The Distinct Role of Cyclooxygenase-2 in Prostate and Bladder Carcinogenesis

Home , FMO2, Leptin receptor

THE DISTINCT ROLE OF CYCLOOXYGENASE-2 IN PROSTATE AND BLADDER CARCINOGENESIS

DISSERTATION

Presented in Partial Fulfillment of the Requirement for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Xingya Wang, M.S.

* * * * *

The Ohio State University 2007

Dissertation Committee: Approved by

Dr. Russell D. Klein, Co-adviser

Dr. Steve K. Clinton, Co-adviser Co-adviser Dr. Mark L. Failla, The Ohio State University Dr. Martha A. Belury Nutrition Graduate Program

ABSTRACT

Overexpression of cyclooxgenase-2 (COX-2) and prostaglandin E2 (PGE2) have been demonstrated to play a significant role in the tumorigenesis of several human cancers. Inconsistent and controversial reports on the expression and activity of COX-2 in prostate cancer raised the question of whether COX-2 plays a pivotal role in prostate carcinogenesis. In contrast, there is more consistent support for a positive link between

COX-2 overexpression and bladder carcinogenesis. However, the exact role of COX-2 overexpression and how changes in gene regulation alter in urinary bladder carcinogenesis has not been defined.

I first examined the effects of COX-2 inhibition on prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) model by either feeding TRAMP mice a diet containing non-steroidal anti-inflammatory drugs or by genetic disruption of the expression of the COX-2 gene in TRAMP mice. Our results demonstrate that neither

NSAIDs nor genetic disruption of COX-2 inhibited tumorigenesis of TRAMP prostate.

Interestingly, I found that the expression of COX-1 and COX-2 mRNA, protein, and activity (ability to synthesize PGE2) dramatically decreased during TRAMP prostate carcinogenesis. These observations suggest a mechanism whereby TRAMP carcinogenesis may be resistant to COX-1/COX-2 changes. I further found that treatment with exogenous PGE2 modulated MAPK/Erk and Akt phosphorylation, and induced

secretion of Vascular Endothelial Growth Factor (VEGF) through PGE2 receptor

EP2-mediated cAMP pathway in prostate cancer cells in vitro.

I also used a gene expression array analysis to determine the effects of COX-2

overexpression on gene expression profile in urinary bladder of a COX-2 overexpression

transgenic mouse model (BK5.COX-2). My results revealed that genes associated with

Immune/Stress Response and Cell Cycle/Proliferation systems were significantly

overexpressed in the BK5.COX-2 transgenic mouse bladder. Upregulated Cell

Cycle/Proliferation genes included growth factors (Ereg and IGF-1) and cell cycle genes

(Mki67, Cdc2a, and Top2a). Relevant downregulated genes included three transforming

growth factor (TGF) beta related genes (Tgfb2, Tgfb3, Tgfbi), and the anti-angiogenic

gene thrombospondin 2 (Thbs2). The growth factor epiregulin was the most highly

induced gene among those validated by qRT-PCR. I further demonstrated that epiregulin

mRNA was directly induced by PGE2 treatment in the wild type mouse bladders. In addition, epiregulin increased cell proliferation and activated MAPK/Erk activity in bladder cancer cells.

In summary, my results suggest that COX-2 may not play a significant role during prostate carcinogenesis in the prostate TRAMP model. However, overexpression of

COX-2 may play a pivotal role in murine bladder carcinogenesis.

iii

DEDICATION

This dissertation is dedicated to my advisor Dr. Russell D. Klein and his family

IN MEMORIUM (1962-2006)

Dr. Russell D. Klein passed away on December 1, 2006 after a year long battle with leukemia at the James Cancer Hospital. He will be greatly missed by his colleagues and students as a superb scientist, mentor, and gentleman.

ACKNOWLEDGMENTS

I deeply thank my advisor, Dr. Russell D. Klein, for his guidance, encouragement, dedication to excellence in research and teaching, his sacrificial commitment to students.

He taught me how to conduct research beyond all my expectations. His faith will always lift me up and encourage me to move on whenever I encounter any difficulties.

I would like to express my sincere gratitude to Dr. Mark L. Failla for his consistent

support and encouragement over the years, for his critical advice and scientific insights

during manuscprit and dissertation preparation, and for his dedication to each one of us in

the Klein laboraotory. I am also very grateful to Dr. Steven K. Clinton for his intellectual

challenge, scientific insights, dedication to teaching in all the Nutritional Biosystems of

Cancer Meeting, and his continued support and care to me and others in the Klein

laboratory during the past years. I am also grateful for all the facilities and reagents Dr.

Clinton provided for me to conduct some experiments. I would like to thank Dr. Martha

A. Belury for giving me valuable advice on my dissertation research, and for her

continued support and care.

I would like to express my sincere gratitude to Dr. Susan M. Fischer, our collaborator at M.D. Anderson Cancer Center, for her contintued guidance and critical advice for my research dissertation, and in particular, for her helps with many things

during the past one year. I am very grateful to Jennifer Colby for conducting the NSAID

feeding experiment and all other hard work she did at M.D.Anderson Cancer Center. I thank Dr. Robert Newman and his postdoc fellow Dr. Peiying Yang for analyzing the level of eicosanoids in the TRAMP/COX-2 study.

