Copyright by Luke Yun-Kong Koong 2014

The Dissertation Committee for Luke Yun-Kong Koong Certifies that this is the approved version of the following dissertation:

THE DIRECT EFFECTS OF ESTRADIOL AND SEVERAL

XENOESTROGENS ON CELL NUMBERS OF EARLY- VS. LATE-

STAGE PROSTATE CANCER CELLS

Committee:

Cheryl S Watson, PhD, Mentor

Darren Boehning, PhD, Chair

Gracie Vargas, PhD

Randall M Goldblum, MD

Nancy Ing, DVM, PhD

______Dean, Graduate School THE DIRECT EFFECTS OF ESTRADIOL AND SEVERAL

XENOESTROGENS ON CELL NUMBERS OF EARLY- VS. LATE-

STAGE PROSTATE CANCER CELLS

by

Luke Yun-Kong Koong, BS

Dissertation Presented to the Faculty of the Graduate School of

The University of Texas Medical Branch in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

The University of Texas Medical Branch December, 2014

Dedication

This dissertation is dedicated to my family, whom has supported me with love and encouragement throughout my life; my friends, who push me to greater heights; and most importantly God, who continues to bless me every day with life and joy.

Acknowledgements

I would like to acknowledge my mentor, Cheryl S Watson, who has guided me through my graduate work. She has shown me how to be a true scientist and a responsible steward of our environment. I am forever grateful for the avenues she has opened up for my life and career. Additionally, I would like to thank my committee members for all of their constructive ideas throughout my project, as well as encouragement along the way. Another key figure in my graduate career is Jennifer Jeng of the Watson laboratory, who helped teach me many of the assays used in this study, and who was also available for advice and suggestions. The National Institutes of Environmental Health Sciences (NIEHS) Environmental Toxicology Training Grant helped me acquire numerous skills toward my career, as well as monetary support, and for that I am truly grateful. Finally, I want to acknowledge the GSBS and the support they provided me in finishing my degree. This work was supported by the NIEHS T32ES007254 Environmental

Toxicology Training Grant.

iv THE DIRECT EFFECTS OF ESTRADIOL AND SEVERAL

XENOESTROGENS ON CELL NUMBERS OF EARLY- VS. LATE-

STAGE PROSTATE CANCER CELLS

Publication No.______

Luke Yun-Kong Koong, PhD The University of Texas Medical Branch, 2014

Supervisor: Cheryl S Watson

Prostate cancer is the most common non-cutaneous cancer among men, and diethylstilbestrol (DES) is an estrogen that has been used clinically to combat advanced tumors. Estrogens can indirectly decrease androgen production by central negative feedback inhibition, but may also have direct tumor killing mechanisms by a less well understood mechanism. To elucidate these mechanisms and provide understanding for potential future therapies, we identified cellular pathways and rapid signaling events that act via estrogen receptors (ERs) and contribute to estradiol (E2) or DES-mediated cell killing/growth arrest. E2 was much more effective than DES at reducing cell numbers of both LAPC-4 early-stage androgen-dependent and PC-3 late-stage androgen-independent prostate cancer cells. Both E2 and DES rapidly (within minutes) activated mitogen- activated protein kinases, generated reactive oxygen species (ROS), induced apoptosis and necroptosis, and regulated the activation and levels of cell cycle proteins. Regulation of cyclin D1 played a major role in blocking cell proliferation, but extracellular signal-

v regulated kinase (ERK) causing ROS generation, phosphorylation of p38, apoptosis, and phosphorylation of p16INK4A also contributed. Our rapid effects suggested the participation of membrane ERs (mERs). ERα and β mediated E2’s ability to increase ROS through sustained ERK activation, and increased p-cyclin D1 levels in LAPC-4 cells. However, ERβ and GPR30 mediated these responses in PC-3 cells. Apoptosis was initiated in LAPC-4 and PC-3 cells by E2 only, and not by DES. Necroptosis was not altered by estrogens in either cell line. We showed for the first time the presence of mERs in both early and late prostate cancer cells. Then several xenoestrogens (XEs) were evaluated for their ability to increase or decrease prostate cancer cell numbers. Coumesterol and genistein unexpectedly stimulated prostate cancer cell growth, while resveratrol minimally increased cell numbers in both early and late-stage cells. Bisphenol A (BPA) slightly, though significantly, increased cell numbers in LAPC-4 cells only. Again, control of cyclin D1 protein levels was a key mediator of the growth change effects. The findings of these studies should be relevant to the clinical treatment of prostate tumors, including development of ER subtype-specific agents to control proliferation and cell death signaling pathways. Our findings regarding XEs should help establish guidelines for prostate cancer patients regarding consumption of safe dietary concentrations of phytoestrogens and acceptable exposure levels to BPA.

vi TABLE OF CONTENTS

List of Tables ...... ix

List of Figures ...... x

List of Abbreviations ...... xiii

Chapter 1: Introduction ...... 15

Prostate tumors and methods of treatment ...... 15 Prostate function and anatomy ...... 17 Prostate cancer treatment regimens ...... 22 Development of androgen-independent tumors ...... 24 Estrogens and estrogen receptor physiology ...... 27 Roles of membrane estrogen receptors in nongenomic responses ...... 31 Estrogen receptor control of cell proliferation or death ...... 33 Estrogen receptors in the prostate and prostatic disease ...... 34 Xenoestrogens and effects on living systems ...... 35 Aims of studies in this dissertation ...... 37 Aim 1 ...... 37 Aim 2 ...... 38

Chapter 2: Direct estradiol and diethylstilbestrol actions on early- vs. late-stage prostate cancer cells ...... 39

Abstract ...... 39 Introduction ...... 40 Materials and Methods...... 43 Results ...... 46 Discussion ...... 58 Conclusions ...... 63

Chapter 3: Rapid, nongenomic signaling effects of several xenoestrogens involved in early- vs. late-stage prostate cancer cell proliferation ...... 64

Abstract ...... 64 Introduction ...... 65 Materials and Methods...... 68

vii Results and Discussion ...... 72 Conclusions ...... 84

Chapter 4: Conclusions and Future Directions ...... 87

Major Conclusions from Our Studies ...... 87 In vivo model ...... 90 Receptor targeted agonists (Acadia ERβ agonists) ...... 94 Other potential studies and directions ...... 96

References ...... 99

Vita 154

viii List of Tables

Table 2.1. Summary of mechanisms contributing to estrogen-induced decline in

numbers of LAPC-4 or PC-3 prostate cancer cells...... 60

ix List of Figures

Figure 1.1. Functional domains of AR...... 19

Figure 1.2. Canonical AR signaling pathway...... 21

Figure 1.3. Summary of roles for membrane and intracellular ERs...... 28

Figure 1.4. ERα and ERβ splice variants...... 30

Figure 1.5. Comparison of the structures of E2, DES, BPA, and three

phytoestrogens...... 36

Figure 2.1. Cell viability after 3 days of E2 or DES treatment...... 46

Figure 2.2 Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after E2 or

DES treatments...... 48

Figure 2.3. ROS measured in LAPC-4 vs. PC-3 cells treated with 0.1 nM E2 or 1

µM DES...... 49

Figure 2.4. Phospho-JNK (pJNK) levels in LAPC-4 and PC-3 after E2 or DES

treatments...... 51

Figure 2.5. Caspase 3 activity levels after E2 or DES treatments...... 52

Figure 2.6. Necroptosis after E2 or DES treatments...... 52

Figure 2.7. Phospho-p16 (p-p16) levels after E2 or DES treatments...... 54

Figure 2.8. Activated p38 (p-p38) levels after treatment with 0.1 nM E2 and 1 µM

DES...... 54

x Figure 2.9. Phosphoryated cyclin D1 (p-cyclin D1) vs. total cyclin levels and their

ratios after treatment with 0.1 nM E2 and 1 µM DES...... 56

Figure 2.10. ER subtype-selective antagonists inhibit E2- or DES-induced ROS

and p-cyclin D1 responses...... 57

Figure 2.11. Direct mechanisms of E2/DES action on prostate cancer cell

survival...... 59

Figure 3.1. Cell number after 3 days of XE treatment...... 72

Figure 3.2. ER subtype (α, β, and GPR30) levels (total vs. membrane) in LAPC-4

and PC-3 prostate cancer cells...... 74

Figure 3.3. Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after XE

treatments...... 76

-10 -6 -9 Figure 3.4. ROS levels after treatment with 10 M E2, 10 M H2O2, 10 M BPA, 10-7M coumestrol, 10-7M genistein, and 10-8M resveratrol, ± ER

subtype-selective antagonists...... 77

Figure 3.5. Cyclin D1 phosphorylation and degradation by XEs, and inhibition

by ER-selective antagonists...... 79

Table 3.1 Summary of XE responses for mechanisms that affect the number of

viable LAPC-4 vs. PC-3 cells...... 84

Figure 4.1. Various components of the prostate tumor microenvironment...... 91

Figure 4.2 Primary steps in establishing a xenograft prostate cancer model in

SCID mice...... 92

xi Figure 4.3 Number of viable prostate cancer cells following Acadia ERβ agonists.

...... 95

Figure 4.4. Improper epigenetic regulation of normal prostate cells may lead to

an increased chance for prostate cancer...... 97

xii List of Abbreviations

AR Androgen Receptor

ARE Androgen Response Element

BPA Bisphenol A cAMP Cyclic Adenosine Monophosphate

DES Diethylstilbestrol

DHT Dihydrotestosterone

E2 Estradiol

EE Ethinyl Estradiol

EGF Epidermal Growth Factor

ER Estrogen Receptor

ERK Extracellular Signal-Regulated Kinase

ERE Estrogen Response Element

FHS Follicle Stimulating Hormone

IGF Insulin-like Growth Factor

JNK c-Jun N-terminal Kinase

LH Luteinizing Hormone

LHRH Luteinizing-Hormone-Releasing Hormone mER Membrane Estrogen Receptor

MAPK Mitogen Activated Protein Kinase

PI3K Phosphoinositide 3-kinase

PSA Prostate Specific Antigen

xiii ROS Reactive Oxygen Species

SCID Severe Combined Immunodeficiency

STAT Signal Transducers and Activators of Transcription

T Testosterone

VEGF Vascular Endothelial Growth Factor

XE Xenoestrogen

xiv

Chapter 1: Introduction

Prostate tumors and methods of treatment

Androgens have been known to stimulate the growth of the prostate gland and tumors derived from them since the late 18th century, but the first written report of prostate cancer did not appear until 1853, when John Adams was able to study the histology of a prostate tumor [reviewed in 1]. Early treatments employed surgical castration as a means to decrease androgen levels and reduce prostate gland size 2. The use of estrogens to treat prostate cancers was first used in the 1940s by Charles Huggins and Clarence Hodges who administered the pharmaceutical estrogen diethylstilbestrol (DES) and estradiol benzoate to patients as a form of androgen ablation via central negative feedback control 3, slowing metastatic prostate cancer cell growth and inhibiting release of acid phosphatase. A study in the 1960s by the Veteran’s Administration

Cooperative Urological Research Group then found that DES administration was as effective as orchiectomy in treating prostate tumors 4. While these estrogens indirectly inhibit prostate cancer cell growth, the rapid, direct mechanisms of estrogen action on prostate tumor cells have not been considered. In recent years, prostate cancer has become the most diagnosed non-cutaneous

15 cancer in men, with close to 200,000 cases diagnosed each year and about 30,000 deaths annually in the United States 5, 6. The incidence rate varies across ethnicities, but has shown a steady overall decline since 1999 7, 8. The occurrence of prostate tumors increases with age, and patient age correlates to the ability to histologically detect cancerous cells in prostate gland biopsies. Most tumors are commonly found in males in their 60-70’s 9. Precancerous lesions are the precursors for eventual prostate tumors and can be found in the prostates of 33% of healthy men, but the development of lesions into actual cancerous cells is significantly less (1 in 9 patients) 9. Additionally, the incidence

of prostate cancer in the US (1 in 7) is higher than in Asian countries (1 in 10,000) 5, 10. This large discrepancy has led many to examine the differences in diets between the US and East Asia and how they might affect prostate cancer 11-15, which has largely focused on the soy-based isoflavones. There is also evidence however that the large difference seen between the two populations is simply due to better reporting and screening practices in the US 16. The susceptibility of developing prostate cancer does not appear to be genetic 17,

18, with the exception of a late-developing chromosomal translocation causing the fusion of the transmembrane protease serine 2 (TMPRSS2) gene to the coding region of the erythroblast transformation-specific (ETS) family of transcription factors, leading to androgen-independent growth. This translocation can be found in 40-70% of prostate cancer patients according to several studies, but these lesions may not be continually selected as the tumor progresses 19-21. However, tumors with this translocation have increased metastatic potential and can be more aggressive, leading to higher grade cancers 19, 22. Common ETS family proteins involved in this gene fusion include ETS- related gene, ETS translocation variant 1, and protein C-ets-1 23. The TMPRSS2-ETS gene fusions are rarely detected in benign prostate tissue. Interestingly, this major genetic mutation shows a reliance on estrogens and estrogen receptors (ERs) for genomic regulation resulting in prostate cancer cell proliferation, specifically via ERβ 19. ERβ binds to an upstream site of the TMPRSS2 promoter, directly regulating the transcription of the TMPRSS2-ETS fusion products. (Because our project instead detects inhibition of prostate cancer cell proliferation by estrogens, this genetic mutation is not a candidate responsible mechanism in our nongenomic studies.) Another common genetic subtype of prostate cancers contains a mutation in the speckle-type POZ protein (SPOP) gene preventing down-regulation of ARs and promoting cell growth, and is found in 15% of patients with prostate tumors 24, 25. Other more rare genetic alterations that have been

16

found are losses in NKX3.1 26 and PTEN 27, and gene amplification of the AR (regions Xq11-Xq13) 28. Prostate cancer cell lines that are the focus of our study have also been reported to have some genetic modifications. The hypotetraploid LAPC-4 androgen-dependent prostate cancer cells possess a deletion of chromosome 12p12 29, while chromosomes of Group A, No.5, No.15, and the entire Y chromosome are absent in androgen-independent PC-3 cells 30. Neither of these cell lines possess the TMPRSS2-ERG gene fusion 31.

Another major gene product, tumor-suppressor p53, is mutated in LAPC-4 cells and not found in PC-3 cells 32. We did not use the commonly used androgen-dependent cell line LnCAP because it has a mutated AR, unlike our choice of LAPC-4 cells that have wild- type ARs; PC-3 cells do not possess ARs, contributing to the difference in androgen requirement for tumor cell growth 32. PC-3 cells are typical of the most advanced cancers due to their hormone insensitivity, level of dedifferentiation, and chromosomal loss.

Prostate function and anatomy

The prostate is a male reproductive gland that provides seminal fluid that protects and sustains sperm. During ejaculation, the prostate releases this fluid into the urethra, as well as prevents urine from mixing with semen 33. The prostate is typically divided into three major zones: the peripheral zone, where about 75% of tumors originate; the central zone, which rarely has tumors; and the transition zone, where benign prostatic hyperplasia occurs 34-37. The human prostate can also be divided into four lobes: anterior, posterior, lateral, and median 38-40.The prostate gland, and tumors that develop from it, rely on androgens for sustained growth, in particular 5α-dihydrotestosterone (DHT) 41. The indirect ability of DES and other estrogens to inhibit the growth of prostate cancer cells has mainly been attributed in the past to a decline in androgen production through negative feedback control via the hypothalamic-pituitary-testicular axis 42. Androgens are

17

synthesized in the testes and adrenal glands from cholesterol 43. The hypothalamus releases gonadotropin-releasing hormone (or luteinizing-hormone-releasing hormone, LHRH), which stimulates the release of luteinizing hormone (LH) and follicle- stimulating hormone (FSH) from the pituitary gland. LH and FSH are responsible for stimulating the production of testosterone (T) and also a small amount of E2 in the Leydig cells of the testes. T can be further converted into DHT by 5α-reductase, or into E2 by the enzyme aromatase, which is found and functional in several tissues throughout the body, including the prostate gland 44-46. Early-stage, androgen dependent LAPC-4 and late-stage, androgen-independent PC-3 prostate cancer cells express aromatase, but PC-3 cells have decreased expression levels 47. DHT is essential to normal growth of the prostate, as well as defining male secondary sex characteristics 48. There are epidemiological studies to suggest that mutations in the 5α-reductase enzyme increases the risk of prostate cancer, particularly in men of African descent 49. The polymorphism at codon 89 increases enzyme activity and

50 increases prostatic levels of DHT . Estrogens, such as E2, are traditionally viewed as female steroid hormones, but have several important functions in mediating the growth and differentiation of the prostate. Its specific and broader roles are highlighted later in this review.

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Figure 1.1. Functional domains of AR. The four major functional domains: N-terminal transactivation domain (NTD); DNA binding domain (DBD); hinge and ligand binding domain (LBD) (Figure from Gao et al., 2005).

