib, NS-398. COX-1 specific inhibitor: SC-560.

36 1.5. Summary: Interrelationships between enzymes

This introductory chapter described the importance of breast cancer research and

the understanding of the enzymes involved in its development and progression.

Estrogen-dependent tumors require both aromatase and sulfatase pathways to produce

growth-stimulating estrogen. Believing such, inhibition of both enzymes, with or without

COX modulation, may well sensitize HDBC cells to either line of endocrine therapy.

The aromatase and sulfatase pathways to estradiol appear to be able to exist

independent of each other. However, exogenous influence on either pathway, by

inhibitors or regulatory species, may bring about their connection. In theory, the inhibition of aromatase and/or COX-2 should not only initially decrease E2 production,

but might also in time increase the cancer cells’ reliance on the sulfatase pathway in

hormone-dependent breast cancer. Likewise, inhibition of STS should favor the

aromatase pathway to estrogen production. Being that the expression patterns of all three

enzymes are elevated in HDBC compared to normal tissue, there may be relationships between these enzymes that has not yet been elucidated.

COX-2 mRNA is present in cancerous tissue and not in normal tissue, and COX- derived prostaglandins are modulating aromatase expression; this explains the inhibiting effects on NSAIDs on hormone-dependent breast cancer tumors. These statements also support the idea that inhibition of COX-2 reduces mammary tumor development at least in part by its ability to inhibit estrogen biosynthesis.

Previous studies within the Brueggemeier laboratory, in association with Drs. Li and Shapiro, proved a positive linear relationship between CYP19 gene expression and

COX-2 expression from 20 human breast cancer samples 37. This evidence further

37 suggests an autocrine and/or paracrine mechanism exists utilizing these two enzymes in

HDBC. Additionally within our lab, we showed that COX-2 short-interfering RNAs

(siRNA) not only silence their intended COX-2 gene expression, but also suppressed

CYP19 expression, and the associated aromatase enzyme activity, in SK-BR-3 breast cancer cells 37. This is further evidence of a causal relationship between these two

species. Not only should the combinations of COX-2 and aromatase inhibitors show

increased effectiveness in HDBC, but inhibitor combinations of other targets, such as

STS, should fully blockade the total biosynthesis of estradiol. Additionally, with the

evolution of genetic testing and pharmacogenomics, those breast cancer patients who test

higher for STS expression may better benefit from STS inhibitors in addition to or after

responding to anti-estrogens or aromatase inhibitors.

38 1.6. References

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6. Vargo-Gogola, T. and Rosen, J. M. Modelling breast cancer: one size does not fit all. Nature Reviews: Cancer 2007, 7, 659-672.

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8. Di Cosimo, S. and Baselga, J. Targeted therapies in breast cancer: Where are we now? European Journal of Cancer 2008, 44, 2781-2790.

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10. Coutts, A. S. and Murphy, L. C. Elevated mitogen-activated protein kinase activity in estrogen-nonresponsive human breast cancer cells. Cancer Research 1998, 58, 4071-4074.

11. Smith,H.J. and Simons,C. Development of Enzyme Inhibitors as Drugs. In Enzymes and Their Inhibition: Drug Development. Smith, H. J. and Simons, C. Eds.; CRC Press: London, 2005; pp 171-250.

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13. Dooley, T. P.; Haldeman-Chahill, R.; Joiner, J.; and Wilborn, T. W. Expression profiling of human sulfotransferase and sulfatase gene superfamilies in epithelial tissues and cultured cells. Biochemical and Biophysical Research Reviews 2000, 277, 236-245.

39 14. Valle, L. D.; Toffolo, V.; Nardi, A.; Fiore, C.; Bernante, P.; Di Liddo, R.; Parnigotto, P. P.; and Colombo, L. Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue. Journal of Endocrinology 2006, 190, 129-139.

15. Billich, A.; Bilban, M.; Meisner, N. C.; Nussbaumer, P.; Neubauer, A.; Jager, S.; and Auer, M. Confocal fluorescence detection expanded to UV excitation: the first continuous fluorimetric assay of human steroid sulfatase in nanoliter volume. Assay and Drug Development Technologies 2004, 2, 21-30.

16. Nussbaumer, P. and Billich, A. Steroid sulfatase inhibitors. Medicinal Research Reviews 2004, 24, 529-576.

17. Stute, P.; Gotte, M.; and Kiesel, L. Differential effect of hormone therapy on E1S- sulfatase activity in non-malignant and cancerous breast cells in vitro. Breast Cancer Research and Treatment 2008, 108, 363-374.

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19. Yoshimura, N.; Harada, N.; Bukholm, I.; Karesen, R.; Borresen-Dale, A.; and Kristensen, V. N. Intratumoroual mRNA expression of genes from the oestradiol metabolic pathway and clinical and histopathological parameters of breast cancer. Breast Cancer Research 2004, 6, R46-R55.

20. Pasqualini, J. R.; Chetrite, G. S.; Blacker, C.; Feinstein, M.-C.; Delansorne, R.; Talbi, M.; and Maloche, C. Concentrations of estrone, estradiol, and estrone sulfate and evaluation of sulfatase and aromatase activities in pre- and postmenopausal breast cancer. Journal of Clinical Endocrinology and Metabolism 1996, 81, 1460-1464.

21. Roberts, K. D.; Rochefort, J. G.; Bleau, G.; and Chapdelaine, A. Plasma estrone sulfate levels in postmenopausal women. Steroids 1980, 35, 179-187.

22. Pasqualini, J. R. and Chetrite, G. S. Estradiol as an anti-aromatase agent in human breast cancer cells. Journal of Steroid Biochemistry & Molecular Biology 2005, 98, 12-17.

23. Dierks, T.; Schmidt, B.; and von Figura, K. Conversion of cysteine to formylglycine: A protein modification in the endoplasmic reticulum. PNAS 1997, 94, 11963-11968.

40 24. Stanway, S. J.; Purohit, A.; Woo, L. W.; Sufi, S.; Vigushin, D.; Ward, R.; Wilson, R. H.; Stanczyk, F. Z.; Dobbs, N.; Kulinskaya, E.; Elliott, M.; Potter, B. V.; Reed, M. J.; and Coombes, R. C. Phase I study of STX 64 (667 Coumate) in breast cancer patients: the first study of a steroid sulfatase inhibitor. Clinical Cancer Research 2006, 12, 1585-1592.

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26. Jackson, T.; Woo, L. W. L.; Trusselle, M. N.; Chander, S. K.; Purohit, A.; Reed, M. J.; and Potter, B. V. Dual aromatase-sulfatase inhibitors based on the anastrozole template: synthesis, in vitro SAR, molecular modelling and in vivo activity. Organic & Biomolecular Chemistry 2007, 5, 2940-2952.

27. Woo, L. W. L.; Bubert, C.; Sutcliffe, O. B.; Smith, A.; Chander, S. K.; Mahon, M. F.; Purohit, A.; Reed, M. J.; and Potter, B. V. Dual aromatase-steroid sulfatase inhibitors. Journal of Medicinal Chemistry 2007, 50, 3540-3560.

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29. Howe, L. R. Cyclooxygenase/prostaglandin signaling and breast cancer. Breast Cancer Research 2007, 9, 210-218.

30. Agrawal, A. and Fentiman, I. S. NSAIDs and breast cancer: a possible prevention and treatment strategy. International Journal of Clinical Practice 2008, 62, 444-449.

31. Richards, J. A. and Brueggemeier, R. W. Prostaglandin E2 regulates aromatase activity and expression in human adipose stromal cells via two distinct receptor subtypes. Journal of Clinical Endocrinology and Metabolism 2003, 88, 2810-2816.

32. Bennett,A.; Berstock,D.A.; and Carroll,M.A. Breast cancer, its recurrance and patient survival in relation to tumour prostaglandins. In Advances in Prostaglandin, Thromboxane and Leukotriene Research. 12 ed.; Raven Press: New York, 1983; pp 299-302.

33. Harris, R. E.; Chlebowski, R. T.; Jackson, R. D.; and et al Breast cancer and nonsteroidal anti-inflammatory drugs: prospective results from the women's health initiative. Cancer Research 2003, 63, 6096-6101.

34. Garcia Rodriguez, L. A. and Gonzalez-Perez, A. Risk of breast cancer among users of aspirin and other anti-inflammatory drugs. British Journal of Cancer 2004, 91, 525-529.

41 35. Ready, A.; Velicer, C. M.; McTiernan, A.; and White, E. NSAID use and breast cancer risk in the VITAL cohort. Breast Cancer Research and Treatment 2008, 109, 533-543.

36. Vane, J. R.; Bakhle, Y. S.; and Botting, R. M. Cyclooxygenases 1 and 2. Annual Reviews of Pharmacology and Toxicology 1998, 38, 97-120.

37. Brueggemeier, R. W.; Diaz-Cruz, E. S.; Li, P. K.; Sugimoto, Y.; Lin, Y. C.; and Shapiro, C. L. Translational studies on aromatase, cyclooxygenases, and enzyme inhibitors in breast cancer. Journal of Steroid Biochemistry & Molecular Biology 2005, 95, 129-136.

38. Dirix, L. Y.; Ignacio, J.; Nag, S.; Bapsy, P.; Gomez, H.; Raghunadharao, D.; Paridaens, R.; Jones, S.; Falcon, S.; Carpentieri, M.; Abbattista, A.; and Lobelle, J.-P. Treatment of advanced hormone-sensitive breast cancer in postmenopausal women with exemestane alone or in combination with celecoxib. Journal of Clinical Oncology 2008, 26, 1253-1259.

39. Mazhar, D.; Ang, R.; and Waxman, J. COX inhibitors and breast cancer. British Journal of Cancer 2006, 94, 346-350.

40. Barnett, J.; Chow, J.; and Ives, D. Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system. Biochimica et Biophysica Acta 1994, 1209, 130-139.

41. Renard, J.-F.; Julemont, F.; de Leval, X.; and Pirotte, B. The use of nimelsulide and its analogues in cancer chemoprevention. Anti-Cancer Agents in Medicinal Chemistry 2006, 6, 233-237.

42. Su, B.; Diaz-Cruz, E. S.; Landini, S.; and Brueggemeier, R. W. Suppression of aromatase in human breast cells by a cyclooxygenase-2 inhibitor and its analog involves multiple mechanisms independent of cyclooxygenase-2 inhibition. Steroids 2008, 73, 104-111.

43. Smith, C. J.; Zhang, Y.; and Loboldt, C. M. Pharmacological analysis of cyclooxygenase-1 in inflammation. PNAS 1998, 95, 13313-13318.

44. Li, W.; Xu, R.; Lin, Z.; Zhuo, G.; and Zhang, H. Effects of a cyclooxygenase-1- selective inhibitor in a mouse model of ovarian cancer, administered alone or in combinatipon with ibuprofen, a nonselective cyclooxygenase inhibitor. Medical Oncology 2008, Epub ahead of print.

42 CHAPTER 2

STATEMENT OF RESEARCH PROBELEM AND SPECIFIC AIMS

2.1. The research problem

Much study is and has been dedicated to understanding breast cancer growth

mechanisms and their potential inhibitory targets. However, breast cancer remains one of

the top causes of cancer-related death in women all over the world. In hormone-

dependent breast cancer, several essential estrogen-producing enzymes have been

identified. First, aromatase is a downstream, relatively specific target in local estrogen

biosynthesis that is responsible for the conversion of androgens to active estrogens.

Second, cyclooxygenase-2 (COX-2) increases aromatase transcription through its function of prostaglandin synthesis. Thirdly, steroid sulfatase (STS) additionally produces active estrogens by hydrolyzing inactive sulfated estrogens, which are highly present in both the plasma and tumor tissue of breast cancer patients. These enzymes have also been shown to be present in hormone-independent cancer cells, where estrogen production is not required for cellular growth.

43 Potent and specific inhibitors have been developed for all three targets, and

clinical trials have existed for the individual effects of the best performing inhibitors.

Some studies have begun to analyze the combination and adjuvant use of an aromatase

inhibitor with celecoxib. However, combinations of different enzyme inhibitors with

steroid sulfatase inhibitors have not been examined to study the shared networks of these estrogen-producing enzymes in a clinical setting. While the roles and regulation of both aromatase and COX-2 have been fleshed out by the significant work of many researchers, we believe that STS deserves a more significant place in the search for breast cancer therapies. We hypothesize that potent steroid sulfatase inhibitors will not only attenuate the enzyme’s action, through suppression of both its activity and expression, but their combinations with either aromatase or cyclooxygenase inhibitors should act synergistically to block the total biosynthesis of estradiol. Additionally, we seek to probe the relationships between these three enzymes in vitro to better understand the effects of the attenuation of one on the others. It was recently demonstrated by Brueggemeier,

Shapiro, and Li (see Figure 2.1., not published) that there exist strong statistical correlations between the levels of aromatase and COX-2, aromatase and steroid sulfatase, and steroid sulfatase and COX-2 in the collected tissue samples of 43 breast cancer patients. These relationships, be them causal, associated through other lurking variables, or simply coincidental, are important for the further understanding of the enzymes’ collective inhibitions for multi-targeted breast cancer therapies.

high low

Figure 2.1. Tissue microarray analysis of biomarkers and molecular targets from fixed tissue specimens from breast cancer patients. Provided by Brueggemeier, Shapiro, and Li (unpublished results).

45 2.2. Specific Aims

1. To treat ER-positive and ER-negative breast cancer cells lines with the following

compounds:

STS inhibitors DU-14 and DU-15

COX (1 and/or 2) inhibitors celecoxib, NS-398, SC-560, indomethacin,

ibuprofen, naproxen sodium, acetylsalicylic acid, piroxicam, niflumic acid

Aromatase/ER Inhibitors letrozole, exemestane, 4-hydroxy tamoxifen

2. To analyze their individual effects on STS, CYP19, and COX-2 gene expression

by qualitative Real-Time RT-PCR to determine if any compounds affect other

enzymes than their intended target.

3. To analyze their individual affects, if any, on E1-STS enzyme activity by an in-

cell radioactivity assay.

4. To investigate their abilities to effect cellular growth proliferation by the

Promega® MTS Assay.

5. To combine STS inhibitors DU-14 and DU-15 with either aromatase or

cyclooxygenase inhibitors to evaluate any combinational effects.

6. To explore the effect of potential regulators on STS expression and activity.

46 CHAPTER 3

MATERIALS AND METHODS

3.1. Chemicals, biochemicals and reagents

Radiolabeled NET-203 estrone sulfate, ammonium salt, [6,7-3H(N)]- was

obtained from Perkin Elmer, Waltham, MA. The following compounds were purchased from Sigma-Aldrich, St. Louis, MO: acetylsalicylic acid, dexamethasone, Bay 11-7082, dibutyryl cyclic AMP (DbcAMP), 17β-estradiol (E2), 17β-estradiol 3-sulfate sodium salt

(E2S), estrone 3-sulfate (E1S), H-89, 4-hydroxytamoxifen, ibuprofen, interleukin-6 (IL-6), indomethacin, naproxen sodium, niflumic acid, NS-398, prostaglandin E2 (PGE2),

phorbol 12-myristate 13-acetate (PMA), piroxicam, SC-560, testosterone, tumor necrosis factor-alpha (TNFα). DU-14 and DU-15 were gifts from Dr. Pui-Kai Li and Dr. Kyle

Selcer (synthesized at Duquesne University, Pittsburgh, USA). Celecoxib and letrozole were gifts from Dr. Ching-Shih Chen, The Ohio State University, College of Pharmacy.

Trypsin, TRIzol, glutamine, gentamycin, FBS, media, PBS and other assay reagent enzymes were obtained from Invitrogen/Gibco, Carlsbad, CA. DNA primers and probes were purchased from Applied Biosystems, sequences having been designed by Dr.

Yasuro Sugimoto. Human albumin and transferrin was provided by OSU Hospital

Pharmacy.

47 3.2. Cell culture

MCF-7, T-47D, and MDA-MB-231 breast cancer cell lines were obtained from

ATCC, Rockville, MD. Cell cultures were maintained in phenol red-free custom media

(MEM, Earle’s salts, 1.5x amino acids, 2x non-essential amino acids, L-glutamine, 1.5x

vitamins, (Gibco BRL, Carlsbad, CA)) supplemented with 10% fetal bovine serum

(FBS), 2 mM L-glutamine, and 20 mg/L gentamycin. FBS was heat-inactivated for 30

min in a 56ºC water bath before use. Cell cultures were grown at 37ºC, in a humidified

atmosphere of 5% CO2. For all experiments, cells were subcultured in either 100 mm

plates, 96-well plates, or 6-well plates. Before treatment, the cell media was changed to

either a defined media for experiments requiring strictly controlled growth factors, or a

dextran-coated charcoal (DCC) stripped-FBS culture media for longer experiments but lacking estrogens. The defined media contained DMEM/F12 (Sigma) supplemented with

1.0 mg/mL human albumin, 5.0 mg/L human transferrin, and 5.0 mg/L bovine insulin.

Cells were treated with chemicals in DMSO (vehicle) for various time points.

3.3. RNA extraction

Twenty-four hours after treatment, cells were harvested from their 100 mm dishes

or 6-well plates and total RNA was isolated by using the TRIzol reagent protocol

provided by the manufacturer. Media/treatment was removed and the cells were rinsed

with warm phosphate-buffered saline solution (PBS). TRIzol (1 mL) reagent was added

to each culture dish to lyse cells (1 mL/10 cm2) and pipetted to mix the cell lysate. The

dishes were incubated for 5 min at room temperature to allow for complete dissociation

of nucleoprotein complexes. One milliliter (1 mL) of TRIzol suspension was collected

48 and transferred to 2 mL RNase-free Eppendorf tubes. To each tube, 0.25 mL of

chloroform (0.2 mL for each mL TRIzol used) was added and the tubes were vortexed for

15 sec and then incubated for 3 min at room temperature. The samples were centrifuged

at 12000xg for 15 min at 4oC. Then, the resulting aqueous layers were transferred to a new set of tubes containing 0.5 mL of 100% isopropanol (0.5 mL for each mL TRIzol used) and vortexed for several seconds for RNA extraction. The samples were either incubated at -20oC overnight or at room temperature for 10 min to allow for RNA

precipitation. The samples were then centrifuged at 12000xg for 10 min at 4oC to pellet

the RNA. The supernatant was discarded and then 1 mL of 75% of EtOH (1 mL for each

mL TRIzol used) was added to wash the RNA pellets. The samples were re-pelleted by

centrifuging again at no more than 7500xg for 5 min at 4oC. The supernatant wash was discarded and the RNA pellets were allowed to air-dry for 5 – 10 min. DNase, RNase- free water or 10 mM Tris buffer (30 – 40 μL) was added to the pellets and was pipetted to mix. The RNA pellets were dissolved in the refrigerator for 3 – 4 h or with heat for 10 min in 60oC water bath. 10X Turbo DNase Buffer at 0.1 volume (3 – 4 μL) and 1 μL

TURBO DNase were added to the RNA solution and mixed gently and incubated at 37oC for 20 – 30 min. Then, 0.1 volumes (3 – 4 μL) DNase Inactivation Reagent were added and mixed well, allowing incubating at room temperature for 2 min, mixing at least 2-3x.

The tubes were then centrifuged at 10000xg for 1.5 minutes. The RNA supernatant was transferred to a fresh tube and either immediately measured absorbance for RNA concentration or frozen at -80oC until use.

49 To determine the concentration of the collected RNA, the RNA samples were

quickly vortexed and pipetted RNA solution to mix completely. Then 98 μL of TE solution (pH 8.0, 10 mM Tris Cl, 1 mM EDTA) was added to each well of UV transparent bottom 96-well plates (Costar 3635). RNase-free water/Tris buffer (2 μL) was added to wells for blank. RNA samples (2 μL each) were added to the plate in duplicate. A GENios plate reader, using the Magellan2 program, with a

260/280excitation filter was used to read the plate, reading both 260 for RNA concentration, and 280 nm for DNA to check purity. RNA (μg/μL) was calculated by taking the average over the two 260 nm readings and converting by the dilution factor.

Greater than 2 μg total RNA were needed to make cDNA. The purity ratio of RNA to

DNA (A260 / A280) needed exceed 1.6.

50 3.4. cDNA synthesis

One to four milligrams of isolated total RNA was reverse-transcribed using

SuperScript II or M-MLV Reverse Transcriptases. The resulting RT templates were used

for subsequent real-time RT-PCR. To begin, the RNA solution and enough distilled,

nuclease-free water to achieve 30 μL were combined. Then, 2.5 μL Random Primer mix

(3 μg/μL) and 1 μL 10 mM dNTP Mix were added to each RNA sample well. The mixture was heated to 65oC for 5 min in a thermocycler and then quickly chilled on ice.

Next, a mix of 10 μL 5X First Strand Buffer, 5 μL 0.1 M DTT, and 1 μL RNaseOUT (an

RNase inhibitor) was added to each sample. The samples were incubated at room

temperature for 10 min, then 37oC for 2 min. Finally, 3 μL distilled, nuclease-free water

and 1 μL (200 units) SuperScript II or M-MLV Reverse Transcriptase were incorporated

into the samples and incubated at 37oC for 50 min, followed by inactivation at 70oC for

15 min in a thermocycler.

3.5. Real-time quantitative RT-PCR

Real-time quantitative RT-PCR was performed by OpticonTM 2 system (MJ

Research). The primers and probes used for STS, COX-2, and CYP19 mRNA analysis are listed in Table 3.1. 18S rRNA primers and probes were used for normalization.

First, the individual primer mixes were prepared using the ratios listed in Table 3.2.

Second, materials were added to the chilled, white PCR plates, combining the Primer

Mix, Probe (10 μM), PCR Universal Master Mix (Applied Biosystems), and water up to

20 μL (see ratios in Table 3.3.). Then 5 μL of cDNA were added in duplicate for each sample, adding 10 mM Tris buffer in the blank wells. The wells were capped with PCR 51 clear, flat-top lid strips. To ensure mixing, the plate was then centrifuged for 2 min at

1200 rpm at 4oC. The MJ Research RT-PCR machine and computer, using the Opticon II program, was set up for a singleplex experiment, measuring the FAM dye only.