I am very grateful to Robert Rengel for his assistance with mouse genotyping, colony maitanence, microdissection, and other hard work. I would like to thank

Kimberly Carter at Dr. Clinton’s laboratory for teaching me histology and immunohistochemistry, and for her friendship and care. Special thanks to Valerie DeGroff for her kindness, friendship, care, generosity and help with all lab-related issues.

I would like to thank members from Klein laboratory: Robert Rengel (again),

Kristin Keatly, Justin Smolinsky and Tiffany Reineman for their continued friendship, care and help through these years.

I would also like to extend my appreciation to many faculty and staff in the

Department of Human Nutrition for their support during the past years. I would like to thank all present and former graduate students in our department, particularly Hong Wang,

Lifen Liu, and Shihua Wang, for their friendship all these years.

I also wish to thank Brenda Klein and her family for their continued care and support, for their faith and strength during the past one and half year.

Finally, I would like to thank my parents for their endless love and support, for taking care of my son during the past four years. I thank my sister and my brother for their care and encouragement. I thank my husband, Jiansen Niu, for his continued love and support with many things, including cooking, shopping and fixing computer problems. I especially thank my beloved son, Yucheng Niu, for being such a wonderful and lovely little boy, for the joy and laugh he brings to our family each day.

vii

VITA

Sep 25, 1975………………… Born in Lanzhou, P. R. China

1994-1998…………………… Bachelor of Science, Beijing Agricultural University

2001-2003…………………… Master of Science, The Ohio State University

2003-2007…………………….Graduate Research Associate, The Ohio State University Interdisplinary program of Nutrition (OSUN)

PUBLICATIONS

1. Wang X, Clubbs E, Bomser, JA. Effects of genistein on extracellular signal regulated kinase signaling and proliferation in human prostate epithelial cells. Journal of Nutritional Biochemistry, 2006 Mar;17 (3):204-10. Epub 2005 Sep 15.

2. Sargeant A, Klein RD, Rengel R, Clinton SK, Kulp S, Kashida Y, Yamaguchi M, Wang X, Chen CS. Chemopreventive and bioenergetic signaling effects of PDK1/Akt pathway inhibition in a transgenic mouse model of prostate cancer. (Accepted, Toxicologic Pathology, Dec., 2006)

3. Wang X and Klein RD. Prostaglandin E2 induces Vascular Endothelial Growth Factor secretion in prostate cancer cells through EP2 receptor mediated cAMP pathwayJun. Mol Carcinog. 2007 Apr 11; [Epub ahead of print].

FIELD OF STUDY

Major Field: The Ohio State University Nutrition Program

viii

TABLE OF CONTENTS

Abstract……………………………………………………………………………. .… ....ii Dedication………...………………………………………………………………... …....iv In memorium (1962-2006)……...…………………………………………………………v Acknowledgements………………………………………………………………… . ..…vi Vita………………………………………………………………………………… ...viii List of tables……………………………………………………………………………xi List of figures……………………………………………………………………………xii

Chapters: 1 Introduction……...…………………………………………………………………...1 2 Literature review…………………………………………………………………...5 2.1 Prostate and bladder cancer……………………………………………………5 2.1.1 Etiology and pathogenesis of prostate cancer……………………………...5 2.1.2 Etiology and pathogenesis of bladder cancer…..………………………....7 2.2 Diet and Cancer……………...……………………………………………….12 2.2.1 Dietary factors and carcinogenesis…..……...……………………………12 2.2.2 Dietary factors and prostate cancer...…..…………………………………13 2.2.3 Dietary factors and bladder cancer...…..…………………………………15 2.2.4 Fatty acids, their metabolites and carcinogenesis………...………………17 2.3 Cancer chemoprevention………………………………………………………21 2.3.1 Prostate cancer chemoprevention…..…………………………………….23 2.3.2 Bladder cancer chemoprevention…..…………………………………….25 2.3.3 The use of NSAIDs as prostate and bladder cancer chemoprevention…...26 2.4 Molecular pathway in human prostate and bladder cancer…………………..27 2.4.1 Hallmarks of cancer.……………………………………………………...27 2.4.2 Molecular pathway in prostate cancer….………………………………...29 2.4.3 Molecular pathway in bladder cancer….………………………………...30 2.5 Cyclooxygenase genes and enzymes……….………………………………..33 2.5.1 Isomers of cyclooxygenase………….….………………………………...33 2.5.2 Cellular localization of COX isozymes...………………………………...34 2.5.3 Regulation and tissue expression of COX isozymes...... ………………...36 2.5.3.1 Tissue expression...... ………………...36 2.5.3.2 Regulation of COX gene expression...... ………………...37 2.6 COX-2 and cancer……………………………………….…………………..39