Androgens exert their hormonal actions through androgen receptors (AR), which are part of the steroid nuclear-receptor superfamily 51, 52. The human AR gene is located at position 12 on the long arm of chromosome X 53. There is currently only one known form of AR 54, 55, unlike ER, which has two functional genes within this family of proteins (although a type of G protein-coupled receptor can also mediate estrogenic responses, see below), as well as several splice variants 56-58. AR has four major domains: the N-terminal transactivation domain (NTD); the DNA binding domain; hinge, and the

C-terminal ligand binding domain (Figure 1.1) 51. ARs function similar to other steroid hormones, inducing transcription upon ligand activation of DNA binding. However, ARs have also recently been associated with nongenomic signaling events 59-62. Genomically inactive AR is found localized in the cytoplasm, bound to a heat shock protein complex, including Hsp90, p23, and FKBP 63. Upon ligand binding (typically with androgens), AR is released from the complex and changes conformation, dimerizes, and becomes

19

activated through phosphorylation 52, 64. The AR homodimer then translocates to the nucleus and binds to androgen-response elements of target genes, leading to cellular growth, differentiation, and the synthesis and release of prostate specific antigen (PSA) 65-67 from the prostate epithelium. PSA is an enzyme found in semen, responsible for cleaving semenogelin I and II in the seminal coagulum 68. This protein is upregulated in prostate cancer, and has consequently been used as a biomarker for prostate cancer screening 69-71 (Figure 1.2), though the usefulness of this biomarker is currently being debated 72. In advanced prostate tumors, AR function is apparently no longer needed to sustain cell proliferation, and is often lost entirely 41. This transition to hormone independence is typical of many types of steroid growth-driven tumors 73-75. However, estrogens, which are not the tumor-sustaining hormone of the prostate, have been shown to have tumor-shrinking effects on these advanced tumors 3, and we have hypothesized that these effects are directly mediated by ERs via nongenomic signaling mechanisms.

20

Figure 1.2. Canonical AR signaling pathway. Androgens enter prostate cells through the membrane, then bind to inactive ARs. Binding induces a conformational change in AR. ARs will then be phosphorylated and dimerize, translocating to the cell nucleus. Transcription begins after binding to AREs and co-activators (ARA70 and general transcription activators (GTA)) are recruited. Biological responses include increased PSA levels, cell growth, and cell survival (Figure from Feldman & Feldman 2001).

21

Prostate cancer treatment regimens

If a prostate tumor is detected early enough, localized prostatectomy and radiation can be effective treatments. One of the first forms of therapy developed for prostate tumors other than tumor resection was orchiectomy, to deprive the tumor of the androgens that early-stage tumors often require to proliferate. Recent pharmaceutical advances have presented chemical castration as a viable option over surgical prostatectomy and castration 1.

Treatment with estrogens, specifically DES, was one of the first regimens discovered to chemically castrate patients 1. High-doses of DES used to be a typical treatment for late-stage cancers, but this treatment is no longer available in the United States. Ethinyl estradiol (EE) given at a dose of 0.05mg/day decreased plasma T serum concentrations below castration levels 76. However, when administered alone, estrogens such as DES (lowest clinical dosage 1mg/day) and EE can induce thromboembolic events. Most DES and EE treatments now are given in conjunction with anti- thromboembolic medication as a result 77, 78. Serum levels of DES resulting from treatment administration can reach as high as 10µM 79 where they work as effective inhibitors of luteinizing hormone secretion from the pituitary. This causes a subsequent decline in synthesis and secretion of testosterone from the testes by up to 95% 80, 81. Clinical use of DES in the United States has declined however as a result of cardiovascular threats and the efficacy of other treatments, such as LHRH agonists, without the need for additional medication 82, 83. Research in the 1970-80’s highlighted the use of synthetic LHRH agonists as a means to decrease serum testosterone levels. In particular LHRH agonists down- regulated the LHRH receptors present in the pituitary gland, as well as the subsequent levels of androgens in the body 84. Some common LHRH analogs currently in use for this

22

treatment include leuprolide (Lupron®), goserelin (Zoladex®), triptorelin (Trelstar®), and histrelin (Vantas®) 85. Both surgical and chemical castration result in 70-80% reductions in tumor growth for patients with androgen-responsive cancers (early-stage tumors) 42. LHRH receptor antagonists such as degarelix (Firmagon®) have also been recently discovered that work equally as well, with the added benefit of no testosterone flare (an initial increase in testosterone levels during the first 1-3 weeks of treatment with LHRH agonist) when first used 86, 87.

Potent anti-androgen therapies were developed between 1960-70 88, and have been shown to reduce tumor size similar to treatments with DES and LHRH agonists 89. Anti-androgenic compounds are now rarely given as the sole means of treatment, and are most often used in combination with chemical/surgical castration. Flutamide (Eulexin ®), bicalutamide (Casodex ®), and nilutamide (Nilandron ®) are currently the most commonly used anti-androgens 90. Anti-androgens can be classified as steroidal or nonsteroidal. Steroidal compounds can also act on progesterone receptors (as many steroids can bind an alternative receptor at high concentrations), and often cause impotence and decreased libido. Nonsteroidal compounds work only on ARs, while maintaining libido and sexual potency 88. In recent decades, radiation therapy has become another of frontline therapy 1. Radiation therapy is most effective when tumor cells are still localized to the prostate gland, and is often administered in conjunction with other therapies, such as localized prostatectomy and anti-androgens 91. However, X-ray radiation therapy can have significant negative side effects such as rectal toxicitiy and bleeding, erectile dysfunction, disruption of proper gastrointestinal function, and urinary incontinence 92. Proton beam therapy has also garnered interest due to the ability to localize high radiation doses on tumorous cells, but has not received full use yet due to concerns over cost, toxicity, and long-term effectiveness 93.

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Late-stage, usually metastatic and androgen-independent cancers are more difficult to treat. Chemotherapy is one of the treatments of last resort at this stage. Docetaxel (Taxotere ®), Paclitaxel (Taxol ®), and Estramustine (Emcyt ®) are some common chemotherapeutics; use of these compounds has shown up to 75% declines in serum PSA 94. Radiation however is not feasible once metastasis has occurred, but use of chemotherapeutics is not specific to the tumor site. Side-effects of chemotherapeutics range in severity, from hair-loss, rashes, gynecomastia, soreness of the mouth, to decreased white blood cell count. Estramustine use can elicit severe cardiovascular side- effects, such as heart attacks, edema, and blood clots 95. Additionally, prostate cancer cells can eventually develop a resistance to chemotherapeutics like docetaxel 96.

Development of androgen-independent tumors

The number of androgen-independent (compared to androgen–dependent) cells in early tumors has been estimated to 1 in 1,000,000 97, so even the most effective androgen ablation treatments will leave surviving cells. Androgen-independence often occurs when tumors return after initial treatment, and these returning tumors are typically more aggressive. Surviving cells from the initial treatment often develop mechanisms to evade detection by cellular processes that result in their death. They include: dysregulation of steroid receptors that sustain growth, like AR; loss of regulatory proteins like glutathione S-transferase π; and alterations in proliferative cell cycle controls. All are typical progressive hallmarks of prostate cancer “evolution” to androgen-independent tumors in particular 98-101. Initial therapies targeting early-stage cancer cells will no longer provide therapeutic benefit once these cellular changes have occurred. There are several mechanisms behind the development of advanced androgen- independent cancers 41. The first mechanism is not actual androgen-independence, but rather the development of hypersensitivity to lower concentrations of serum androgens

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102. Just following castration the majority (~70%) of the tumor cells will die due to lower androgen levels, but a small subset of androgen-dependent prostate cancer cells continue to grow 41 because they utilize low concentrations of androgens more effectively. It is important to note that this subset of cells is still androgen-dependent, as they die with complete removal of androgens. Lingering serum androgens in minute, detectable levels are still produced by the adrenal glands, even after chemical/surgical orchiectomy 42. Additionally, these tumor cells may also show an increase in AR levels. It is reported that close to 30% of prostate tumors have increased duplications of AR gene exons, such as exon 3 in 22Rv1 cells, as well as subsequent increases in AR production and expression 28, 103-105. These higher levels of AR may increase the sensitivity of the tumor to minute amounts of androgens. Interestingly, this phenomenon also occurs in breast cancers, suggesting that this is a common exploit for endocrine tumors 106. Another mechanism of androgen-independent growth is the ability of ARs to bind other ligands, which then mimic regular androgen/AR activity. Mutations in the AR ligand binding domain allow for higher levels of promiscuous binding of other hormones, such as estrogens, or small molecules resembling androgens 107, 108. The commonly used prostate cancer cell line LnCAP harbors an alanine to threonine substitution at residue 877, and allows estrogens, and other steroid hormones to bind readily 107. The MDA prostate cancer cell line also has a promiscuous mutated AR, which can bind glucocorticoids, but deceases the ability of androgens to bind 109, 110. Aberrant binding of non-androgenic ligands then leads to inappropriate AR activity and signaling.

Other mechanisms supporting androgen-independent growth include the ability of proliferative cellular pathways to bypass hormonal control and activate without any androgenenic ligand or AR present. These pathways are stimulated by or utilize growth factors and kinases, which then stimulate AR activity 111, 112. Insulin-like growth factor (IGF) stimulates cell proliferation by activating the phosphoinositide 3-kinase (PI3K) signaling pathway and inhibiting of FOXO1’s suppressive activity on AR-DNA binding,

25

increasing AR transactivation 113. Epidermal growth factor (EGF) increases signal transducers and activators of transcription 3-AR (STAT3-AR) complex formation and AR activity, in conjunction with IL6 114. Vascular endothelial growth factor C (VEGF-C) is up-regulated in tissues with low androgen concentrations, such as with androgen- independent prostate cancer cells, and increases AR activity by stimulating the expression of BAG-1L, an AR co-activator 115. Improper AR activity in the absence of androgens may be mediated by the NTD of the receptor - amino acids 294-556 were found to be required for androgen independent localization to the nucleus 116. Nongenomic signaling through ARs has been relatively unstudied to date, but has been shown to mediate growth-stimulatory effects. These signals can emanate from caveolin-1 through Akt and ERK, preventing apoptosis 117. Nongenomic signaling through ARs can also rapidly activate L-type calcium channels through an inhibitory G- protein, stimulating PKC activity and increasing gene transcription 118. In the nervous system, androgens can rapidly activate the ERK signaling pathway, serving as a neuroprotective against against β-amyloid 119 and serum deprivation 120. Another way to increase the number of cells in a tumor is to block the process of apoptosis. The anti-apoptotic bcl-2 gene in androgen-responsive cells is suppressed by androgens, but is overexpressed in androgen-independent tumors, allowing for uncontrolled growth 121, 122. Endothelin-1, which is produced from cells of the prostate epithelium, downregulates pro-apoptotic proteins, through phosphorylation of Akt and PI3K 123. On the other hand, some androgen-independent prostate cancer cells have also developed a resistance to TRAIL-induced apoptosis through constitutively active Akt phosphorylation 124. This immunity to apoptosis then allows the surviving androgen- independent cells to grow unchecked. Evading destruction by the body’s immune system is another key hallmark of cancer 125. Cells that are androgen-dependent will die when androgen-ablation therapy is given, while the subset of cells that are androgen-independent will survive or “lurk”,

26

evading immune cells that would otherwise kill them 126. The cells employ mechanisms such as recruiting immune cells that inhibit effector T-cells 127, 128 and preventing maturation of dendritic cells via increased VEGF secretion 129. These surviving “lurker” cells then slowly repopulate the tumor 130.

Estrogens and estrogen receptor physiology

As described above, estrogens such as DES have shown some beneficial indirect effects by controlling androgen production that supports prostate tumor growth.

However, the potential direct effects of estrogens on prostate cancer cells are not well understood 131. In particular, the nongenomic effects of estrogens, as mediated by mERs, is entirely unknown in this type of tumor cell. Estrogens are traditionally viewed as a female hormone, and are responsible for the regulation of the female reproductive cycle and proper development of secondary sexual characteristics. Estrogens also have many regulatory functions in males though, such as proper storage of mature sperm cells 132, differentiation of the reproductive organs 133-135, cardiovascular health 136, and potentially mental health 137. Estrogen deficiency in males can lead to impaired reproductive fecundity, decreased libido, uncontrolled bone growth, and brittle bones 138.

27

ERα or ERβ

GPR30

Figure 1.3. Summary of roles for membrane and intracellular ERs. mERs initiate rapid, nongenomic signaling cascades after estrogenic stimulation, leading to such signaling changes as second messengers (ion flux, lipid signals, cyclic nucleotides) cell cycle protein posttranslational changes, and kinase and other enzyme activations. Examples of endpoints are shown such as release of secretory vesicles, efflux, or, trafficking of transporters. Stimulation of nuclear ERs leads to long-term transcriptional effects. (Figure from Watson 1999).

There are currently three known ERs - α, β, and GPR30 139, 140. ERα and β are members of the same (nuclear receptor) gene superfamily and the product of two separate genes, ESR1 and ESR2 139. They each have their own splice variants (Figure 1.4) 141-143 that can still be active, such as in the activation of MAPKs by the truncated ERα36 144, or regulation of proliferative transcription factors by ERβ2 145. These truncated forms lack the transcriptional transactivation capabilities of full-length ERs, but can still mediate nongenomic responses by coordinating the activities of other signaling molecules.

28

Truncated forms of ERα have typically shown activity at or near the plasma membrane 146 147 in multiple cell lines. ERα46, but not ERα36, has been shown to bind the same ligands (including several phytoestrogens and antagonists used in our studies) as full-length ER, though at much lower binding affinities 148. ERα46 is involved in rapid,

149 E2-induced eNOS activation in endothelial cells . ERα36 in endometrial cells is involved in rapid, membrane-initiated ERK and PI3K/Akt pathway signaling in response to testosterone stimulation 150. Truncated forms of ERα have not been reported in prostate cells yet, but many antibodies used in studies (such as the MC-20 antibody) do not distinguish between full-length ERα and its truncated forms 151. ERβ1 (wild-type) and ERβ2 have currently been identified and studied for their roles in proliferation and metastasis in PC-3 cells and other cell lines, but not in LAPC-4 cells 145, 152, 153. ERβ antibody clone 9.88 has not currently been shown to distinguish between wild-type or ERβ2, but other researchers have developed antibodies specific to truncated forms 154, 155. ERβ2 has been reported to not bind ligands, but may instead serve to repress transcriptional activity of full-length ERβ 156, 157. GPR30 is an entirely different type of protein -- a seven-transmembrane receptor G-protein coupled receptor so far known to be involved only in nongenomic actions of estrogens 158.

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AF-1 & AF-2 (Activation Function 1 & 2); DBD – DNA-Binding Domain; LBD – Ligand- Binding Domain

Figure 1.4. ERα and ERβ splice variants. Domains of the ERs and their splice variants. From left to right: the N-terminal A/B domain that encodes AF-1; DNA binding domain; the hinge domain; and the C-terminal E/F domain that encodes the ligand binding domain and AF-2. Many of the splice variants are still functional ERs, such as ERα36, ERβ2, and ERβ5. Also listed are sites of post-translational modification. Red triangles indicate the epitopes that are bound by the ER antibodies used in this study (Figure modified from Thomas & Gustafsson 2011).

Various post-translational modifications can control ER function 159 (Figure 1.4).

Acetylation is mediated by E2 and cAMP responsive element protein, which can help to stimulate or inhibit the transcriptional activity of ERα 159. Glycosylation influences the cellular fate and destination for ERα and ERβ and many other proteins 160-162. Nitrosylation of ERα impairs or inhibits the receptor from binding to EREs 163, 164. Myristoylation and palmitoylation can also direct ERs or their variants to the cell

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membrane 165-167. Membrane ERs are localized to the membrane via palmitoylation, tethering them close to the inner surface, mainly in association with caveolae rafts 168. Disruption of palmitoylation or the interaction with caveolae has negatively affected cell proliferation 159. It is currently still unknown if mERs are able to span the entire plasma membrane, due to difficulties studying the subcellular location 169, but this is unlikely due to the exposure of many epitopes all along the protein on the outside of the cells 170-173. These post-translational modifications may also offer a rapid form of ER regulation without the need to increase or decrease receptor levels.

ERs are expressed in the prostate gland, and contribute to proper function and development 174. ERs are found both on the membrane and intracellularly in several cell types, but this has not yet been fully characterized in prostate cancer cells. The canonical nuclear pathway of action involves estrogenic ligands passing through the lipid bilayer of the cell and binding to intracellular receptors 139. The receptors are released from heat shock protein complexes 175, then dimerize and bind to estrogen response elements (ERE) to initiate transcription while partnering with coregulator proteins 139, 176.

However, membrane initiated responses do not invoke genomic actions, at least initially 177. They rapidly activate signaling pathways that cause second messenger enzymatic synthesis (cyclic nucleotides, phospholipids) or movement (ion flux 178-180, resulting in functional changes such as peptide hormone release 170, 181, activity or location of transporters or channels 182, 183, apoptosis 178, 184, 185, or cell proliferation 186, 187.