Additionally, the TaqMan Standard protocol was selected for a 25 μL reaction volume

(Step 1: 50oC for 2 min, Step 2: 95oC for 10 min, Step 3: 95oC for 15 sec, Step 4: 60oC

for 1 min, Step 5: repeat steps 3 and 4 for 49 cycles). Upon completion of the run, the

data was viewed in Log Scale and the blank well values were subtracted. Then, the

appropriate cycle ranges for each gene were selected (e.g. 18S = 10, CYP19 = 21). The

thresholds for the curves were manual set at the inflection point (dx/dy = 0). The C(T)

values were then evaluated for relative expression by first normalizing to 18S (positive

control), then to DMSO-treated control.

52 Gene Oligonucleotide Sequences STS Primer (S) 5’-GAT CAT TCA GCA GCC CAT GT-3’ Primer (A) 5’-GAG GTA GGA CAA GAC AAG CAG G-3’ Probe 5’-6FAM-TTC ATA CAG CGG AAC ACT GAG ACT CC- TAMRA-3’ COX-2 Primer (S) 5’-GAA TCA TTC ACC AGG CAA ATT G-3’ Primer (A) 5’-TCT GTA CTG CGG GTG GAA CA-3’ Probe 5’-6FAM-TGG CAG GGT TGC TGG TGG TAG GA-TAMRA-3’ CYP19 Primer (S) 5’-TGT CTC TTT GTT CTT CAT GCT ATT TCT C-3’ Primer (A) 5’-TCA CCA ATA ACA GTC TGG ATT TCC-3’ Probe 5’-6FAM-TGC AAA GCA CCC TAA TGT TGA AGA GGC AAT- TAMRA-3’ 18S Primer (S) 5’-CGG CTA CCA CAT CCA AGG AA-3’ Primer (A) 5’-GCT GGA ATT ACC GCG GCT-3’ Probe 5’-6FAM-TGC TGG CAC CAG ACT TGC CCT C -TAMRA-3’

Table 3.1. Oligonucleotide primer and probe sequences for real-time PCR. All genes were analyzed using TaqMan technology and sequence-specific fluorogenic probes. (S) = Sense; (A) = Antisense.

18S STS COX-2 CYP19 S-primer (100 μM) 0.025 0.075 0.075 0.225 A-primer (100 μM) 0.050 0.225 0.225 0.150 H2O 0.925 0.700 0.700 0.625

Table 3.2. Primer Mix ratios for use in RT-PCR experiments.

18S STS COX-2 CYP19 Master Mix 12.5 12.5 12.5 12.5 Primer Mix * 1 1 1 1 Probe (10 μM) 1 1 0.75 1 H2O 5.5 5.5 5.75 5.5

Table 3.3. Mix ratios for use in RT-PCR experiments. (* from Table 3.2.)

53 3.6. STS enzyme radioactivity assay

Cells cultured in 6-well plates were treated with 1 μM E1S and radioactive

substrate in the presence or absence of various inhibitors (total volume 1 mL, 1:1000

DMSO solvent ratio to media) using defined media containing DMEM/F-12 media, 1.0

mg/ml human albumin, 5.0 mg/L human transferrin, and 5.0 mg/L bovine insulin.

o Twenty-four hours of 37 C/5% CO2 incubation following treatment, STS activity was determined by measuring the amount of water-soluble [3H] estrone-3-sulfate converted

into ether-soluble [3H] estrone. Plates were first cooled for 15 min at 4oC before

converted substrate in the medium (1 mL) was extracted with chilled ether (3 mL) and

centrifuged for 5 min. Ether supernatant (1.5 mL) was added to 4.5 mL scintillation fluid

and counted for tritium radioactivity. The amount of [3H] estrone radioactivity in the supernatant was corrected for blanks and normalized for cell contents with total DNA concentration (Section 3.7.). Results were expressed in dpm of substrate formed per microgram DNA per hour incubation time (dpm/μg DNA/h).

3.7. DNA assay

The amount of [3H] estrone radioactivity in the supernatant was normalized with

the total DNA concentration determined using the double-stranded DNA-binding

HOECHST 33258 fluorescent dye, and standardized by known amounts of calf thymus

DNA. 8 mM NaOH (1960 μL) and 40 μL 1 M HEPES were added to the cells layers remaining each treatment well. For a standard curve reading, 100 μL DNA standard (100

μg DNA/mL) was serially diluted in wells of 100 μL NaOH/HEPES solution. Then, for treatments, 100 μL cell solutions were added in duplicate to the white assay plates, 54 leaving several wells empty for blanks. Then, 100 μL dye solution (1:20 volume Hoechst

33258 dye solution in 2xTNE) was added to every well of the plate for a final 200 μL

volume per well. The amount of blue fluorescence of the dye’s binding was then

measured at 461 nm on a Tecan GENios Plus plate reader.

3.8. Cell proliferation assay

MCF-7 and MDA-MB-231 were harvested, counted, and plated at various

concentrations depending upon time desired to reach subconfluency in 150 μL total

volume/well in 96 well plates. After 24 hours, the culture medium was removed and the

wells (N = 6) were treated with control vehicle (DMSO) or compounds in DMEM/F12

media supplemented with 10% charcoal-stripped FBS every two days for two to seven

days. Following the last treatment, a 20:1 solution of 3,(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxy phenyl)-2- (4-sulfophenyl) -2H-tetrazolium (MTS) and phenazine methosulfate (PMS) was prepared and 20 μL of this reagent were added to each well.

After 1 – 2 h of incubation at 37°C, absorbance at 490 nm was measured using a

SPECTRAmax plate reader. Decreased absorbance compared to control indicates

reduced cellular metabolism.

3.9. Statistical analysis

Statistical and graphical data was determined using GraphPad Prism and

Microsoft Excel. Statistically significant differences were calculated using two-tailed student’s t-test. P values are reported at 95% confidence intervals.

55 CHAPTER 4

EFFECTS OF STEROID SULFATASE INHIBITORS ON mRNA EXPRESSION,

STS ENZYME ACTIVITY AND CELLULAR PROLIFERATION

4.1. Introduction

Studies in breast cancer cells and tissues have shown that the sulfatase pathway

can be between 40 and 500 times more important to the production of estradiol (E2) than the aromatase pathway, making steroid sulfatase (STS) the more important target in the estrogen biosynthetic cascade. STS activity and mRNA expression are very high in intact hormone-dependent cell lines, such as MCF-7 and T-47D cells 1. However, STS activity

appears low in intact hormone-independent cells lines like MDA-MB-231 and MDA-

MB-436. But, upon homogenization of these cells, STS activity become intensely

detectable, suggesting there may be inhibitory factors in these cell lines limiting the

appearance of their STS activity. In a study using RT-PCR, STS mRNA expression was

shown to be variable over the different breast cancer cells lines tested. STS mRNA levels

were shown to be highest in T-47D (ER-positive) and MDA-MB-231 (ER-negative) cell

lines 1. Though these separate reports seem in conflict, the increase seen in STS mRNA

correlates with the STS enzyme activity found in the homogenized cells. Poor prognosis,

lymph node metastases, and tumor histological grade were all positively associated with

56 STS mRNA levels, but not with aromatase or 17β-HSD levels in patients with estrogen

receptor (ER)-positive tumors 2. Furthermore, STS mRNA was a significant prognostic

indicator of shorter relapse-free survival, independent of either tumor grade or lymph

node status. By this theory, any cancerous cells that escape tumor removal surgery (i.e.

lumpectomy of the breast) will maintain their ability, via their high STS activity, to

quickly grow and thus reduce the time to tumor reoccurrence.

As previously described in Chapter 1, STS inhibitors have been developed and

optimized to achieve potent and sustainable modulation of the enzyme’s action. Some

compounds, such as that seen with synthetic progestin Promegestone (R-5020), can

decrease STS mRNA as well as inhibiting the enzyme activity 1. It is possible that these

types of anti-sulfatase compounds not only affect the enzyme itself, but may also affect

transcriptional factors. The STS inhibitors DU-14 and DU-15 (see Figure 4.1.) studied in the following research were developed by investigators at Duquesne University in

Pittsburgh, Pennsylvania, USA and are named “DU” as such.

DU-14 H iPr N O 12 CH3 N iPr O H2NO2SO

DU-15 H2NO2SO

Figure 4.1. STS sulfamate inhibitors designed by P.-K. Li and K. Selcer at Duquesne University. 57 The steroidal DU-15 compound was found in an attempt to synthesize analogs of

estrone 3-O-sulfate, one of the enzyme natural substrates. Before the substitution of the

3-position with a sulfamate group, Li et al first attempted such substitutions as

2- - phosphates (-OPO3 ), methylsulfonates (-OSO2CH3), methylenesulfonates (-CH2SO3 ),

3-5 amines (-NH2), and thiols (-SH) at the 3-position of the estrone-based steroid nucleus .

However, it was found at that time that their STS inhibitors required an oxygen (or similar) link between the steroid and a sulfonate moiety, which needed an oxygen anion or other electronegative group at the sulfur, to achieve high affinity similar to E1S

enzyme binding; these findings eliminated most of their compounds for further

development. The phosphate compounds, though having good binding and electronic

structure consistent with the desired template, were not worth pursuing due to the

inability of STS to hydrolyze the compounds.

The DU-14 compound was discovered upon investigation into non-steroidal STS

inhibitors. Being that hydrolysis of the steroidal compounds released potentially

estrogenic species, Li et al developed a series of (p-O-sulfamoyl)-N-alkanoyl tyramines,

which were found to be potent and irreversible inhibitors of STS 6;7. The

phenylsulfamate portion of the compounds mimicked the A-ring of E1S, whereas the remaining N-alkanoyl group served to provide hydrophobic bulk to occupy the rest of the enzyme’s active site. Additionally, the amide functionality of the side chain is essential for the compounds’ activity, possibly taking advantage of an available hydrogen bonding site within the enzyme. The two-carbon spacer between the ring systems and the amide provides the optimal distance. The best compound of this group, (p-O-sulfamoyl)-N- tetradecanoyl tyramine and later termed DU-14, showed an IC50 against human placental

58 microsomes (a source of high STS levels) of 55.8 nM and an IC50 of 350 nM in MDA-

MB-231 cells 8. It dose-dependently inhibited STS activity as well as MCF-7 cell

proliferation over 7 days in the presence of 1 μM E1S (IC50 of 38.7 nM). DU-14 has also

been examined in vivo in nude mice transplanted with engineered STS-over producing

MCF-7 cells (“MCS-2”); the compound decreased tumor size by 75% after 18 days in the

9 absence of E1S as well as blocking E1S-stimulated tumor growth .

Upon further understanding of sulfamate nonsteroidal inhibitors, Li et al continued to optimize their steroidal compounds since they remained the most potent inhibitors to date (see estrogenic EMATE in Chapter 1). Addition of long or bulky alkyl

chains at the C17 position of the steroid nucleus appeared to allow the compound to

insert itself into cellular membranes, anchoring the inhibitors near the known location of

the STS enzyme. Two series of compounds, 17β-(N-alkylcarbamoyl)-estra-

1,3,5(10)trien-3-O-sulfamates and 17β-(N-alkanoyl)estra-1,3,5(10)-trien-3-O-sulfamates,

were created as potent non-estrogenic inhibitors with variable alkyl chain length 10. This method was a success, since the freshly designed steroidal inhibitors did not stimulate cell proliferation and still had potent anti-STS activity (0.5 nM in MDA-MB-231 cells) exceeding that of EMATE. DU-15 came out of the carbamoyl series, having an IC50 of approximately 5 nM in the MCS-2 cell line (data not published). DU-15 is currently being developed outside academia for pharmaceutical use.

Li et al found that steroidal STS inhibitors were also able to inhibit breast cancer cellular growth, including estrogen-independent MDA-MB-231 cells, but not to the same extent as in ER-positive MCF-7 cells. This implies that MCF-7 cells may be more sensitive to STS inhibitor growth inhibition, being that their growth is dependent upon 59 the cell’s estrogen production. The (p-O-sulfamoyl)-N-alkanoyl tyramines also were able

to inhibit MDA-MB-231 sulfatase activity irreversibly. It is interesting to note here that

MDA-MB-231 cells tested 7-fold higher than MCF-7 cells for intact cellular STS

activity. This STS activity in MDA-MB-231 cells was additionally shown in an

immunoreactivity assay using a designed steroid sulfatase antibody 11. Both MDA-MB-

231 and MCF-7 cells showed high levels of STS protein in culture. In patient samples,

however, ER+/PR+ human breast carcinomas had high STS immunoreactivity, but ER-

/PR- samples had little. Though seemingly contradictory, this data actually confirms

previous studies showing that STS expression is variably distributed in different types of

breast cancer tissues. Larger sample sizes may be needed to create a more general

consensus, but since tumors themselves are not homogeneous, conflicting data may likely

be observed many times over.

We hypothesize there are sophisticated networks between the three major

enzymes in breast cancer estrogen biosynthesis. Therefore, we analyzed multiple enzyme

inhibitors in the attempt to identify the details of those relationships. In the following

studies, STS inhibitors DU-14 and DU-15 were analyzed in MCF-7 and MDA-MB-231

cells, looking for their effects on STS enzyme activity, on STS, COX-2, and CYP19 mRNA expression, and on breast cancer cell proliferation.

60 4.2. Results and discussion

4.2.1. Steroid sulfatase enzyme activity

We first confirmed the anti-sulfatase activity of DU-14 and DU-15 in our cell

3 lines. Using a radiometric assay to determine the amount of tritium labeled [ H]-E1S

3 conversion to [ H]-E1 in intact cells, DU-14 significantly inhibited STS activity by

approximately 80% at 100 nM and 70% at 250 nM (Figure 4.2.) in MCF-7 cells. DU-14

was slightly more effective in ER-negative MDA-MB-231 cells, inhibiting STS activity

88% at 100 nM and 93% at 250 nM. This nonsteroidal compound is very potent at

eliminating nearly all STS activity after 24 hours.

Steroidal DU-15 appeared to be even more effective than DU-14. DU-15

inhibited all STS activity in MCF-7 cells, with the results actually showing a negative 3H dpm count value. Though it is not possible to inhibit an enzyme more than is actually available, this data is represented in its raw form normalized only to the control activity with DMSO vehicle and 1 μM supplemented E1S. Nevertheless, one can observe that

DU-15 abolished all available STS enzyme activity at 10 and 25 nM. In MDA-MB-231

cells, DU-15 inhibited STS by 84% at 10 nM and completely at 25 nM.

The IC50 values of DU-14 were previously shown to be it the range of 25 to 350

nM, whereas DU-15 had 50% inhibitory concentrations of approximately 0.5 to 5 nM.

These results support the claims of the highly potent inhibitory activities of both DU

compounds. Based upon the wide range given for DU-14, we determined the IC50 concentration for the drug (Figure 4.3.). We confidently found an IC50 of 8.3 nM in

61 MDA-MB-231 cells, and a less statistically confident IC50 of 12.6 nM in MCF-7 cells.

Due to high error in the lower concentrations in MCF-7 results, two points were removed from the IC50 calculation. However, even with these considerations, we were able to show that DU-14’s inhibition in our experiments tended towards the more potent end of the given range.

STS Enzyme Activity in Breast Cancer Cells 200 MCF-7 175 MDA-MB-231 150 # 125 # # n = 3 100 * * 75 H in toluene (dpm) layer

3 50

25 Raw

0 No cells No No cells No S (1 uM) S (1 S (1 uM) S (1 1 1 E E DU-15 (10 nM) (10 DU-15 nM) (25 DU-15 DU-15 (10 nM) DU-15 (25 nM) DU-14 (100nM) DU-14 (250nM) DU-14 (100nM) DU-14 (250nM)

Figure 4.2. Suppression of steroid sulfatase activity in MCF-7 and MDA-MB-231 breast cancer cells by DU-14 and DU-15 at multiple concentrations in the presence of 1 μM E1S. The results were normalized agents a control with vehicle (DMSO). Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 4, unless otherwise shown. MCF-7: 100% control = 181 dpm/24 h. MDA- MB-231: 100% control = 171 dpm/24 h.

100

75 MCF-7 IC50 = 12.6 nM

50 dpm/ug DNA/h MDA-MB-231 24 h Sulfatase Activity 25 IC50 = 8.3 nM

0 -5 -4 -3 -2 -1 0 1 2 3 log [DU-14] (μM)

Figure 4.3. DU-14 suppression of steroid sulfatase activity in MCF-7 and MDA-MB-231 breast cancer cells. The results were normalized against vehicle control (DMSO) as well as total DNA. IC50 values were calculated using nonlinear regression analysis. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6 for each data point. MCF-7: 100% control = 39.9 dpm/μg DNA/h. MDA-MB-231: 100% control = 8.9 dpm/μg DNA/h.

63 4.2.2. Gene expression

After solidifying the DU compounds inhibition of STS in our laboratory, we next looked to see if these compounds had any affect on STS mRNA expression (Figure 4.4

(A)). DU-14 decreased STS mRNA production (non-significantly) in MCF-7 cells at 100

nM, but did not have an effect at the higher concentration tested (250 nM). In the same

cell line, non-steroidal DU-15 significantly decreased STS mRNA at a low concentration

(10 nM), but did not have an affect at the higher 25 nM concentration. Letrozole, a

potent aromatase enzyme inhibitor, had no effect on STS mRNA in MCF-7 cells at 10

nM, but significantly decreased STS mRNA at 1 μM.

The addition of 1 μM E1S had no significant effect alone or when combined with the higher doses of the DU compounds and 10 nM letrozole. It is interesting that the STS substrate did not up-regulate the gene, but we saw later that the addition of E1S to

estrogen-deprived MCF-7 cells causes a significant increase in STS mRNA (see Chapter

5). The addition of 10 nM estradiol, however, significantly decreased the sulfatase

expression by 40%, and its combinations with the higher doses of the DU compound and

10 nM letrozole also inhibited STS mRNA. However, the inhibition seen of E2 +

inhibitor was not significantly different than with E2 alone. This inhibitory effect of

estradiol can be explained by a negative feedback loop to control its own production.

Inhibition of STS would limit the amount of E2 produced by that pathway. However, this

does conflict with a reported study of differential effects of hormone treatment on STS,

64 which said they observed an increase of STS activity and expression with E2. However, multiple other studies also showed contradictory effects of estrogen on STS 12;13.

Therefore, we present our results in support of estradiol as a potential anti-STS and anti- aromatase agent.

MDA-MB-231 cells had similar STS expression patterns with DU-14 and DU-15

(Figure 4.4. (B)). The lower dose of DU-14 (100 nM) reduced STS mRNA about 25%, whereas the higher dose (250 nM) had no effect. DU-15, on the other hand, inhibited

MDA-MB-231 STS expression by 20 and 30%, with the larger dose significantly eliciting the higher inhibition. Though the compounds showed less overall inhibition of STS mRNA in the ER-negative cells than the ER-positive cells, the important factors appear to be that (1) the STS inhibitors can inhibit STS mRNA but (2) the inhibition is neither dose-dependent nor dependent on ER status. Therefore, in future experiments it will be important to look for the effects of the STS inhibitors on other cellular targets potentially mediating their inhibitory action on gene expression. Also to note, 1 μM letrozole also significantly inhibited STS mRNA in the ER-negative cell line greater than 50%. We will later look into aromatase inhibitors’ actions on STS (Chapter 7).

The addition of 1 μM E1S in MDA-MB-231 cells significantly inhibited STS

mRNA alone and when treated with both DU compounds, though the combinations were

not statistically different than with E1S alone. Likewise, estradiol also inhibited STS

mRNA. Here, the combinations with inhibitors may be relevant. The combination of E2 with DU-14 (250 nM) significantly increased its inhibition on STS mRNA, even more than with E2 alone. However, the addition of E2 to steroidal DU-15 (25 nM) and

letrozole (10 nM) completely eliminated their previous inhibitions when used alone. 65 Though MDA-MB-231 cells are estrogen-independent, they have been shown to have

significantly high STS expression and activity. Therefore, the modulating effects on E1S and E2 on STS expression may require further investigation. Since STS is also the

enzyme used to convert adrenal DHEAS to DHEA, it is possible the hydrolysis of

androgens or even other sulfated steroids may be playing a role in the ER-negative cell

lines.

Due to DU-15’s significant inhibition of STS mRNA in both MCF-7 and MDA-

MB-231, we decided to test for a 50% inhibitory concentration value (Figure 4.5).

Except for one outlier at 500 nM in the MDA-MB-231 study, the inhibitor appeared to,

contradictorily so, inhibit STS mRNA production dose-dependently. However, an IC50 value could not be obtained for DU-15 in MCF-7 cells due to the fact that even the lowest concentration tested (5 pM) inhibited sulfatase expression near 50% and thus an appropriate curve fit could not be created. The drug’s inhibition compared to the initial study is much greater and with possible dose-dependency. Conditions were not changed between experiments, except for passage of the cell lines (different “ages” of cells), use of a different stored collection of MDA-MB-231 cells, or the preparation of new drug dilutions. Therefore, human error could be responsible for the contradictory data.

Otherwise, variability in cell samples and passages may explain the sensitivity and varying response of the cells to DU-15.

66 (A) MCF-7

150

125 n = 8

100

# 75 # # # # (STS / 18S) (STS # 50

25 RelativeExpression mRNA STS

0 ) O O S S S O 2 2 2 M) M) M) 1 1 1 E E nM) μ μ E E 0 nM) 0 0 n 5 nM 0 nM) + ) + ) + E DMS 0 1 (1 DMS + + DMS ) + E (10 nM) M 5 (2 le S (1 M) 2 nM) 15 (1 o 1 nM) E 14 (1 - z E 5 nM 25 nM - U-1 10 250 DU D trozole ( tro 250 n 5 ( DU DU-14 (25 5 (2 Le Le 14 ( 14 ( - U-1 U-1 D U- D rozole ( DU D Letrozole (10 n Let

(B) MDA-MB-231

125

100

n = 8 # # 75 # # n = 8 # # 50 # (STS / 18S)

25 Relative ExpressionmRNA STS

O O S S S 2 2 2 M) 1 1 1 E E nM) M) M) MS 0 nM) μ MS μ E + + E D 50 n (25 1 D DMSO 2 (10 nM)nM) nM) (100 nM) ( 5 (10 nM)5 e ( e (1 S (1 2 4 1 1 ol 1 E 5 14 - E 5 nM) + 2 - 2 U DU- DU 5 ( e (10 nM) + DU-1 D ( 1 Letroz Letrozol 15 - zol -14 (250U o U D tr DU- D DU-14 (250 nM) + E Le Letrozole (10 nM) + E

Figure 4.4. Effect of steroid sulfatase inhibitors with estrogens on STS mRNA expression. (A) MCF-7 and (B) MDA-MB-231 cells were treated with each of the agents at the indicated concentrations and relative STS mRNA expression was measured as described in Chapter 3. The results were normalized agents a control with vehicle (DMSO). Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown. .