2.6.1 COX-2 and prostate cancer…………..…………………………………...42 2.6.2 COX-2 and bladder cancer…………..…………………………………...44 2.7 Family of PGs and their role in cancer…….…………….…………………..46 2.7.1 Family of PGs………………………..…………………………………...46 2.7.2 PGE2 and prostate cancer…...………..…………………………………...47 2.7.3 PGE2 and bladder cancer…...………..…………………………………...48 2.8 Mechanism of PGE2 action………….….……………….…………………..49 2.8.1 PG receptors...………………………..…………………………………...49 2.8.2 EP receptor and cancer…..…………..…………………………………...52 3 Prostaglandin E2 induces vascular endothelial growth factors secretion in prostate cacner cells through EP2 receptor mediated pathway………….……………………57 3.1 Abstract…..…………………………………………………………………58 3.2 Introduction…………………………...………………………………………59 3.3 Materials and methods………………………………………………………..62 3.4 Resutls……….………………………………………………………………..67 3.5 Discussion………………………………………………………………...…..84 4 Prostate tumorigenesis in TRAMP mice is not inhibited by NSAIDs or genetic disruption of cyclooxygenase-2 expression…………………...……………………91 4.1 Abstract…..…………………………………………………………………92 4.2 Introduction…………………………...………………………………………93 4.3 Materials and methods………………………………………………………..96 4.4 Resutls……….………………………………………….……………………103 4.5 Discussion…………………………………………………….…………...…127 5 Overexpression of cyclooxygenase-2 in the mouse urinary bladder induces the expression of immune and cell proliferation related genes…….…………………137 5.1 Abstract…..………………………………………………………………138 5.2 Introduction…………………………...……………………………………139 5.3 Materials and methods……………………………………………………….142 5.4 Resutls……….…………………………………………………….…………149 5.5 Discussion………………………………………………………….……...…168 Epilogue……………………….…………………………………………..175 Bibliography……………………………………………………………………………178 Appendix………………………………………………………………………………205

LIST OF TABLES

Table Page

2.1 Cyclooxygenase 2 is overexpressed in several premalignant and malignant conditions…………………………………………………………………………...40

2.2 Prostaglandin receptor agonists and antagonists………………….………………...56

4.1 Incidence of poorly-differentiated (PD) carcinomas and metastases (Mets) in TRAMP mice fed diets containing cyclooxygenase inhibitors……………………111

4.2 Incidence of poorly-differentiated carcinomas (PD) and metastases (Mets) in TRAMP mice either wild-type (WT) or homozygous mutant (KO) for the COX-2 gene.…………………………………………………………………………...... 120

5.1 Histopathologic analysis of the phenotype of urinary bladders in BK5.COX2 transgenic mice and wild-type mice at different ages.…………………....………153

5.2 Expression Analysis Systematic Explorer (EASE) analysis of top 100 upregulated genes that associated with Biologic Process ontology.…………..……………….158

5.3 Expression Analysis Systematic Explorer (EASE) analysis of top 100 upregulated genes that associated with Molecular Function ontology.………… …………….159

5.4 Expression Analysis Systematic Explorer (EASE) analysis of top 100 downregulated genes.…………...…………………………………………………160

LIST OF FIGURES

Figure Page

2.1 Contemporary model of prostate cancer progression…….………………………....8

2.2 T staging of bladder cancer…………………….....………………………………...10

2.3 Dual tract concept of human bladder carcinogenesis………………………………11

2.4 Age-adjusted prostate cancer death rates per 100, 000 population in various nations around the world……………………………………………………………………14

2.5 The arachidonic acid cascade……………………….…….………………………...19

2.6 Multistage carcinogenesis: processes and prevention strategies…………………...22

2.7 The hallmarks of cancer- six essential pathways underling the molecular mechanisms of tumorigenesis in the world…………………………………………28

2.8 Model for bladder cancer progression showing the molecular pathways of tumorigenesis in the world…………………………….……………………………31

2.9 Crystallographic structures of ovine COX-1 (left) and murine COX-2 (right) homodimers...………………….……………………………………………………35

2.10 Regulation of cyclooxygenase 2 (COX-2) in cancer………………………..……...38

2.11 Possible mechanisms of COX-2-derived PGE2 contributions to tumor development.revention strategies…………………………………………………...41

2.12 Prostaglandin synthesis and actions……...…………………………………………50

3.1 Relative mRNA expression of EP2 and EP4 receptors in PC-3, DU145, LNCaP and PrEC cells determined by qRT-PCR…………..………………………68

3.2 PGE2 and the EP2 receptor agonist CAY10399 induce VEGF protein xii

secretion in human prostate cancer cells……………………………...…………….70

3.3 PGE2 and the EP2 receptor selective agonist CAY10399 increases cAMP concentrations in PC-3, DU145 and LNCaP cells………………………………….72

3.4 PGE2 at 1 nM (A) and 10 μM (B) induces cAMP production in PC-3 cells in a time-dependent manner……..……………………………………………………73

3.5 PGE2 and CAY10399 induce phosphorylation of Erk in time-dependent manner in PC-3 cells……………………………..…………………………………75

3.6 PGE2 induces phosphorylation of Akt only in EP2- expressing PC-3 and DU145 cells………………………………………………………………………...77

3.7 Comparison of the effects of PGE2 and CAY10399 at 1 nM or 10 μM on Erk and Akt phosphorylation in PC-3, DU145 and LNCaP cells……………………....78