Roles of membrane estrogen receptors in nongenomic responses

ERα and ERβ are localized to the plasma membrane through palmitoylation, while the location of GPR30 has been debated between the endoplasmic reticulum 188 and the plasma membrane 140. mERα and mERβ do not appear to be different from their

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nuclear counterparts 189, 190, other than their localization to the plasma membrane through post-translational modifications 165. Palmitoylation at Cys-447 is necessary for ERα membrane localization and to induce nongenomic signals via the MAPKs – alteration of this site can lead to cellular and tissue disfunction (loss of signaling pathways, infertility, disrupted vasculature) 191, 192. Nongenomic signals as elicited by estrogens can have a variety of functions. Our laboratory has shown a host of these signaling events and outcomes using several assays and cell lines. Cell survival and death are usually tied to transcriptional events that take hours to days to manifest. However, estrogenic compounds working through mERs initiate the signaling pathways leading to these outcomes in a matter of seconds to minutes. These signals may also accumulate and have sustained effects, or modify (often phosphorylate) downstream molecules involved in transcription 193. In benign prostatic hyperplasia, E2 in conjunction with serum hormone binding globulin rapidly increases (<15 min) cyclic adenosine monophosphate 194, a signaling molecule that can lead to cell proliferation and transcriptional regulation of genes 195. While nuclear and membrane ERs are quite similar, they can initiate different cellular outcomes in response to the same ligand because they partner with different other proteins. The genomic and nongenomic signaling pathways are not always independent of each other. Transcriptional events initiated by nuclear receptors can by influenced by signaling pathways that are activated by membrane receptors. For instance, the Watson laboratory previously reported that the transcription factors Elk and ATF-2 are rapidly

193 phosphorylated following stimulation by E2 or xenoestrogens (XEs) . ERs also regulate transcription without binding to EREs in DNA. Instead, nuclear ERs can form complexes with proteins such as AP-1 196, Sp1 transcription factor, or NF-κβ transcription factor,

197 after stimulation with E2 or another estrogenic ligand . These complexes then regulate transcription of IGF-I 198, cyclin D1 199, c-fos 200, and IL-6 201. Thus, multiple immediate and delayed cellular outcomes can result from a single ligand binding event.

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Estrogen receptor control of cell proliferation or death

Estrogens and ERs regulate cell fates in a variety of cell types. E2 can phosphorylate and activate mitogen-activated protein kinases (MAPKs) in tumor cells originating from the pituitary, neurons, heart, and breast 181, 202-204. MAPKs include ERK, c-Jun N-terminal kinase (JNK), and p38. ERK activities are often associated with cell proliferation 205, 206, JNK activities are often associated with cell death and apoptosis. Phosphorylated JNK promotes apoptosis by activating the intrinsic caspase cleavage pathway of cell death and the release of cytochrome c from mitochondria. JNK can also further promote cell death by deactivating suppressors of TNF-α driven apoptosis 207. JNK can also be activated by reactive oxygen species (ROS) after inhibition of MAP kinase phosphatase activity, leading to eventual apoptosis 208. Phosphorylated p38 participates in control of cell differentiation 209, proliferation 205, and death 210 in response to cellular stress 211, inflammation 212, and UV radiation 213. Estrogens can also use other signaling cascades such as activation of the PI3K/Akt signaling pathway 214, or can even inhibit cell death in some cases by regulating the anti-apoptotic Bcl-2 protein 215. Our laboratory and others have also shown that cyclic nucleotides can be increased in response to estrogens, through adenylyl cyclase and protein kinase A, in a rapid, nongenomic manner 186, 195 stimulating gene transcription and regulating cell proliferation. GPR30 can inhibit cell proliferation, such as with breast cancer cells 216, or stimulate proliferation, as with thyroid cancer cells 217. Therefore, there are many different signaling interactions initiated at the membrane in which ERs of different types can be involved.

Estrogens and ERs have also been shown to be important regulators of cell number in a host of organs. In breast tissue, estrogens via ERs stimulate cell proliferation at various life stages (puberty, pregnancy, menopause) by increasing

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production of several growth factors and altering expression of a host of genes (amphiregulin, VEGF, loss of Slit2, frizzled-related genes FRP1/FRZB) 218, 219. Bone mass (including cells and their products) is regulated by estrogens, which drive the proliferation of osteoblasts and or death osteoclasts through anti- and pro-apoptotic gene products 220. The ER subtypes in ovarian cells may be a determinant of sensitivity to agents that cause ovarian cancer to develop, as well respond to treatments 221, 222. The prostate requires estrogens for proper development, including cell proliferation, while ER status may also influence further tumor development and metastatic potential 223, 224. Estrogens in the colon are necessary for cell growth and division, and may play a protective role against tumor development, specifically through ERβ 225, 226. Normal lung function is maintained by estrogens and their control of cell number, such as formation of alveoli during development and repair in adulthood 227.

Estrogen receptors in the prostate and prostatic disease

The effect of estrogens on prostate cancer are thought to be mediated by ERβ, the dominant form of the receptor in the tissue, but this conclusion must await further examination of all receptors that are present. ERβ is regularly expressed at high levels in the healthy prostate 228, and is mainly found in the epithelial cells vs. the stroma, compared to ERα 142. During the progression of prostate cancer, the levels of ERα increase throughout the gland, while ERβ levels decline (yet remain functional), even in advanced cases of the disease 19, 229, 230. The expression of splice variants ERβ2 and ERβ5

223, 231 increases metastatic potential . Additionally, exposing neonatal rats to E2 and DES severely stunted the regular growth of the prostate gland. Interestingly, ERα was shown to be responsible for this inhibitory effect 232, which is different given ERα is often associated with proliferative effects in other cell types 233. ERβ is not initially expressed in the development of the prostate, and ERα is responsible for regulating cell growth.

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Androgen signaling decreases ERα levels, removing the regulation of ERβ expression, allowing ERβ to rise, and prostate maturation and differentiation to occur 234. Selective estrogen receptor modulators for ERα did not work as a beneficial treatment for advanced prostate cancer patients, 235, 236, though they did inhibit proliferation of PC-3 prostate cancer cells in vitro 237, perhaps suggesting an additional compensatory mechanism present in the actual tumor environment. GPR30 can inhibit prostate cancer cell growth after treatment with the agonist G-1, through sustained activation of ERK, up-regulation

238 of the cyclin-dependent kinase p21, and cell cycle arrest in G2 . It is apparent though that the overall role of the separate ER subtypes in the prostate may be reversed, and also depend on male life-stage.

Xenoestrogens and effects on living systems

ERs often bind other estrogenic compounds besides physiological estrogens.

Phytoestrogens, such as coumestrol, genistein, and resveratrol, are estrogens found in plants. Pharmaceutical estrogens include the aforementioned DES, used clinically, while EE is used in contraceptives. Synthetic estrogens are man-made compounds found in many every-day products and are pervasive in the environment. Examples are bisphenol A (BPA) in plastic products, nonylphenol in paints and oil dispersants, and dichlorodiphenyldichloroethylene, a breakdown product of the pesticide dichlorodiphenyltrichloroethane. These estrogen mimetics/XEs can function similarly to physiological estrogens and activate the same cellular pathways (often inappropriately) 239, 240, but they are not identical in their actions. Phytoestrogens have been touted as potential preventatives or therapeutics for diseases 241, while environmental estrogens are pervasive and are associate with negative effects on the health of wildlife, humans, and the ecosystem 242-244. A further review of XEs is in Chapter 3, highlighting the overall ability of XE, at dietary- or environmentally-relevant concentrations, to stimulate cell

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growth by utilizing signaling pathways differently from E2 and DES, as well as the varied ER requirements to elicit such cellular responses.

E2 BPA Coumestrol

Genistein DES

Resveratrol

Figure 1.5. Comparison of the structures of E2, DES, BPA, and three phytoestrogens.

Our laboratory has shown that E2 induces rapid, non-genomic signaling as well as cell death in MCF-7 breast cancer cells expressing high levels of mERα 186, 204. Our laboratory has also reported that XEs, agonistic estrogen-like compounds (synthetic chemicals/phytoestrogens) induce cell proliferation or apoptosis, dopamine transporter efflux, and prolactin release in MCF-7 breast cancer, PC-12 pheochromocytoma, and

182, 193, 245 GH3 pituitary cells, respectively . The underlying, rapid and direct mechanisms that contribute to cell proliferation or cell death, in response to estrogens/XEs, have not been examined in prostate cancer cells. Therefore, we hypothesized that estrogens directly inhibit prostate cancer cells, rapidly initiating signaling cascades (MAPKs/caspases) that lead to inhibition of cell proliferation or induction of cell death. In addition, we hypothesized that XEs also utilized these same pathways,

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although not necessarily with the same signaling magnitude or receptor signature of endogenous estrogens, and that mERs were required in influencing their anti- proliferative vs. proliferative effects on prostate cancer cells.

Aims of studies in this dissertation

The work and data for this dissertation can be divided into two parts. The first part focuses on identifying key cellular pathways that are involved in regulating cell proliferation or death using E2 or DES, a physiological estrogen vs. a pharmaceutical mimic. While there are innumerable mechanisms to look at, this work focuses on the rapid activation or phosphorylation of MAPKs and cell cycle proteins and their consequences. We also looked at the induction of ROS, apoptosis via caspase 3 activation, and necroptosis (a relatively novel form of programmed cell death). The second part of this dissertation focuses on the effects of XEs on prostate cancer cells, with a particular interest in the specific mERs that are present and necessary to elicit or prevent cell proliferation or death.

Aim 1. To identify and characterize rapid and direct cell mechanisms involved in E2- or DES-initiated cell proliferation or death of androgen-dependent

LAPC-4 or androgen-independent PC-3 prostate cancer cells. We will examine several rapid (nongenomic) signaling pathways that have an influence over cell proliferation or death of prostate cancer cells, as initiated by E2 or

DES. We anticipate that E2 or DES at physiologically or clinically relevant concentrations have direct and likely rapid effects on prostate cancer cells that could provide therapeutic benefits besides central inhibition of androgen production. Multiple mechanisms, such as MAPK activation, cell cycle protein phosphorylations, and ROS induction, will be examined to determine if they contribute to the overall balance between

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cell proliferation and death of prostate cancer cells. The ERs important for regulating these pathways will also be elucidated.

Aim 2. To determine if XEs exert anti-proliferative/proliferative effects, using the same signaling pathways and ERs identified with E2 and DES, in LAPC-4 and PC-3 prostate cancer cells. We will identify and compare the membrane and intracellular levels of ERs (α, β,

GPR30) that are present in both LAPC-4 and PC-3 cells. Then we will study the XEs BPA, coumestrol, genistein and resveratrol for their effects on cell viability/proliferation and cell death, as well as the key cellular pathways identified from Aim 1. The XEs will be evaluated at environmentally or dietary-relevant concentrations (10-14 M to 10-6 M).

The results of this research should contribute to the current body of knowledge by showing a direct mechanism through which estrogens and XEs initiate proliferative/anti- proliferative effects via mERs in prostate cancer cells. Additionally, the results of this project should aid other researchers and clinicians in developing much more specific therapeutic compounds that target the relevant ERs or cell signaling pathways. At the same time, expounding the proliferative/anti-proliferative effects of XEs binding to mERs will elucidate the risks or benefits of chronic exposure to environmental contaminants or dietary phytoestrogens in existing tumors. These results will also potentially lead to changes in regulating environmental concentrations of estrogenic compounds or recommendations about dietary intake of phytoestrogens for cancer patients.

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Chapter 2: Direct estradiol and diethylstilbestrol actions on early- vs.

late-stage prostate cancer cells1

Abstract

Diethylstilbestrol (DES) and other pharmaceutical estrogens have been used at ≥µM concentrations to treat advanced prostate tumors, with successes primarily attributed to indirect hypothalamic-pituitary-testicular axis control mechanisms. However, estrogens also directly affect tumor cells, though the mechanisms involved are not well understood. LAPC-4 (androgen-dependent) and PC-3 (androgen-independent) cell viability was measured after estradiol (E2) or DES treatment across wide concentration ranges. We then examined multiple rapid signaling mechanisms at 0.1 nM

E2 and 1µM DES optima including levels of: activation (phosphorylation) for mitogen- activated protein kinases, cell-cycle proteins, and caspase 3, necroptosis, and reactive oxygen species (ROS). LAPC-4 cells were more responsive than PC-3 cells. Robust and sustained extracellular-regulated kinase activation with E2, but not DES, correlated with

ROS generation and cell death. c-Jun N-terminal kinase was only activated in E2-treated PC-3 cells and was not correlated with caspase 3-mediated apoptosis; necroptosis was not involved. The cell-cycle inhibitor protein p16INK4A was phosphorylated in both cell lines by both E2 and DES, but to differing extents. In both cell types, both estrogens activated p38 kinase, which subsequently phosphorylated cyclin D1, tagging it for degradation, except in DES-treated PC-3 cells. Cyclin D1 status correlated most closely with disrupted cell cycling as a cause of reduced cell numbers, though other mechanisms also contributed. As low as 0.1 nM E2 effectively elicited these mechanisms, and its use could

1 Chapter 2 taken from Koong LY & Watson CS (2014). Direct estradiol and diethylstilbestrol actions on early- vs. late-stage prostate cancer cells. The Prostate. 74(16):1589-1603.

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dramatically improve outcomes for both early- and late-stage prostate cancer patients, while avoiding the side effects of high-dose DES treatment.

Introduction

Prostate tumors are usually androgen-dependent in the beginning, so front-line therapies are initially aimed at reducing the amount of free androgens that sustain these tumors. Traditional therapies to treat androgen-dependent tumors have included radiation and surgical removal of tumors, followed by androgen receptor antagonists, or pharmaceutical castration with luteinizing-hormone-releasing hormone (LHRH) analogs that block androgen production 246. However, these treatments become ineffective against tumors that have previously regressed with treatment, and have then escaped these controls to grow again. These recurrent or advanced prostate tumors often develop androgen-independence, and as an alternative, synthetic estrogens like diethylstilbestrol

(DES) have been used as treatments 77. Estrogen treatment is thought to indirectly decrease androgen production by negative feedback control on the hypothalamic- pituitary-testicular axis 42, though with the proven androgen-independence of these tumors, this may not provide much additional therapeutic benefit. Current clinical practice employs high doses of DES (≥µM), which can cause many unwanted side-effects in patients 77 including erectile dysfunction, decrease in sex drive, weight gain, gynecomastia, and cardiovascular problems.

Estrogens (or their mimics) could control cellular proliferation or death via a variety of mechanisms which we examined in these studies. They bind to both intracellular and membrane receptors that transduce extracellular signals to downstream effectors 186, 247-249. Downstream mitogen-activated protein kinases (MAPKs) are responsive to a large number of external stimuli and are nodes of signaling integration for estrogenic signals, as well as those from other classes of ligands, via their receptors 250,

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251. There are three primary MAPK subclasses: extracellular signal-regulated kinases (ERKs); c-Jun N-terminal kinases (JNKs); and p38. ERKs 1 and 2 are well known for being involved in controlling cell proliferation 251 by integrating upstream signals to regulate cell cycle proteins by post-translational modifications 252. ERKs also propagate the signaling cascade to other important cellular response molecules such as c-Myc 253, Elk-1 254, eIF4E 255 and cyclin D1 256. On the other hand JNK is better known for triggering cellular apoptotic responses, for instance after UV radiation or other types of

DNA damage 208, 257, 258. The MAPK called p38 has some functions similar to JNK, but is more typically reported to be involved in cytokine, cellular stress, and escalating inflammatory responses. The p38 MAPK can also control cell number by cyclin D1 phosphorylation at Thr-286 which ubiquitin-tags it for rapid degradation through proteasomes 259, cyclin-dependent kinase (CDK) regulation at major checkpoints 256, 260, or inducing apoptosis through cellular stress mechanisms 261. Therefore, there are multiple mechanistic pathways via which MAPKs are important regulators of cell numbers. A novel role of ERK phosphorylation is the generation of high levels of ROS, which in turn further sustain ERK activation through the inactivation of dual-specificity phosphatases. This prolonged activation of ERK will then produce even higher ROS levels, creating a positive feed-forward mechanism for ramping up this destructive response quickly. Generation of ROS can lead to cell death via the initiation of apoptosis, cell senescence mechanisms, or autophagy 262.

As cell cycle proteins drive cell division, they directly cause increased cell numbers; therefore inhibition of these mechanisms can cause decreases in cell numbers as cells die and are not replaced. The cell cycle is tightly regulated, being controlled at each major checkpoint by cyclins and CDKs, as well as inhibitory proteins acting on them. Cyclin D1 controls the G1/S transition with its partners CDK4/CDK6 263. Cyclin D1 protein levels can be changed via p38 phosphorylation (resulting in degradation),

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depending on the stage of the cell cycle. CDK4/CDK6 can be regulated by the cell cycle inhibitor p16INK4A 264, which when phosphorylated binds to CDK4/CDK6, preventing the formation of the cyclin D1-CDK4/6 holoenzyme required for successful cell cycle progression. Therefore, signals that activate or inhibit this cascade have profound effects on cell numbers. Programmed cell killing can also play a major role in controlling cell numbers. Several effector caspases feed into executioner caspases, represented in our studies by caspase 3 265, that dismantle DNA. Necroptosis is an alternative, relatively newly described method of programmed cell death. Cells undergoing this process have morphologies similar to necrotic cells (plasma membrane integrity loss, increases in cell and organelle volume), but do not require the activation of caspases 266. Though necroptotic cells are best known to initiate the death process after Fas ligand (FasL) binding, tumor necrosis factor α (TNFα), or TNF-related apoptosis-inducing ligand (TRAIL) stimulation 267, a growing number of other receptors and stressors are being reported to be involved in this mechanism 268. Receptor-interacting protein kinase 1 (RIPK1) serves as a scaffold for this process 269, and the selective inhibitor necrostatin-1 keeps it in an inactive conformation, thus preventing necroptosis 270. We and others have previously shown that rapidly initiated (nongenomic) steroid- induced signaling events are involved in the modulation of tissue size/cell number changes (involving both cell proliferation and cell killing) in tumor cell types that contain membrane estrogen receptors 249, 271, 272. We now predict that these mechanisms could also be active in prostate cancer cells, where the expected direct response to estrogens would be to mediate rapid cellular signaling leading to cell killing, or the slowing of cell proliferation; direct mechanisms of action of estrogens on prostate tumor cells are still relatively understudied. Better understanding of such a direct effect could result in significant improvements in treatment strategies to suppress tumor growth while reducing harmful side effects due to current high dose DES treatments.