MCF-7 MDA-MB-231 1.50

1.25 n = 6 n = 3

1.00

0.75 n = 6 # * * * * # (STS / 18S) / (STS

Expression * 0.50

Relative mRNA STS * 0.25

0.00 5 uM 5 nM 5 pM 5 uM 5 nM 5 pM 50 nM 50 pM 50 50 nM 50 pM 50 DMSO DMSO 500 nM 500 pM 500 nM 500 pM

[DU-15]

Figure 4.5. DU-15 suppression of STS mRNA expression in MCF-7 and MDA-MB-231 cells. The results were normalized agents a control with vehicle (DMSO). Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown.

68 Upon analyzing the DU compounds in MCF-7 cells, only the 10 nM dose of DU-

15 and 1 μM dose of letrozole had any effect on CYP19 expression, both inhibiting aromatase greater than 50% (Figure 4.6 (A)). In fact, though not significant, the 250 nM treatment of DU-15 appeared to stimulate CYP19 to 150% of the control expression.

Addition of E1S eliminated all inhibition in MCF-7 cells and even caused its combination

with DU-14, previously inactive against CYP19, to stimulate CYP19 expression nearly 2-

fold. However, while estradiol treatment alone slightly yet statistically inhibited CYP19 mRNA by 25%, its combination with both DU compounds and letrozole had no affect at all on aromatase expression. Therefore, the addition of E2 also eliminated DU-15 and

letrozole’s inhibition. Since MCF-7 cells rely on estrogens for growth, the addition of E2

would have a negative feedback on its own synthesis, as previously discussed, and E1S

should not affect aromatase since it is not one of its enzyme substrates. However, their

blockade of two compounds’ solo inhibition suggested that neither compound is eliciting

their action without some other estrogen-regulated mediator.

Aromatase expression in MDA-MB-231 cells was inhibited by all three inhibitors, except for DU-14 at its higher 250 nM dose and letrozole at its lower 10 nM dose

(Figure 4.6. (B)). Like STS, it is possible these MDA-MB-231 cells have significant

CYP19 levels, and if the DU compounds like letrozole have anti-aromatase activities,

they could affect its expression even in an ER-negative cell line. Additional estrogens

E1S and E2 both abolished all inhibitory abilities of the inhibitors as was seen in MCF-7

cells. E1S (1 μM) even appeared to stimulate CYP19 in these cells.

69 (A) MCF-7

250 n = 8 # 200

n = 8 150

n = 6 n = 7 mRNA Expression

100

(CYP19 / 18S) / (CYP19 # CYP19

50 # # Relative 0 ) ) O M) M ) S S S 2 2 2 S nM) n n M M) 1 1 1 M M μ μ + E D 0 0 DMSO 1 + E + E + E DMSO 5 (10 1 (1 ) M) + E M) (2 ( (10 n n n 5 le le S ( M) M) 2 1 o o 1 n n E - z z E 0 -14 o o 1 (25 (10 U DU DU-15 (25r nM) r ( 5 DU-14 (100D nM) (250 1 le Let Let 4 le o 1 zo -14 (250 nM) + E - o U DU- troz DU-15 (25tr nM D e DU e L L

(B) MDA-MB-231

250

n = 8 200 #

n = 8 150 n = 7 mRNA Expression

100 (CYP19 / 18S)

CYP19 # # # 50 # Relative 0 ) O ) O S S S O 2 2 2 M M M) 1 1 1 E nM) n μ μ E E nM) + MS 0 MS + + MS D 50 nM) (10 (1 D D (2 (1 (10 M) M) + E e S 2 n n 4 E 1 E 0 0 -14 (100 -1nM) U-15 zol (1 U D DU-15 (25ro nM) (25 e D DU et Letrozole (1 L -15 (25 nM) zol -14 (250U nM) U-14 DU-15 (25ro nM) + E U D D et D L Letrozole (10 nM) + E

Figure 4.6. Effect of steroid sulfatase inhibitors with estrogens on CYP19 mRNA expression. (A) MCF-7 and (B) MDA-MB-231 cells were treated with each of the agents at the indicated concentrations and relative STS mRNA expression was measured as described in Chapter 3. The results were normalized agents a control with vehicle (DMSO). Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown.

70 Finally, inhibitor effects were tested on COX-2 mRNA. In MCF-7 cells, none of the inhibitors inhibited COX-2 mRNA, but the higher concentrations of DU-14 and DU-

15 may stimulate COX-2 expression (Figure 4.7 (A)). It is possible that the inhibition of

the sulfatase pathway may additionally activate the regulation of the aromatase pathway.

The increase of COX-2 would result in an increase of aromatase-stimulating PGE2. The aromatase pathway could then compensate for the blocked sulfatase pathway. Another possibility is that other COX-related signaling pathways may be activated to overcome the initial stall of cellular growth created by the STS inhibitors. Pathways including NF-

κB or MAPK signaling have been associated with non-estrogen-related COX-2

14-16 expression . The addition of E1S did not affect COX-2 expression, regardless of inhibitor presence. However, E2 blocked any inhibitor-stimulated COX-2 expression. In

MCF-7 cells, E2 could act through COX-2 to regulate its own synthesis, as seen with the

other genes tested here.

In MDA-MB-231 cells (Figure 4.7 (B)), both of the lower doses of the DU

compounds inhibited COX-2 mRNA (by 30% for 100 nM DU-14; by 50% for 10 nM

DU-15). Letrozole (1 μM) also inhibited COX-2 expression by 50%, which may be a

high-dose, non-specific action (like that seen for STS) or the result of an additional

specific binding site on a non-aromatase target. The addition of both E1S and E2 eliminated the inhibitory effects of all three compounds, though E2 alone showed

minimal independent suppression of COX-2 mRNA. Due to DU-15’s apparent inhibitory activity against COX-2, we tested the compound for dose-response in MCF-7 cells

(Figure 4.8). Though DU-15 effectively inhibited COX-2 mRNA at various concentrations, no specific trend was observed and no IC50 value could be calculated. 71 (A) MCF-7

200 n = 8 # n = 8

n = 4

150 n = 7

n = 8 n = 8 100 (COX-2 / 18S) / (COX-2

50 Relative COX-2mRNA Expression 0 ) ) S S S O ) 2 2 2 M) M) 1 1 1 μ μ ) + E DMSO DMSO (1 DMS 10 nM 250 nM) ) + E ( S 2 1 E -14 (100 -nM14 ( E U DU-15 (10DU-15 nM) (25 nM) trozole (1 DU D Le Letrozole (10 nM -15 (25 nM -14 (250 nM) + E DU-15 (25 nM) + E DU DU DU-14 (250 nM) + E Letrozole (10 nM Letrozole (10 nM) + E

(B) MDA-MB-231

# 150 #

125

100

75 #

Expression #

(COX-2 / 18S) / (COX-2 50 Relative COX-2 mRNA COX-2 Relative 25

M) S S S 2 E 2 E 2 nM) nM) n nM) M) M) 1 E 1 E 1 0 μ μ nM) + + DMSO 00 DMSO + + DMSO 0 (1 (1 (1 (1 e S M) M) (1 5 (25 n n 2 4 (250 ole (10 nM) E 1 E 5 nM) 0 nM) -14 -1 U-1 z 5 (2 (1 U DU-15 D 250 nM) +(2 E 5 D DU etrozol ( 5 e (10 Letro L -1 zol DU DU-1 ro DU-14 (250 nM) + E DU-14 t Letrozole Le

Figure 4.7. Effect of steroid sulfatase inhibitors with estrogens on COX-2 mRNA expression. (A) MCF-7 and (B) MDA-MB-231 cells were treated with each of the agents at the indicated concentrations and relative STS mRNA expression was measured as described in Chapter 3. The results were normalized agents a control with vehicle (DMSO). Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown.

1.5

n = 8 1.0 n = 6 # # * * Expression

(COX-2 / 18S) / (COX-2 0.5 Relative COX-2mRNA

0.0 M M M M μ n nM p p MSO 0 0 5 nM 0 0 5 pM 5 5 5 D 50 50

[DU-15]

Figure 4.8. Dose-response of DU-15 on COX-2 mRNA expression in MCF-7 cells. Representated as a bar chart since no IC50 could be calculated. The results were normalized agents a control with vehicle (DMSO). Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown. .

73 4.2.3. Breast cancer cell growth

Weak growth inhibition was observed in ER-positive MCF-7 cells treated with

DU-14 as low as 50 nM (Figure 4.9 (A)). However, the most effective and consistent

dose was 50 μM over 7 days. A different mechanism of action other than STS inhibition

may occur at concentrations greater than 50 μM, shown by a non-dose dependent trend.

In ER-negative MDA-MB-231 cells, minimal growth inhibition by DU-14 occurred at all concentrations tested as low as 50 nM (Figure 4.9 (B)).

In general, DU-15 treatment produced dose-dependent growth inhibition in MCF-

7 cells (Figure 4.10 (A)), with significant growth inhibition as low as 5 nM. However, the most effective and consistent doses over 7 days ranged between 25 and 50 μM. In

MDA-MB-231 cells (Figure 4.10 (B)), significant growth inhibition occurred at higher concentrations tested up to 50 μM. At lower concentrations, an inconsistent inhibitory pattern was observed over the time points tested.

Overall, both STS inhibitors are capable of inhibiting cellular growth in ER- positive and ER-negative cell lines in vitro. Whether this cytotoxicity is due to STS inhibition or another action is for future discovery.

74 (A) MCF-7 2 days 4 days 125 7 days n = 5 th n = 5 100 # # # # # # # n = 5 # # n = 5 # #

grow * l 75 * *

ro * * t 50 con f * o 25 * %

0 5 uM 5 uM 1 uM 5 uM 1 uM 50 uM 25 uM 10 uM 50 nM 50 uM 25 uM 10 uM 50 nM 50 uM 25 uM 10 uM 50 nM 75 uM 75 uM DMSO DMSO DMSO 100 uM 100 nM 500 nM 500 nM 500 100 uM 100 uM 100 FudR (10 ng/ml) FudR (10 ng/ml) FudR (10 ng/ml)

(B) MDA-MB-231 2 days 4 days 125 7 days th n = 5 100 n = 5 # # # # # # * # # # * n = 4

grow * * # l * # 75 * ro t 50 con f * o 25 % * * 0 5 uM 5 uM 1 uM 5 uM 1 uM 50 nM 50 uM 25 uM 10 uM 50 nM 50 uM 25 uM 10 uM 75 uM 50 uM 25 uM 10 uM 50 nM 75 uM DMSO DMSO DMSO 500 nM 500 uM 100 uM 100 100 uM 100 nM 500 500 nM 500 FudR (10 ng/ml) FudR (10 ng/ml) FudR (10 ng/ml)

Figure 4.9. Proliferation assay of DU-14 in (A) MCF-7 and (B) MDA-MB-231 cells over 2, 4, and 7 days of treatment. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

75 (A) MCF-7

2 days 150 4 days 125 7 days th

100 grow l n = 5 # n = 11 # ro * # # t 75 * * * n = 11 * * # * * * * * con f 50 * o *

% * * * 25 * *

n/a 0 5 nM 5 nM 5 nM 5 uM 5 uM 1 uM 5 uM 1 uM 50 uM 25 nM 50 nM 50 uM 35 nM 25 nM 50 nM 50 uM 35 nM 10 uM 10 uM 25 uM 10 uM 50 nM DMSO DMSO DMSO 100 nM 100 nM 100 500 nM 500 nM 500 nM 500 FudR (10 ng/ml) FudR (10 ng/ml) FudR (10 ng/ml)

(B) MDA-MB-231

400 n = 5 2 days * 4 days 300 * 7 days 200 n = 5 th 175 # * 150 grow l

ro 125 t n = 5 n = 11 # 100 n = 11 ### #

con #

f * * * n = 5 o 75 * # # * n = 5 % n = 5 * # * 50 * * * * 25 * * 0 5 uM 5 uM 5 uM 5 nM 5 nM 5 nM 1 uM 1 uM 50 uM 35 uM 25 uM 10 uM 50 nM 25 uM 50 nM 10 uM 50 uM 50 uM 35 uM 25 uM 50 nM 10 uM 10 DMSO DMSO DMSO 100 nM 100 nM 500 nM 500 nM 500 nM FudR (10 ng/ml) FudR (10 ng/ml) FudR (10 ng/ml)

Figure 4.10. Proliferation assay of DU-15 in (A) MCF-7 and (B) MDA-MB-231 cells over 2, 4, and 7 days of treatment. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

76 4.3. Conclusions

The two steroid sulfatase inhibitors provided by Li and Selcer were shown to be

effective inhibitors of STS activity, breast cancer cell proliferation, and potentially STS,

CYP19, and/or COX-2 mRNA expression. However, it remains to be seen if the compounds act directly or through other modulators to achieve these effects. Estrogens estrone sulfate and estradiol are able to block or potentiate other inhibitors’ actions while often having actions of their own on STS, CYP19, and COX-2 gene expression.

Acquired resistance to existing anti-estrogen therapies necessitates the development of novel inhibitors, including those for novel targets. This is especially observed in the Purohit, Woo, Potter and Reed research groups, investigating the efficacies of dual aromatase and sulfatase inhibitors (DASIs) 17;18. Their methodology

includes the inclusion of a sulfamate group on an existing aromatase inhibitor base,

allowing for the initial sulamoylation of the STS active site FGly residue and the

subsequent release of a complete aromatase inhibitor to leave the STS active site to find

the active site of an aromatase enzyme and exert its reversible action (coordination with

the P450 heme iron) on its secondary target.

As hypothesized by this group, the inhibition of both estrogen-producing

pathways should completely block estradiol synthesis. This same methodology may also be applied to other enzyme inhibitors to include STS inhibition. Further research may

investigate the incorporation of a sulfamate-based COX-2 dual inhibitor. Being that

77 COX inhibitors indirectly (or directly if in the SAM category of compounds) affect

aromatase transcription and downstream activity, as well as exhibiting their own pro-

apoptotic activities, the duality of an STS-COX-2 inhibitor may actually be able to target all three enzymes implicated in the growth of hormone-dependent breast cancer.

78 4.4. References

1. Pasqualini, J. R.; Chetrite, G.; and Nestour, E. L. Control and expression of oestrone sulphatase activities in human breast cancer. Journal of Endocrinology 1996, 150, S99-S195.

2. Miyoshi, Y.; Ando, A.; Hasegawa, S.; Ishitobi, M.; Taguchi, T.; Tamaki, Y.; and Noguchi, S. High expression of steroid sulfatase mRNA predicts poor prognosis in patients with estrogen receptor-positive breast cancer. Clinical Cancer Research 2003, 9, 2288-2293.

3. Li, P. K.; Pillai, R.; and Dibbelt, L. Estrone sulfate analogs as estrone sulfatase inhibitors. Steroids 1995, 60, 299-306.

4. Selcer, K. W. and Li, P. K. Estrogenicity, antiestrogenicity and estrone sulfatase inhibition of estrone-3-amine and estrone-3-thiol. Journal of Steroid Biochemistry & Molecular Biology 1995, 52, 281-286.

5. Selcer, K. W.; Jagannathan, S.; Rhodes, M. E.; and Li, P. K. Inhibition of placental estrone sulfatase activity and MCF-7 breast cancer cell proliferation by estrone-3-amino derivatives. Journal of Steroid Biochemistry & Molecular Biology 1996, 59, 83-91.

6. Li, P. K.; Milano, S.; Kluth, L.; and Rhodes, M. E. Synthesis and sulfatase inhibitory activities of non-steroidal estrone sulfatase inhibitors. Journal of Steroid Biochemistry & Molecular Biology 1996, 59, 41-48.

7. Chu, G.-H.; Milano, S.; Kluth, L.; Rhodes, M. E.; Boni, R.; Johnson, D. A.; and Li, P. K. Structure-activity relationship studies of the amide functionality in (p-O-sulfamoyl)-N-alkanoyl tyramines as estrone sulfatase inhibitors. Steroids 1997, 62, 530-535.

8. Selcer, K. W.; Hegde, P. V.; and Li, P. K. Inhibition of estrone sulfatase and proliferation of human breast cancer cells by non-steroidal (p-O-sulfamoyl)- N-alkanoyl tyramines. Cancer Research 1997, 57, 702-707.

9. Nakata, T.; Takashima, S.; Shiotsu, Y.; Murakata, C.; Ishida, H.; Akinaga, S.; Li, P. K.; Sasano, H.; Suzuki, T.; and Saeki, T. Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients. Journal of Steroid Biochemistry & Molecular Biology 2003, 86, 455-460.

10. Li, P. K.; Chu, G.-H.; Guo, J. P.; Peters, A.; and Selcer, K. W. Development of potent non-estrogenic estrone sulfatase inhibitors. Steroids 1998, 63, 425-432.

11. Selcer, K. W.; Difrancesca, H. M.; Chandra, A. B.; and Li, P. K. Immunohistochemical analysis of steroid sulfatase in human tissues. Journal of Steroid Biochemistry & Molecular Biology 2007, 105, 115-123. 79 12. Pasqualini, J. R. and Chetrite, G. S. Estradiol as an anti-aromatase agent in human breast cancer cells. Journal of Steroid Biochemistry & Molecular Biology 2005, 98, 12-17.

13. Tobacman, J. K.; Hinkhouse, M.; and Khalkhali-Ellis, Z. Steroid sulfatase activity and expression in mammary myoepithelial cells. Journal of Steroid Biochemistry & Molecular Biology 2002, 81, 65-68.

14. Jobin, C.; Morteau, O.; Han, D. S.; and Sartor, R. B. Specific NF-kB blockade slectively inhibits tumuor necrosis factor-a-induced COX-2 but not constitutive COX-1 gene expression in HT-29 cells. Immunology 1998, 95, 537-543.

15. Takada, Y.; Bhardwaj, A.; Potdar, P.; and Aggarwal, B. B. Nonsteroidal anti- inflammatory agents differ in their ability to suppress NF-kB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene 2004, 23, 9247-9258.

16. Yamamoto, Y. and Gaynor, R. B. Therapeutic potential of inhibition of the NF- kB pathway in the treatment of inflammation and cancer. Journal of Clinical Investigation 2001, 107, 135-142.

17. Woo, L. W. L.; Bubert, C.; Sutcliffe, O. B.; Smith, A.; Chander, S. K.; Mahon, M. F.; Purohit, A.; Reed, M. J.; and Potter, B. V. Dual aromatase-steroid sulfatase inhibitors. Journal of Medicinal Chemistry 2007, 50, 3540-3560.

18. Woo, L. W. L.; Sutcliffe, O. B.; Bubert, C.; Grasso, A.; Chandler, S. K.; Purohit, A.; Reed, M. J.; and Potter, B. V. L. First dual aromatase-steroid sulfatase inhibitors. Journal of Medicinal Chemistry 2003, 46, 3193-3196.

80 CHAPTER 5

EFFECTS OF POTENTIAL REGULATORY FACTORS ON mRNA

EXPRESSION, STS ENZYME ACTIVITY, AND CELL PROLIFERATION

5.1. Introduction

5.1.1. STS promoters

Though steroid sulfatase has proven to be an interesting and important target for

the study of breast cancer treatment, in that it plays a major role in the production of estrogen precursors, the regulatory control of its gene transcription (via transcription factors and cis-acting elements of DNA) as well as its intracellular control remain inconclusive. However, to this date, promoter and 5’ upstream regions of STS have only been characterized in human placental cells 1. There STS activity is high, but the

promoter has low basal activity; it therefore appears that additional regulation is needed

to achieve high STS levels in other tissues.

Typically in eukaryotic cells, gene promoters include a TATA box or other cis-

acting initiator elements at the transcription start site for basal transcription. However, most genes additionally have sites for the binding of transcription enhancers and/or

silencers, to either increase or decrease transcription. These transcription factors act by

mediating the binding of the RNA Polymerase II transcription complex to the promoter

81 region. General transcription factor species include TFIIA, TFIIB, TFIID, etc., which are

all associated with RNAPoly II in the preinitiation complex. Other more specific but

commonly known transcription factors utilized in cancer processes include c-Fos, c-Jun,

c-Myc, p53, nuclear and steroid hormone receptors, CREB, and STAT. Though differing

in their induction and target DNA sequences, they all are proteins with one or more

DNA-binding domains. Additionally, many genes will have multiple promoter regions,

evidenced by CYP19’s tissue-specific promoters 2;3. These separate promoters are

regulated by different regulatory species and can vary greatly from the gene’s basal

transcription.

The human STS gene is located on the short arm of the X chromosome and

contains 10 exons corresponding to 583 amino acids and a signal peptide 4. In the Valle

group, 5’-rapid amplification of STS cDNA showed that its transcription in adipose tissue

is regulated by two promoters, not including the main one used for placental

transcription 5. Adipose transcripts have a distinct untranslated first exon, 0a and 0b,

followed by a commonly translated exon 1b, and nine other exons shared with the

placental transcripts. The resulting proteins have slightly different N-terminal regions; but, since the N-terminus is later cleaved during enzyme maturation, these dissimilarities do not affect any of the enzymes’ catalytic domains. Nevertheless, the regulation of these different promoters is still not understood. The placental promoter (a putative 110 bp region) lacks any binding sites for known transcription factors and contains no TATA box or sequences for other initiator elements, such as SP1 sites or CG rich TATA-less regions 6.

82 STS, like other genes, has multiple mRNA variants from numerous splicing and

promoter options. Alteration of the 5’ region of the STS gene (using four different leader

exons) was found to produce four distinct STS products in MCF-7 cells by Zaichuk et al

(see Figure 5.1.) 1. It is likely the use of these alternate exons confers tissue-specificity.

The largest isoform contains the exon previously identified in placental cells (named

exon 1a). The next two products share an identical 131 bp sequence (exon 1b) identified in adipose tissue. The longer of the two contains an additional sequence, which has the same sequence as adipose-specific exon 0b. The fourth and shortest product is derived from the first intron (termed exon 1c). These experiments showed that STS is comprised of both a variable and constant region, with a total length of 318 kb. The ATG initiation codon is located at various positions in the different transcripts. Placental STS mRNA

was mainly 1a transcripts. However, the highest levels of tissue-specific STS mRNA

expression were found to use transcript 1b. These tissues included the brain (where STS

is used to produce neuroactive steroids), liver, ovary, heart, adipose tissue, and muscle.