3.8 The MAPK/Erk and Akt signaling pathways are not involved in PGE2- induced VEGF protein secretion in PC-3 cells……………………………………..81

3.9 VEGF protein secretion is significantly increased by the adenylate cyclase activator forskolin in a dose-dependent manner in PC-3 cells….………………….82

3.10 The adenylate cyclase inhibitor 2’5’-dideoxyadenosine significantly blocks PGE2-induced cAMP production .………………………………………….83

3.11 Working model of the major signaling pathways elicited by PGE2 in human prostate cancer cells …………………...…………………………………………...90

4.1 Expression of cyclooxygenase enzymes in dorsal prostate (DP) tissue is reduced in TRAMP mice compared to wild-type (WT) mice…….………………104

4.2 COX-2 immunohistochemical staining of serial sections from TRAMP dorsal prostate (DP) tissue with prostatic intraepithelial neoplasia (PIN), moderatelydifferentiated carcinoma (MD), and poorly differentiated (PD) tumors. ………………………………………………………….….………………………106

4.3 Prostaglandin E2 (PGE2) levels in dorsal prostate (DP) tissue are reduced in 10 wk old TRAMP mice compared to wild-type (WT) mice…………………..…107

4.4 Histopathologic criteria used to score prostatic glandular ducts and metastases in tissues from TRAMP tumor studies. ………..……………………..109

4.5 Kaplan-Meier survival analysis for the incidence of palpable tumors..…………..112

xiii

4.6 Treatment of TRAMP mice with cyclooxygenase inhibitors does not significantly alter prostate wet weights of prostate lobes. …………….………….113

4.7 Treatment of TRAMP mice with cyclooxygenase inhibitors does not significantly alter prostate carcinogenesis. ………………..…………...………....115

4.8 PGE2 levels in dorsal prostate (DP) tissue from 10 wk old TRAMP mice fed an AIN-76A semi-purified diet (control), or 500 ppm celecoxib, 1500 ppm celecoxib, or 4 ppm indomethacin for two weeks. ……...……………………….…………..117

4.9 PGE2 levels are significantly reduced in 10 wk old dorsal prostate (DP) tissues of the COX-2 knockout TRAMP mice (COX-2 KO) compared to COX-2 WT TRAMP mice (COX-2 WT)…………………………….….………………..119

4.10 Kaplan-Meier survival analysis for the incidence of palpable tumors. ...………..121

4.11 Wet weights of prostate lobes in COX-2 knockout TRAMP (KO) compared to COX-2 WT TRAMP (WT) mice.sied. ………………..……………………….124

4.12 Incidence of pathologic lesions in the glandular ducts of prostatic lobes from TRAMP mice with the indicated genotypes (COX-2 WT and COX-2 KO).……………………………………………………………….………………125

5.1 Expression of cyclooxygenase enzymes and PGE2 synthesis in bladder tissues of 10 wk old BK5.COX2 (Tg) mice compared to wild-type (Wt) mice. …………150

5.2 Transitional Cell hyperplasia (TCH) in 10 wk old BK5.COX2 transgenic mouse urinary bladder………………………………….….………………………154

5.3 Histopathology of mouse urinary bladders from older (20-30 wk) BK5.COX2 mice………………………………………………………………..…155

5.4 qRT-PCR validation of six genes of interest that were significantly changed in 10 wk old BK5.COX2 transgenic mice (Tg) compared to wild-type (Wt) mice by Gene Array analysis……………………………… ………..……………..………162

5.5 Expression of the six validated genes in 30 wk old BK5.COX2 (Tg) transgenic mice and age matched wild-type (Wt) mice by qRT-PCR analysis…...163

5.6 Immunohistochemistry analysis of the proliferation marker Ki67 10 wk old BK5.COX2 (Tg) transgenic mice and wild-type (Wt) mice bladders....…….……164

5.7 PGE2 at 10μM induces epiregulin mRNA expression in a time-dependent manner in bladder tissues from 10 wk old FVB wild-type mice. …….………...... 165

xiv

5.8 Recombinant hEpiregulin induces proliferation and Erk phosphorylation in bladder cancer UMUC-3 cells. ……………...……………………………………167

A.1 EP2, EP3 and EP4 are expressed in different pattern in examined human prostate cell lines……………………………………………...….………………206

A.2 Non-specificity of antibody against EP4 receptor that from Cayman Chemical….207

A.3 PGE2- and CAY10399-induced Erk and Akt activation in DU145 cells……....…208

A.4 Adenylate cyclase inhibitor 2’5’-dideoxyadenosine significantly blocks 1nM PGE2-induced secretion of VEGF in PC-3 cells. ……………..……………….....209

A.5 PGE2 levels in three prostate lobes of 10 wk old wild-type mice. …………...…..210

A.6 COX-2 immunohistochemical staining of the same section in the dorsal prostate (DP), lateral prostate (LP), and ventral prostate (VP) tissues of a 10 wk old Pb.COX-2 mouse. . ………………………………………………….………….211

A.7 PGE2 levels in three prostate lobes of 10 wk old Pb.COX-2 and wild-type mice..212