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Materials and Methods

Cell Lines and Reagents: We chose cell lines representing the two main types of human prostate cancers -- androgen-dependent vs. androgen-independent. LAPC-4 androgen-dependent prostate cancer cells 29 were maintained to sub-confluence in phenol red-free Iscove’s Modified Dulbecco’s Medium (IMDM, MediaTech - Manassas, VA) with 10% fetal bovine serum (FBS) (Atlanta Biologicals – Lawrenceville, GA), 4 mM L- glutamine (Sigma-Aldrich – St. Louis, MO), and 10 nM dihydrotestosterone (Sigma-

Aldrich). PC-3 androgen-independent prostate cancer cells 30 were maintained by growth in phenol red-free RPMI 1640 (Sigma-Aldrich) with 10% FBS and 2 mM L-glutamine.

Both cell lines were propagated at 37°C and 5% CO2. E2 and DES (Sigma-Aldrich) were dissolved in ethanol to a stock concentration of 10 mM (final concentration of EtOH 0.001%) before serial dilution into IMDM or RPMI 1640 at concentrations ranging from

10-14 M to 10-6 M for our studies. MTT Cell Viability Assay: Cells were plated at 5,000 cells/well in poly-D-lysine- coated 96-well assay plates (BD Biosciences – Bedford, MA; 96-well assay plates: Corning – Tewksbury, MA), and then allowed to attach overnight. The next day, 100 µl of medium containing 1% four times charcoal-stripped FBS, plus either vehicle, E2, or DES was added. After three days, treatment solutions were removed and 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) was added for 1 hr. Cells were then lysed and the signal read at 590 nm in a Wallac 1420 plate reader (Perkin Elmer – Waltham, MA) . Plate Immunoassays: Phosphorylated proteins were assayed by recognition with antibodies (Abs) specific for these post-translationally modified epitopes: pERK1/2

(Thr202/Tyr204), pJNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), phospho- cyclin D1 (Thr286) (all from Cell Signaling – Danvers, MA), or phospho-p16INK4A

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(Ser152) (Thermo Scientific – Rockford, IL). Changes in cyclin D1 levels were measured by using an Ab to the total cyclin D1 (recognizing both modified and unmodified protein; Cell Signaling Cat. No. 2922). A plate immunoassay developed previously by our laboratory 273, was adapted for use with the BIOMEK FXP workstation (Beckman Coulter – Brea, CA) to automate the majority of the liquid handling, thereby reducing experimental variability and increasing experimental output. Prostate cancer cells were plated at 10,000 cells/well in 96-well assay plates, allowed to attach overnight, and weaned from steroids and other small molecules in the growth media by treatment with 100 µl of media with 1% four times charcoal-stripped

FBS for 48 hr. Cells were then treated with E2 or DES for up to 60 min on the workstation, followed by simultaneous fixation and permeabilization (2% paraformaldehyde, 1% glutaraldehyde, 0.5% Nonidet P-40, 0.15 M Sucrose). Primary Ab to the phosphorylated epitopes was then added and incubated with the cells overnight. The next day, biotinylated anti-mouse/anti-rabbit IgG secondary Ab (Vector Labs –

Burlingame, CA) was added for 1 hr. Next, cells were incubated with avidin-biotinylated conjugated alkaline phosphatase (ABC-AP, Vector Labs) for 1 hr, then for 30 min with para-nitrophenylphosphate substrate (Sigma Aldrich), allowing the yellow color of the para-nitrophenol product to accumulate. Plates were read at 405 nm in a Wallac 1420 plate reader. Readings were then normalized to cell number, estimated by the crystal violet dye (Sigma-Aldrich) assay, as described previously 274. ER Antagonist Assays: To investigate the involvement of different ERs and their potential role in altering ROS formation or cyclin D1 phosphorylation, the following ER antagonists were used at their most selective concentrations: for ERα, 10-7 M 1,3-bis(4- hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP, Tocris Bioscience – Minneapolis, MN); for ERβ, 10-6 M 4-[2- phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl] phenol PHTPP (Tocris);

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and for GPR30, 10-6 M G15 (Tocris). Cells were pre-incubated with antagonists for 30 min prior to estrogen treatments. Caspase 3 Assays: To determine estrogen-induced activations of caspase 3, cells were seeded into black optical 96-well plates (Corning) at a density of 5,000 cells/well and allowed to attach overnight. Estrogen treatments were started the next day in media with 1% four times charcoal-stripped FBS, for times ranging from 2-24 hr (only the 8 hr optimum time point data are shown in Fig. 2.5). After treatment, plates were centrifuged at 300 g for 5 min, and treatment-containing media were suctioned off. Cells were then lysed with 50 µL of lysis buffer (10 mM HEPES; 2 mM EDTA; 0.1% CHAPS; 1 mM DTT; pH 7.4) and stored at -20°C until assay. Assay buffer (50 µL of 50 mM HEPES; 100 mM NaCl; 0.1% CHAPS; 1 mM EDTA; 10% glycerol; 10 mM DTT; pH 7.4) containing a 50 µM final concentration of Ac-DEVD-AFC caspase-3 assay substrate (Enzo Life Sciences – Farmingdale, NY) was added. The cellular enzyme-catalyzed release of 7-Amino-4-trifluoromethylcoumarin was monitored using a FlexStation 3 microplate reader (Molecular Devices – Sunnyvale, CA) at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Staurosporine at 1µM was used as a positive control for inducing caspase activity. Necroptosis Assays: This mechanism was defined by the use of a selective necroptosis-inhibitor in the MTT assay (described above). Cells were treated for three

-10 -6 days with EtOH (0.0001%) vehicle or 10 M E2 or 10 M DES; TNFα (10 ng/ml, Millipore) plus cyclohexamide (10 µg/ml, Sigma-Aldrich) were used together to provide a positive control for necroptosis. Necrostatin-1 (20 µM, Millipore – Billerica, MA) was used to specifically define necroptosis by the inhibition of RIP1 kinase 275. ROS Assays: Cells were plated at 10,000 cells/well in a 96-well plate, allowed to attach overnight, and then treated with 100 µl of media containing 1% four times charcoal-stripped FBS for 48 hr. Cells were loaded with 15µM 2’,7’- Dichlorodihydrofluorescein diacetate (DCDHF) (Enzo Life Sciences) for 1 hr. Then the

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production of ROS was measured in cells after E2 or DES treatment for 15 min. Hydrogen peroxide (1µM, Fisher Scientific – Pittsburg, PA) and ethanol (0.0001%) were used as positive and negative controls, respectively. For both E2 and DES treatments, the concentrations spanned 10-14 to 10-6 M. Dichlorofluorescein production, formed as a result of ROS/DCDHF interaction, was measured at an excitation of 485 nm, and an emission of 538 nm in a SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices). For studies with MEK inhibitor U0126 (Promega – Madison, WI), cells were co-incubated with 10-7M inhibitor during the last 30 min of the DCDHF incubation. Statistics: One-way analysis of variance was conducted for each experiment. A Holm-Sidak post hoc test was used to measure the significance of each treatment vs. the vehicle control. Significance was set at P<0.05.

Results

Figure 2.1. Cell viability after 3 days of E2 or DES treatment. LAPC-4 and PC-3 prostate cancer cells were treated with either estrogen, and cell viability was measured by the MTT assay. In all figures throughout the manuscript white symbols denote LAPC-4 cells and black symbols PC-3 cells; triangles represent E2 treatments, and circles represent DES treatments. *denotes significance from vehicle (V) controls at P<0.05, and shaded gray bars represent the response to vehicle ± SEM.

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Cell Viability: E2 treatment (represented by triangles in this and all subsequent line graphs) for three days effectively decreased the number of viable cells by 20-30% below the vehicle treatment level at all concentrations tested (10-14 to 10-6 M) in LAPC-4 androgen-dependent (early-stage) prostate cancer cells (represented by white symbols and bars in all figures) (Fig. 2.1A) and at 10-10 to 10-8 M concentrations in PC-3 androgen-independent (late-stage) prostate cancer cells (represented by black symbols and bars in all figures). DES treatment (represented by circles in all line graphs) decreased cell viability by as much as 20% in LAPC-4 cells only at 10-14 to 10-12 M and 10-6 M, while the cell viability of PC-3 cells was not significantly decreased by DES at any concentration (Fig. 2.1B). For comparison (positive control), when these cells were grown in complete serum-containing media, LAPC-4 cell numbers increased by 27%, while PC-3 increased by 19% (not shown). Phospho-ERK Driven ROS Accumulation and Phospho-JNK Driven Caspase

Activation: In both LAPC-4 and PC-3 cells, most concentrations of E2 and DES elicited similar rapid ERK and JNK activations and deactivations in a typical oscillating time pattern 181, 204, 276 (Figs. 2.2 & 2.4). However, there were some instances where values for

ERK activations at some concentrations did differ (for E2 at 1 and 10 min, and for DES at 1 and 15min); we chose the non-variable 5 min time point for subsequent measurements at single time points. To better visualize the composite temporal pattern, the insets in these figures show the averages of these changes for all concentrations. Because of these largely similar responses (most not significantly different from one another due to concentration), we chose conditions for all further assays to mimic physiologically (10-10

-6 M E2)- and clinically (10 M DES)-relevant concentrations that were also active in reducing cell viability (see Fig. 2.1). The response for those chosen concentrations is shown in bold lines for each in Figs. 2.2 and 2.4. At these selected concentrations E2- treated LAPC-4 cells showed a rapid and significant increase in phospho-ERK levels (16%) after 5 min (Fig. 2.2A), and a maximal response (26%) after 15 min of treatment.

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After falling to control levels, activation was seen again at 60 min, as we have often observed in other cell lines 181, 204, 273. A similar but less robust response was seen in PC-3 cells [ERK activated by 8% after 10 min of E2 treatment, with another rise to 18% at 60 min (Fig. 2.2B)]. In both cell lines, DES at most concentrations moderately, though significantly, decreased phospho-ERK levels rapidly (Fig. 2.2C & D). In LAPC-4 cells, the ≤20% decrease in phospho-ERK levels was mostly maintained throughout the time course. The activations of ERK in our studies were not consistent with the traditional role for ERK in causing cell proliferation, as our cells numbers instead declined.

Figure 2.2 Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after E2 or DES treatments.

Cells were treated with E2 or DES at different concentrations (different color of symbols and lines). pERK was measured by plate immunoassay for up to 60 min. Insets show the average of all [E2] (A & B) or all [DES] (C & D) treatments. * denotes significance from vehicle response (shown as 0 time) at P<0.05. The shaded gray horizontal bars represent the response to vehicle ± SEM.

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Therefore, we next investigated the role of sustained ERK in the generation of ROS. The production of ROS and associated sustained activation of ERK can lead to cell death, via a positive feed forward mechanism 262. To test this possibility we treated

LAPC-4 and PC-3 cells with E2, which increased ROS across all concentrations after 15 min (the optimum in the time course, not shown) in both cell lines (Figs. 2.3A & C). Addition of MEK inhibitor U0126 inhibited ROS increases by ~50% in both cells lines, indicating ERK1/2 is involved in this E2-mediated pathway, but not exclusively responsible for ROS generation. However, DES did not significantly alter the levels of ROS at any concentration in either cell line (2.3B & D), consistent with the more robust cell-killing effect by E2.

Figure 2.3. ROS measured in LAPC-4 vs. PC-3 cells treated with 0.1 nM E2 or 1 µM DES. ROS levels were measured after 15 min of each estrogen treatment (A-D). Black stars represent response to 1 µM H2O2, a positive control for ROS generation. White symbols denote LAPC-4 cells, black symbols for PC-3 cells; triangles represent E2, and circles DES treatments. * denotes significance from vehicle control at P<0.05. The shaded gray horizontal bars represent the response to vehicle ± SEM.

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E2 treatment evoked different JNK responses from androgen-dependent compared to androgen-independent cell lines (Fig. 2.4). In LAPC-4 cells, JNK was slightly deactivated after 1 min, and was intermittently deactivated thereafter (Fig. 2.4A).

However, in PC-3 cells, JNK was activated (20%) at 1-10 min of E2 treatment, (Fig. 2.4B) followed by a return to control levels and a 10% decrease at 30 min, rising again to baseline at 1 hr. In contrast, DES decreased JNK activation in both cell lines by 5-10% as early as 5 min, an effect that mostly persisted through the remainder of the time course

(Fig. 2.4C & D). This is mostly inconsistent with the typical role of JNK in decreasing cell viability compared to the cell line-specific viability responses we saw (Fig. 2.1). Therefore, we next examined caspase activation to determine if apoptosis contributed to any of the cell number declines observed in our viability assays (using staurosporine as a positive control) 277. A time-course study at 2-24 hrs showed that the highest levels of caspase 3 activity were seen after eight hrs in both cell lines (entire time course not shown); this time was therefore chosen for the comparative studies. Caspase 3 was significantly activated in LAPC-4 cells by both E2 and DES, but not in PC-3 cells (Fig. 2.5). Though not entirely consistent with the cell-killing effects we saw in our viability assays, nor with the traditional role of JNK in activating caspases, these data could contribute some mechanistic explanations for cell-killing to the balance of multiple mechanisms affecting cell number.

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Figure 2.4. Phospho-JNK (pJNK) levels in LAPC-4 and PC-3 after E2 or DES treatments.

Cells were treated with different concentrations of E2 or DES (each line) and then pJNK was measured by plate immunoassay for up to 60 min. Insets show the average response values of all [E2] (A & B) or all [DES] (C & D) treatments. * denotes significance from the vehicle response (time 0) at P<0.05. The shaded gray horizontal bars represent the response to vehicle ± SEM.

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Figure 2.5. Caspase 3 activity levels after E2 or DES treatments.

LAPC-4 and PC-3 cells were treated with 0.1 nM E2 or 1 µM DES and caspase 3 activity measured after 8 hr (which is the response optimum; time course not shown). White bars denote LAPC-4 cells, and black bars PC-3 cells. * denotes significance from vehicle (V) at P<0.05. The shaded gray horizontal bars represent the response to vehicle ± SEM. Staurosporine (Stauro) at 1µM was the positive control for caspase 3 activation.

Necroptosis: We next examined the possibility that this alternative mechanism of cell death contributed to these reductions in cell viability caused by estrogens. This form of programmed cell death was not observed in either cell line after either estrogen treatment (Fig. 2.6). The combination of TNFα and cyclohexamide served as a positive control for necroptosis. Addition of the necroptosis inhibitor necrostatin-1 blocked the cell-killing effects of the positive control, but did not alter the cell number decreases caused by the estrogens. Therefore, necroptosis did not appear to be involved in the killing of these cells by estrogens.

Figure 2.6. Necroptosis after E2 or DES treatments. LAPC-4 and PC-3 cells were treated with 0.1 nM E2 or 1 µM DES and cell viability was measured via MTT assays after 3 days. White bars denote LAPC-4 cells; black bars are PC-3 cells. * denotes significance from vehicle (V) at P<0.05. The shaded gray horizontal bars represent the

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response to vehicle ± SEM. TNFα plus cyclohexamide (TNF/Cyclo) were positive controls for inducing necroptosis in these cells which is defined by necrostatin (NecS) reversal of the response. The cell viability in complete growth medium is shown for comparison at this 3 day time point (Complete).

Phospho-p16INK4A: Next we turned to modulation of the cell cycle through phosphorylation of the CDK inhibitor, p16INK4A. We hypothesized that activation of p16INK4A would interfere with CDK‘s partnering with cyclin D1, and thereby hinder progression through the cell cycle, leading to reduced cell numbers. In LAPC-4 cells, the

INK4A levels of phospho-p16 rapidly increased ~45% in1-5 min after both E2 and DES treatment (Fig. 2.7), and then fell to 20-25% increase for the duration of the time course

(Figs.2.7A & B). In PC-3 cells, E2 initially activated p16 less robustly, but increasing to about 37% by 15 min and sustaining this level throughout the time course (Fig. 2.7C). However, DES activation of p16 in PC-3 cells though significant, was severely blunted (Fig. 2.7D). Therefore, these data could help explain in part the data of Fig. 1 where DES-treated PC-3 cell numbers did not decline.