Breast tissue was not tested in this particular experiment. The 1c transcript was always

expressed, but in much lower quantities. However, those tissues with the highest levels of 1c transcripts were the spleen, lung, and liver.

Figure 5.1. Schematic representation of STS exon structure. Open boxes indicate alternative exons in the different STS mRNA isoforms. Gray exon 2 box is common to all indicated transcripts 1.

Upon analysis of 5 normal breast sample and 21 human breast carcinomas with

their adjacent tissue, it was determined that ERα-positive tumors had significantly higher

total STS expression than ERα-negative tumors by immunohistochemistry 1.

Additionally, ERα-negative tumors had even lower levels of STS transcripts than their

surrounding benign tissue as well as normal breast samples. Transcript 1b was the highest

found in normal breast tissue. In general, levels of transcripts 1a and 1c were higher in

the cancerous tumors than in the corresponding adjacent tissue.

84 Valle et al more recently demonstrated six total alternative first exons in human

STS, including 1a in placenta and 0a and 0b in adipose tissue 4. The 1b variant was

additionally expressed in the ovary and 1c in the thyroid. A sixth variant, with another

untranslated first exon termed 1d, was only found in mononuclear leukocytes.

There was further study to correlate STS isoform expression with ER status 1. In

the adjacent benign tissues of ERα-positive tumor patients, the expression of 1a STS

transcript positively correlated to ERα expression, as well as ERβ RNA to total STS

expression. However, neither correlation was found in the tumor tissues themselves. An

additional significant correlation was the expression of ERβ and 1c STS transcript in

ERα-negative tumor samples.

5.1.2. Previous examinations of potential regulatory factors

Inflammatory cytokines such as interleukins IL-6 and IL-1β are secreted by

inflammatory cells in breast cancer tissue 7. IL-6 and IL-1β are able to stimulate

aromatase and sulfatase cellular activities, and they both are able to stimulate MCF-7

7 cellular proliferation when co-incubated with E1S . Like IL-6, tumor necrosis factor α

(TNFα) also stimulates STS activity, but neither affects STS mRNA expression 8.

Therefore, they must modify the enzyme post-transcriptionally. Nevertheless, there are likely other mediators or interactions (cytokines, steroids, growth factors, etc.) that are affecting STS expression because the intracrine and paracrine systems established in the close-quartered HDBC model are so complex.

85 With age, there is a general decrease of DHEAS and a corresponding increase of

glucocorticoids 9. Therefore, there is an immune response to the presence of additional

glucocorticoid levels and a shift to excess IL-6 production 10. Obesity additionally can cause increased levels of IL-6 and TNF-α, promoting the production of aromatase in adipose tissue. IL-6 and TNF-α are selective aromatase modulators (SAMs) at promoter

1.4 and require co-stimulatory glucocorticoids (i.e. dexamethasone, DEX) 11.

Dexamethasone stimulates normal breast aromatase expression via glucocorticoid

receptor-mediated transcription 2. Promoters PII and P1.3, on the other hand, are

10 regulated by cAMP, thus explaining the effect of PGE2 on CYP19 transcription .

Aromatase expression is also stimulated by cytokines IL-1β, IL-6, and TNFα 2.

9 Aromatase activity is regulated by cytokines IL-6 and TNFα, as well as PGE2 . In fact,

a circulatory effect exists in stromal cells in that PGE2 can increase IL-6 production and

TNFα can increase PGE2.

Tetradecanoyl phorbol acetate (TPA), a natural product with known tumor-

promoting activity, stimulates PKC pathways and induces COX-2 levels by 75% in

MDA-MB-231 cells 12. TPA also stimulates aromatase by means of PKA and PKC

pathways in breast cancer cells 2. Epidermal growth factor (EGF), which stimulates

tyrosine kinase activity, and TPA, which stimulates protein kinase C activity, both

increased aromatase activity and mRNA levels in MCF-7 cells. Transforming growth

factor-β (TGFβ), which suppressed growth using SMAD proteins, and TPA increased aromatase activity in MDA-MB-231 cells.

86 COX-2 expression is likely regulated by both NFκB and MAPK pathways.

Specific inhibitors of p38 MAPK inhibited TNFα-mediated COX-2 expression 13.

Exogenous stimulation of TNFα up-regulated COX-2 mRNA and protein expression and

14 PGE2 production in HT-29 cells, but no effect was observed in COX-1 levels .

However, in repressed NF-κB cells, PGE2 production was significantly inhibited. Both

IL-1β and TNFα induce COX-2 expression, and their potencies are enhanced by TGFβ1.

NFκB also stimulates COX-2 and TNFα gene expression, showing interconnectivity

between TNFα’s duplicate actions on COX induction.

NF-κB comprises a protein family (including p50, p52, p65) of inducible

transcription factors that regulate inflammatory responses and protect cells from

apoptosis. In cancers, high levels of NF-κB are produced in tumor cell nuclei. In an

unstimulated cell, NF-κB is bound in an inhibitory complex with an IκB protein in the

cytoplasm (see Figure 5.2.). Upon stimulation, IκB kinases (IKKs) phosphorylate IκB,

tagging it for proteasomic degradation and freeing NF-κB units to translocate into the

nucleus. There, NF-κB activates the expression of genes for inflammation (COX-2,

15 TNFα, and IL-6) and survival (ex. BCL-XL) .

Cytokines IL-1β and TNFα and phorbol esters can all stimulate and are activated by NF-κB in an autoregulatory loop 15. TNFα also stimulates IKK activity, promoting

the degradation of IκB and the release of intracellular NF-κB. Glucocorticoids (i.e.

DEX) and several NSAIDs are able to inhibit the NF-κB pathway. Dexamethasone

induces IκB mRNA expression to promote the cytosolic retention of NF-κB. DEX can

also repress IL-6 expression without affecting IκB levels. NSAIDs inhibit NF-κB 87 through the suppression of TNFα-induced NF-κB activation and NF-κB-induced gene products and in some cases inhibiting ATP-binding to IKKs (and thus their ability to phosphorylate IκB) 15;16. They also were able to specifically suppress NF-κB-regulated

COX-2 protein expression and TNFα-induced PGE2 synthesis. However, none of the

NSAIDs were shown to interact directly with NF-κB or its DNA targets 16.

Figure 5.2. NF-κB cellular activation by TNFα, IL-1, and phorbol esters. Taken from EMD Calbiochem Technical Resources. http://www.emdbiosciences.com/html/cbc/other_inhibitors_NF-kB_activation.htm

88 89 5.1.3. Estrogen deprivation

Human breast cancer cells can adapt to long-term estrogen deprivation (LTED)

and actually develop enhanced sensitivity to estradiol. Some possible mechanisms for

"adaptive hypersensitivity" include the up-regulation of several rapid and non-genomic

estradiol-induced pathways. These include MAP kinase signaling from enhanced HER2 expression, PI3-kinase signaling from insulin-like growth factor-1 receptor action, and

mTOR pathways from increased growth factor binding 17-19. LTED also corresponded to

elevated levels of phosphorylated ERα, a job typically performed by estrogen binding.

Increased ER phosphorylation, even in the absence of E2, allows for ER dimerization and

promotion of estrogen target genes 20.

ER-positive MCF-7 cells which underwent LTED were able to gain the ability to

grow without E2. After 6 months, LTED cells had a basal growth rate equal to that of E2- stimulated wild-type MCF-7 cells 21. Additionally, the deprivation appeared to hypersensitize the LTED cells to E2, in that the cells required much lower levels of

supplemented E2 for growth stimulation. Furthermore, the basal aromatase activity of the

LTED cells was 4 – 5-fold higher than in the wild-type cells, and was still able to be

highly stimulated by phorbol esters. Not surprisingly, the LTED cells showed a 5-fold

increase in estrogen receptor sites 22. These observations are further confirmations that

breast cancer cells can adapt to low levels of estrogen by simply enhancing their

sensitivity to estradiol.

90 Adaptive hypersensitivity mechanisms have important clinical implications. The

efficacy of aromatase inhibitors in patients relapsing on tamoxifen could be explained by

this mechanism and inhibitors of growth factor pathways should be able to reverse the

hypersensitivity phenomenon and prolong of the efficacy of hormonal therapy for breast

cancer 18.

5.1.4. Conflicting reports with estrogen treatment

Stute et al showed the addition of estradiol (concentrations between 10-8 and 10-6

M) to MCF-7 cells stimulated STS activity and increased the production of more estradiol, without forming more estrone, after 24 hours 23. Estradiol (10-10 and 10-8 M) also increased the production of STS mRNA 1.34 – 1.52 fold in the same cells. Evans et al treated MCF-7 cells with 10-8 M estradiol, which showed an increase in STS mRNA

levels 24. However, they were able to terminate this increase with the subsequent addition

of tamoxifen. Though tamoxifen is not believed to inhibit STS itself, it may modulate the

expression and activity of STS by another mechanism.

Pasqualini and Chetrite, on the other hand, experimentally found that estradiol

(concentrations of 5x10-5, 5x10-7, 5x10-9 M) inhibited its own formation in MCF-7 and T-

47D cell lines through inhibition of both sulfatase and aromatase activities after 24 hours

25 of exposure . Additionally, Tobacman et al showed in MCF-7 cells that over 7 days, E2

(10-8 M) gradually but significantly decreased STS activity to a 70% reduction by

day 7 26. These latter two findings fit with the idea of estradiol having a negative

feedback on its own production in those cells requiring E2 for growth.

91 Multiple estrogen-3-sulfates (estrone-3-sulfate, estradiol-3-sulfate, estriol-2-

sulfate) can provoke biological responses in human breast cancer cells, such as

stimulating progesterone receptor levels 27. However, estrogens with other sulfated carbons such as estrogen-17-sulfates do not have any significant effects due to there being no STS hydrolysis activity for C17-sulfates. Also, estrogen sulfates are unable to bind the estrogen receptor; they require STS hydrolysis to elicit their biological responses.

92 5.2. Results and discussion

5.2.1. STS promoter search

Using the Genomatrix Model Inspector search engine, several promoter module sequences were found to possibly match promoter elements. These included potential transcription binding sites for NF-κB, CREB, SMAD, and STAT amongst others.

However, these sequences were located throughout the STS gene and may or may not be involved in the promoter region of the gene. The Model Inspector also identified the most common occurring transcription factor sequences in STS. These are listed in Table 5.1.

Though not investigated further at this time, future research might focus on probing these potential targets in human breast cancer cells expressing varying levels of STS.

Number Transcription Description Factor 4 E2FF E2F-myc activator/cell cycle regulator 4 CREB cAMP-response element binding proteins 4 RORA ERB and RAR-related orphan receptors 4 PAX5 PAX-5 B-cell-specific activator protein 4 MYT1 C2HC zinc finger protein 4 LEFF LEF1/TCF 4 EVI1 Myeloid transforming protein

Table 5.2. Genomatrix Model Inspector search results for most commonly occurring sites for potential STS promoter transcription factors.

93 5.2.2. Steroid sulfatase expression

Though not tested here, progestins (i.e. promegestone) and anti-estrogens have

been reported to inhibit the mRNA expression and activity of STS in breast cancer cell lines. Additionally, FGF and IGF-1 were both shown by Selcer to increase STS activity in dose- and time-dependent manners.

In our experiments we tested species that have shown themselves to be regulators of aromatase or COX expression and/or activity. Those tested as potential STS regulators here were synthetic glucocorticoids dexamethasone (at 100 nM and 200 nM), inflammatory cytokines IL-6 (50 ng/mL) and TNFα (20 mg/mL), COX-2 product PGE2,

phorbol ester phorbol-12-myristate-13-acetate (PMA, 10 nM), estradiol sulfate (1 μM) as

another sulfated steroid, and testosterone (1 μM) as an example androgen and aromatase

substrate.

IL-6 and TNFα were previously shown to stimulate STS activity, but not mRNA

expression, in MCF-7 and T-47D cells 7;9. Therefore, it is believed they do not use any

promoter elements to produce their response. Here we demonstrate (Figure 5.3. (A)) that

IL-6 can repress STS mRNA in MCF-7 cells near 40%, though it was not statistically

significant in these experiments. TNFα on the other hand slightly stimulated STS mRNA

to 135% of control, but this again was not significant. DEX, which has been shown to

increase CYP19 expression via promoter I.4, stimulated STS mRNA at its lower dose, but

significantly inhibited STS at the higher dose used. Dexamethasone has also been shown

to decrease STS activity in rat granulose cells, but it has not been reported to affect STS

in breast cancer cells.

94 Like TNFα, PGE2 and E2S both weakly and insignificantly increased STS mRNA.

The COX product so far as not been tied to STS regulation, but its affect on cAMP or IL-

6 levels may be the mechanism of action occurring in this case. E2S acts like E1S, instead of E2, to stimulate the sulfatase gene (see Chapter 4). PMA, a phorbol ester like TPA,

typically induces enzymes like aromatase to stimulate carcinogenesis. Here however,

PMA slightly decreased STS. Testosterone, and androgen not in the sulfatase pathway like that of DHEAS, had no effect on STS levels in MCF-7 cells.

In MDA-MB-231 cells (Figure 5.3 (B)), DEX (200 nM) slightly increased STS

mRNA, but otherwise most other species elicited some degree of suppression of sulfatase expression. Only PGE2 and PMA showed statistical inhibition (close to 40%), but IL-6,

TNFα, E2S, and testosterone all insignificantly reduced STS mRNA in the ER-negative

cell line. None of these actions have been previously reported as such.

95 (A) MCF-7

175

n = 8 150

125

100

(STS / 18S) (STS # 50

25 RelativemRNA Expression STS 0 ) ) ) M) M n nM mL) M) n M) M μ μ μ DMSO 0 ng/mL) ng/ (1 (10 (1 (1 (20 2 A S (20 E 2 α G E Dex (100 Dex P PM erone IL-6 (50 NF T ost est T (B) MDA-MB-231

150

125

100

75 # # (STS / 18S) (STS 50

25 Relative Expression mRNA STS 0

M) M) M) n n M) M) μ g/mL) μ μ DMSO 00 00 n ng/mL) (1 1 2 (1 0 2 A (10 nM) S (1 x ( x ( (5 (20 E 2 ne e e α M E ro D D PG P te IL-6 s TNF o st e T

Figure 5.3. STS mRNA expression in (A) MCF-7 and (B) MDA-MB-231 cells treated with potential growth effectors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown.

96 5.2.3. Cyclooxygenase-2 expression

In MCF-7 cells, DEX (200 nM) and IL-6 significantly inhibited COX-2 mRNA expression (Figure 5.4 (A)). However, other interleukins IL-1β and TNFα have been previously shown to increase COX-2 potentiated by TGFβ. While DEX is known to increase IL-6 production, neither of these species appears to follow previous trends.

TNFα did, however, significantly stimulate COX-2 expression greater than 10-fold.

PMA, following suit, increased COX-2 expression by nearly 25-fold. TNFα can stimulate NF-κB signaling, amongst others, while phorbol esters activate PKC signaling.

None of the remaining effectors influenced COX-2 expression in MCF-7 cells except for a minimal increase with PGE2

In MDA-MB-231 cells (Figure 5.4 (B)), TNFα and PGE2 showed slight

stimulation of COX-2, increasing it to approximately 130% of control expression. PMA

again stimulated COX-2 nearly 25-fold. However, in these cells, testosterone significantly inhibited COX-2 mRNA by almost 50%, which was not evidenced in MCF-

7 cells or in either cell line with STS expression.

97 (A) MCF-7

3000 * 2000 * 1000

200

n = 7 150

n = 8 n = 8 Expression

(COX-2 / 18S) / (COX-2 100 # Relative mRNA COX-2 50 *

0 ) ) ) SO M) M M) M μ μ μ D ng/mL) 10 nM 200 nM (1 ( (1 (100 nM) ( 2 S 50 (20 ng/mL) 2 ex α E D Dex PGE PMA IL-6 ( TNF Testosterone (1 (B) MDA-MB-231

3000 * 2000 1000

150 #

n = 8 100 n = 8 Expression (COX-2 / 18S) / (COX-2 * 50 Relative COX-2 mRNA

0 O M) M) nM) /mL) M) n M) μ g/mL) μ 0 μ DMS 00 n (1 100 nM) 2 0 (1 (1 ( ( 2 2 S (1 x (50 ng ( 2 α GE MA E Dex De F P P terone IL-6 N T tos s Te

Figure 5.4. COX-2 mRNA expression in (A) MCF-7 and (B) MDA-MB-231 cells treated with potential growth effectors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown.

98 5.2.4. Aromatase expression

In MCF-7 cells, dexamethasone did not have its expected stimulatory effect at

either concentration tested (100, 200 nM) on CYP19 expression. Cytokines inteleukin-6 and TNFα significantly inhibited CYP19 expression, opposite of what reports predicted, by 38% and 35%, respectively. Reed and Purohit showed that several cytokines including IL-6 and TNF-α were capable of inducing CYP19 expression in breast cancer cells. Testosterone (1 μM) insignificantly increased aromatase mRNA expression, likely increasing the amount of aromatase enzyme for the available substrate. Expected but not observed, neither PGE2 nor PMA had any effect on CYP19 gene expression. This is inconsistent with previously reported data; phorbol esters are known as and experimentally used as tumor promoters via their activation of protein kinase C (PKC).

And, PKC signaling should be able to increase aromatase expression through cAMP- responsive promoter PII.

Interestingly, the addition of exogenous STS substrate E2S (though not the natural

substrate) slightly increased, though not significantly, aromatase mRNA expression. E2S may act as a competitive inhibitor of estrone sulfate for steroid sulfatase, thus producing estradiol (E2). Depending on the enzyme rate constant for E2S, the aromatase pathway

may compensate for the lack of action by the sulfatase pathway. However, E2S is known

to be hydrolyzed to E2 by STS and free E2 was shown to inhibit CYP19 mRNA

expression (see Chapter 4).

In ER-negative cells, DEX has a slight stimulatory effect, consistent with

previous reports. IL-6, TNFα, and testosterone all had significant inhibition of aromatase

expression, which, like that just seen in MCF-7 cells, is opposite of other reports. 99 (A) MCF-7

200 RNA

m 150

n = 8

CYP19 100 ve Expression ti (CYP19 / 18S) / (CYP19 # a l e

R 50

0 L) M) M) n /m M) n M) M) MSO g μ μ μ D 0 nM) g/mL) 00 0 n (1 (1 (1 (1 (2 0 2 S x x (5 (20 n E 2 e α E rone D De PG PMA (10 e IL-6 NF st T o st e T (B) MDA-MB-231

200

150

100 # Expression (CYP19 / 18S) 50 Relative mRNA CYP19

0 ) L nM) M) nM) M) M) μ μ μ DMSO (1 (1 (1 (200 2 S x (50 ng/mL) (20 ng/m E 2 ne α E ro Dex (100 nM)De PG PMA (10 IL-6 ste TNF o Test

Figure 5.5. CYP19 mRNA expression in (A) MCF-7 and (B) MDA-MB-231 cells treated with potential growth effectors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown.

100 5.2.5. NF-κB and cAMP pathway regulators

Though the probing of STS regulation provided some helpful clues, the

contradictions found by known regulators on CYP19 and COX-2 mRNA expression justified the experimentation of our own MCF-7 and MDA-MB-231 cells for other known pathway mediators. Therefore, we selected Sigma compounds dibutyryl cyclic

AMP (DbcAMP) and H-89 to investigate cAMP stimulatory processes.

DbcAMP is a cell-permeable cAMP analog that activates cAMP-dependent protein kinases (PKA). DbcAMP is typically growth-stimulatory in many normal systems. It was shown twenty years ago that DbcAMP stimulates the rate of P450arom in

human adipose stromal cells; this stimulation was also potentiated by phorbol esters 11.

H-89 (an isoquinolinesulfonamide) is a selective, potent inhibitor of PKA, and somewhat but less so of PKC. In theory, the inhibition of PKA by H-89 should counteract the stimulation by DbcAMP. However, overactive PKA can actually inhibit human breast cancer cell proliferation because it can phosphorylate ERα on a serine residue within the

ER’s DNA-binding domain that prevents ER dimerization in the absence of estradiol 20.

Therefore, cAMP signaling through PKA can actually decrease estrogen-regulated cell growth. In these cases, PKA inhibition has been shown to promote ERα-dependent growth in MCF-7 cells.

Upon treatment with H-89, only MDA-MB-231 cells had increased STS enzyme activity (Figure 5.6 (A)). Dose-response testing of H-89 (Figure 5.7.) in both cell lines produced an EC50 value (50% effective concentration) of 1 nM in MDA-MB-231 cells,

and an IC50 value of 42 pM for MCF-7 cells. Additionally, only MDA-MB-231 cells had

101 decreased STS activity with DbcAMP. These simple tests imply that our MDA-MB-231 cells have active PKA cell signaling, whereas the MCF-7 cell do not. However, STS mRNA expression (Figure 5.6 (B)) was inhibited only in MCF-7 cells by H-89, though both cell lines had slightly reduced STS with DbcAMP treatment.

102 (A) 300

200

(dpm/ug DNA/h) 100 Relative STS Radioactivity STS Relative

0 ) M) M MSO μ μ D 0 (1 (50 P 89 M H- A bc D (B) 125

100

50 Expression / 18S) (STS

Relative STS mRNA 25

M) M) μ μ 0 DMSO 1 ( 9 H-8 cAMP (50 b D

Figure 5.6. Effect of selective pathway effectors and key hormones on (A) STS enzyme activity and (B) STS mRNA expression. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. For figure (A), N = 9 – 12 and MCF-7 100% control = 84.6 dpm/μg DNA/h and MDA-MB-231 100% control = 4.0 dpm/μg DNA/h. N =6 for figure (B). 103 H-89 IC50 Determination

250

MCF-7 200 MDA-MB-231 MDA-MB-231 EC = 1 nM 150 50

100

MCF-7 IC50 = 42 pM 24 h Sulfatase24 Activity 50 % ofcontrol (dpm/ug DNA/h)

0 -6 -5 -4 -3 -2 -1 0 1 2 log Concentration (μM)

Figure 5.7. IC50 calculation of H-89 on STS enzyme activity. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6 for each data point. MCF-7: 100% control = 39.9 dpm/μg DNA/h. MDA-MB-231: 100% control = 8.9 dpm/μg DNA/h.