A.8 H&E stained section of ventral prostate (VP) from one mouse of a founder line of Pb.COX-2 mouse model.……………………..……………………….…………..213

A.9 Eexpression of COX-1 and COX-2 mRNA are significantly reduced in both anterior prostate (AP) and dorsal prostate (DP) tissues of the 10 wk old conditional PTEN knock mice (Tg) compared to wild-type (WT) mice.)……………………..……..214

A.10 Western blot analysis of COX-1 and COX-2 protein levels in seven prostate cell lines …………………………..………………………………………………….215

A.11 Prostaglandin A2 (PGA2), PGD2, 15-d-PGJ2 and 12-HETE levels in dorsal prostate (DP) tissues of 10 wk old wild-type (WT) and TRAMP mice. …….…………….216

A.12 Transgene construct of BK5.COX2 mouse model.……………………………….217

CHAPTER 1

INTRODUCTION

Prostate cancer and bladder cancer are the two most common malignancies in the

adult male urogenital system. Prostate cancer represents the most common malignancy

and the second leading cause of cancer death in US men. Bladder cancer is the fourth

most common malignancy in men and the ninth most common type of cancer in women

in the US. The identification of new molecular targets for the prevention and treatment of prostate and bladder cancer is a potential means to reduce the development and progression of both malignancies. Cyclooxygenase enzymes, COX-1 and COX-2, catalyze a rate limiting step in the synthesis of prostaglandins from arachidonic acid.

COX-1 is generally considered to be constitutively expressed in nearly all tissues and has

functions in maintaining tissue homeostasis, whereas COX-2 is inducible by a number of

factors (1). Overexpression of COX-2 in several cancers, including colon, breast, lung,

bladder, skin and pancreas has been observed in numerous studies (1). Prostaglandins

have been reported to induce cell proliferation, enhance angiogenesis, and promote

invasion and metastasis of these cancers (2, 3). NSAIDs that inhibit COX activity have

received much attention as potential chemopreventive agents for these cancers. The

primary mechanism by which NSAIDs are thought to prevent cancer is through inhibition

of the synthesis of prostaglandins.

1 Numerous studies have demonstrated that COX-2 is overexpressed in transitional

cell carcinoma (TCC) of human urinary bladder (4-7). The degree of COX-2 expression

is significantly correlated with the higher tumor grade and greater depth of invasion of

TCC (8-10). A substantial number of studies demonstrated that NSAIDs inhibit the growth of bladder cancer cells in vitro (11-14). We recently demonstrated that forced expression of COX-2, under the control of a keratin 5 (K5) promoter, is sufficient to cause urinary bladder transitional cell hyperplasia (TCH), which progresses to TCC in a small percentage of transgenic mice (3). These results suggest that there is a strong association between COX-2, prostaglandin synthesis and the development and progression of urinary bladder cancer. However, the exact role and mechanisms of action for COX-2 overexpression during bladder carcinogenesis have not been well defined.

In contrast to the above, accumulating studies on the role of COX-2 in prostate carcinogenesis are inconsistent and controversial. Retrospective case-control and prospective cohort epidemiologic analyses examining the risk of prostate cancer in men consuming NSAIDs have provided mixed findings. Results from some studies suggest no protective effect of NSAIDS (15-19), whereas there are several reports of a significant reduction in prostate cancer risk attributable to frequent NSAID use (20-23). Initial reports from several research groups indicated that COX-2 was also overexpressed in human prostate cancers (24-29). More recent studies have found that COX-2 is only minimally expressed in most prostate cancers, and that elevated expression is only observed in areas of chronic inflammation or proliferative inflammatory atrophy (30, 31).

There have been relatively few reported animal studies that have tested the level of COX-2 expression and the effect of NSAIDs on prostate carcinogenesis. The TRAMP

2 model for prostate cancer is now well established and demonstrates several characteristics that make it advantageous for chemoprevention studies (32, 33). Two

groups of investigators recently reported that the COX-2 selective inhibitor celecoxib

decreased prostate tumorigenesis in the TRAMP transgenic model (34, 35). Celecoxib,

however, has been shown to have multiple non-COX-2 mediated activities that could

account for its effect on tumorigenesis in this model (36, 37). Until now, investigations

into the role of COX-2 in prostate cancer were largely hindered by the lack of prostate

specific COX-2 overexpression and knockout mouse models. The potential for non-COX mediated effects of NSAIDs, combined with the lack of expression of COX-2 in human prostate cancers (27, 30, 31), led us to question whether COX-2 inhibition was actually responsible for the reported chemopreventive activity of celecoxib in the TRAMP model.

In addition, it is well established that PGE2, the most common and ubiquitously

produced PG produced by COX enzymes, may contribute to tumorigenesis via induction

of cell proliferation, angiogenesis, invasion and metastasis (1-3). In particular, PGE2 has been shown to induce the production of VEGF, a key mediator of angiogenesis in several cancers (38-43). The physiological effects of PGE2 are mediated by four G protein

coupled membrane receptors (EP1, EP2, EP3 and EP4) that are encoded by different

genes (44). EP receptors have been implicated in promoting carcinogenesis in different

types of tumors, including lung (45), ovarian (46), colorectal (47, 48), breast (49), skin

(50, 51), and prostate (52). Unfortunately, the study of the expression of EP receptors in

prostate and bladder tissues and their direct role in carcinogenesis of these diseases is

limited.