Phospho-p38: Cyclin D1 can be directly phosphorylated by p38 at a site that directs its degradation 278, so we next examined whether p38 was activated by estrogens in our model systems. In both LAPC-4 cells and PC-3 cells, both E2 and DES phospho- activated p38 rapidly (within 1 min) with similar patterns (Fig. 2.8). The most robust response was In LAPC-4 cells, where DES activated p38 by almost 100% after 5 min of treatment (Fig. 2.8B). All other increases were in the ~50% ranges (Figs. 2.8A, C, and D). Interestingly, these activations were sustained for at least 60 min under all conditions, and could therefore be available to direct phospho-activation of cyclin D1.

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Figure 2.7. Phospho- p16 (p-p16) levels after E2 or DES treatments. LAPC-4 and PC-3 cells were treated with 0.1 nM E2 or 1 µM DES and p-p16 was measured for up to 60 min by plate immunoassays. White symbols denote LAPC-4 cells, and black symbols PC-3 cells. Triangles represent E2, and circles DES. * denotes significance from vehicle (at time 0) at P<0.05. The shaded gray horizontal bars represent the response to vehicle ± SEM.

Figure 2.8. Activated p38 (p-p38) levels after treatment with 0.1 nM E2 and 1 µM DES. LAPC-4 and PC-3 cells were treated with E2 or DES and p- p38 was measured for up to 60 min via plate immunoassays. White symbols denote LAPC-4 cells, black PC-3 cells. Triangles represent E2, and circles DES. * denotes significance from vehicle (time 0) at P<0.05. The shaded gray horizontal bars represent the response to vehicle ± SEM.

Phospho-cyclin D1 and Total Cyclin D1: As p38-mediated phosphorylation of cyclin D1 leads to its degradation, this could in turn slow or halt progress through the cell cycle 259,

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278, causing cell numbers to decline. Both estrogen treatments of LAPC-4 cells caused a rapid rise in cyclin D1 phosphorylation (Fig. 2.9A and B), as did E2 treatment of PC-3 cells (Fig. 2.9C), though the latter was not as well sustained. However, DES treatment of PC-3 cells instead caused a dephosphorylation of cyclin D1 (Fig. 2.9D). Although this decrease in phosphorylation fluctuated, it lasted at least 30 min. If these cyclin phosphorylations affect the levels of cyclin proteins as expected, they could explain our estrogen-driven changes in cell numbers. Therefore, we next examined total cyclin D1 levels to determine if phosphorylation did indeed correlate with degradation shortly thereafter. To assess this, we treated LAPC-4 and PC-3 cells with E2 or DES over a time course of 5-950 min (16 hrs.). Decreases in total cyclin D1 were observed in each scenario where cyclin D1 was phosphorylated (Fig. 2.9E, 2.9F, 2.9G). E2 caused a significant decrease in total cyclin D1 within an hr in both LAPC-4 and PC-3 cells, and DES acted similarly in LAPC-4 cells. Maximum reductions at 16 hrs in these three cases were ~25%. However, there was no significant decrease in total cyclin D1 levels in PC-3 cells after DES treatment (Fig. 2.9H), which correlated with its lack of phosphorylation and its inability to reduce the numbers of viable cells in this case. The ratio of p-cyclin D1 to total cyclin D1 shows a very rapid change in cyclin D1 kinase “activity” which in this case is a marking of the protein for degradation, except in DES-treated PC-3 cells (Fig. 2.9I-2.9L). This last view of the data best correlates with the pattern of estrogen- induced decrease in cell number.

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Figure 2.9. Phosphoryated cyclin D1 (p- cyclin D1) vs. total cyclin levels and their ratios after treatment with 0.1 nM E2 and 1 µM DES. LAPC-4 (early) and PC-3 (late) cells were treated with E2 or DES. The p-cyclin D1 levels (A-D) were measured for up to 60 min and the total cyclin D1 levels (E-H) were measured up to 16 hrs via plate immunoassays. The ratio of p- cyclin D1 to total cyclin D1 was calculated for overlapping time points at 5, 15, and 60 min (I-L). White symbols denote LAPC-4 cells and black PC-3 cells. Triangles represent E2 and circles DES. * denotes significance from vehicle (time 0) at P<0.05. The shaded gray horizontal bars represent the response to vehicle ± SEM.

ER-selective Antagonists: Next we examined the involvement of different estrogen receptor subtypes (Fig. 2.10), focusing on the mechanisms that had given the most robust

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of our explanations for estrogen-induced declines in cell numbers -- ROS generation and cyclin D1 phosphorylation/degradation. For this we blocked each receptor subtype with a selective antagonist at a very selective concentration, and then measured the estrogen- induced responses at time points selected as optimal for each mechanism. We found that the ROS production caused by 15 min of E2 treatment in androgen-dependent LAPC-4 cells was antagonized by both ERα- and ERβ-selective inhibitors (Fig. 2.10A), while the GPR30 antagonist had no effect. Androgen-independent PC-3 cells also had increased

ROS levels after E2 treatment, but in this case antagonizing only ERβ and GPR30 decreased the amount of ROS produced (Fig. 2.10B). DES was not active in generating ROS, and so was not tested for receptor participation.

Figure 2.10. ER subtype-selective antagonists inhibit E2- or DES-induced ROS and p-cyclin D1 responses. LAPC-4 and PC-3 cells were pretreated with antagonists for each of the three ER subforms: α (MPP), β (PHTPP), and GPR30 (G15) and then treated at the response time optima with 0.1 nM E2 or 1 µM DES. ROS were measured after 15 min (A & B). P-cyclin D1 levels were measured after 1 min for E2-treated PC-3 cells and 15 min for all others (C & D). White bars denote LAPC-4 cells, and black bars PC-3 cells. * denotes significance from vehicle (V) response at P<0.05. # denotes significance from the E2 response. The shaded gray horizontal bars represent the response to vehicle ± SEM.

The roles of the three known estrogen receptors in altering p-cyclin D1 levels after 15 min of E2 treatment were dictated both by the cell type in which the tests were done and the estrogen mediating the response. That is, in LAPC-4 cells, E2 again

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operated via ERα and ERβ to increase cyclin D1 phosphorylation (Fig. 2.10C), while in

PC-3 cells, a 1 min E2 treatment acted via ERβ and GPR30 to increase p-cyclin D1 levels (Fig. 2.10D). DES was also active in cyclin D1 activation, but only in LAPC-4 cells, where it required ERβ and GPR30. Strikingly, a 15 min DES treatment in PC-3 cells reduced cyclin D1 phosphorylation in a way that was independent of any of these three ERs (Fig. 2.10D), suggesting that at this high concentration this synthetic estrogen works via non- receptor-mediated mechanisms in ways that did not reduce cell number.

Discussion

Our results indicate that when several mechanisms of estrogen action are considered using cell lines that represent different stages of human prostate cancer, DES at a concentration achieved by typical clinical treatments (1 mg, three times daily leading to serum concentrations measured at ≥1 µM) 79 does not kill or otherwise reduce the numbers of cells that represent androgen-independent late-stage tumors (PC-3 cells). Ironically, these late-stage tumors are the ones usually treated with DES. While cell- killing effects have been observed at concentrations 10-100 fold higher than those used in our studies 279, such high concentrations can cause many unwanted side-effects in patients, and are unlikely to act via receptors. However, in our studies, E2 was much more potent in decreasing the numbers of both androgen-dependent and -independent prostate cancer cells in a concentration range (optimal at 0.1 nM) that may be far better tolerated by patients. The mechanisms that we examined for participation in estrogen-induced cell number decline in prostate cancer cell number are depicted in Figure 2.11 and their effects summarized in Table 2.1.

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Figure 2.11. Direct mechanisms of E2/DES action on prostate cancer cell survival.

E2/DES initiate rapid signals at mERs and through the activation or deactivation of MAPKs. These signals can also lead to apoptosis, ROS increases, or phosphorylation of cell-cycle proteins that delay the cycle. Each of these mechanisms can contribute to the estrogenic control of cell number.

MAPKs are important signal integrators of external stimuli leading to cell proliferation or death. The most commonly cited mechanisms explain cell number decreases by ERK inactivation (halting proliferation) or JNK activation (inducing apoptosis via caspases). Instead, we demonstrated that E2 rapidly increased pERK levels in both early- and late-stage prostate cancer cell lines. The activation of JNK did not in any case correlate with the expected effect on caspase activity, so it did not explain any of our cell viability results. Therefore, we had to entertain other explanations and pathways for the therapeutic (tumor cell-killing) actions of these estrogens via MAPKs.

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Table 2.1. Summary of mechanisms contributing to estrogen-induced decline in numbers of LAPC-4 or PC-3 prostate cancer cells.

E2/DES activate differing pathways (listed) in LAPC-4 vs. PC-3 cells, which can lead to cell death (apoptosis/necroptosis, ROS increases) or lack of cell proliferation (↓ pERK, phosphorylation of cell cycle proteins). Each of the mechanisms listed in red or green font were able to contribute to the estrogenic control of cell numbers in our studies. Mechanisms in the gray font indicates a pathway that was tested but does not contribute. These number of mechanisms contributing to each outcome were summed. The red text mechanisms and summary numbers indicate participation of mechanisms that will kill cells or stop their growth; the green text mechanisms and summary numbers indicate mechanisms that will cause cells to proliferate or survive. We counted changes in ERK and ROS as separate mechanisms. Only a 0.5 score is awarded to the p-p16 result for DES-treated PC-3 cells because it was a very low response compared to the others. For non-color print versions: These are the mechanisms that decrease cell number: ↑↑ERK→ROS, ↓ERK, ↑JNK, ↑ caspase 3, ↑p-p-16, ↑p-cyclin→↓total cyclin. They are summed by the top number in each panel. Mechanisms that increase cell number: ↓JNK, ↓p- cyclin→↑total cyclin -- are summed by the bottom number in each panel.

Sustained ERK activation (often throughout 60 min) associated with ROS

262 generation-mediated cell killing demonstrated in our studies did correlate with E2- induced declines in cell numbers. In this type of response ROS activates even more ERK, likely participating in a positive feed-forward loop 262. The increased ROS production we observe may not be due solely to sustained pERK, as use of MEK inhibitor U0126 only

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abrogated the response by about 50%. Other possible sources of E2-generated ROS include E2-induced DNA damage or intercalation of E2 into the plasma membrane, where it can undergo redox cycling 280, 281. Activated JNK levels did not correlate with nor explain decreased cell numbers via apoptosis. Apoptosis, apparently not controlled by JNK, played a role only in the cell model of early-stage prostate cancer (LAPC-4) where this type of killing was caused by both estrogens. Late-stage cancer cells represented by the PC-3 cell line were unaffected by either of these mechanisms. Neither cell type underwent estrogen-induced necroptosis. Again, such a lack of response to estrogens in cells representing late-stage prostate tumors was surprising, given that this is the type of patient who is most often treated with the estrogen DES 282. We showed that blocking the actions of proteins that drive the cell cycle was also involved in these estrogen-induced declines in cell numbers. The major cell-cycle protein CDK inhibitor p16INK4A and the cyclin D1-phosphorylating kinase p38 were both induced by estrogens in all cases, regardless of whether these treatments actually reduced cell numbers; so while these proteins may participate (be permissive), they do not alone or primarily control a critical step in determining the cell viability outcome. However cyclin D1 phosphorylation mediated by p38 283, leading to its rapid degradation, was directly correlated with estrogen-induced cell number declines in all cases. Cyclin D1 availability and activity are major deciding factors for cell-cycle progression, and a diminished ability to move through this checkpoint readily decreases cell numbers. E2 performed much better in this pathway endpoint, providing a treatment advantage for both early- and late-stage cancer cells. It is unknown why activated p38 cannot mediate DES-induced phosphorylation of cyclin D1 in PC-3 cells, and this perhaps points to the involvement of another unknown factor. Changes in cell viability and caspase activation were measured over a course of several hours to days, and therefore the involvement of both genomic and nongenomic signals is likely. However, for our signal transduction assays, the mechanisms we

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examined were activated rapidly (within minutes) after estrogen treatments, and likely were mediated by a membrane form of these estrogen receptors, as we have shown in other cancer cell types (GH3 pituitary, MCF-7 breast, PC-12 pheochromocytoma) 284-286. However, we have also previously seen estrogen-mediated rapid phosphorylation/activation of Elk-1 and ATF-2 transcription factors as early as 10-15 min 287. This illustrates how signaling initiated early via nongenomic mechanisms can progress to downstream genomic mechanisms. Our previous studies demonstrated rapid activation via mERs for the three MAPK activations demonstrated here. In addition, our previous work on glucocorticoid-induced killing of T lymphoma cells showed dependence on the presence of a membrane form of the glucocorticoid receptor 288. Our demonstration here of novel non-genomic actions of estrogens on prostate cancer cells should open new avenues of thinking about therapeutic approaches using estrogens of many types. Estrogen receptors play a major role in normal physiological regulation and development of many types of cells including the prostate, but also in cell survival and cancer development 289. The primary ER of the prostate gland is ERβ, with lower levels of ERα and GPR30. However, each is a possible target for cancer therapy 290. Our results using ER subtype-selective antagonists suggest that estrogens mediate specific mechanisms (increased phosphorylation of cyclin D1 resulting in its degradation, ROS generation via ERK activation) via some but not all ERs -- and not always the same ones

-- to affect cell viability. E2 required ERβ to operate the most important contributory mechanisms related to cell number declines (increasing ROS and p-cyclin in both cell types), but ERα and GPR30 participated variably, especially depending upon early- vs. late-stage tumor cell type. However, the high concentration DES effects in late-stage PC- 3 cells required no receptor involvement at all, were in the opposite direction from those mechanisms associated with the decline in cell numbers, and were therefore in agreement with the inability of DES to reduce cell numbers. It has previously been observed that at such high concentrations DES can bypass estrogen receptor-mediated mechanisms, and

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instead alter such chemical properties as fluidity, lipophilicity, and polarity of the lipid bilayer 291, 292, so perhaps DES is operating on these cells via these other less productive mechanisms.

Conclusions

The degree to which these treatments are more effective because they engage more of the mechanisms that can reduce tumor cell numbers are summarized in Table 1; we listed the active mechanisms in color, and then added up how many mechanisms were active in each case toward increasing (green) or decreasing (red) cell number. Only by examining all of these responses together were we able to comprehensively consider which pathways contributed to the therapeutic cell-killing effects of estrogens on prostate cancer cells of different stages. These studies are an important example of how systematic examination of multiple mechanisms can elucidate the extent to which a therapeutic agent will be effective on tumor cell stages with distinct characteristics. We showed that estrogens have a rapid and direct effect on prostate tumor cells, and that multiple, but not all cell-killing mechanisms contribute to the therapeutic response. E2 was much more potent and efficacious than DES, suggesting that E2 could be a better form of treatment for men with all stages of prostate cancer. This represents a particularly important opportunity for treatment of advanced prostate cancers where treatment options are limited. However, these results also suggest that very early-stage developing tumors in high-risk cancer-susceptible men could benefit from low-dose E2 treatment, the natural levels of which may decline in men during aging 293, 294.

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Chapter 3: Rapid, nongenomic signaling effects of several xenoestrogens

involved in early- vs. late-stage prostate cancer cell proliferation

Abstract

Xenoestrogens (XEs) are exogenous mimics capable of binding to estrogen receptors (ERs), competing with/disrupting the actions of physiological estrogens, and promoting tumor growth in the prostate and other endocrine tissues. Humans are exposed to numerous XEs including environmental contaminants such as plastics monomer bisphenol A (BPA), and dietary phytoestrogens such as coumestrol and genistein from soy, and resveratrol, highest in red grapes. There is growing interest in the ability of phytoestrogens to prevent or treat tumors. We previously reported that multiple cellular mechanisms influence the number of prostate cancer cells after estradiol or diethylstilbestrol treatment. We now examine the effect of these XEs on signaling mechanisms that alter the number of LAPC-4 (androgen-dependent) and PC-3 (androgen- independent) cells at environment- and diet-relevant concentrations. Coumesterol and genistein both increased the number of LAPC-4 and PC-3 cells dramatically. Rapid alterations of phospho- and total-cyclin D1 levels most closely correlated with the XE- induced changes in cell numbers. Sustained activation (phosphorylation) of the extracellular signal-regulated kinases 1 and 2 as a prelude to generation of reactive oxygen species also partially contributed to the XE’s effects on cell numbers. Early-stage cells expressed higher levels of all three ERs (including those in membranes) than did late-stage cells; ER subtypes were variably involved in the signaling responses. Taken together, these results show that each XE can elicit its own signature constellation of signaling responses, highlighting the importance of managing exposures to both

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environmental and dietary XEs for existing prostate tumors. These mechanisms may offer new cellular targets for therapy.

Introduction

Prostate cancers are well-known for their initial androgen responsiveness, which diminishes with the progression of disease stage. While the corresponding decrease in androgen receptor (AR) levels that accompanies this decline in responses has been well documented 295, little is known about the relationship of tumor progression with the estrogen receptor (ER) types that might be involved, such as those that are thought to mediate the therapeutic effects of the pharmaceutical estrogen diethylstilbestrol (DES).

There are many types of xenoestrogens (XEs) – exogenous estrogen-like compounds that bind to ER ligand binding pockets 58, 296, 297. In normal or cancer cells, they imitate, compete with, or disrupt the actions of physiological estrogens 298, 299. Some XEs are known to promote tumor development in many tissues by stimulating inappropriate endocrine responses via ERs, promoting angiogenesis, increasing DNA adducts, or altering the epigenome 300-304. Actions of XEs via ERs have also been shown to cause the proliferation of established endocrine tumors or tumor cell lines of many types, including those from brain, breast, kidney, lung, pancreas, prostate, and testis 287, 305-311.