To then probe the NF-κB signaling pathway in our cell lines, we used Bay 11-

7082 to inhibit NF-κB cellular activity. Bay 11-7082 is an inhibitor of cytokine-induced

IκB-α phosphorylation produced by Sigma-Aldrich. In other words, it is an NF-κB

activity inhibitor, with IC50 values approximately 10 μM in multiple cancerous cell systems 28. To note, while the inhibition of NF-κB is useful in a laboratory setting, its

therapeutic use is questionable due to the potential effects on other tissues of the immune

system.

104 Bay 11-7082 increased STS enzyme activities dose-dependently in MCF-7,

MDA-MB-231, and an MDA-MB-231 cell line stably transfected with ERα1 (Figure

5.8.). This third cell line was included to see if estrogen receptor status was a mitigating factor in the variable results. The stark increase of STS activity with even 10 μM Bay

11-7082 shows (1) NF-κB signaling is active, (2) ER state does not appear to mediate the activity, and (3) STS activity is conversely related to NF-κB activity.

MCF-7 MDA-MB-231 ERα1 6000 MDA-MB-231

5000

4000

3000

2000 Relative STS Radioactivity STS Relative

% of control (dpm/ug DNA/h) 1000

M M M SO μ μ μ M 1 0 D 2 1 50 8 2 0 8 7 0 - -7 1 1 11-7082 BAY 11 BAY BAY

Figure 5.8. STS enzyme activity of Bay 11-7082 in MCF-7, MDA-MB-231, and MDA- MB-231-ERα1 cells. The results were normalized agents a control with vehicle (DMSO). Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown. MCF-7: 100% control = 17.1 dpm/μg DNA/h. MDA-MB-231-ERα: 100% control = 5.0 dpm/μg DNA/h. MDA-MB- 231: 100% control = 5.6 dpm/μg DNA/h. 105 5.2.6. Estrogen deprivation

MCF-7 cells were grown in media with FBS stripped of all hormones (estimated

99% efficiency) to mimic the conditions of long-term estrogen deprivation. STS mRNA steadily increased to 2.7-fold after 11 weeks; when 1 μM E1S was added, STS increased

2.9-fold after 14 weeks (see Table 5.2.). STS enzyme activity increased 177-fold after 8 weeks, but dropped to 0.3-fold after 11 weeks. Activity increased 55-fold after 11 weeks in E1S, but dropped to 1.2-fold after 14 weeks total. We attribute this initial peek to the cells’ developing their adaptive hypersensitivity to growth stimuli. STS activity became overactive, correlating to the increased mRNA production, to produce estradiol by any means available. However, after additional time in the absence of estrogen, the cells may shift from relying on the sulfatase and aromatase pathways for growth, and instead find available stimuli from non-estrogen-related COX-2 pathways.

COX-2 mRNA initially decreased by 0.5-fold, but by 11 weeks increased 28-fold; when E1S was added over 14 weeks, increased only 1.7-fold. When the STS substrate was available, the cells may not have needed to depend as much on non-estrogen sources.

CYP19 mRNA decreased to 0.2-fold after 11 weeks; when 1 μM E1S was added, CYP19 still decreased 0.2-fold after 14 weeks. This is evidence that the LTED cells are no longer relying on the aromatase pathway for estrogen production.

106

Cell treatment COX-2 CYP19 STS mRNA mRNA mRNA STS activity FBS-supplemented B 1 1 1 1 media (normal growth)

Dextran-Coated 2.7 0.5 (8 wks) 0.2 (11 wks) 177 (8 wks) Charcoal (DCC) (11 wks) 28 (11 wks) 0.3 (11 wks) stripped serum B media DCC media + 1 μM E1S 2.9 1.7 (14 wks) 0.2 (14 wks) 55 (11 wks) (14 wks) 1.2 (14 wks)

Table 5.3. mRNA expression and STS activity ratios of LTED cells / normal MCF-7 cells.

MCF-7 cells were also grown in DCC-stripped media supplemented with only 1

μM E1S to see how gene expression would change if the only available steroid for

estrogen-dependent growth was the STS substrate (Figure 5.9.). STS mRNA was

significantly increased 2-fold in MCF-7-S cells compared to normal MCF-7 cells. This

effect was expected due to the cells adaptation to the need for E1S conversion by STS to

produce the necessary E2 for cellular growth. CYP19 expression, on the other hand, was cut to 50% of the normal value in MCF-7-S cells. Again, this is evidence of the ER- positive cells’ reliance on E1S for their estrogen source; the cells are not using as much

aromatase for the conversion of androgens to estrogens. COX-2 mRNA was unchanged by the different growth media, showing no change between the two cells line. This result is not surprising in that the MCF-7-S cells appear to be growth-dependent on the sulfatase pathway and would not need COX-2 expression to stimulate aromatase production.

107 3 MCF-7 MCF-7-S #

(gene / 18S) (gene 1 # Relative mRNA Expression Relative 0 STS COX-2 CYP19

Figure 5.9. Basal mRNA expression in STS, COX-2 and CYP19 in normal MCF-7 cells and MCF-7 cells grown in E1S-supplemented DCC-strippped-media (MCF-7-S). Each gene was normalized to its own normal MCF-7 expression. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 9, unless otherwise shown.

108 5.3. Conclusions

In summary, we found no new STS regulatory factors in our laboratory tests, but

the analysis of factors that have been reported, one way or another, to influence

aromatase and COX-2 provided some helpful clues. The cytokines and dexamethasone

tested often had effects on our MCF-7 and MDA-MB-231 cells that were not predicted

by previous research (see Table 5.1.). For example, IL-6 and TNFα have been shown to

stimulate CYP19 and COX-2 mRNA expression in MCF-7 cells, but in our experiments,

both had suppressive effects. This may be explained by several reasons. First, some

results published were tested in different cell lines than those chosen here, and even two

ER-positive cell lines can have vastly different responses to exogenous treatments.

Secondly, even when the same cell line is used, cells can often change characteristics depending on how they are cultured in the media used for growth and treatment, by their

age of how many times they have been passed (sub-cultured), and by their cell density

upon treatment and analysis. In essence, the MCF-7 and MDA-MB-231 cells used on our

group may no longer share the same traits as those originally produced by ATCC or those grown in other laboratories.

Nevertheless, these response differences to “known” cytokines and glucocorticoids regulators are what led us to investigate PKA and NF-κB signaling in our cell lines. Additionally, using MDA-MB-231 cells expressing estrogen receptor α showed that the ER is not likely involved in NF-κB signaling, though NF-κB and PKA

pathways were strongly present in our ER-negative MDA-MB-231 cells. Finally, we examined the effects long-term estrogen deprivation would have on STS activity and

mRNA expression. After 11 weeks, both STS and COX-2 levels had significantly 109 increased in both LTED ER-positive MCF-7 and T-47D cell lines, as well as in the MCF-

7 cells grown in the presence of E1S, though there was only an initial burst in STS enzyme activity.

Although there is still much left to learn about STS regulation, studies such as these and others investigating the gene’s promoter region are making headway in understanding its effectors and tissue-specific expression. Hopefully we can soon learn about the enzyme’s regulation so that we may be able to control it in human breast cancers.

110 5.4. References

1. Zaichuk, T.; Ivancic, D.; Scholtens, D.; Schiller, C.; and Khan, S. A. Tissue- specific transcripts of human steroid sulfatase are under control of estrogen signaling pathways in breast carcinoma. Journal of Steroid Biochemistry & Molecular Biology 2007, 105, 76-84.

2. Su, B.; Diaz-Cruz, E. S.; Landini, S.; and Brueggemeier, R. W. Suppression of aromatase in human breast cells by a cyclooxygenase-2 inhibitor and its analog involves multiple mechanisms independent of cyclooxygenase-2 inhibition. Steroids 2008, 73, 104-111.

3. Brueggemeier, R. W.; Hackett, J. C.; and Diaz-Cruz, E. S. Aromatase inhibitors in the treatment of breast cancer. Endocrine Reviews 2005, 26, 331-345.

4. Valle, L. D.; Toffolo, V.; Nardi, A.; Fiore, C.; Armanini, D.; Belvedere, P.; and Colombo, L. The expression of hte human steroid sulfatase-encoding gene is driven by alternative first exons. Journal of Steroid Biochemistry & Molecular Biology 2007, 107, 22-29.

5. Valle, L. D.; Toffolo, V.; Nardi, A.; Fiore, C.; Bernante, P.; Di Liddo, R.; Parnigotto, P. P.; and Colombo, L. Tissue-specific transcriptional initiation and activity of steroid sulfatase complementing dehydroepiandrosterone sulfate uptake and intracrine steroid activations in human adipose tissue. Journal of Endocrinology 2006, 190, 129-139.

6. Li, X.-M.; Alperin, E. S.; Salido, E.; Gong, Y.; Yen, P.; and Shapiro, L.-J. Characterization of the promoter region of human steroid sulfatase: a gene which escapes X inactivation. Somatic Cell and Molecular Genetics 1996, 22, 105-117.

7. Honma, S.; Shimodaira, K.; Shimizu, Y.; Tsuchiya, N.; Saito, H.; Yanaihara, T.; and Okai, T. The influence of inflammatory cytokines on estrogen production and cell proliferation in human breast cancer cells. Endocrine Journal 2002, 49, 371-377.

8. Newman, S. P.; Purohit, A.; Ghilchik, M. W.; Potter, B. V. L.; and Reed, M. J. Regulation of steroid sulphatase expression and activity in breast cancer. Journal of Steroid Biochemistry & Molecular Biology 2000, 75, 259-264.

9. Purohit, A. and Reed, M. J. Regulation of estrogen synthesis in postmenopausal women. Steroids 2002, 67, 979-983.

10. Purohit, A.; Newman, S. P.; and Reed, M. J. The role of cytokines in regulating estrogen synthesis: implications for the etiology of breast cancer. Breast Cancer Research 2002, 4, 65-69.

111 11. Evans, C. T.; Corbin, C. J.; Saunders, C. T.; Merrill, J. C.; Simpson, E. R.; and Mendelson, C. R. Regulation of estrogen biosynthesis in human adipose stromal cells. Journal of Biological Chemistry 1987, 262, 6914-6929.

12. Richards, J. A.; Petrel, T. A.; and Brueggemeier, R. W. Signaling pathways regulating aromatase and cyclooxygenases in normal and malignant breast cells. Journal of Steroid Biochemistry & Molecular Biology 2002, 80, 203- 212.

13. Chen, C.-C.; Sun, Y.-T.; Chen, J.-J.; and Chang, Y.-J. Tumor necrosis factor- alpha-induced cyclooxygenase-2 expression via sequential activation of cermide-dependent mitogen-activated protein kinases, and I-kappaB kinase 1/2 in human alveolar epithelial cells. Molecular Pharmacology 2001, 59, 493-500.

15. Yamamoto, Y. and Gaynor, R. B. Therapeutic potential of inhibition of the NF- kB pathway in the treatment of inflammation and cancer. Journal of Clinical Investigation 2001, 107, 135-142.

16. Takada, Y.; Bhardwaj, A.; Potdar, P.; and Aggarwal, B. B. Nonsteroidal anti- inflammatory agents differ in their ability to suppress NF-kB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene 2004, 23, 9247-9258.

17. Tobe, M.; Isobe, Y.; Tomizama, H.; Nagasaki, T.; Takahashi, H.; Fukazawa, T.; and Hayashi, H. Discovery of quinazolines as a novel structural class of potent inhibitors of NF-kB activation. Bioorganic & Medicinal Chemistry 2003, 11, 383-391.

18. Santen, R. J.; Song, R. X.; Masamura, S.; Yue, W.; Fan, P.; Sogon, T.; Hayashi, S.; Nakachi, K.; and Eguchi, H. Adaptation to estradiol deprivation causes up- regulation of growth factor pathways and hypersensitivity to estradiol in breast cancer cells. Advances in Experimental Medicine and Biology 2008, 630, 19-34.

19. Martin, L.-A.; Farmer, I.; Johnson, S. R. D.; Ali, S.; Marshall, C.; and Dowsett, M. Enhanced estrogen receptor (ER) alpha, ERBB2, and MAPK singla transduction pathways operate during the adpatation of MCF-7 cells to long term estrogen deprivation. Journal of Biological Chemistry 2003, 287, 30459- 30468.

112 20. Al-Dhaheri, M. and Rowan, B. G. Protein kinase A exhibits selective modulation of estradiol-dependent transcription in breast cancer cells that is associated with decreased ligand binding, altered estrogen receptor alpha promotor interaction, and changes in receptor phosphorylation. Molecular Endocrinology 2007, 21, 439-456.

21. Yue, W.; Santen, R. J.; Wang, J.-P.; Hamilton, C. J.; and Demers, L. M. Aromatase within the breast. Endocrine-Related Cancer 1999, 6, 157-164.

22. Masamura, S.; Santner, S. J.; Heitjan, D. F.; and Santen, R. J. Estrogen deprivation causes estradiol hypersensitivity in human breast cancer cells. Journal of Clinical Endocrinology and Metabolism 1995, 80, 2918-2925.

23. Stute, P.; Gotte, M.; and Kiesel, L. Differential effect of hormone therapy on E1S- sulfatase activity in non-malignant and cancerous breast cells in vitro. Breast Cancer Research and Treatment 2008, 108, 363-374.

24. Evans, T. R.; Rowlands, M. G.; Luqmani, Y. A.; Chander, S. K.; and Coombes, R. C. Detection of breast cancer-associated estrone sulfatase in breast cancer biopsies and cell lines using polymerase chain reaction. Journal of Steroid Biochemistry & Molecular Biology 1993, 46, 195-201.

25. Pasqualini, J. R. and Chetrite, G. S. Estradiol as an anti-aromatase agent in human breast cancer cells. Journal of Steroid Biochemistry & Molecular Biology 2005, 98, 12-17.

26. Tobacman, J. K.; Hinkhouse, M.; and Khalkhali-Ellis, Z. Steroid sulfatase activity and expression in mammary myoepithelial cells. Journal of Steroid Biochemistry & Molecular Biology 2002, 81, 65-68.

27. Pasqualini, J. R.; Gelly, C.; Nguyen, B. L.; and Vella, C. Importance of estrogen sulfates in breast cancer. Journal of Steroid Biochemistry & Molecular Biology 1989, 34, 155-163.

28. An, J.; Sun, Y.; Fisher, M.; and Rettig, M. B. Antitumor effects of bortezomib (PS-341) on primary effusion lymphomas. Leukemia 2004, 18, 1699-1704.

113 CHAPTER 6

EFFECTS OF CYCLOOXYGENASE INHIBITORS ON mRNA EXPRESSION,

STS ENZYME ACTIVITY AND CELLULAR PROLIFERATION

6.1. Introduction

COX-2 is overexpressed in multiple breast cancer cells lines, including the invasive estrogen-independent cell line MDA-MB-231 1. COX inhibitors can be

selective for one COX isoform over the other or can be non-selective, as seen with many

non-steroidal anti-inflammatory drugs (NSAIDs) (see Figure 6.1.). Additionally,

multiple COX inhibitors have COX-independent activities. For example, celecoxib, a

potent COX-2 selective inhibitor, inhibits human breast cancer cell proliferation in vitro

1. It has also shown significant reduction of tumor burden, decreased proliferation, and

increased tumor cell apoptosis in an in vivo mouse mammary tumor study 2. These

results were additionally correlated to a decrease in cell survival protein kinase B, also known as Akt. Furthermore, celecoxib treatment correlated to decreased antiapoptotic

Bcl-2 protein and increased levels of proapoptotic Bax protein 2. These results confirm

celecoxib’s ability to promote apoptosis in a metastatic breast cancer model. For further

information about COX enzymes and their inhibition, please see the introductory chapter

of this dissertation (Chapter 1).

114

Table 6.1. Selectivity of various NSAIDs for COX-1 and COX-2 isoforms 3.

Multiple COX inhibitors are able to dose-dependently reduce aromatase activity in breast cancer cells (see Figure 6.1.) 4. The NSAIDs tested, however, required high micromolar doses to achieve this effect. Indomethacin was the most effective NSAID, with an IC50 value of 157 μM in SK-BR-3 breast cancer cells, followed by piroxicam and

ibuprofen. SC-560, a COX-1 selective inhibitor, also inhibited aromatase enzyme

activity at a low IC50 of 5.8 μM. Of the COX-2 selective inhibitors, NS-398 was the

most potent against aromatase (IC50 1.0 μM), followed by agents celecoxib, nimesulide, and niflumic acid all less potent, but still showing noticeable aromatase suppression. NS-

398, in particular, suppressed aromatase activity by decreasing PKA/PKC driven transcription. These NSAIDs and selective inhibitors all also proved to decrease CYP19

115 mRNA expression in the same SK-BR-3 cells, indicating their effect on aromatase is actually occurs at the transcriptional level. However, because these COX inhibitors modulate aromatase expression to different degrees, this suggests they may use different

mechanisms, possibly targeting various promoters of the CYP19 gene or influencing the

expression of other pathway mediators, to achieve their inhibitory effects 5. Therefore, some have termed NSAIDs as the first generation of selective aromatase modulators

(SAMs) in HDBC cells.

Figure 6.1. Effects of NSAIDs and COX inhibitors on aromatase activity in breast cancer cells. Adapted from the dissertation of Dr. Edgar Diaz-Cruz.

116

Aspirin has been shown to dose-dependently decrease COX-2 mRNA and protein,

as well as inhibit cell proliferation and increase cytoplasmic IκB protein, in esophageal

cancer cells 6. Due to its inhibition of NF-κB protein, and thereby increase of IκB

protein, aspirin is thought to be able to induce apoptosis through inactivation of the NF-

κB pathway. This pathway typically prevents apoptosis upon nuclear damage or the

presence of cytotoxic cytokines. NF-κB can regulate COX-2 expression in multiple cells

lines, including gastric and colon cancer cells 7;8. NSAIDs not only inhibit COX

enzymes directly, but they can also affect COX mRNA expression through their indirect inhibition of NF-κB-stimulated gene products. Aspirin, salicyclic acid, and others were able to inhibit IκΒ kinases (IKKs), keeping NF-κB stuck in its IB inhibitory complex and

preventing its nuclear translocation 8;9. Indomethacin, for example, was only able to

inhibit COX-2 expression, but aspirin and sulindac were able to inhibit both COX-2 and

NF-κB expression.

These various activities of COX inhibitors towards other targets in inflammation

and cancer progression give rise to the idea that they may as well have affects, direct or

otherwise, on the sulfatase pathway in human breast cancer. In the following studies, we

analyzed selective and nonselective COX inhibitors on STS enzyme activity, on STS,

COX-2, and CYP19 mRNA expression, and on breast cancer cell proliferation. Not only

may we find additional activities of NSAIDs, but we may also be able to examine further

the interplay between the estrogen-producing enzymes.

117 6.2. Results and discussion

6.2.1 Gene expression

To analyze the general actions of COX inhibitors on gene expression, all

compounds were tested in cells at 25 μM concentrations. Though the compounds have different levels of potency against COX enzymes, we chose 25 μM as a screening concentration for all the compounds again other targets. As seen in Figure 6.2., none of the COX “selective” inhibitors (celecoxib, NS-398, SC-560) affected STS mRNA in

MCF-7 cells. However, both piroxicam and niflumic acid (which is actually COX-2

selective) stimulated STS expression by 1.5 and 2-fold increases. In MDA-MB-231, celecoxib significantly increase STS mRNA, whereas ibuprofen, acetylsalicyclic acid, and

niflumic acid decreased sulfatase expression.

In Figure 6.3., we see that COX inhibitors have quite variable affects on COX-2

mRNA expression in MCF-7 cells. COX-1 specific SC-560 slightly decreased COX-2

mRNA, but ibuprofen, naproxen sodium, acetylsalicylic acid, piroxicam, and niflumic

acid all significantly decreased COX-2 expression. Therefore, indomethacin was the only

NSAID in the study to not affect COX-2 mRNA expression. Additional variability was observed on COX-2 modulation in MDA-MB-231 cells. Here, all of the non-specific

NSAIDs except piroxicam, stimulated COX-2 mRNA in the ER-negative cell line.

Celecoxib, NS-398, SC-560, and piroxicam all had slight but insignificant (except in the case of piroxicam) suppression of COX-2.

118

Similar to that seen in STS gene expression, no COX inhibitors affected CYP19 expression (Figure 6.4.) in MCF-7 cells except for niflumic acid, which stimulated aromatase mRNA 1.5-fold. MDA-MB-231 cells showed little (but significant) to no inhibition of CYP19 for all inhibitors except for SC-560. The COX-1 selective inhibitor increased CYP19 expression 1.3-fold. Indomethacin, ibuprofen, and acetylsalicylic acid significantly inhibited CYP19 mRNA, but no greater than 20% reductions.