3 The goal of my dissertation research has been to a) define the role of COX-2

overexpression and PGE2 production in prostate and bladder cancer tumorigenesis, b) to

determine the molecular mechanism involved in prostaglandin pathway and carcinogenesis in prostate and bladder cancers, and c) to determine the molecular targets

(biomarkers) of COX-2 overexpression for future chemopreventive studies.

CHAPTER 2

LITERATURE REVIEW

2.1 Prostate and bladder cancer

Prostate cancer and bladder cancer are the two most common malignancies in the adult male urogenital system. Prostate cancer is the most commonly diagnosed cancer in men in the US aside from skin cancer and accounts for about 30% of new cancer cases annually. It is the second leading cause of cancer death in US men, after lung cancer. An estimated 218,890 new cases will occur in the US during 2007, and about 27,050 men will die from the disease this year (53). Bladder cancer is the fourth most common malignancy in men and the ninth most common type of cancer in women in the US. An estimated 67, 160 new cases are expected to occur in the US during 2007, and about 13,

750 deaths will occur this year (53). Worldwide, both prostate and bladder cancers are significant health problems, and health care costs are great for both the individual and society.

2.1.1 Etiology and pathogenesis of prostate cancer

Despite the clinical importance of prostate cancer, the multiple interacting mechanisms underlying the development and progression of this disease remain speculative (54). There are several genetic and environmental factors that contribute to

5 the development of prostate cancer. Age is the most significant risk factor for prostate cancer. The disease is rare in men under age 45, but incidence rates rise rapidly with age.

As the proportion of elderly men continues to increase in the US population, this major public health problem is expected to worsen. Men with a family history of prostate cancer are at increased risk for this disease. African-American men have significantly higher incidence rates than Caucasians, and mortality rates among Africa-American men are twice as high as those of Caucasians (55). Other factors such as androgen levels, diet and lifestyle factors have been proposed to be directly involved in the development of prostate cancer (56, 57). More recently, chronic inflammation has been linked to the development of prostate cancer by several lines of evidence (30). For example, evidence from epidemiologic studies have established an association between prostatitis and sexually transmitted infections, which all cause chronic inflammation, perhaps leading to increased prostate cancer risk (58, 59). Genetic studies have suggested that polymorphisms and mutations in genes regulating the inflammatory process play an important role in the increased risk of prostate cancer (59, 60). De Marzo et al. proposed that chronic inflammation that caused by infection, dietary carcinogens, hormonal imbalances and other factors can lead to early steps in prostate carcinogenesis, which is termed as proliferative inflammatory atrophy (PIA) (61). Lesions of PIA contains activated inflammatory cells and proliferation epithelial cells, which may serve as precursors of prostatic intraepithelial neoplasia (PIN) lesions and prostatic carcinoma

(59). However, the exact mechanism by which inflammation influences the development and progression of prostate cancer has not yet been firmly established.

6 The progression of prostate cancer is characterized by the gradual loss of regulation of cell growth within the prostate epithelium due to accumulating genetic defects. PIN is thought to be the precursor of invasive prostate cancer. PIN is characterized by cellular proliferation and is associated with progressive abnormalities in phenotype and genotype that are intermediate between normal prostatic epithelium and dysplasia. PIN is generally divided into two grades, i.e., low grade and high grade. High- grade PIN (HGPIN) will further develop into superficial cancers, and finally to invasive carcinoma. Clinical studies suggest that PIN predates carcinoma by 10 years or more, and clinically significant cancer may not occur for another 3-15 years (62).

Although the mechanism underlying the development and progression of this disease remain elusive (54), recent scientific discoveries have led to better understanding of the molecular and genetic changes associated with prostate cancer carcinogenesis (63).

Figure 2.1 shows the contemporary model of prostate cancer progression. The genetic and molecular changes associated with prostate carcinogenesis will be discussed in detail below.

2.1.2 Etiology and pathogenesis of bladder cancer

There are several genetic and environmental factors that contribute to the development of bladder cancer. Age is one of the most significant risk factor for bladder cancer. As for prostate cancer, bladder cancer will also become a greater health concern with the increasing proportion of elderly in the US. Caucasians are about twice as likely to develop bladder cancer as African Americans. However, the mortality rates are similar

Figure 2.1 Contemporary model of prostate cancer progression. Genetic predisposition, oxidative damage and inflammatory changes are associated with earliest steps of prostate cancer development (63).

8 for the two populations (64). The greatest environmental risk factor for bladder cancer is cigarette smoking (65). Occupational exposures to chemical carcinogens and bladder infections have also been associated with the occurrence of this disease (65). In addition, dietary factors have been related to the risk of developing bladder cancer (66), which will be discussed in detail below. More recently, the role of inflammation in the development and progression of bladder cancer has been increasingly recognized (67, 68).