Alternatively, some XEs, especially dietary compounds, have been credited with preventing tumors in some of these tissues 312-315, highlighting a broad range of XE response profiles. Genistein is a phytoestrogen found in soy products, fava beans, and some coffee bean preparations 316 that can cause cell cycle arrest and growth inhibition at concentrations within the ranges achieved by the diets of some cultures (10-8 M to 10-6

M), via the down regulation of cyclin B 317, 318. Coumestrol (found in red clover, alfalfa

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sprouts, and also some soy products) can kill breast and colon cancer cells by producing reactive oxygen species (ROS) 319. Resveratrol, found in grapes, can decrease cell numbers by increasing intracellular calcium levels, disrupting G1/S progression, and stimulating apoptosis 320-324.

XEs also include environmental contaminants from the manufacture, use, or leachates of consumer products (e.g. plastics, chlorinated pesticides, alkylphenol surfactants). Bisphenol A (BPA) is a component of many plastic products [such as water bottles, food containers, receipt paper, and the inner coatings of food cans 325] and leaches from these products more readily with heat or acidity. As a result, BPA is a common environmental and human/animal contaminant 326 that has been shown to alter cell proliferation 327, cell signaling through the activation of mitogen-activated protein kinases (MAPKs), 327-329 and intracellular calcium levels 330, 331, prostate cancer cell migration 332, and increase susceptibility to certain diseases 300, 333.

Epidemiological studies generally support an association between diets high in phytoestrogens and low cancer incidence 12, 334, 335. African-Americans have a higher incidence of prostate cancer 336, and dietary differences are being investigated as a possible factor in tumor development and progression within that population. East Asians consume high amounts of phytoestrogens, and their incidence of many types of cancer, including prostate cancer, is much less 12. Asian diets contain high levels of soy ingredients, with the best-known active estrogenic components being daidzein, genistein, and coumestrol 15. Genetics may also play a role in the sensitivity of various cancer- relevant mechanisms to estrogens, including the ability to metabolize dietary

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phytoestrogens to more active compounds 337, though this can also be due to the type of gut microbiome present 338.

Until recently, most studies on XEs were focused on the gene expression

(genomic) consequences of exposures 339-341. Even at high concentrations, XEs generally elicited only low levels of transcriptional responses, so these compounds were thus labeled as weak estrogens 339, 342. However, XEs can also initiate non-genomic responses, so classifying them as weak without taking these more rapid cellular responses into consideration may be misleading 287, 307, 343-347. Some endogenous estrogen metabolites such as estriol were formerly labeled as weak because of their limited ability to activate specific transcription, but have recently been found to have profound effects on disease expression 348, 349. Estriol, like XEs can have quite potent effects on nongenomic responses 350. Because of this belief that XEs were weak, many past studies did not evaluate estrogens at environmentally relevant low doses [reviewed in 351]. Dose responses to estrogens are typically non-monotonic and therefore must be assessed over a wide and detailed range of concentrations (reaching down to the femtomolar to nanomolar range) to predict their ability to act, especially at relevant environmental and dietary levels 287, 302, 350, 352.

We have previously shown that XEs can rapidly activate cellular signaling pathways in tumor cells of other tissues (pituitary, breast, adrenal), and when in combination with them can modify the actions of physiological estrogens 328, 329, 344, 353-

355. MAPKs can be rapidly activated or deactivated by XEs, leading to alterations in such functional end points as proliferation, apoptosis, and prolactin release 287, 356. Moreover, these MAPK phosphorylations [such as those for the extracellular signal regulated

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kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 kinase] often occur at low physiological estrogen and XE concentrations 287, 328, 357.

We also recently demonstrated that estradiol (E2) and diethylstilbestrol (DES) can rapidly stimulate or deactivate ERKs in LAPC-4 and PC-3 prostate cancer cells 358, and when sustained, cause ROS generation, contributing to a decrease in viable cells 262, 358.

In addition, estrogen-induced rapid phosphorylation of cyclin D1 led to its subsequent prompt degradation, which in turn was correlated to the ability of E2 and DES to inhibit growth of these cells 358. We will now investigate if some XEs also alter the viability of prostate cancer tumor cells via these mechanisms. Elucidating how these XEs function in early- vs. late-stage prostate tumor cells could lead to selective advice for patients about diet and exposure to environmental estrogens.

Materials and Methods

Cell lines and hormones: We chose cell lines representing the two main types of prostate cancers – androgen-dependent vs. androgen-independent. LAPC-4 androgen- dependent prostate cancer cells (passages 45-50) 359 were maintained to sub-confluence in phenol red-free Iscove’s Modified Dulbecco’s Medium (IMDM; MediaTech,

Manassas, VA) with 10% fetal bovine serum (FBS, Atlanta Biologicals, Lawrenceville,

GA), 4 mM L-glutamine (Sigma-Aldrich, St. Louis, MO), and 10-9M dihydrotestosterone

(Sigma-Aldrich). PC-3 androgen-independent prostate cancer cells (passages 18-23) 30 were maintained by growth in phenol red-free RPMI 1640 (Sigma-Aldrich) with 10%

FBS and 2mM L-glutamine. Both cell lines were propagated at 37°C in 5% CO2. BPA, coumestrol, genistein, and resveratrol (all from Sigma-Aldrich) were dissolved in ethanol to a stock concentration of 10mM before serial dilution into IMDM or RPMI 1640 at

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concentrations ranging from 10-14M to 10-6M (and a final EtOH concentration of

0.0001%).

MTT Cell Viability Assay: Cells were plated at 5,000 cells/well in poly-D-lysine- coated (BD Biosciences, Bedford, MA) 96-well assay plates, (Corning, Tewksbury, MA), and then allowed to attach overnight. The next day, XE treatments were added in 100µL of medium with 1% 4x charcoal-stripped FBS. Extensive charcoal stripping of serum was done to remove and thus minimize the effect of any steroid hormones already present; these conditions were previously optimized to demonstrate effects of steroids and mimics on cell proliferation for these cell lines. After three days, treatments were removed and 3-

(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich) was added for 1 hr. Cells were then lysed and the signal read at 590nm in a Wallac 1420 plate reader (Perkin Elmer, Waltham, MA).

Plate Immunoassays: Phosphorylated proteins were recognized by antibodies

(Abs) specific for these post-translationally modified epitopes: pERK1/2

(Thr202/Tyr204) and phospho-cyclin D1 (Thr286) (both from Cell Signaling, Danvers,

MA). Changes in total cyclin D1 levels were measured by using an Ab to cyclin D1 recognizing both modified and unmodified protein cyclin D1 (Cell Signaling Cat. No.

2922). ER Abs used included ERα (MC-20, Santa Cruz Biotechnology, Dallas, TX); ERβ

(clone 9.88, Sigma-Aldrich); and GPR30 (Cat. No. NLS4271, Novus Biologicals,

Littleton, CO). Membrane and total ER levels were measured by controlling for permeabilization of the cell membrane. A plate immunoassay developed in our lab 284 and used in many of our past studies was recently adapted 360 for use with the BIOMEK FXP

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workstation (Beckman Coulter, Brea, CA) to automate the majority of the plate assay’s liquid handling, decreasing experimental variability and increasing experimental output.

Prostate cancer cells were plated at 10,000 cells/well in 96-well assay plates, allowed to attach overnight, and given 100µL of medium with 1% 4x charcoal-stripped

FBS for 48 hr. Cells were then treated with XEs for up to 60 min on the workstation, followed by fixation (2% paraformaldehyde, 1% gluteraldehyde) ± permeabilization

(0.15M sucrose, 0.5% Nonidet P-40; to access internal vs. extracellular epitopes). The primary Ab to the phosphorylated epitopes was then added and incubated with the cells overnight. The next day, biotinylated anti-mouse/anti-rabbit IgG secondary Ab (Vector

Labs, Burlingame, CA) was added for 1 hr. Next, cells were incubated for 1 hr with avidin-biotinylated conjugated alkaline phosphatase (ABC-AP, Vector Labs), then for 30 min with para-nitrophenylphosphate substrate (Thermo Scientific, Rockford, IL), allowing the yellow color of the para-nitrophenyl product to accumulate. Plates were read at 405nm in a Wallac 1420 plate reader. Readings were then normalized to cell number, estimated by the crystal violet dye (Sigma Aldrich) assay as described previously 284.

Subtype-selective ER Antagonist Assays: To further determine ER subtype involvement in altering ROS formation or cyclin D1 phosphorylation, the following ER antagonists were used at their receptor-selective concentrations: for ERα, 10-7M MPP; for

ERβ, 10-6M PHTPP; and for GPR30, 10-6M G15 (all from Tocris Bioscience,

Minneapolis, MN). All were dissolved in ethanol to a stock concentration of 10mM, then serially diluted into culture medium. Final ethanol concentrations were 0.0001%, which was used as vehicle control for all studies. Cells were incubated with antagonists for 30 min before XE treatments.

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ROS Assays: Cells were plated at 10,000 cells/well in a 96-well assay plate, then allowed to attach overnight. Cells were then treated with 100µL of medium containing

1% 4x charcoal-stripped FBS for 48 hr. 2’,7’-Dichlorodihydrofluorescein diacetate

(DCDHF, Enzo Life Sciences, Farmingdale, NY; 15µM) was loaded into cells for 1 hr, and XE treatments were then administered for 15 min. Hydrogen peroxide (Fisher

Scientific, Pittsburg, PA) and ethanol (0.0001%) were used as positive and negative controls, respectively. E2 (1nM) was a positive control for previously determined estrogenic responses 358. Dichlorofluorescein production, formed as a result of

ROS/DCDHF interaction, was measured at an excitation of 485 nm, and an emission of

538 nm in a SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices).

Statistics: All experiments were conducted a minimum of three times. One-way analysis of variance was conducted for all experiments except ER quantification, which was analyzed using a Student’s t-test. A Holm-Sidak post hoc test was used to measure the significance of each treatment versus the vehicle control. Significance was set at p<0.05, unless otherwise stated.

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Results and Discussion

Figure 3.1. Cell number after 3 days of XE treatment. LAPC-4 and PC-3 prostate cancer cells were treated with XEs and viable cells were measured by the MTT assay. In all figures throughout the manuscript white symbols denote LAPC-4 cells and black symbols PC-3 cells. *denotes significance from vehicle (V) controls at p<0.05, and shaded horizontal bars represent the response to V ± SEM. In this and other graphs, where error bars are not visible, they were within the size of the symbol. Dietary or environmentally relevant concentration ranges are shown by the solid horizontal bars below the graphs for each XE. The 358 insets show cell numbers after three days of E2 treatment, for comparison (and see )

XE Effects on the Number of Viable Cells: XEs at environment- or diet-relevant concentrations caused some increases in the numbers of LAPC-4 and PC-3 prostate cancer cells, observed here after three days of exposure in media containing 1% charcoal- stripped serum (Figs. 3.1A & B). Coumestrol increased viable cell numbers at all but the lowest concentration assessed (10-14M) in both cell lines (by >200% in LAPC-4 cells,

>400% in PC-3 cells). There was a strikingly different response to genistein between cell lines representing different tumor stages; genistein did not affect LAPC-4 cells, while it strongly stimulated the growth of PC-3 cells (by ~4-fold at concentrations from 0.1nM to

1µM, all levels achievable by some diets). BPA caused a small stimulatory effect in

LAPC-4 cells, at concentrations >10-12M (maximal increases of ~40-50%), but had no

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effect on PC-3 cells. Resveratrol showed minimal, but significant stimulation compared to vehicle in both cell lines at most concentrations tested. For comparison to other estrogens assessed in our previous studies 358 (see insets), even at physiological (10-10-10-

8 M) concentrations E2 instead caused a significant decrease in cell viability for both cell lines by ~20-30%. The actions of these dietary estrogens in causing prostate cancer cell growth are perhaps unexpected, given the epidemiological evidence that cultures with diets rich in genistein and coumestrol show decreased levels of prostate cancer 14, 361.

However, some other studies have also shown a prostate cell proliferation effect by these phytoestrogens 362-364.

Subcellular Location and Levels of ERs: We next asked which types of ERs were present in LAPC-4 and PC-3 cell lines that could mediate these changes in viable cell number. We had previously noted a variable dependence of rapid responses on all three

ER subtypes (determined by using selective antagonists) in our studies on E2 vs. DES treatment of these cells 358. Plasma membrane versions of estrogen receptors (mERs) are thought to mediate rapid signaling involved in cell number changes in other cell types

[reviewed in 186, 353, 365], so we examined the subcellular location of these receptors here.

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Figure 3.2. ER subtype (α, β, and GPR30) levels (total vs. membrane) in LAPC-4 and PC-3 prostate cancer cells. The negative control samples used no primary antibody (Ab) for any of the ER subtypes, as indicated by the first bar and the shaded bar extending horizontally across the graph (average ±SEM). *denotes significance from controls at p<0.05.

Using our plate immunoassay ± cell permeabilization with detergent (Fig. 3.2), we observed that late-stage tumor cells (panel B) had much lower expression of ERs than did early-stage cells (panel A; note the ~6-fold vertical scale difference between panels A and B), a frequent finding among steroid receptors in endocrine cancers of multiple types

366-368, although this evaluation for mERs in prostate tumor cells is novel. We also saw that membrane receptor populations were much lower than total (and thus intracellular) receptor forms in early-stage cells; the levels of mERs α and β were about 20% and 24% of their total receptor populations, respectively, as we have seen previously for the proportion of membrane versions of these receptors in other tumor cell types 171, 286, 369.

Although much lower, we detected significant levels of all three ER types in PC-3 cells. In these late-stage tumor cells the membrane receptor population was a much larger percentage of the total receptor numbers, perhaps in keeping with their more

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undifferentiated state, as we have seen with membrane glucocorticoid receptors in human lymphoma cells compared to normal circulating lymphocytes 288. ERβ predominated in

LAPC-4 cells (and to a lesser extent in PC-3 cells), as expected based on the literature regarding the dominance of this receptor type in normal prostate tissues and the early- stage tumors that arise from them 57, 223. However, there were also significant levels of

ERα and GPR30, suggesting that they might also play a role in mediating estrogenic mechanisms. Interestingly, we found that the sizable amount of GPR30 was largely intracellular in LAPC-4 cells. GPR30 has been identified in other prostate cancer studies, but the subcellular location was not elucidated 206, 238, 370. The subcellular location of

GPR30 in other tissues and their cancers has been a point of contention; different groups have demonstrated this receptor form as either primarily in the plasma membrane or in the endoplasmic reticulum 371, 372.

Phospho-ERK: Our next goal was to identify pathways and mechanisms responsible for any changes in numbers of viable cells, and ERK phosphorylation is one mechanism that has traditionally been associated with cell proliferation 262. We selected an effective and environment- or diet-relevant concentration for each XE studied (10-9M

BPA, 10-7M coumestrol, 10-7M genistein, and 10-8M resveratrol; see Fig. 3.1 for relevant ranges), and measured their ability to elicit ERK phosphorylation in both cell lines over

60 min (Figs. 3.3A & B). We observed activation for all compounds except genistein, but found that a sustained (60 min) pERK response did not predict a XE’s positive influence on cell number, as has been a long-held association 373, 374.

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Figure 3.3. Phospho-ERK (pERK) levels in LAPC-4 and PC-3 cells after XE treatments. LAPC-4 and PC-3 cells were treated with 10–9M BPA, 10-7M coumestrol, 10-7M genistein, and 10-8M resveratrol. pERK was measured up to 60 min via the plate immunoassay. *denotes significance from vehicle (shown at time 0) controls at p<0.05, and horizontal shaded bars represent the response to vehicle ± SEM.

The most striking result was the difference between cell line-specific responses after resveratrol treatment, which caused a strong ERK deactivation (40%) in LAPC-4 cells, while it had slightly increased the number of viable cells (Fig. 3.1). In PC-3 cells resveratrol rapidly activated and sustained pERK (at 60 min), and caused modest cell proliferation. BPA and coumestrol rapidly stimulated ERK phosphorylation in both cell lines (Figs. 3.3A & B), all with sustained levels at 60 min, but had no proliferation effects in late-stage cells. Genistein rapidly though modestly deactivated ERK in both cell lines, but substantially increased viable PC-3 cell numbers. Only the activation of ERK by coumestrol in both cell types correlated with its ability to cause these cells to proliferate. Therefore, these XEs elicited unique patterns of ERK activation/deactivation,

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which could contribute to cell survival, cell death, or proliferation 262, 373, 375, but clearly more mechanisms needed to be considered.

-10 -6 -9 -7 Figure 3.4. ROS levels after treatment with 10 M E2, 10 M H2O2, 10 M BPA, 10 M coumestrol, 10-7M genistein, and 10-8M resveratrol, ± ER subtype-selective antagonists. Antagonists (Antag) were 10-7M MMP for ERα; 10-6M PHTPP for ERβ; and 10-6M G15 for GPR30. ROS levels were measured after 15 min of each XE treatment (the optimal response time). *denotes significance from vehicle (V) controls at p<0.05, while # denotes significance from paired XE treatment values (p<0.05). ERα inhibition was significantly different vs. resveratrol alone in PC-3 cells ($) at p<0.09. The shaded horizontal bars represent the response to vehicle (V) ± SEM.