119 (A) MCF-7

300

200 n = 4 # Expresion (STS / 18S) (STS 100 n = 4 Relative STS mRNA

0 O ) M) M) M) M) M) M) M) M) M MS μ μ μ μ μ μ μ μ μ 5 D 2 ( b (25 n (25 m (25 oxi aci diu icam (25 ec profen (25o c Acid el NS-398 (25SC-560 (25 meth bu S mi C I licylic AcidPirox (25 xen a Indo o Niflu r tyls ap ce N A

(B) MDA-MB-231

150 # n = 4 n = 4 n = 5 100

n = 4 Expresion (STS / 18S) (STS 50 Relative STS mRNA

0 ) ) M ) ) ) μ M M) M) M M) M M M) μ μ μ μ μ μ μ μ 5 5 5 DMSO 25 2 2 ( ( (25 en (25 f ium (25 S-398 od lic Acid ( N SC-560 (25 upro S y Celecoxib (25 methacin Ib Piroxicam do iflumic Acid (2 In oxen N apr N Acetylsalic

Figure 6.2. Effects of COX inhibitors on STS mRNA expression in (A) MCF-7 cells and (B) MDA-MB-231 cells. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

120 (A) MCF-7 200

150

100

Expresion n = 4 ## (COX-2 / 18S) (COX-2 # 50 n = 4 # * Relative COX-2 mRNA Relative COX-2

0 ) ) ) ) SO M) M) M M M) M M M) M) μ μ μ μ μ μ μ μ μ 5 5 5 DM 2 25 2 25 ( (25 ( n ( m xib ( 398 fe cid (25 ro A p ic Acid (25 leco NS- SC-560 (25 Sodiu roxicam (2 Ce omethacin Ibu Pi lum d en f In ox Ni pr Na Acetylsalicylic

(B) MDA-MB-231 400

* 300

n = 4 #

200 # # # (COX-2 / 18S) (COX-2 n = 5 100 # Relative COX-2 mRNA Expresion 0 ) ) ) ) M M) M) M) M) M M) M M μ μ μ μ μ μ μ μ μ 5 DMSO 25 25 2 25 25 25 25 25 ( ( ( ( ( ( n ( id d ( 398 560 um - haci ofen di Ac S- pr c ic Aci N SC li roxicam met Celecoxib (25 Ibu licy Pi um a fl Indo oxen So s Ni apr N Acetyl

Figure 6.3. Effects of COX inhibitors on COX-2 mRNA expression in (A) MCF-7 cells and (B) MDA-MB-231 cells. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

121 (A) MCF-7

200 #

100 Expresion (CYP19 / 18S) Relative CYP19 mRNA

0 ) ) ) M) M) M) M M) M) M M) M μ μ μ μ μ μ μ μ μ 5 5 DMSO 25 2 25 (25 ( (25 (2 (25 ( ib in 560 ac um ox - h Acid (25 Acid ec C profen ic NS-398 (25S ylic el Ibu c C en Sodi Piroxicam (lum x ali Indomet ls Nif ety c Napro A (B) MDA-MB-231 200

150

n = 5 n = 4 n = 4 100 # # # (CYP19 / 18S)

50 Relative CYP19 mRNA Expresion 0 ) ) ) ) SO M) M) M M M) M) M M M) μ μ μ μ μ μ μ μ μ 5 5 DM 25 2 25 25 ( (25 ( ( (2 (25 8 n m id 9 fe ium (25 c ro A coxib (25 thacin ( p xica c e NS-3 SC-560 e o bu Sod r Cel I n Pi Indom Niflumi tylsalicylic Acid e Naproxe c A

Figure 6.4. Effects of COX inhibitors on CYP19 mRNA expression in (A) MCF-7 cells and (B) MDA-MB-231 cells. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

122 6.2.2. Steroid sulfatase enzyme activity

While the effects of the COX inhibitors were consistent in the RT-PCR results, studying their inhibitory abilities of steroid sulfatase was quite complex. In Figure 6.5.

(A), Celecoxib at 25 μM significantly inhibited STS enzyme activity and NS-398, a weak

COX-2 inhibitor as well as an aromatase inhibitor, had no effect. SC-560 strongly stimulated STS in MCF-7 cells, but due to large experimental error, it was not considered significant. Then, upon analysis of the remaining NSAID compounds (Figure 6.5. (B)), significantly variable activities were found for several compounds in the same MCF-7 cell line, each data set only differing by passage number (age) of the cells tested.

Indomethacin and niflumic acid consistently did not affect STS activity, and ibuprofen significantly and consistently inhibited STS activity. But, naproxen sodium, acetylsalicylic acid, and piroxicam all had statistically significant and drastic changes in

STS activity in the time of one cell passage (typically 6-7 days, depending on initial culturing density). These three NSAIDs were all found to strongly stimulate STS activity one week, and then have little to no stimulation in another.

The same affect was observed in MDA-MB-231 cells, this time tested over three sequential passages of cells. First, in Figure 6.6. (A), we see that celecoxib, NS-398, and

SC-560 all had STS activity stimulation, with SC-560 being the strongest inducer, following by significant drops in affect on STS activity in a later passage of the same

MDA-MB-231 cells. Then, considering NSAID effects, indomethacin, naproxen sodium, acetylsalicylic acid, and niflumic acid all generally showed increasing STS stimulation over two to three passages. But, ibuprofen and piroxicam both displayed drops from initial STS stimulation. Ibuprofen actually significantly inhibited STS activity in MDA-

123 MB-231 cells. Several IC50 value calculations were attempted for those inhibitors

seemingly more consistent, but the data were so variable that calculations could not be performed (data not shown).

124 (A) 400

350

300

250

200

(dpm/ug DNA/h) 150 Relative STS Radioactivity 100

# 50

0 ) ) ) M) M M M μ μ μ μ 5 25 25 2 ( ( ( S (1 8 0 E 1 9 6 -5 C NS3 S Celecoxib (B) Contradicting NSAID Effects in MCF-7s 1000 # MCF-7 p.9 900 MCF-7 p.10

800

700 #

600

500

400 (dpm/ug DNA/h) 300 # Relative STS Radioactivity STS Relative 200

100 #

0 ) ) M) M) M) M) M M) M μ μ μ μ μ μ μ 25 S (1 (25 ( (25 1 n d (25 d E fe ium ci ci ro A p od ic A ic u S m Ib n cyl Piroxicam (25 domethacin (25 xe iflu In ro N Nap Acetylsali

Figure 6.5. Effect of (A) COX inhibitors and (B) NSAIDs on STS activity in MCF-7 cells. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 3, unless otherwise shown. 100% control = 4.4 to 9.8 dpm/μg DNA/h. 125 (A) # 800 early passage 600 later passage 400 400

300

200 (dpm/ug DNA/h) Relative STS Radioactivity STS Relative

100

M) M) M) M) μ μ μ μ 1 ( 25 25 S ( ( E 1 60 oxib (25 -5 c C NS398 S Cele (B) Contradicting NSAIDs in MDA-MB-231s 1100 231 p.8 231 p.9 1000 231 p.10 900

800

700 # 600

500

# (dpm/ug DNA/h) 400

Relative STS Radioactivity 300

200

100 * not not not 0 tested tested tested ) ) ) M M) M) M) M) M M μ μ μ μ μ μ μ 5 5 25 25 25 25 S (1 ( ( (2 ( (2 ( id id E 1 cin c m c ha A ica A lic ic Sodium irox Ibuprofen en P lum lsalicy Nif Indomet rox y ap N Acet

Figure 6.6. Effect of (A) COX inhibitors and (B) NSAIDs on STS activity in MDA-MB- 231 cells. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 3, unless otherwise shown. 100% control = 3.4 to 19.0 dpm/μg DNA/h. 126

6.2.3. Breast cancer cell growth

Regardless of complications in STS activity assessments, COX inhibitors showed

significant and time-dependent growth inhibition of MCF-7 and MDA-MB-231 cells

(Figure 6.7.). In MCF-7 cells, none of the compounds showed much if any growth inhibition by 2 days. But, by 4 and 7 days post-treatment, all COX inhibitors tested inhibited cell growth 35 to 65% of control. Indomethacin, piroxicam, and niflumic acid

were the most cytotoxic agents tested in this series.

In MDA-MB-231 cells, celecoxib, acetylsalicylic acid, and niflumic acid initially

demonstrated a small stimulation of cell growth statistically significant compared to

control by 2 days. However, all compounds, including those three, time-dependently

inhibited MDA-MB-231 cell growth by 4 and 7 days. In this group, indomethacin,

ibuprofen, and naproxen sodium were the most potent growth inhibitors.

127 (A) MCF-7

150 2 days 4 days 125 7 days

n=10 # # 100 n=10 # n=11 * * * * 75 * # * * * # # 50 # #

% ofcontrol growth * 25

0 ) ) )) ) M) M) M) M) M) M M M M μ μ μ μ μ μ μ μ 5 μ DMSO 25 25 25 5 ( (25 (25 2 0 ( ( (2 98 6 n id (25 cin um m ( oxib (25 -3 ofe a c C-5 ha r di Acid t xic ic Ac ele NS S So C me Ibup n cylic ro li Pi do xe iflum In lsa N ro ty ap ce N A (B) MDA-MB-231

150 2 days 4 days 125 # 7 days # * 100 # # * * * * # 75 * * * * 50 * * * % ofcontrol growth 25

0 ) ) ) ) ) ) ) M) M M M M M M M μ μ μ μ μ μ μ M)) μ μ DMSO 5 5 5 5 5 5 (2 (25 (25 2 2 (25 (2 (2 b 98 60 id ( xi fen (2 o -3 -5 o ium am S C d Ac ic Acid ( lec N S thacin pr o c c e e u S ox C Ib ir en licyli P x Indom ro lsa Niflumi p y Na Acet

Table 6.7. Proliferation assay of (A) MCF-7 and (B) MDA-MB0231 cells over 2, 4, and 7 days of treatment of COX-inhibitors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 12, unless otherwise shown.

128 6.3. Conclusions

In this chapter, we analyzed multiple known cyclooxygenase inhibitors against

gene expression and STS activity in breast cancer cells, as well as measuring their

inhibition of cell growth over a week. The COX-2 selective inhibitors used were celecoxib, which has ongoing clinical applications, and NS-398, which is actually a weak inhibitor of its intended target. SC-560 is a COX-1 selective inhibitor used not for desired COX-1 inhibition but to collect a full picture of the various effects on the COX isozymes. NSAIDs indomethacin, ibuprofen, naproxen sodium, acetylsalicylic acid, piroxicam, and niflumic acids were all analyzed like the isoform-specific inhibitors to see if any trends arose related to their COX-1/COX-2 preference.

Of the inhibitors effects on STS expression, niflumic acid stood out as a significant enhancer of STS mRNA in MCF-7 cells. In MDA-MB-231 cells, ibuprofen, acetylsalicylic acid and niflumic acid showed mid-level inhibition of STS expression.

Most of the inhibitors affected COX-2 mRNA expression, but not in manner consistent with their COX selectivities. Generally, the NSAID compounds were inhibitory in MCF-

7 cells and in MDA-MB-231 cells on COX-2 expression. Only two compounds made notice on CYP19 mRNA modulation. Niflumic acid stimulated aromatase in MCF-7 cells, and SC-560 stimulated in MDA-MB-231 cells.

SC-560, an apparent activator of STS enzyme activity, did not have related effects on STS mRNA in MCF-7 cells, nor did acetylsalicylic acid or piroxicam. In MDA-MB-

231 cells, most compounds had to ability to stimulate STS activity, but could not do so consistently.

129 Regardless of their actions in these categories, previous research has shown just as conflicting data in their studies of NSAIDs and cancer progression and chemoprevention. The same compound in multiple studies will show chemopreventative properties, whereas in others show that they can increase the risk of breast cancer incidence 10-12. No firm conclusions can be drawn out of our performed experiments

except (1) COX inhibitors can affect multiple targets involved in breast cancer

development, (2) that NSAIDs can inhibit cancer cell growth but by yet understood mechanisms, and (3) that those mechanisms require probing within multiple pathways if ever to find a common link.

130 6.4. References

1. Mazhar, D.; Ang, R.; and Waxman, J. COX inhibitors and breast cancer. British Journal of Cancer 2006, 94, 346-350.

2. Basu, G. D.; Pathangey, L. B.; Tinder, T. L.; LaGioia, M.; Gendler, S. J.; and Mukherjee, P. COX-2 inhibitor induces apoptosis in breast cancer cells in an in vivo model of spontaneous metastatic breast cancer. Molecular Cancer Research 2004, 2, 1-11.

3. Vane, J. R.; Bakhle, Y. S.; and Botting, R. M. Cyclooxygenases 1 and 2. Annual Reviews of Pharmacology and Toxicology 1998, 38, 97-120.

4. Brueggemeier, R. W.; Diaz-Cruz, E. S.; Li, P. K.; Sugimoto, Y.; Lin, Y. C.; and Shapiro, C. L. Translational studies on aromatase, cyclooxygenases, and enzyme inhibitors in breast cancer. Journal of Steroid Biochemistry & Molecular Biology 2005, 95, 129-136.

5. Su, B.; Diaz-Cruz, E. S.; Landini, S.; and Brueggemeier, R. W. Suppression of aromatase in human breast cells by a cyclooxygenase-2 inhibitor and its analog involves multiple mechanisms independent of cyclooxygenase-2 inhibition. Steroids 2008, 73, 104-111.

6. Liu, J.-F.; Jamieson, G. G.; Drew, P. A.; Zhu, G.-J.; Zhang, S.-W.; Zhu, T.-N.; Shan, B.-E.; and Wang, Q.-Z. Aspirin induces apoptosis in oesophageal cancer cells by inhibiting the pathway of NF-kappaB downstream regulation of cyclooxygenase-2. Surgical Research 2005, 75, 1011-1016.

7. Yamamoto, Y. and Gaynor, R. B. Therapeutic potential of inhibition of the NF- kB pathway in the treatment of inflammation and cancer. Journal of Clinical Investigation 2001, 107, 135-142.

8. Takada, Y.; Bhardwaj, A.; Potdar, P.; and Aggarwal, B. B. Nonsteroidal anti- inflammatory agents differ in their ability to suppress NF-kB activation, inhibition of expression of cyclooxygenase-2 and cyclin D1, and abrogation of tumor cell proliferation. Oncogene 2004, 23, 9247-9258.

9. Hardwick, J. C. H.; van den Brink, G. R.; Offerhaus, G. J.; van Deventer, S. J. H.; and Peppelenbosch, M. P. NF-kappaB, p38 MAPK and JNK are highly expressed and active in the stroma of human colonic adenomatous polyps. Oncogene 2001, 20, 819-827.

10. Agrawal, A. and Fentiman, I. S. NSAIDs and breast cancer: a possible prevention and treatment strategy. International Journal of Clinical Practice 2008, 62, 444-449.

131 11. Garcia Rodriguez, L. A. and Gonzalez-Perez, A. Risk of breast cancer among users of aspirin and other anti-inflammatory drugs. British Journal of Cancer 2004, 91, 525-529.

12. Ready, A.; Velicer, C. M.; McTiernan, A.; and White, E. NSAID use and breast cancer risk in the VITAL cohort. Breast Cancer Research and Treatment 2008, 109, 533-543.

132 CHAPTER 7

EFFECTS OF ER ANTAGONISTS AND AROMATASE INHIBITORON mRNA

EXPRESSION, STS ENZYME ACTIVITY AND CELLULAR PROLIFERATION

7.1. Introduction

7.1.1 Aromatase

Endocrine therapy is a method of treatment that inhibits the cell proliferative

effects of hormones (i.e. estrogen) by either blocking access of the hormone to its

receptor or by reducing the synthesis of the hormone itself 1. A third method, destroying the ovaries to eliminate their major estrogen production, is only relevant to pre-

menopausal patients and will not be discussed here.

The major method of blocking the estrogen receptor is with anti-estrogen

tamoxifen, other selective estrogen receptor modulators (SERMs), aromatase inhibitors,

and selective aromatase modulators (SAMs) 1. Tamoxifen and SERMs block estrogen

from binding the ER and this inhibits the downstream transcription of estrogen-activated, cell proliferative genes. Aromatase inhibitors, however, inhibit one of the final steps of estrogen biosynthesis and thus reduce the amount of estrogen present to stimulate cell growth. Denying hormone-dependent cancer cells of estrogen eliminates the energy source for their continued growth and the cells should thus halt growth upon treatment.

133 Third generation aromatase inhibitors letrozole and exemestane, discussed in

Chapter 1, were chosen for use in the following experiments to observe any affects these

inhibitors may have on STS and COX-2 species. Letrozole is a nonsteroidal triazole ring

compound with an IC50 value against aromatase activity of 11.5 nM in human placental

microsomes 2. Steroidal exemestane, on the other hand, is a potent aromatase inactivator, with a Ki of 26 nM against the enzyme.

7.1.2. Estrogen receptors

There are two forms of the estrogen receptor (ER), α and β, that share the

functional structure of most steroid nuclear receptors 3. They are located in the uterus,

vagina, liver, bone, and breast. Most female mammary ERs are the alpha form, which

uses estrogen to stimulate proliferation and gene expression of hormone-dependent breast

cancer cells 2.

Selective estrogen receptor modulators bind the ER to form a drug-receptor

complex incapable of promoting DNA transcription. SERMs are nonsteroidal

compounds that have tissue-specific efficacy. In some tissues they can act as agonists,

whereas they can act as antagonists in others. Tamoxifen blocks the ER in MCF-7 cells

(ER-positive cell line), but has no effect in MDA-MB-231 cells (ER-negative cell line).

The most active form of tamoxifen is actually its metabolite 4-hydroxy tamoxifen (4-

OHT), which has an affinity for the ER comparable to estradiol itself. However, a major

limitation to tamoxifen therapy is that long-term exposure results in drug-resistant

cancers. Another SERM in clinical use is ICI 182,780, also known as fulvestrant. Since

they act similarly in mechanism and potency, only 4-OHT was used in the following

134 experiments to examine its effects on expression and activity patterns of the estrogen-

producing enzymes. We did not test for aromatase enzyme activity inhibition because (1)

these compounds have already been analyzed in our and other labs, and (2) because our

labs have repeatedly positively correlated CYP19 mRNA expression to aromatase

enzyme activity 4-6. Therefore, we decided to only measure CYP19 expression and not

activity in the following experiments.

7.2. Results

7.2.1. Gene expression

In MCF-7 cells (see Figure 7.1.), both aromatase inhibitors had significant

inhibition of STS mRNA after 24 hours, with 10 nM letrozole suppressing STS to 56% of control expression, and 50 μM exemestane to only 36%. Since these inhibitors potently inhibit the aromatase enzymes, one might expect to observe a delayed stimulation of STS mRNA in the cells shift to favoring the sulfatase pathway for estradiol production.

However, their significant suppression of STS mRNA may be evidence of them directly or indirectly affecting STS transcription. 4-OHT showed slight STS mRNA inhibition (to

75% of control), but it was not statistically significant. The same inhibition results were not observed in either COX-2 or CYP19 mRNA experiments. For COX-2 expression, neither 4-OHT nor exemestane had any effect, though 10 nM letrozole suppressed COX-

2 mRNA statistically by 28% (to 72% of control expression). For the aromatase gene, 4-

OHT had an insignificant stimulatory effect, and neither aromatase inhibitor had any effect at the concentrations tested.

135 The same compounds had more activity on gene expression in MDA-MB-231 cells (Figure 7.2.). Though only statistically significant for exemestane (with 33% suppression), all inhibitors in this series mildly suppressed STS mRNA. 4-OHT and letrozole both inhibited STS mRNA by 10 – 15% compared to control. Again, one might have assumed the blockage of the aromatase pathway to positively feed back to STS for its estrogen production. However, this may be explained by their effect on COX-2 expression. All three compounds inhibited COX-2 mRNA approximately 30 – 40%, with letrozole (10 nM) showing the largest inhibition. There appears to be no positive feedback to COX-2 for additional production of aromatase. The compounds are more likely to be influencing cellular transcription factors that modulate these related enzyme networks; this possibility in itself may hint at shared regulatory factors between STS and

COX-2. Finally, 4-OHT and letrozole had no affect on CYP19 expression, but exemestane surprisingly had a 1.7-fold stimulatory effect after 24 hours of incubation.

136 (A) STS (B) COX-2

150 150

100 100 n = 5 # # n = 4 Expression

Expression (STS / 18S) 50 50 # 18S) / (COX-2 Relative STS mRNA Relative COX-2 mRNA

0 0 ) ) ) ) O M M M M S n μ n nM) μ M 0 0 D 10 DMSO 00 ( (1 (5 (1 e e le n n o ife sta troz x e etrozol Le mo L em a x Exemestane (50 T E OH 4-OH Tamoxifen (100 nM) 4-

150

n = 5 100

Expression 50 (CYP19 / 18S) Relative CYP19 mRNA

0 ) O M S μ M D ne (50 ifen (100 nM) esta Letrozole (10 nM) Tamox Exem H 4-O

Figure 7.1. mRNA expression in MCF-7 cells with ER and aromatase inhibitors of (A) STS, (B) COX-2, and (C) CYP19. Experiments performed using real-time RT-PCR as described in Chapter 3. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

137 (A) STS (B) COX-2

150 150

100 100 # # # # Expression (STS / 18S) 50 Expression 50 (COX-2 / 18S) (COX-2 RelativemRNA STS RelativemRNA COX-2

0 0 O ) ) ) M) M) M) M n n μ nM μ DMS 0 0 0 (1 (5 DMSO 50 e n (10 (100 e (10 nM e an ol ozole st en tane ( tr e troz es moxif L oxif a Le em am x Exeme T E H -OH T 4 4-O

200 #

150 RNA m

100 CYP19 ve Expression ti (CYP19 / 18S) / (CYP19 a l

e 50 R

0 ) ) O M M nM) n μ 0 0 0 DMS 0 (5 (1 le (1 e n o n e z sta ro e Let m amoxif xe T E H 4-O

Figure 7.2. mRNA expression in MDA-MB-231 cells with ER and aromatase inhibitors. Experiments performed using real-time RT-PCR as described in Chapter 3. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

138 7.2.2. Steroid sulfatase enzyme activity

STS enzyme activity was determined after 24 hours of incubated with the 4-OHT and aromatase inhibitors (Figure 7.3.). However, the drugs’ individual mechanisms of action may play a role in their affects on this non-intended target. In MCF-7 cells, 4-

OHT (100 nM) inhibited STS activity to 62% (+/- 13% SEM), whereas both aromatase inhibitors stimulated STS activity. Letrozole (10 nM) increased radioactivity to 178% and exemestane (50 μM) increased it to 167% of control. This stimulation coincides with the needed STS activity to compensate for the inhibited aromatase pathway. However, as we have seen, this is not associated with an increase of STS mRNA expression.

In MDA-MB-231 cells, 4-OHT still inhibited STS activity (to 47% control).

However, letrozole also inhibited STS activity to 50% of control. Exemestane had no discernable effect due to high variability in the results. Being that this cell line does not depend on estrogen for growth, no stimulation of STS activity was predicted.

Nevertheless, the inhibition of enzyme activity seen in MDA-MB-231 cells most likely is an indirect effect on STS though other cellular factors.

139 (A) MCF-7

300

200

100 (dpm/ug DNA/h) Relative STS Radioactivity STS Relative

O M) M) n μ MS 0 0 D 5 (1 ( le e o n fen (100 nM) z ta xi o s o tr e me L e am x T E -OH 4 (B) MDA-MB-231

125

100

50 (dpm/ug DNA/h)

25 Relative STS Radioactivity STS Relative

M) nM) μ DMSO (50 (100 ne a ifen st e m Letrozole (10e nM) Ex H Tamox O 4-

Figure 7.3. STS enzyme activity in (A) MCF-7 and (B) MDA-MB-231 cells with ER and aromatase inhibitors. Radioactivity experiments performed with [3H]-estrone sulfate conversion as described in Chapter 3. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 3, unless otherwise shown. MCF-7 100% control = 103.1 dpm/μg DNA/h. MDA-MB-231 100% control = 29.0 dpm/μg DNA/h. 140 7.2.3. Breast cancer cell growth

Treatment of MCF-7 cells (Figure 7.4 (A)) with letrozole (10 nM) had no effect

on their proliferation after 48 hours, and only had a slightly increasing inhibitory effect

(approximately 10%) on cell growth by 4 and 7 days. Steroidal exemestane (50 μM), on

the other hand, initially stimulated cell growth compared to control after 2 days to 138%.