Inflammation can be directly or indirectly caused by smoking, chronic irritation, or infection.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma (UC), is

the most common histologic type of bladder cancer, accounting for about 90% of all bladder cancers (66). At presentation, 80% of bladder cancers (TCC) are “superficial” and involve low-grade papillary tumors in that they are confined in the epithelium

without invading into the surrounding muscle, or known as Ta and T1 tumors (69, 70)

(Figure 2.2). The remaining superficial cancers are known as carcinoma in situ (CIS) that involve non-papillary growth. Invasive cancers extend into or through muscle layers, and

are called T2-T4 tumors (69). Figure 2.3 shows the two pathways of human bladder carcinogenesis, which are distinct but also overlap each other (71). The pathway to development of invasive TCC seems to start with dysplasia, progressing to CIS, followed by invasion of the lamina propria (72). Despite the high percentage of the papillary pathway in bladder cancer, the most important clinical challenges of bladder cancer are recurrence and progression. Although several advances have been made in the treatment of superficial bladder cancer, the recurrence rate reaches as high as 70% (67).

Figure 2.2 T staging of bladder cancer. Tis, carcinoma in situ; Ta, tumor involves mucosa only; T1, tumor invades through lamina propria; T2a, tumor invades into superficial muscle; T2b, tumor invades into deep muscle; T3a, tumor microscopically invades perivesical tissue; T3b, tumor macroscopically invades perivesical tissue and forms extravesical mass; T4a, tumour invading prostate/uterus/vagina; T4b, tumour fixed to pelvic wall (69).

Figure 2.3 Dual tract concept of human bladder carcinogenesis (71).

11 2.2 Diet and Cancer

2.2.1 Dietary factors and carcinogenesis

In a 1981 landmark review on the causes of cancer in US, Doll and Peto estimated

that about 35% of deaths from cancer might be due to, and thus preventable, by changing

in diet (73, 74). Thereafter, many investigations including epidemiological observations,

clinical trials, animal and in vitro cell experiments have addressed the potential role of

diet. The effects of factors such as total fat, dietary fat, total energy intake, red meat,

fruits and vegetables, phytochemicals, vitamins and minerals, on the incidence and

progression of cancers have been examined. Although these studies have generated

mixed findings, epidemiological evidence continues to support the general conclusion of

Doll and Peto in 1981 (74). Numerous studies have examined the mechanisms underlying

the initiation and promotion or prevention of human cancers. The hypothesis that dietary

factors may affect the risk of cancer is supported by their biologic properties. Some dietary components are carcinogens and cause DNA mutations and others are growth factor stimulators, or antioxidants. A number of nutrients prevent DNA damage, support

DNA repair, suppress the expression of oncogenes, or affect hormone levels, the immune system and inflammatory responses (75, 76). More recently, the role of dietary factors in the modification of epigenetics during carcinogenesis, such as alterations in histone acetylation and methylation, have gained increased attention (77).

12 2.2.2 Dietary factors and prostate cancer

Epidemiological evidence suggests that the incidence and mortality of prostate cancer is considerably lower in Asian populations compared to those in North America and northwest Europe (78-80). Figure 2.4 shows the age-adjusted death rates from prostate cancer per 100, 000 population in various countries (81). There is at least a 5- fold difference in prostate cancer mortality between western and Asian populations.

Interestingly, migration studies indicate that a marked increase in prostate cancer

incidence occurs in the migrants to the United States from low-risk geographical areas

who abandon their traditional culture and dietary patterns (82, 83). Shimizu et al. found

that Asian men who immigrate at young ages have the same risk as American men,

suggesting that the earlier in life are migrants arrive to US, the more closely their risk

approaches that of American men (84). Another intriguing fact is that although the

incidence and mortality of prostate cancer varies greatly among populations, the

incidence of pre-cancerous lesions is similar (85). Early autopsy studies reveal that the

incidence of latent carcinoma of the prostate is the same in men of all races worldwide

(86). By age 50, 15 to 30% of men have cancer cells present within the prostate gland

regardless of where they reside. In high risk areas, these cancer cells will undergo

promotion and progression with aging and cause permanent lesions. However, in low risk

areas these cancer cells tend to remain dormant and do not undergo promotion and

progression to a clinically significant life-threatening malignant state. These associations

indicate that environmental factors and lifestyle, such as dietary factors, are directly

involved in the development of prostate cancer.

13 Norway Denmark Cuba Ireland New Zealand Netherland Chile France Finland Austria United Germany Australia Portugal Hungary United States Canada Mexico Czech Lithuania Estonia Croatia Colombia Spain Slovakia Israel Poland Greece Bulgaria Romania Russia Kyrgyzstan Japan Kazakhstan Turkmenistan China 0 5 10 15 20 25 30 Age-adjusted Prostate Death Rates (Per 100, 000)

Figure 2.4 Age-adjusted prostate cancer death rates per 100, 000 population in various nations around the world (81).

14 Numerous studies have demonstrated that diets high in fat, particularly animal fats,

can promote prostate cancer (87-90). High intake of red meat is also associated with the

increased prostate cancer risk (91). Recently, 2-amino-1-methyl-6-phenylimidazo [4,5-b]

pyridine (PhIP), a heterocyclic amine, has been identified as a dietary carcinogen (92).