ROS generation: Others have recently associated sustained ERK activations with ROS generation leading to cell killing 262, and we recently extended this to the ability of a

358 physiological estrogen (E2) to induce cell death in early-stage prostate cancer cells . Therefore, we examined if XEs (at optimal concentrations for such ERK responses) could be linked to any ROS elevations (measured at the peak time of 15 min, time course not shown). The positive controls for ROS generation, including both H2O2 and E2, caused robust ROS generation (Figs. 3.4A and B), and as we saw previously, cell death 358. Most XE treatments generated significant ROS levels regardless of whether they had caused

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sustained ERK activation (Fig. 3.3), though BPA and genistein did so only in one cell line each. These ROS increases were all modest compared to those caused by E2. Therefore, ROS elevation due to sustained ERK activation was not considered to be a primary mechanistic determinant of viable cell number in these studies. Perhaps higher levels of ROS need to be generated to kill this cell type.

Therefore, these MAPK, and potentially linked ROS responses, did not individually predict cell proliferation vs. cell killing effects. Possibly a more traditional route of ROS generation not involving ERKs was involved in these cells. Estrogens have been shown to damage DNA 281, which can also cause ROS generation 376, 377. The ability

358 of these XEs to induce ROS was different from that of E2 , further highlighting the imperfect mimicry of physiological estrogens by XEs. Overall, the pERK and ROS responses to XE treatments in both cell lines do not appear to be lone driving mechanisms that elicit changes in cell numbers. Therefore, we have to consider the combined contribution of these responses to an overall balance of competing mechanisms

(see below).

We observed previously that E2 required different ER subtypes to elicit ROS responses in LAPC-4 vs. PC-3 cells 358. Here XEs also demonstrated unique ER-use signatures for this response (Fig. 3.4). In LAPC-4 (early-stage) cells, BPA caused these modest ROS increases independent of any known ERs, and genistein did not raise ROS levels (Fig. 3.4A). The increases due to coumestrol required ERα and GPR30, while resveratrol required only ERα. A somewhat different profile was evident in PC-3 (late- stage) cells, where coumestrol and resveratrol both increased ROS via ERβ, while genistein stimulation of ROS did not require any known ERs, and BPA did not cause a

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response (Fig. 3.4B). Clearly, the regulation of this pathway via ERs became quite different as cell types progressed to a less differentiated state with far lower receptor numbers (Fig. 3.2). The involvement of more than one ER subtype in some XE-generated responses also suggests the participation of multiple pathways.

Figure 3.5. Cyclin D1 phosphorylation and degradation by XEs, and inhibition by ER- selective antagonists. Cyclin phosphorylation was measured at 1-60 min, and total cyclin D1 levels over 16 hr of XE treatment. For 5A & 5B, LAPC-4 and PC-3 cells were pretreated with antagonists (Antag) for each of the three ER subtypes: α (MPP), β (PHTPP), and GPR30 (G15,) and then treated with 10- 9 -7 -7 -8 -10 -6 M BPA, 10 M coumestrol, 10 M genistein, 10 M resveratrol, 10 M E2 or 10 M DES. Shaded horizontal bars represent V ± SEM. * denotes significance compared to vehicle (V) at p<0.05. # denotes significance from paired XE treatment responses at p<0.05. For 5C & 5D, LAPC-4 and PC-3 cells were treated with each XE for the times indicated and total cyclin D1 levels were measured with a plate immunoassay.

Total and Phosphorylated Cyclin D1: Control of cyclin D1 levels 378, 379 is a mechanism we previously identified as being a significant contributor to E2- or DES- evoked changes in prostate cancer cell survival 358. Both estrogens caused rapid cyclin D1

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phospho-activation leading to swift degradation of this cell-cycle protein. This response to E2 and DES is also shown here in Fig. 3.5A & B. Others have also shown that phytoestrogens can affect other cell-cycle protein levels 317, 318, 322, 323, 380, which in turn affect the number of cells. Of the four XEs studied here, the three phytoestrogens

(coumestrol, genistein, and resveratrol) all affected cyclin D1 phosphorylation levels, though in distinctly different directions, and differently for early- vs. late-stage cells

(Figs. 3.5A & B). In each case where cyclin D1 was phosphorylated, the corresponding expected rapid decline in total cyclin D1 levels occurred (panels C and D). Interestingly, we observed significant declines in total cyclin D1 as early as 5 min after genistein or resveratrol treatment (~10%), but the largest decreases for most compounds were seen after 4 hr. Coumestrol caused cyclin dephosphorylation, resulting in cyclin D1 level increases, correlating very well with its ability to increase cell numbers. Resveratrol signaling significantly phosphorylated cyclin D1 in both cell lines, driving total cyclin D1 levels down, yet while eliciting very small increases in cell proliferation, a less perfect correlation. Genistein was the only compound that caused opposing effects on these mechanisms in the two cell lines. In LAPC-4 cells, it increased phosphorylated cyclin D1, causing its degradation, but that did not correlate with measured changes in viable cell numbers. However, in PC-3 cells, genistein depressed cyclin phosphorylation, allowing increases in cyclin levels and correlating with a strikingly robust cell proliferative response. BPA did not affect cyclin D1 phosphorylation in either cell line, nor did it change levels of total cyclin D1, in keeping with its minimal effects on cell numbers.

While these correlations generally go in the expected direction, they do not entirely predict the degree of the functional (cell number-changing) responses. Therefore, we

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ultimately considered all of these mechanisms together (see summation in Table 3.1), to see if other mechanisms in some cases modified this dominant response to cyclin D1 changes (see Conclusions).

Other signaling pathways that we have not examined here may also contribute to the net change in cyclin D1 phosphorylation and consequent total protein levels.

Glycogen synthase kinase 3β, which is regulated by the phosphoinositide 3- kinase/protein kinase B pathway (PI3K/Akt), has also been shown to phosphorylate cyclin D1 on Thr286, as well as regulate the protein’s subcellular location in mouse fibroblasts 381. However, the exact role of GSK-3β/PI3K/Akt in driving phosphorylation of cyclin D1 has been debated, as Guo et al., found that the activity of those pathways did not change (during the relevant S-phase), nor decrease cyclin D1 protein levels in mouse or human fibroblasts 382. In addition, inhibition of GSK-3β in MCF-7 breast cancer cells did not completely disrupt cyclin D1 protein degradation 383. Other pathways that can cause degradation of cyclin D1 include p38, which we previously studied for activation

358 by DES and E2 and did not include here because it was activated for all cell types and treatments. Cyclin D1 phosphorylation through p-p38 seems to be especially prevalent in response to the damage to DNA caused by environmental agents which require rapid cellular responses to prevent propagation of genomic mistakes 384, 385. The Mirk/Dyrk1b kinase, active during G0/G1, has also been shown to regulate cyclin D1 protein levels through phosphorylation at Thr288 386. Therefore, multiple pathways can cause phosphorylation of cyclin D1, but they may influence degradation of cyclin D1 to varying degrees, and may be tissue-selective.

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We next examined which ER subtypes (α, β, or GPR30) might be involved in the cyclin D1 phosphorylations, again using selective antagonists for each receptor subtype.

For genistein, either the ERβ or GPR30 antagonist reversed the cyclin D1 phosphorylation in LAPC-4 cells, but only the ERβ antagonist decreased p-cyclin D1 levels in PC-3 cells. Coumestrol apparently did not utilize any of the known ER subtypes in LAPC-4 cells to decrease p-cyclin D1 levels, but ERα was required in PC-3 cells.

Coumestrol’s lack of dependence on any known receptor subtype in LAPC-4 cells is surprising, given the plentiful expression of all of these receptors in that cell line. It is possible that the low p-cyclin levels and thus larger errors in the measurement, caused by coumestrol treatment made antagonist reversals difficult to detect. Resveratrol’s induction of p-cyclin D1 levels in both cell lines showed a dependence on ERs α and

GPR30 in LAPC-4 cells, and on ERα in PC-3 cells. Therefore, each XE showed a dependence on a different ER subtype or subtype combination in the two cell lines. These dependencies are consistent with what has been previously shown about receptor subtype binding preferences for these XE compounds. For example, resveratrol has a higher binding preference for ERα than for ERβ 296, while coumestrol and genistein are strong

ERβ agonists 297, but are still capable of binding to ERα 299, 387, 388. The predominance of

ERβ in these prostate cell lines may influence their responses to these XEs that affect cell number. However, mostly genomic pathways have been examined in the past, such as the ability to activate ER reporter constructs, with differences between cell types for different

XEs 389-391. Few comparisons for nongenomic responses are available, though we previously observed different MAPK activation patterns and mostly positive proliferative

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276, 329, 392 responses to E2 and various XEs in GH3/B6/F10 pituitary cells that have high mERα and low mERβ levels

Also consistent with our results are known XE effects that do not involve these

ERs. An example is the well-known direct inhibitory effect of genistein on tyrosine kinases 393-395. In other instances, small lipophilic compounds like these can intercalate into cell membranes and as “border lipids” influence the actions of proteins embedded in them. Lipophilic estrogens can change membrane fluidity 291, 292, especially when they are present at relatively high concentrations (as is true for most effective phytoestrogen concentrations resulting from dietary exposures). Because changes in cell numbers are best observed after three days, the nuclear-localized receptors forms involved in slower transcriptional regulation 396, 397 may also be relevant to these effects, which we did not examine in our studies of these more novel rapid actions.

Another possible contributor to tumor cell behavior in prostate cell lines is the tumor-suppressor p53 32. It is mutated in LAPC-4 cells, and not present in PC-3 cells, and therefore unlikely to drive estrogen-mediated prostate tumor viability in our present studies. We also chose cell models for our studies that do not present the added complication of mutant ARs (such as in LnCaP cells 398) to which estrogens can more readily bind and elicit effects, especially at high concentrations. LAPC-4 cells have wild- type ARs and PC-3 cells do not express ARs 32.

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Conclusions

Table 3.1 Summary of XE responses for mechanisms that affect the number of viable LAPC-4 vs. PC-3 cells. Estradiol is shown for comparison, summarizing the data from our previous publication 358. Mechanisms in red text contribute to decreases in viable cell numbers, while mechanisms in green text increase the number of viable cells. Gray text indicates mechanisms that did not make any contribution to changes in cell numbers. These mechanistic contributions are summed in the red and green numbers in the upper right-hand corner of each box. ∆ = change.

The estrogen-induced mechanism that dominated our effective predictions of cell growth behavior in these studies was the rapid phosphorylation of cyclin D1, followed shortly thereafter by its degradation. This mechanism largely predicted the primary response to each XE in terms of cell number changes (except in the case of resveratrol).

However, no single mechanism entirely predicted the degree of these XE-induced changes, so we also examined the balance of the effects of other pathways (summarized in Table 3.1). In this table each of the mechanisms examined in these studies for each cell type due to each XE is summarized in colored text: red denotes actions causing decreases in viable cells, while green represents mechanisms that increase cell numbers,

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and gray indicates no effect. These mechanistic contributions are summed in the red and green numbers in the upper right-hand corner of each box. The modest effects of these

XEs are in contrast to the strong cell growth inhibitory/cell killing effects we saw

358 previously with E2; these previous conclusions are shown in the first column of the table for the sake of comparison. E2 engaged a total of seven signaling responses in

LAPC-4 cells and PC-3 cells -- though in this table we list only the 4 that were examined here that gave the best predictions of therapeutic responses (decreases in cell numbers) to compare with XEs. Coumestrol is a robust stimulator of cell growth in both early- and late-stage prostate cancer cells, matching its strong positive effects on cyclin D1 status and levels. The ability to activate sustained ERK, and through it to increase ROS levels, did not decrease the cyclin D1-driven outcome. Genistein’s different effects on early- vs. late-stage cells could also be largely explained by the cyclin D1 changes. Resveratrol had a very small effect in both cell lines, though the altered cyclin D1 levels and the ROS generation should have predicted a large decrease in cell viability, which was not observed. BPA showed the smallest changes in these responses that we examined, corresponding to only minimal growth stimulation, in only LAPC-4 cells.

Once again, mechanistic responses (phosphorylations, cyclin level changes, and

ROS generation) to these varied estrogens were documented to be very rapid, supporting the notion that important tumor-altering effects can occur, or at least be initiated, via nongenomic signaling mechanisms. This was supported by our demonstration that membrane versions of these receptors are present in these tumor cells. Our present and recent 287, 328, 329, 331, 343, 344, 353, 356, 399 studies continue to support conclusions about the ability of XEs to imperfectly mimic physiological and pharmaceutical estrogens, as well

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as their unique patterns of mechanism engagement and ER requirements 286, 287, 328, 329, 343,

344, 353, 356, 399, 400. Because of these differences, it is important to consider the potential effects of each XE individually at its typical culturally- or environmentally-relevant levels, to determine what exposure advice these mechanistic studies may point to. Given that some of these compounds can have profound stimulatory effects on prostate cancer cell numbers (particularly the soy-related phytoestrogens coumestrol and genistein, and especially in late-stage tumor cells), it may be prudent to advise such patients against consuming foods that contain these phytoestrogens. On a lower priority level, resveratrol and BPA exposures may warrant similar warnings (Table 3.1). Because BPA and genistein had different effects on the number of viable cancer cells, depending on prostate cancer stage, patients having recurring or long-term tumors may need different exposure advice. Because cell growth-promoting mechanisms receive stimulation via different ER subtypes depending upon the compound, it may be prudent to recommend blocking of these effects via all of these receptors. Alternatively, testing of a patient’s individual tumor receptor profiles may allow for tailoring of therapies with antagonists for individual receptor subtypes.

Our initial hypothesis was that some of these alternative dietary estrogens might fulfill the hoped for anti-tumor signaling and cell growth effects. It appears that this is not the case, and the best estrogen to mediate tumor cell killing effects is E2, profiled in more detail for these mechanisms (and others) in our previous report 358. Taken together, these findings should have profound implications for dietary recommendations for prostate cancer patients, as well for as the development of ER-specific treatments to shrink tumors or slow tumor progression.

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Chapter 4: Conclusions and Future Directions

MAJOR CONCLUSIONS FROM OUR STUDIES

 Showed for the first time membrane estrogen receptors (α, β, GPR30) in early- and late-stage prostate cancer cell lines  Identified several direct, non-genomic cellular pathways that are rapidly activated by estrogens, both physiological and exogenous, which contribute to the overall

control of prostate cancer cell numbers  Delineated the differences in signaling and proliferative responses between early- and late-stage prostate cancer cells to estrogenic therapies, dietary estrogens, and environmental contaminants  Characterized the membrane estrogen receptors involved in propagating non- genomic signals after stimulation with estrogens  Identified potential cellular signaling targets and estrogenic compounds for future

study and prostate cancer therapies

The interconnected web of rapid, estrogen-induced cell signaling is capable of producing a myriad of responses in prostate cancer cells. We have also seen that the numerous mechanisms and pathways activated in our two cell lines must be taken into consideration as a whole, not as individual causes of actions. There were no single pathways that completely dominated the fate of the prostate cancer cells, but there were several signals that strongly indicated whether a cell would survive, proliferate, or die. This was true regardless of the stage of the prostate cancer cells (LAPC-4 vs PC-3), though the ER controls governing later stage cancers exhibit less influence than their early counterparts. Most importantly, we have shown novel mechanisms for direct controls over prostate cancer cells, independent of central control alterations in serum

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hormone levels. These findings should be useful in the identification and development of effective therapies for prostate tumors, as well as the recognition of potential risks to patients with existing carcinomas.