However, this stimulation abated by 4 days, with the cellular growth returning to the level of control, and is even inhibited by 7 days to 51% of control. These effects on cellular

growth inhibition have been previously observed 7.

In MDA-MB-231 cells (Figure 7.4. (B)), both letrozole and exemestane had weak

initial suppression of cellular growth after 2 days to 86% and 77%, respectively.

However, with increased incubation time, cell growth normalizes for both compounds,

returning to that of control by 7 days. At these concentrations, neither compound is

generally cytotoxic nor do they exhibit any growth suppression by their assumed

mechanisms of action.

141 (A) MCF-7

2 days 150 * 4 days 7 days 125

100

75 * 50 % of controlgrowth 25

O M) M) n μ 0 DMS 1 ( (50 e ne zol o ta etr mes L e Ex (B) MDA-MB-231

2 days 150 4 days 7 days 125

100 # * 75

50 % of controlgrowth 25

M) M) n μ 0 0 DMSO 1 (5 ne zole ( a o st e Letr m xe E

Figure 7.4. Cellular growth proliferation in (A) MCF-7 and (B) MDA-MB-231 cells with ER and aromatase inhibitors. The experiments were performed over 2, 4 and 7 days post- treatment using the MTS assay as described in Chapter 3. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. N = 6, unless otherwise shown.

142 7.3. Conclusions

In this chapter we evaluated the effects of aromatase inhibitors letrozole and

exemestane, as well as anti-estrogen metabolite 4-hydroxy tamoxifen. While their

general effects on cellular proliferation are not outstanding, they have marked

suppression of STS mRNA in both MCF-7 and MDA-MB-231 cell lines. Additionally,

the aromatase inhibitors and 4-OHT suppressed COX-2 mRNA in MCF-7 cells.

However, the aromatase inhibitors showed no obvious effects on CYP19 expression, except in the one case of stimulation by exemestane (50 μM) in ER-negative MDA-MB-

231 cells.

Nevertheless, the aromatase inhibitors did not suppress the corresponding STS enzyme activity. In fact, both letrozole and exemestane stimulated STS activity in MCF-

7 cells, but had different effect in MDA-MB-231 cells; letrozole inhibited STS in the ER- negative cells and exemestane was not active. In both cell lines, 4-OHT inhibited STS activity to approximately 50%. Inhibition activities of 4-hydroxy tamoxifen on both STS mRNA and enzyme activity are very interesting in vitro findings. Evans et al found that

tamoxifen can inhibit stimulated STS activity and mRNA expression in MCF-7 cells;

however, they believe the compound does not directly interfere with STS itself 8.

In a clinical study by Stanway et al, neither aromatase inhibitor therapy (using anastrozole) nor tamoxifen therapy affected patient DHEAS or DHEA serum concentrations or the DHEAS:DHEA ratio, indicating neither therapy inhibited STS activity 9. Though this clinical study further displays the likely genetic difference

between our cell lines and primary tumors, it does give evidence for the therapeutic use

of a specific STS inhibitor, alone or in combination with aromatase inhibitors.

143 7.4. References

1. Smith,H.J. and Simons,C. Development of Enzyme Inhibitors as Drugs. In Enzymes and Their Inhibition: Drug Development. Smith, H. J. and Simons, C. Eds.; CRC Press: London, 2005; pp 221-240.

2. Brueggemeier, R. W. Aromatase inhibitors: new endocrine treatment of breast cancer. Seminars in Reproductive Medicine 2008, 22, 31-43.

3. Smith,H.J. and Simons,C. Development of Enzyme Inhibitors as Drugs. In Enzymes and Their Inhibition: Drug Development. Smith, H. J. and Simons, C. Eds.; CRC Press: London, 2005; pp 171-250.

4. Masri, S.; Lui, K.; Phung, S.; Ye, J.; Zhou, D.; Wang, X.; and Chen.S. Characterization of the weak estrogen receptor alpha agonistic activity of exemestane. Breast Cancer Research and Treatment 2008, Epub ahead of print.

5. Brodie, A.; Sabnis, G. J.; and Jelovac, D. Aromatase and breast cancer. Journal of Steroid Biochemistry & Molecular Biology 2006, 102, 97-102.

6. Diaz-Cruz, E. S.; Shapiro, C. L.; and Brueggemeier, R. W. Cyclooxygenase inhibitors suppress aromatase expression and activity in breast cancer cells. Journal of Clinical Endocrinology and Metabolism 2005, 90, 2563-2570.

7. Macedo, L. F.; Zhiyong, G.; Tilghman, S. L.; Sabnis, G. J.; Yun, Q.; and Brodie, A. Role of androgens on MCF-7 breast cancer cell growth and on the inhibitory effect of letrozole. Cancer Research 2006, 66, 7775-7782.

8. Evans, T. R.; Rowlands, M. G.; Luqmani, Y. A.; Chander, S. K.; and Coombes, R. C. Detection of breast cancer-associated estrone sulfatase in breast cancer biopsies and cell lines using polymerase chain reaction. Journal of Steroid Biochemistry & Molecular Biology 1993, 46, 195-201.

9. Stanway, S. J.; Purohit, A.; Ward, R.; Palmieri, C.; Coombes, R. C.; and Reed, M. J. Effect of aromatase inhibitor or tamoxifen therapy on steroid sulfatase activity and DHEA-DHEAS concentrations in postmenopausal women with breast cancer. Journal of Clinical Oncology 2008, 26, 1134.

144 CHAPTER 8

EFFECTS OF STEROID SULFATASE INHIBITOR COMBINATIONS WITH

AROMATASE AND CYCLOOXYGENASE INHIBITORS

8.1. Introduction

In the previous chapters, we have discovered multiple actions of the DU steroid

sulfatase enzyme inhibitors, affecting STS mRNA expression as well as that of COX-2

and CYP19 in certain breast cancer cell lines. We have also seen that COX-2 and

aromatase inhibitors can affect STS expression and enzyme activity. Due to their various actions within the estrogen-producing enzyme networks, we have postulated that dual inhibition of two (or more) of these targets at a time should exhibit additive, if not

synergistic, inhibition of estrogen production in post-menopausal breast cancer.

Though other groups have designed dual action inhibitors, we have decided the

best way to probe these potential combinational effects is to examine the grouping of two

or more drugs in a cellular based system. Here we look for any effects upon treatment of

MCF-7 and MDA-MB-231 cells with combinations of DU-14 and DU-15 with three

COX-selective inhibitors (celecoxib, NS-398, SC-560), two aromatase inhibitors

145 (exemestane and letrozole), or SERM metabolite 4-hydroxy tamoxifen. Because of the multiple mechanisms of action of these compounds, we expected to observe multiple combinational effects.

However, as reported in previous chapters of this dissertation, some investigations with several of the inhibitors or antagonists have yielded considerable variability in the experimental data. For example, this variability was seen in the inconsistent effects of

COX inhibitors against STS enzyme activity in breast cancer cell lines from different passages. In this chapter, the experimental data reported for individual treatments were collected at the same time and with the same cells as the data collected for the combinations of inhibitors.

8.2. Results

8.2.1. COX inhibitors combinations

In the combinational studies the STS inhibitors with the selective COX inhibitors, we found no additive effects against STS mRNA expression in MCF-7 cells upon co- treatment of either STS inhibitor with celecoxib (25 μM) (Figure 8.1. (A)). Though not statistically significant, there may be a slight combinational effect of NS-398 and DU-15.

STS mRNA was not affected by either drug alone, but a 20% reduction of STS expression with the combination was observed. Likewise, there may be a weak combinational effect with SC-560 and DU-14, in that the combination statistically differed from DU-14 treatment alone.

In MDA-MB-231 cells (Figure 8.1. (B)), the combination of celecoxib and DU-

15 differed from DU-15 treatment alone, but was not statistically different than the

146 individual effect on STS mRNA by the COX-2 inhibitor. Co-treatment of NS-398 and

DU-14 stimulated STS expression greater than 150% of control, while NS-398 alone had no effect. However, this combination was not unique compared to DU-14’s solo stimulation of STS mRNA in this case. The only actual combinational effect against STS expression was the combination of SC-560, which alone did not affect expression, with

DU-15. The combination statistically differed from both treatments alone, but still only displayed minimal inhibition of STS expression in MDA-MB-231 cells.

COX-2 mRNA expression analysis did not produce any significant additive

effects either (Figure 8.2.). In MCF-7 cells, celecoxib treatment alone slightly stimulated

COX-2 expression, and with the addition of DU-14 (but not DU-15), this stimulation increased to 159% of control, greater than either inhibitor alone (both increased COX-2

expression to approximately 125%). Combinations of the STS inhibitors with NS-398,

on the other hand, reduced COX-2 expression more than any of the inhibitors alone. NS-

398 + DU-15 even inhibited COX-2 25% below control expression. In MDA-MB-231

cells, both celecoxib, NS-398, and SC-560 all had mildly suppressive effects on COX-2

mRNA. However, the combinations of celecoxib + DU-14 and NS-398 + DU-15 both

stimulated COX-2 expression greater than any of the inhibitors alone. Yet, combinations

of either DU compound with SC-560 appeared to reduce COX-2 statistically below SC-

560’s solo effect as well as DU-15’s, suggesting there may be a small additive effect

between these inhibitors.

CYP19 expression in MCF-7 cells was not significantly altered by any

combinations of COX inhibitors with DU-14 or DU-15 (Figure 8.3. (A)). Addition of

DU-15 to either NS-398 or SC-560 may slightly reduce CYP19 mRNA compared to the

147 drugs alone. Also, combination of DU-14 to celecoxib eliminated its solo suppression of

CYP19, with expression returning to the level of DU-14 alone. Therefore, no additive effect exists in this instance. In MDA-MB-231 cells (Figure 8.3. (B)) only the combinations of celecoxib + DU-14 and NS-398 + DU-15 showed obvious changes in

CYP19 expression. Both COX-2 inhibitors alone exhibited small suppressions, but upon the indicated combinations, CYP19 mRNA increased to 135% and 225% of control, respectively. Addition of either DU compound to SC-560 mimicked the treatment of the

STS inhibitors alone.

148 (A) MCF-7

150

n = 4 100 n = 4 n = 5 #

Expression / 18S) (STS 50 Relative STS mRNA

0 O ) 5 ) 4 5 4 M M) -1 M 1 M) -1 nM) μ μ U-1 μ 0 n 5 DU D 5 DU DMS 5 (25 2 DU- 2 ( + + + ( + 8 0 15 xib xib 9 6 U- -3 D co co DU-14 (2 le le e elecoxib +e DU-14 NS-398 (25NS NS-398 SC-560 SC-5 SC-560 + DU-15 C C C

(B) MDA-MB-231

300

# # # 200 # # # n = 5 # # Expression / 18S) (STS 100 * Relative STS mRNA

0 ) 5 4 ) 5 M) 15 14 n M M) -1 - M - -1 μ U-1 μ μ 0 5 DMSO 5 D 2 DU DU DU + ( + (2 8 8 0 + 14 xib 9 9 6 oxib (25 -3 -3 -560 (25 -560 + U- DU-15 (25c nM) co D le le S C C e NS N NS-398 +S DU S SC-5 C CelecoxibCe + DU-14

Figure 8.1. STS mRNA expression in (A) MCF-7 and (B) MDA-MB-231 after 24 incubation with combinations of COX inhibitors with STS inhibitors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the COX inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 6, unless otherwise shown.

149

(A) MCF-7

200

n = 5

100 Expression (COX-2 / 18S) / (COX-2 Relative COX-2 mRNA

0 O 5 S M) -14 -15 M) -14 -15 M) 1 μ μ μ DU DU DU DU 5 DM (25 nM) 25 (25 + + ( (2 + DU-14 + DU- 0 ib ib ib 98 -56 U-14 (250DU-15 nM) C D NS-3 NS-398 +NS-398 +S SC-560 SC-560 Celecox Celecox Celecox (B) MDA-MB-231

150

100

# #

Expression 50 (COX-2 / 18S) (COX-2 Relative COX-2 mRNA COX-2 Relative

0 5 4 5 ) 5 SO M) M) M) M) 1 1 M -1 n μ μ 0 n μ U-1 DM 5 25 D DU- DU- 5 DU 2 ( 25 + + + (2 + DU-14 + ( ( 4 15 8 (25 8 0 1 xib 9 9 98 6 U- -3 -3 D co DU- le elecoxib e NS-3 NS NS SC-560 SC-560 SC-5 C CelecoxibC + DU-14

Figure 8.2. COX-2 mRNA expression in (A) MCF-7 and (B) MDA-MB-231 after 24 incubation with combinations of COX inhibitors with STS inhibitors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the COX inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 6, unless otherwise shown. .

150 (A) MCF-7

150

n = 5

n = 4 100

Expression 50 (CYP19 / 18S) Relative CYP19 mRNA

0 ) ) ) 5 4 5 5 M M M M) -1 1 M) -1 n n μ μ 0 5 μ U-14 U-1 U U-14 U DMSO 5 2 D D 5 D DU- D D 2 ( 2 + ( 5 + ( + 4 1 b 8 8 + 0 (25 0 1 - xi 98 9 9 6 - U o -3 3 -3 -5 -56 U D coxib (25coxib + c S D e e le S- C-560 + e N N NS SC S SC Cel Cel C

(B) MDA-MB-231

300

200 Expression

(CYP19 / 18S) 100 Relative CYP19 mRNA

0 5 4 M) 14 n M) - M) -1 M) MSO nM) μ U μ U U-15 μ U-15 5 D 5 D D D D 50 (2 DU-1 (2 + (2 + b + b 8 8 4 xi 9 9 -3 -560 + DU-15 coxib (25co S DU-1 e e lecoxi S-3 el e N N NS-398 +SC-560 (25SC-560 +SC DU-14 Cel C C

Figure 8.3. CYP19 mRNA expression in (A) MCF-7 and (B) MDA-MB-231 after 24 incubation with combinations of COX inhibitors with STS inhibitors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the COX inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 6, unless otherwise shown.

151 There were no combinational effects observed in MCF-7 cells against STS

enzyme activity with co-treatment of the COX inhibitors (Figure 8.4.). In fact, the DU

compounds were so effective at inhibiting STS activity alone that the combinations with celecoxib, NS-398, and SC-560 were all dominated by the STS inhibitors. Even the stimulatory effects SC-560 were knocked down with the addition of either DU-14 or DU-

15. Similar results were found in MDA-MB-231 cells (Figure 8.5.) Again, the STS inhibitors strongly reduced any stimulation of STS activity by the COX inhibitors, returning the STS enzyme activity close to the level of STS inhibitor treatment alone. In this cell line, there was one statistical difference between the inhibition profiles of DU-15 alone and with SC-560, in that the DU compound’s STS inhibition was slightly abated in the presence of any of the COX inhibitor.

152

400

350

300

250

200

150 (dpm/ug DNA/h) 100 Relative STS Radioactivity STS Relative # 50

# # # # # # # # # # # # 0

-50 ) ) M M) -14 M) 15 M) 15 μ μ μ U-14 μ U-14 DU D D DU- (1 (25 + S b + 1 -15 (25 nM) 98 + E -14 (250 nM 560 + U DU coxib (25 S398 (25 C-560 D N NS3 NS398 + DU-S SC- SC-560 Cele Celecoxi Celecoxib + DU-15

Figure 8.4. STS enzyme activity in MCF-7 cells after 24 incubation with combinations of COX inhibitors with STS inhibitors. Cells were treated with 1 μM E1S and radioactive substrate in the presence or absence of various inhibitors. Twenty-four hours after treatment, STS activity was determined and the amount of [3H]estrone radioactivity was normalized with the total DNA concentration. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the COX inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 3, unless otherwise shown. 100% control = 1.5 to 4.9 dpm/μg DNA/h.

153 # 800 600 400 400

300

200 (dpm/ug DNA/h) Relative STS Radioactivity STS Relative

100

# # # # # # # # # # # # # # 0 ) ) 4 ) 4 5 5 M -15 M -1 M) -14 M) nM μ μ μ μ 0 U U U (1 DU-1 D DU-1 D D DU-1 S + l + + + 4 (25 e 0 + E 1 -15 (25 nM) 98 60 (25 6 60 -1 U Cel C 5 5 5 U S3 - - D NS398 (25NS398 +N C D elecoxib (25 SC- SC S C

Figure 8.5. STS enzyme activity in MDA-MB-231 cells after 24 incubation with combinations of COX inhibitors with STS inhibitors. Cells were treated with 1 μM E1S and radioactive substrate in the presence or absence of various inhibitors. Twenty-four hours after treatment, STS activity was determined and the amount of [3H]estrone radioactivity was normalized with the total DNA concentration. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the COX inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 3, unless otherwise shown. 100% control = 1.6 to 2.5 dpm/μg DNA/h.

154 The three COX enzyme inhibitors alone all had less than 40% growth inhibition in MCF-7 cells by 7 days (Figure 8.6.). Addition of DU-15 increased the growth inhibitory effects of celecoxib, NS-398, and SC-560 in MCF-7 cells by 7 days, but no better than that seen for DU-15 alone. The combinations with DU-14 had no additive effects in MCF-7 cells. In MDA-MB-231 cells, combinations of DU-15 with celecoxib and SC-560 again appeared to increase the COX inhibitors’ growth inhibition, but really did not exceed the solo inhibition of the STS inhibitor. No changes were observed for the additions of DU-14 or DU-15 to NS-398.

155 (A) MCF-7

2 days

150 4 days 7 days 125

# # 100 # # * # # n=11 * * # # * * n=11 * 75 * * * # **** ** # * ** ** n=10 * ** ** 50 ** # ** # #

% ofcontrol growth * ** ** 25 * ** 0 ) ) 4 ) SO M M M) 1 15 M 14 M) 15 M μ μ μ μ μ 5 D 2 25 ( + DU- (25 + DU- ( 0 0 + DU- 15 98 6 398 3 coxib + DU- coxib - - DU-14 (50 DU- NS NS NS-398 + DU-15SC-56 SC-560 + DU-14SC-5 Celecoxib (25Cele Cele

(B) MDA-MB-231

2 days 150 n=11 4 days # # 7 days 125 # n=11 # # 100 # # # * ** * * * # ## 75 * * * # n=9 ** *#* * * ** # * * #* **** # ** 50 * *

% of controlgrowth ** 25 ** # 0 4 M) M) M) -1 M) -15 M) μ μ μ U U-15 μ U-14 U μ U-14 0 DMSO 5 D D 5 D (25 + (2 + 4 ( 0 0 1 xib o oxib 398 + coxib + D c -398 (25 -56 DU- DU-15 (25 le S S- C ele N N NS-398 + D S SC-56 SC-560 + DU-15 Celec Ce C

Figure 8.6. Proliferation assay of (A) MCF-7 and (B) MDA-MB0231 cells over 2, 4, and 7 days of COX inhibitor treatment. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the aromatase/ER inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 12, unless otherwise shown.

156 8.2.2. Aromatase/ER inhibitor combinations

The combinations of either DU-14 or DU-15 with 4-OHT and letrozole all showed increased suppression of STS mRNA compared to the individual treatments in

MCF-7 cells (Figure 8.7.). The combination of exemestane and DU-15 was also greater than exemestane alone, but to a lesser extent. However, since the individual effects of the

DU compounds were not tested in this particular experiment series, difference from the

STS inhibitors alone cannot be determined; therefore no conclusive additive effects can be concluded. However, we can conclude that the combinations decrease STS mRNA greater than the aromatase/ER inhibitors alone. Similar trends were observed in MDA-

MB-231 cells, in that the presence of the DU compounds, particularly DU-15, increase the suppression of STS mRNA. However, in this study we were able to compare these combinations to the effects of the STS inhibitors alone. Statistically, no significant differences were found between the drug combinations and the DU compounds alone against STS expression.

Against COX-2 mRNA expression in MCF-7 cells (Figure 8.8.), the combinations of either DU compound with 4-OHT and letrozole also showed increased suppression of COX-2 mRNA compared to the individual treatments. As for STS expression, the combination of exemestane and DU-15 was also greater than exemestane alone, but not with DU-14. In MDA-MB-231 cells, the addition of DU-14 or DU-15 to the aromatase inhibitors had no combinational effect, though generally they all suppressed COX-2 mRNA. Only a weak additive effect compared to either drug alone was seen in the combination of 4-OHT and DU-15.

157 Several stimulatory combinational effects were observed in CYP19 expression

(Figure 8.9.) upon combination treatments. First, in MCF-7 cells, the addition of DU-14 or DU-15 to letrozole significantly increased CYP19 expression to 211% and 265% of control, respectively. Stimulation was also seen with exemestane and DU-14, but due to high standard error, no conclusions can be drawn. There were no changes in expression with 4-OHT treatment, with or without STS inhibitors. In MDA-MB-231 cells, the combination of 4-OHT and DU-15 stimulated CYP19 mRNA to 255% of control and compared to either treatment alone. There was also a small increase in CYP19 mRNA upon co-treatment with letrozole and DU-14; however, this stimulation was only to 136% of control expression. Combinations of the STS inhibitors with exemestane slightly decreased its apparent stimulation of CYP19 expression in MDA-MB-231 cells.