Many past studies have also observed opposing effects of E2 and DES, mainly on fetal development 401, but these opposing effects could also still be applicable to disease progression. Several assay results from chapter 2 showed opposing effects due to E2 or

DES treatment. This may be due to E2 and DES having different binding affinities toward ERs - DES and other similar synthetic estrogens show greater binding affinities (2-3

299, 402 times higher) for ERs than E2 . In addition, DES showed a very slow dissociation

403 from ERs, similar to E2 . The different strength and stability of DES’s interactions with ERs may therefore convey a different message to the cellular machinery and signaling pathways, compared to E2. Rapid effects initiated from the membrane ERs have the ability to influence longer, genomic actions of the cell (although not all nongenomic effects have a nuclear endpoint). This division of cellular responses affords multiple layers of control to the cell in response to external stimuli. Duration, strength, and localization of signals can help determine the type of cellular response elicited, such as with the activation of ERK and apoptosis 404. The length of these activations can then influence the genes that are induced in responses 114. In addition, many transcriptionally active proteins are activated and regulated through signals from mERs, thus providing a junction between nongenomic and genomic signals 197. It is likely that these same categories of events are occurring in our two cell lines. Our experiments do not directly investigate the long-term effects of nongenomic signals, so further experiments would be necessary. A possible target to study would be Elk-1, which we have observed to be rapidly activated in GH3 cells 193. An effective assay to test this hypothesis would be to develop a transcription factor- specific blocker, such as those for STAT3 and STAT5 405, 406, disrupt the activity of a

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rapidly activated transcription factor, such as Elk-1, and then measure the outcome on viability. The data from our xenoestrogen studies provided insight into possible dietary considerations for prostate cancer patients, but some results between assays appeared to conflict. For example, resveratrol slightly increased prostate cancer cell numbers in both cell lines, yet results from the p-cyclin and total cyclin assays would have suggested that this XE had the ability to kill or prevent the proliferation of cells. It is clear therefore that the scope of our experiments is limited, and does not fully examine all the rapid signaling events initiated by XE stimulation that can affect cell number. As we have mentioned before, other pathways that we did not examine are likely to be contributing to the overall ability of resveratrol to slightly increase cell numbers, and that our current assay results alone do not explain how the XE works. Additional pathways, such as through Akt/PKC, may offer additional insight. The decline in male estrogen levels with age has been well documented lately 293,

294, and this decrease in hormone may account for the increased chance for prostate cancer development. Additionally, the development of the prostate in fetuses is heavily

407 influenced by estrogens . Our results from chapter two indicate that E2 is capable of controlling prostate cancer cell number at physiological concentrations. However, the

-16 lowest concentration of E2 (10 M) had no effect on cell numbers, suggesting a lower limit to possible sub-physiological E2 levels. Further studies to investigate this regulatory role of E2 are warranted. One of the largest drawbacks to our results is that the findings are currently limited to in vitro cell line models. Future studies would have to be extended to an in vivo model, to determine if other regulatory factors in an animal will influence estrogens’ actions on prostate tumors. Cell cultures are limited to a single type of cell, and do not represent the complexity of cell types in the prostate or other forms of regulation present in the body. Our findings are also currently only applicable to already developed prostate

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cancer cells. There may be differences in responses and signaling pathway stimulation regarding initial development of prostate tumors (carcinogenesis). Targeted drug therapy is an emerging interest in the clinical and pharmaceutical industry, and identification of cellular targets, such as ERs or signaling nodes, may provide an additional alternative to radical surgery or chemotherapy. Additionally, it will be interesting to study the interaction between phytoestrogens and environmental contaminants with physiological estrogens in inhibiting or enhancing tumor growth. Some of these potential future studies are discussed as follows.

In vivo model

The results of this study have helped develop a clearer picture for direct estrogenic effects on prostate cancer cells. However, these findings must currently be limited to in vitro systems of prostate cancer. The tumor microenvironment of the originating or metastatic site contains many supporting cells that are necessary for the development and maintenance of cancerous cells 408-410 (Figure 4.1). Therefore, expanding upon these findings in an in vivo model is necessary. We have shown that estrogens have a direct, nongenomic effect on prostate cancer cells, and it is likely they can also affect neighboring non-cancerous cells of the tumor microenvironment. In fact, the interplay between tumor cells and microenvironment has been observed to be necessary for many tumor functions, particularly in metastasis and migration 411, 412.

Skeletal metastasis in prostate cancer patients accounts for 90% of reported deaths, suggesting that the microenvironment of the skeletal system is amenable to metastatic prostate cancer cells 413, 414. Typical components of the tumor microenvironment include endothelial cells, fibroblasts secreting extracellular matrix proteins, muscle cells, immune cells, and of course tumor cells 415. The cellular responses of these non-cancerous cells in

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the tumor microenvironment may induce paracrine signaling that can increase or decrease the growth of tumor cells.

Figure 4.1. Various components of the prostate tumor microenvironment. Numerous types of cells support a prostate tumor cell and its ability to proliferate and eventually metastasize. (1) Support cells that develop from prostate epithelial stem cells. (2) Connexins may play a role in the further development of prostate tumors (Figure from Czyz et al., 2012).

Estrogenic effects on individual components of the tumor microenvironment have already been shown. E2 modulates the levels of nitric-oxide synthase and nitric oxide in endothelial cells, which is vital in angiogenesis 416, 417, as well as endothelial cell

418 migration . Fibroblast migration and proliferation can be controlled by E2 as well, in

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benign and tumor cells 419, 420. ERα in the fibroblasts of the prostate cancer microenvironment also regulate the ability of prostate cancer cells to invade the surrounding tissue, as well as the amount of extracellular matrix molecules produced 421.

The smooth muscle cells of the prostate stroma proliferate in response to E2 and show an increase in cell cycle proteins like cyclin D1 422, which can be mediated by ERα 423. Immune cells, such as macrophages, are also associated with the prostate tumor stroma, and proinflammatory or anti-inflammatory cytokine production is usually decreased by

228, 424 E2 . Therefore, understanding the interplay of these cells and molecules should be the next step toward elucidating rapid, direct estrogenic responses, and in particular, through mERs.

Figure 4.2 Primary steps in establishing a xenograft prostate cancer model in SCID mice. The major steps in developing a prostate cancer xenograft model in SCID mice, as developed by Lawrence and colleagues. The addition of stromal cells and location of implanted cells closely mimics normal tumor physiology (Figure from Lawrence et al., 2013).

Our laboratory has previously done similar in vivo experiments in female F344 Sprague-Dawley rats, measuring the induction of pituitary tumors after exposure to phytoestrogens over an eight week period 425. The use of severe combined immunodeficiency (SCID) mice with prostate cancer xenografts and silastic implants containing E2/DES/XEs is of particular interest. Mice do not develop prostate cancer naturally, and thus provide a solid platform to study prostate cancer, without concerns

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about background 426. Prostate cancer models using SCID mice and estrogen treatments have already been developed, but those studies focus on the systematic alteration of hormone levels and metastatic potential, as opposed to the localized signals initiated by tumor cell ERs 427, 428. The method developed by Lawrence and colleagues provides an optimal platform upon which to study estrogenic responses on tumor cells within a tumor microenvironment and with supporting cells (Figure 4.2). In short, an in vivo model for our study would entail xenografts of LAPC-4 or PC-3 prostate cancer cells implanted subcutaneously or orthotopically 429, 430 into male SCID mice. Alternatively, cells could be injected via the tail vein, though that model would be more appropriate for studying metastasis 431. Tumor cells introduced to the mouse in this protocol maintain their regular characteristics and show the ability to proliferate normally. Additionally, prostate tumor cells are supplemented with the appropriate stromal cells, further mimicking physiological conditions and signaling 432. This model is particularly strong due to the limitation of studies to localized tumor cell interactions with the microenvironment. This however means that studying the ability of cells to metastasize in this model is not viable. Also, due to a lack of or severe depression of the immune system function in SCID mice, studying the interaction of tumor cells with macrophages and immune cells is not possible 433. LAPC-4 prostate cancer cells growth would be supplemented with DHT during the seeding period, as they are androgen-dependent cells. Additionally, LAPC-4 cells can become androgen-independent if deprived of androgens 29.

Following initial seeding and implantation of prostate cancer cells, the mice are left untreated for 6-8 weeks, allowing growth of the xenograft and supporting cells. Once the xenografts have been given the chance to establish, measurements in tumor size would be made before subcutaneous silastic implants, containing cholesterol, E2, and DES, would be inserted. Mice would be treated for an additional three weeks, after which

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tissue would be harvested and analyzed for changes in tumor size, ER levels, and cell cycle proteins levels, such as cyclin D1 and p16INK4A. Live tissue can also be assayed using our laboratory designed plate immunoassay, as long as assay conditions are properly optimized, as we have developed for rat brain slices, human blood cells, and rat hemi-pituitary gland (unpublished protocols). The tissue and cells obtained from the mouse would still harbor many components of the normal tumor microenvironment. Prostate tumor tissue obtained in this manner could be assayed for rapid phosphorylation of MAPKs and cell cycle proteins. An alternative to mimic tumor and paracrine signaling would be to collect media from an estrogen-treated subset of cells (tumor associated fibroblasts for example) and then treat tumor cells with this conditioned media, in conjunction with an estrogen 434.

Receptor targeted agonists (Acadia ERβ agonists)

Direct targeting of specific ER subtypes has become an area of interest in estrogen-mediated/affected cancers. In prostate cancer, ERβ has drawn a lot of attention due to its predominance in the prostate gland and the tumors arising from it, as well as the potential inhibitory role in prostate cancer cell growth 176, 435. To this effect, we evaluated five ERβ specific agonists from Acadia Pharmaceuticals (San Diego, CA) and measured prostate cancer cell viability. LAPC-4 and PC-3 prostate cancer cells were treated with each ERβ agonists for three days, then a MTT cell viability assay was conducted.

Agonizing ERβ in PC-3 cells did not have any cell killing effects, similar to what we have seen when the same cells are treated with DES (Figure 4.3). In fact, there were several concentrations where all agonists except 74131 increased viability, suggesting that agonizing ERβ in PC-3 cells is not a viable therapeutic. On the other hand, LAPC-4 cells showed different responses to the five agonists. Agonist 269623 may be of particular interest due to the 15-20% decrease in viable cells (10-13-10-9M), while 270957

94

-6 at 10 M may also warrant further study (E2 elicited a 25-30% decline in viable cells in LAPC-4 cells). At lower concentrations, 270957 stimulated growth, and the three other remaining agonists also showed the ability to increase cell viability. Further studies into the key signaling pathways and cell proliferation mechanisms are warranted to identify if these compounds interact with prostate cancer cells similarly to E2 or DES.

Figure 4.3 Number of viable prostate cancer cells following Acadia ERβ agonists. MTT assay results after 3 day treatment with agonists in LAPC-4/PC-3 cells.

Given the ERβ rich environment of the prostate and prostatic tumors 435, it is surprising that only two of the agonists were able to reduce cell numbers, and that those actions were limited to early-stage prostate cancer cells. As discussed in Chapter 3, this increased ability to kill LAPC-4 cells may be due to the significant differences in ER levels. Higher expressions levels of ERβ may then provide estrogens and estrogen mimetics a better opportunity to induce cell killing effects 436. Estrogenic stimulation of

95

supporting tumor cells may also play a role in the effectiveness of ERβ agonists, as discussed previously. Based on these preliminary results, the clinical application of using selective ERβ agonists could provide therapeutic options for prostate cancer patients. Targeting the ERβ-rich cells of the prostate may prove a more direct and effective measure, as opposed to the standard therapy of LHRH agonists and antagonists, anti-androgens, and surgery, which can have profound effects on a patient’s hormone levels. Further investigations are necessary however, particularly with an in vivo model, to study the potential side-effects. ERβ is found in various other tissues of the body, including serving major regulatory roles in the nervous system and heart 437, 438.

Other potential studies and directions

Our studies focused on the effects of estrogens on established prostate cancer cell lines. While our results reveals the mechanisms involved in killing or promoting growth of tumor cells, it does not explain the progression of prostate cells from a nonmalignant state to a malignant state. There are many studies focused on the prevention of prostate tumors, particularly through the use of phytoestrogens like genistein 439, 440. Studies on prostatic stem cells could also further elucidate how prostatic tumors form. Epigentic modifications to these pioneer cells has become an emerging field of study (Figure 4.4).

96

Figure 4.4. Improper epigenetic regulation of normal prostate cells may lead to an increased chance for prostate cancer. Improper regulation of DNA methylation may cause the suppression of important regulatory genes (Figure from Cooper & Foster, 2008).

Increased environmental exposure to estrogen-like compounds, such as BPA may be increasing the susceptibility of prostate diseases or deformities via inappropriate methylation patterns 300, 441, 442. There are numerous genes that show hypermethylation in prostate cancers, including INK4A and APC, both of which are known tumor suppressors

443. Methylation of the glutathione S-transferase-pi gene is found in over 90% of prostate cancers, but not in normal prostate cells 444. Hypermethylation of the E-cadherin gene may even promote the further development of cancers to a metastatic state 445. Interestingly, ESR1 and ESR2 are hypermethylated, ,preventing ER expression, and strengthening the regulatory role for estrogens and ERs in the prostate and associated diseases 443.

97

The estrogenic signals exerted on prostate cancer cells are not restricted to only one compound, as in the current paradigm for our in vitro model. As already discussed above, many signals are propagated to prostate cancer cells from the tumor microenvironment and the endocrine system. Specifically, there are other estrogens and mimetics circulating throughout the body, and they often affect the same targets, such as ERs. Therefore, a more physiological and comprehensive study would be to elucidate the effects of estrogenic mixtures on prostate cancer cell survival, both in vitro and in vivo.

Endocrine responses exerted by other physiological hormones or estrogens (physiological or XE) on the tumor and entire body could interact or interfere with the signals initiated at membrane and nuclear ERs of prostate cancer cells. These combinations of estrogens and their responses may not always cause additive or subtractive responses, and may in fact be synergistic, as we have previously seen in our laboratory 239, 276, 328. In conclusion, the application of data from this dissertation can be expanded upon by investigating tumor microenvironment and systems that closely mimic physiological conditions, particularly in live animals. Further knowledge on the important estrogen- regulated cell signals and mechanisms may increase and improve the arsenal of therapeutics available for patients and clinicians to choose from. Improving and creating more specific therapies to target prostate tumor cells, such as ERs or their downstream targets, may also help reduce unwanted side-effects from more traditional approaches such as radical surgery (prostatectomy and orchiectomy) and broad target chemotherapeutics. Furthermore, developing an understanding of how prostate tumors develop, as well as the other cellular messages they require will provide a platform for preventative, rather than reactionary measures.

98

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Vita

Luke Yun-Kong Koong was born January 19, 1986 in Starkville, Mississippi to Kai Siak Koong, Ph.D. and Lai C. Liu, Ph.D. He graduated from McAllen Memorial High School (McAllen, Texas) in 2004, then attended The University of Texas – Pan American in Edinburg, Texas. He graduated in 2008 with a B.S. in Biology, minoring in chemistry and communication-journalism. He entered the graduate program at The University of Texas Medical Branch in 2008 and joined the laboratory of Cheryl S.

Watson, Ph.D. in 2009. His research focuses on the direct effects of estrogens on the cell numbers of early- and late-stage prostate cancer cells, the role of membrane estrogen receptors in prostate cancers and the mechanisms by which xenoestrogens can utilize the same cellular pathways. This research has been applied to toxicology and environmental health in regards to prevalence of exposure to exogenous estrogens.

Education

July 2014-Ph.D.-Cell Biology Graduate Program, The University of Texas Medical Branch, Galveston, TX Dissertation: The Direct Effects of Estradiol & Several Xenoestrogens on Cell Numbers of Early- vs. Late Stage Prostate Cancer Cells

May 2008-B.S. Cum Laude, Biology, Minor in Chemistry & Communication-Journalism,

The University of Texas – Pan American, Edinburg, TX

Publications

154

Koong LY & Watson CS (2014). Rapid, nongenomic signaling effects of several xenoestrogens on early- vs. late-stage prostate cancer cell proliferation (In Submission). Endocrine Disruptors.

Koong LY & Watson CS (2014). Direct estradiol and diethylstilbestrol actions on early- vs. late-stage prostate cancer cells. The Prostate. 74(16):1589-603.

Watson CS, Jeng Y-J, Bulayeva NN, Finnerty CC, Koong LY, Zivadinovic D, Alyea RA, Midoro-Horiuti T, Goldblum RM, Anastasio NC, Cunningham KA, Seitz PK & Smith TD. (2014). Steroid Receptors, Methods in Molecular Biology. 1204:123-33.

Motamedi S, Shilagard T, Edward K, Koong LY, Qui S, & Vargas G (2011). Gold nanorods for intravital vascular imaging of preneoplastic oral mucosa. Biomedical Optics Express. 2(5): 1194-1203.

Koong KS, Koong LY, Liu LC, & Yu M. (2005) An examination of selected drug availability at online pharmacies. International Journal of Electronic Healthcare. 1(3), 291-302.

Proceedings and Abstracts

Koong LY & Watson CS. Direct and rapid estrogenic signaling mechanisms that reduce cell numbers in early- vs. late-stage prostate cancer cells. Endocrine Society. Chicago, IL. June 2014.

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Koong LY & Watson CS. Reduction in cell numbers in early- vs. late-stage prostate cancer cells via direct and rapid estrogenic signaling mechanisms. Cell Biology Graduate Program Symposium, UTMB. Galveston, TX. March 2014.

Koong LY & Watson CS. A Direct Signaling Mechanism Involved in Estradiol and Diethylstilbestrol Effects on Androgen-Dependent and Androgen-Independent Prostate Cancer Cell Viability. Endocrine Society. Houston, TX. June 2012.

Koong LY & Watson CS. Estrogen-mediated signaling differences related to cell viability in androgen-dependent and androgen-independent prostate cancer cells. Society of Toxicology. San Francisco, CA. March 2012.

Koong LY & Watson CS. Rapid, non-genomic signaling in prostate cancer cells via membrane estrogen receptors. Society of Toxicology. Washington, DC. March 2011.

Motamedi S, Shilagard T, Koong LY, & Vargas G. (2010). Feasibility of using gold nanorods for optical contrast in two photon microscopy of oral carcinogenesis. Proc. SPIE Vol. 7576, 75760Z. San Francisco, CA. Jan. 2010.

Jeng Y-J, Guptarak J, Koong LY, & Watson CS. Effects of physiological and nonphysiological estrogenic compounds on proliferation mechanisms in GH3/B6/F10 rat pituitary tumor cells. UTMB Cancer Center Day. Galveston, TX. May 2010.

Permanent address: 7424 El Cielo, Galveston, Texas 77551 This dissertation was typed by Luke Yun-Kong Koong.

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