158 (A) MCF-7

150

100 n = 5

# # # n = 4 # Expression / 18S) (STS 50 # # # # # # #

Relative mRNA STS * #

0 O M) M) M) nM) nM) μ nM) 0 nM) DMS 00 n 25 10 n 25 0 5 25 (5 (1 -15 ( -15 ( -15 ( zole ( -14 (2 stane etro amoxifen L xeme ne + DU T E OH trozole + DU stane + DU 4- e mesta amoxifen + DU Le e T Letrozole + DU-14 (250 nM) x E Exem -OH 4 4-OH Tamoxifen + DU-14 (250 nM)

(B) MDA-MB-231

150

100 # # #, # # #, # # #,* Expression / 18S) (STS 50 RelativemRNA STS

0 ) ) O 4 5 4 ) M M) M -1 M) -1 -15 M -15 n U-1 U n μ DMS 00 n D DU (25 1 (10 + (50 5 + D + DU le le le + DU 14 (250 n o ne - U-1 fen z D xi ozo DU tro trozo estane Le e etr mesta amo L L m xe Exe ExemestaneE + DU-14 OH T 4-OH Tamoxifen + 4-OH Tamoxifen4- (

Figure 8.7. STS mRNA expression in (A) MCF-7 and (B) MDA-MB-231 after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the aromatase/ER inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 6, unless otherwise shown. 159

(A) MCF-7 150

100

#, # #, # # #, # #, #

Expression 50 * # (COX-2 / 18S) (COX-2 Relative COX-2 mRNA

O M) M) M) M) M) n n n nM) nM) n n 0 0 5 DMS 50 1 2 50 2 (25 ( ( 2 (25 5 (25 1 4 zole 1 -14 ( U- o -14 ( U D tr U- U D e D L + n + le + DU-15 + D e + DU-15 n e le + o e n xif o z Exemestane (50 mM) ro tan sta roz s 4-OH Tamoxifen (100 nM) t me amo e Let e T L x E OH Exeme 4- 4-OH Tamoxife

(B) MDA-MB-231

150

100 # # # # # # #, # # #, #

Expression 50 (COX-2 / 18S) / (COX-2 Relative COX-2 mRNA COX-2 Relative

0 4 5 M) M) M) -1 -1 -15 M) n n n 0 5 U U μ DMSO 00 D D DU 1 + + (50 5 (2 ( 1 n n n le + e 14 (25 - e e e o - U if if if z tan x x x o s D tr DU mo mo etrozole (10 nM) mo L LetrozoleLe + DU-14 Ta Ta xemestane + DU-15 H Exeme ExemestaneE + DU-14 OH -O -OH Ta - 4 4 4

Figure 8.8. COX-2 mRNA expression in (A) MCF-7 and (B) MDA-MB-231 after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the aromatase/ER inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 6, unless otherwise shown. 160 (A) MCF-7 350 #, # 300

250 #, #

200

150 (CYP19 / 18S) n = 5 100

50 Relative CYP19 mRNA Expression mRNA CYP19 Relative

0 O ) ) ) ) ) ) ) ) ) S M M M M M M M M M n n n n n n n n M 0 0 5 0 0 5 m 0 5 D 5 2 1 2 0 2 2 ( ( ( (5 ( (10 ( 5 e (25 5 (25 5 n 4 l 4 4 1 o 1 ne 1 fe - z - - U-1 sta oxi DU-1 tro DU-1 D DU + e DU + DU + m + L me n + le + e Ta n le o xe e n fe xife o z E n H o z ta -O tro s esta 4 tro e e m e L m Tam L e xe Tamoxi x E E -OH -OH 4 4 (B) MDA-MB-231

350

#, # 300

250

200 # #

# 150 n = 5 n = 5 (CYP19 / 18S) #, # 100

50 Relative CYP19 mRNA Expression mRNA CYP19 Relative

0 O ) 5 ) S M) 14 M M n nM U- nM) μ D 0 100 DU-1 5 (25 + 1 n ( n + D U- D rozole + DU-15 stane + DU-15 DU-14 (250 nM) moxife moxifen e a a LetrozoleLetrozole (1 Let + DU-14 T xem Tamoxife ExemestaneExemestane (50 E + DU-14 OH -OH 4 4- 4-OH T

Figure 8.9. CYP19 mRNA expression in (A) MCF-7 and (B) MDA-MB-231 after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the aromatase/ER inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 6, unless otherwise shown. 161 In the analysis of combinational effects of inhibitors on STS enzyme activity

(Figure 8.10.) , we found that 4-OHT inhibited STS by 40%, but the addition of either

DU compound further inhibited the enzyme similarly to the levels of those compounds alone. Letrozole- and exemestane-stimulated STS activities were completely eliminated upon co-treatment with DU-14, but DU-15 was only able to return their stimulatory effects to the level of control activity. In this case, DU-14 is blocking aromatase inhibitor effects, where we have thus far typically seen DU-15 exert the dominate effects and mediate the actions of other drugs.

In Figure 8.11., DU-15 appears to be weaker than DU-14 at mediating the effects of other inhibitors in MDA-MB-231 cells. The combinations of 4-OHT, letrozole, and exemestane all inhibit STS activity to varying degrees and the addition of DU-14 further reduces STS activity. Regardless, there were no observed additive effects since the inhibitions seen in combination were similar to the STS inhibitors’ individual effects.

162

250

200

150 # 100 #

50 (dpm/ug DNA/h)

# # # # # # # # # # # # # #

Relative STS Radioactivity STS Relative 0

-50 S ) ) ) 1 -14 M) -15 E nM nM nM μ DU DU (25 nM) 50 (250 nM) (100 nM) + ( + e (10 e en -15 ol ol if tane ane U-14 DU-15 (25ox DU troz rozole + DU-15 D + troz t est + DU-14 (250 Le Le Le en xemes em Tam en if x H E ExemestaneE + DU-14 O ox 4- amoxif Tam T H O -OH 4- 4

Figure 8.10. STS enzyme activity in MCF-7 cells after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors. Cells were treated with 1 μM E1S and radioactive substrate in the presence or absence of various inhibitors. Twenty-four hours after treatment, STS activity was determined and the amount of [3H]estrone radioactivity was normalized with the total DNA concentration. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the aromatase/ER inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 3, unless otherwise shown. 100% control = 103.1 dpm/μg DNA/h.

163 125 n = 6

100

50 # # (dpm/ug DNA/h) # # 25 # # Relative STS Radioactivity STS Relative

0 ) ) 4 4 SO M -1 -14 M) nM) nM) -1 M 5 nM U U μ U D 2 D DU-15 10 D ( + ( (50 D 5 e + + + DU-15 n (100 n n le e e e 14 (250 -1 e fen + zol - U xi zo U D stan stan D mo etro tro e e L Le Letrozole + DU-15 Ta xem xem E E Exemestan -OH 4-OH Tamoxif4 4-OH Tamoxife

Figure 8.11. STS enzyme activity in MDA-MB-231 cells after 24 incubation with combinations of 4-OHT or aromatase inhibitors with STS inhibitors. Cells were treated with 1 μM E1S and radioactive substrate in the presence or absence of various inhibitors. Twenty-four hours after treatment, STS activity was determined and the amount of [3H]estrone radioactivity was normalized with the total DNA concentration. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the aromatase/ER inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 3, unless otherwise shown. 100% control = 29.0 dpm/μg DNA/h.

164 Cellular growth proliferation was also monitored for the combinations of the

aromatase inhibitors and the DU compounds (Figure 8.12.). In MCF-7 cells, letrozole

alone did not significantly suppress cell growth over 7 days. Exemestane initially

stimulated growth of the ER+ cells, but by day 7, significant inhibition was observed.

The combination of either aromatase inhibitor with DU-14 increased their growth inhibition, but not to the same extent as seen for DU-14 alone. In other words, the

presence of either letrozole or exemestane actually reduced the STS inhibitor’s solo

inhibitory effect. However, the addition of DU-15 to either aromatase inhibitor

mimicked DU-15’s individual growth inhibition over 7 days. DU-15’s effects were able

to dominate over the growth profiles of letrozole and exemestane, whereas DU-14

appeared to average the individual inhibitors’ results.

In MDA-MB-231 cells, growth inhibition effects of all treatments generally

decreased with exposure time, suggesting the cells are recovering or adjusting to the

chemical exposure. The addition of DU-14 to letrozole or exemestane had no affect on

their growth effects. But, the addition of DU-15 to either aromatase inhibitor increased

the initial inhibition of cellular growth. However, by day 7 almost all treatments had

returned to normal/untreated cell growth. The only combinational effect observed was

the co-treatment of exemestane and DU-15. By day 7, this combination was more

inhibitory than either compound alone.

165 (A) MCF-7

2 days 150 * ** 4 days 7 days 125

100 # # # 75 # # ** ** * *# # * *# 50 ** # ** # * * % of controlgrowth ** *# 25

0 4 5 4 5 M) M) M) MSO μ μ U1 U1 μ U1 U1 D D 0 D + + D (5 + + D e e e e 5 (25 l l n 14 (50 zo a U U1 ozo tane stan t D D tro r e Letrozole (10 nM) L Let mes me e e x x E E Exemes

(B) MDA-MB-231

2 days 150 4 days 125 th 7 days

100 n = 5 grow # l # # #

ro * ## *

t # 75 * # # * # #*

con * # f 50 **

o ** ** *** % 25

0 O 5 4 M) M) M) M) μ μ n μ 0 DMS DU-1 DU-1 DU-15 + 5 (25 e (1 e + 1 le + n -14 (50 ta rozol ozo s DU DU- t tr Le Letrozole + DU-14 Le me xe Exemestane (50 E Exemestane

Figure 8.12. Proliferation assay of (A) MCF-7 and (B) MDA-MB0231 cells over 2, 4, and 7 days of aromatase/ER inhibitor treatment. Statistical analysis performed by student t test. Significance from control: *, p < 0.0001; #, p < 0.05. Red indicates difference from control; blue indicates different from the aromatase/ER inhibitor in the mixture; green indicates difference from the STS inhibitor in the mixture. N = 12, unless otherwise shown.

166 8.3. Conclusions

The inhibition of both the aromatase and sulfatase estrogen-producing pathways

should completely blockade estradiol synthesis. Selcer and Li et al, upon success of their potent sulfamate STS inhibitors, developed their own “dual action” inhibitors, attaching

the sulfamate pharmacophore onto 4-hydroxy tamoxifen nuclei 1. Like the dual

aromatase-sulfatase inhibitors (DASIs) designed by Woo et al 2;3, these compounds

should irreversibly inhibit STS through the transfer of the covalently binding sulfamate group and then release the potent metabolites of the anti-estrogen tamoxifen available to block estrogen binding the estrogen receptor. This same methodology may also be applied to other enzyme inhibitors to include STS inhibition, combining the actions of

COX-2 or other NF-κB inhibitors. Being that COX-2 regulates aromatase, the duality of a STS-COX-2 inhibitor may actually be able to target all three enzymes implicated in the growth of hormone-dependent breast cancer.

However, the lack of combinational effects in our studies, particularly in the cellular growth proliferation experiments, was surprising. In both MCF-7 and MDA-

MB-231 cells, the DU compounds’ enzyme inhibition dominated the effects of the COX inhibitors against STS enzyme activity, to the extent that no additive effects could be seen, even when the COX inhibitors stimulated STS activity (SC-560 in MCF-7 cells; all three inMDA-MB-231 cells). DU-14 and DU-15 also exhibited greater cellular growth inhibition over 7 days than COX inhibitors alone in both ER+ and ER- cell lines; combinations of the inhibitors did not exceed the effects of the STS inhibitors alone.

Only several minor additive effects were observed upon combining DU-15 with the aromatase inhibitors. In general, DU-15 was better capable of combined treatments with

167 aromatase inhibitors, as evidence by the combination’s effects on STS mRNA expression

and cellular growth inhibition. However, in combinations with COX inhibitors, the DU

compounds had multiple effects depending on cell line and identity of inhibitor tested.

For example, DU-14 stimulated COX-2 mRNA expression compared to control with

celecoxib in MCF-7 and MDA-MB-231 cells, when DU-15 would have no combinational

effect. Then, in other cases, DU-15 would stimulate CYP19 expression with NS-398

treatment in MDA-MB-231 cells, while DU-14 had elicited no change from NS-398

treatment alone.

The lack of additive effects in our combinations is not uncommon as it has been

seen in Angela Brodie’s research examining several combinations of anti-estrogens and

aromatase inhibitors. Brodie et al injected ovariectomized female mice with Ac-1 xenografts (cells transfected with aromatase) with antiestrogen toremifene, steroidal

aromatase inhibitor atamestane, tamoxifen, and the combination of toremifene and

atamestane 4. Their results indicated that the combination of toremifene plus atamestane

was as effective as either antiestrogen alone and did not provide any additional benefits.

In the combination of aromatase inhibitor 4-hydroxyandrostenedione (4-OHA) and

tamoxifen on the growth of human breast cancer cells in the same mouse model, Brodie

et al found no significant benefit in combining these compounds over 4-OHA treatment

alone 5. They also used this mouse model to test the combination of two aromatase inhibitors, letrozole and anastrozole, with tamoxifen 6. Again, the combination of

letrozole or anastrozole with tamoxifen was no more effective than either aromatase

inhibitor alone. Furthermore, their intratumoral aromatase xenograft mouse model has accurately predicted the outcome of similar combinations in clinical trials 7.

168 Due the very complex nature of these networks, as well as the continuing evidence of multiple modes of action of these tested inhibitors (see Chapters 4, 6, and

7), it is challenging to find specific conclusions from this array of data. It has been shown that experiments can be affected by the behavior of the cultured cell lines from passage to passage. However, the major conclusion we drew from the set of studies presented here is that STS inhibitor combinations, with either COX or aromatase inhibitors, do not result in significant additive or synergistic effects on mRNA expression, STS activity, or cell proliferation in vitro. This is evidence that dual treatment of breast cancer cells through simultaneous inhibition of both the sulfatase and aromatase pathways may not be as efficacious as predicted. Better success for the STS inhibitors may be seen in animal models or patient tissue samples, or with singular multi- functionality drugs.

169 8.4. References

1. Chu, G.-H.; Peters, A.; Selcer, K. W.; and Li, P. K. Synthesis and sulfatase inhibitory activities of (E)- and (Z)-4-hydroxytamoxifen sulfamates. Bioorganic & Medicinal Chemistry Letters 1999, 9, 141-144.

2. Woo, L. W. L.; Sutcliffe, O. B.; Bubert, C.; Grasso, A.; Chandler, S. K.; Purohit, A.; Reed, M. J.; and Potter, B. V. L. First dual aromatase-steroid sulfatase inhibitors. Journal of Medicinal Chemistry 2003, 46, 3193-3196.

3. Woo, L. W. L.; Bubert, C.; Sutcliffe, O. B.; Smith, A.; Chander, S. K.; Mahon, M. F.; Purohit, A.; Reed, M. J.; and Potter, B. V. Dual aromatase-steroid sulfatase inhibitors. Journal of Medicinal Chemistry 2007, 50, 3540-3560.

4. Sabnis, G. J.; Macedo, L. F.; Goloubeva, O.; Schayowitz, A.; Zhu, Y.; and Brodie, A. Toremifene-atamestane: alone or in combination: predictions from the preclinical intratumoral aromatase model. Journal of Steroid Biochemistry & Molecular Biology 2008, 108, 1-7.

5. Yue, W.; Wang, J.; Savinov, A.; and Brodie, A. Effect of aromatase inhibitors on growth of mammary tumors in a nude mouse model. Cancer Research 1995, 55, 3073-3077.

6. Brodie, A.; Lu, Q.; Liu, Y.; Long, B.; Wang, J.-P.; and Yue, W. Preclinical studies using the intratumoral aromatase model for postmenopausal breast cancer. Oncology 1998, 12, 36-40.

7. Brodie, A.; Jelovac, D.; and Long, B. The intratumoral aromatase model: studies with aromatase inhibitors and antiestrogens. Journal of Steroid Biochemistry & Molecular Biology 2003, 86, 283-288.

170 CHAPTER 9

CONCLUSIONS AND FUTURE DIRECTIONS

In the post-menopausal state, there are low levels of circulating estrogens.

However, these concentrations are elevated in breast cancer tissue, indicating tumor-

specific hormone biosynthesis and accumulation. Steroid sulfatase (STS),

cyclooxygenase-2 (COX-2), and aromatase (CYP19) are enzymes critical for estrogen biosynthesis and have been shown to be overexpressed in breast cancer cells. Much previous research within and outside of this laboratory has focused on the gene and

enzyme of aromatase. However, here we seek to more fully understand STS and its

relationships to the other estrogen-producing enzymes.

The two steroid sulfatase inhibitors, DU-14 and DU-15, were shown to be

effective inhibitors of STS activity, breast cancer cell proliferation, and potentially

mRNA expression (Chapter 4). The STS inhibitors decreased STS and COX-2 mRNA

levels in an estrogen receptor positive cell line MCF-7 but not in ER-negative MDA-MB-

231 cells. However, due to a lack of clear dose-dependence, it remains to be seen if the

compounds act directly or through other modulators to achieve their effects on gene

expression.

171 Upon studying potential regulatory factors on STS expression and activity

(Chapter 5), we found no new STS regulatory factors. The analysis of some factors that

have been previously reported to influence aromatase and COX-2 (IL-6, TNFα, DEX)

displayed unpredicted effects in some cases. Nevertheless, these response differences to

“known” cytokines and glucocorticoids regulators are what led us to investigate PKA and

NF-κB signaling in our cells lines. We were able to show that NF-κB and PKA

pathways were strongly present in our ER-negative MDA-MB-231 cells, but low in the

MCF-7 cells. Additionally, long-term estrogen deprivation of MCF-7 cells was shown to effect STS activity and mRNA expression differently over time of deprivation.

In Chapter 6, we analyzed multiple known cyclooxygenase inhibitors against gene expression and STS activity in breast cancer cells, as well as measuring their inhibition of cell growth over a week. The COX selective inhibitors celecoxib and NS-

398 were chosen for their COX-2 selectivity, whereas SC-560 was selected as a COX-1 selective inhibitor. NSAIDs indomethacin, ibuprofen, naproxen sodium, acetylsalicylic acid, piroxicam, and niflumic acid were all additionally analyzed to see if any trends arose related to their COX-1/COX-2 preference.

Of the inhibitors effects on STS expression, niflumic acid stood out as a significant enhancer of STS mRNA in MCF-7 cells. In MDA-MB-231 cells, ibuprofen,

acetylsalicylic acid and niflumic acid showed mid-level inhibition of STS expression.

Most of the inhibitors affected COX-2 mRNA expression, but not in manner consistent

with their COX selectivities. Generally, the NSAID compounds were inhibitory in both

MCF-7 cells and MDA-MB-231 cells on COX-2 expression. Only two compounds

172 notably modulated CYP19 mRNA. Niflumic acid stimulated aromatase in MCF-7 cells, and SC-560 stimulated it in MDA-MB-231 cells.

SC-560 activated STS enzyme activity, but did not have corresponding effects on

STS mRNA in MCF-7 cells, nor did acetylsalicylic acid or piroxicam. In MDA-MB-231 cells, most COX compounds had the ability to stimulate STS activity, but could not do so

consistently over cell passages. Therefore, no firm conclusions could be drawn except

that (1) COX inhibitors can affect multiple targets involved in breast cancer development,

(2) that NSAIDs can inhibit cancer cell growth by yet understood mechanisms, and (3)

that those mechanisms require probing within multiple pathways to elucidate their

common link.

Next, we evaluated the effects of aromatase inhibitors letrozole and exemestane,

as well as anti-estrogen metabolite 4-hydroxy tamoxifen (Chapter 7). While the compounds did not greatly affect cellular proliferation, they markedly suppressed STS mRNA in both MCF-7 and MDA-MB-231 cell lines. Additionally, the aromatase inhibitors and 4-OHT suppressed COX-2 mRNA in MCF-7 cells. Nevertheless, the aromatase inhibitors did not suppress the corresponding STS enzyme activity. In fact, both letrozole and exemestane stimulated STS activity in MCF-7 cells, but had different effects in MDA-MB-231 cells; letrozole inhibited STS in the ER-negative cells and exemestane was not active. In both cell lines, 4-OHT inhibited STS activity to approximately 50% of control, similar to that seen by Evans et al for the actions of tamoxifen against STS activity and mRNA expression in MCF-7 cells 8.

In the final experimental Chapter 8, we looked for any combinational effects from treating MCF-7 and MDA-MB-231 cells simultaneously with two different enzyme

173 inhibitors. However, the lack of combinational effects in our studies, particularly in the

cellular growth proliferation experiments, was surprising. In both MCF-7 and MDA-

MB-231 cells, the DU compounds’ enzyme inhibition dominated the effects of the COX

inhibitors against STS enzyme activity, to the extent that no additive effects could be

seen. DU-14 and DU-15 also exhibited greater cellular growth inhibition over 7 days

than COX inhibitors alone in both ER+ and ER- cell lines; the combinations of inhibitors

did not exceed the effects of the STS inhibitors alone. Only several minor additive

effects on STS mRNA expression and cellular growth inhibition were observed upon

combining DU-15 with the aromatase inhibitors. However, in combinations with COX

inhibitors, the DU compounds had inconsistent effects depending on cell line and identity

of secondary inhibitor tested. The major conclusion drawn from these studies is that STS

inhibitor combinations, with either COX or aromatase inhibitors, do not result in

significant additive or synergistic effects on mRNA expression, STS activity, or cell

proliferation in vitro. This is evidence that dual treatment of breast cancer cells through

simultaneous inhibition of both the sulfatase and aromatase pathways may not be as

efficacious as predicted.

In the end, we were able to successfully achieve the specific aims set out in

Chapter 2. First, we treated MCF-7 and MDA-MB-231 cells with STS inhibitors, COX

1/2 inhibitors, and aromatase/ER inhibitors. Then, we assayed these treated cells for

individual and combinational effects on STS, CYP19, and COX-2 gene expression by

qualitative Real-Time RT-PCR, E1-STS enzyme activity by an in-cell radioactivity assay, and cellular growth proliferation by the Promega® MTS Assay. Additionally, we explored the effects of potential regulators on STS expression and activity, treating both

174 ER-positive and ER-negative cells with inflammatory cytokines, steroids, and signaling pathway inhibitors.

However, our central hypothesis could not be confirmed by our studies. We

predicted that inhibition of aromatase and/or COX-2 should not only initially decrease E2

production, but would increase the cancer cells’ reliance on the sulfatase pathway in

hormone-dependent breast cancer cells. As such, inhibition of STS would act in concert

with aromatase and/or COX inhibition to block the total biosynthesis of estradiol. But

due the very complex nature of these networks, as well as the evident multiple modes of

action of the tested inhibitors, further experimentation is required to fully elucidate the

interrelationships between these estrogen-producing enzymes.

Acquired resistance to existing anti-estrogen therapies necessitates the

development of novel inhibitors, including those for novel targets. Depending on the

genetic profiling of individual cancer patients, we may better find treatments suited for

the tumor and surrounding tissue levels of STS, aromatase, and COX-2. Therefore, STS

inhibition may still be a beneficial hormone therapy in addition to or following the

success of first-line anti-estrogens or aromatase inhibitor therapies.

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