Studies from Bogen and Keating demonstrate that higher intake of PhIP among African-

Americans may partially explain the increase in prostate cancer incidence in this group

(93). Excessive energy intake which results in altered hormone status has been linked to

increased prostate cancer risk (94). Insulin-like growth factor-1 (IGF-1) has been

demonstrated to play a key role in prostate carcinogenesis upon changes in energy intake.

In contrast, diets rich in fruits, vegetables and legumes is associated with a reduced risk

of prostate cancer (89, 95, 96). Dietary factors, including lycopene, vitamins E and D,

isoflavones, selenium and zinc, have been identified in epidemiological, animal and

cellular studies to be associated with the prevention of prostate cancer (56, 57, 94, 97).

2.2.3 Dietary factors and bladder cancer

Evidence from numerous studies suggests that diet may also play an important

role in the development and progression of bladder cancer. Like organs in the GI tract,

the bladder is unique in that many compounds contained in foods and their metabolites,

including carcinogens, are excreted through the urinary tract (66). Therefore, dietary compounds and their metabolites could directly interact with the epithelial cells of the bladder to elicit either carcinogenic or anti-carcinogenic effects on the urinary bladder.

Although there have been fewer studies on the role of fat intake in bladder carcinogenesis compared to prostate carcinogenesis, a positive link between fat and bladder cancer has

15 been suggested in some studies. A meta-analysis of 38 studies found a significant association between bladder cancer and high fat intake with a RR of 1.4 (95% CI 1.2-1.6) after adjusting for smoking (98). A multi-center, case control study from Spain demonstrated that men with the highest intake of saturated fat had a significant increased risk of bladder cancer with a relative risk of 2.25 (99). Other studies have reported this association and suggested a dose response relationship between fat intake and bladder cancer incidence (100). However, no significant association was found between meat consumption (RR=1.0) and bladder cancer incidence in this meta-analysis. More recently, results from two large prospective cohort studies, the Health Professionals Follow-UP study (HPFS) and the Nurse’s Health Study (NHS), demonstrate that meat consumption, especially bacon, was associated with an elevated risk of bladder cancer (101). The high contents of nitrosamines and/or heterocyclic amines (such as PhIP) in bacon were speculated to be responsible for its association with increased risk of bladder cancer. In contrast, consumption of fruits and vegetables has been associated with a reduced risk of bladder cancer in many epidemiological studies (102, 103). Evidence from studies on the possible role of micronutrients, including vitamin A, carotenoids, vitamin E and vitamin

C in bladder carcinogenesis, is inconsistent and controversial (66). However, selenium does seem to have an inverse association with the risk of bladder cancer (66).

16 2.2.4 Fatty acids, their metabolites and carcinogenesis

Epidemiological, case-control studies, and cohort studies have examined the

correlation between fat intake and the development of human cancers, such as prostate

and breast cancer. Although some studies suggest a positive link between fat intake and

increased risk of prostate cancer, other studies failed to find such a relationship (104-107).

A recent case-control study even suggested that obesity is inversely associated with

prostate cancer risk in middle-aged men (108). Despite the inconsistency of such

findings, studies generally suggest that the type of dietary fat rather than the amount of

total fat intake may play a more important role in the development of prostate cancer.

Accumulating evidence from both epidemiological and cohort studies suggests that intake

of fish oil that are rich in n-3 polyunsaturated fatty acids eicosapentaenoic (EPA) and

docosahexaenoic (DHA), are associated with a lower prostate cancer risk (109-111). In

contrast, high intake of the n-6 polyunsaturated fatty acids (PUFA), including α-linoleic,

linoleic, and arachidonic acid (AA), may promote prostate tumorigenesis. An early study

demonstrated that high intake of red meat, which is the major source of AA, is associated

with increased prostate cancer risk (91). In general, these studies suggest that higher n-

3/n-6 PUFA ratio is inversely associated with prostate cancer incidence and progression.

However, at present, the mechanisms through which dietary fats modulate carcinogenesis of prostate and other cancers are not fully elucidated.

A common feature of most of the proposed mechanisms by which dietary fats modulate carcinogenesis is through the regulation of eicosanoid production and their downstream signaling pathways (112-114). It has been proposed that n-3 PUFA may suppress eicosanoid biosynthesis derived from AA, resulting in altered immune response

17 to cancer cells and modulation of inflammation, cell proliferation, apoptosis, metastasis,

and angiogenesis (114).

The majority of the AA from diet or synthesized from linoleic acid is incorporated

into the secondary position of the membrane phospholipids. Phospholipases, such as

PLA2, release AA from membrane phospholipids. Upon release, AA serves as substrate

for cyclooxygenase (COX-1 and COX-2), lipoxygenase (LOX 5, 12, 15), or cytochrome

P450 monoxygenase (MOX) to produce eicosanoids (111). Figure 2.5 shows the AA cascade (115). Both n-3 and n-6 fatty acids can be substrates for production of human eicosanoids. The eicosanoids include prostaglandins (PG), prostacyclins (PGI2),

thromboxanes (TX), leukotrienes (LT), and hydroxyl acids. Eicosanoids influence numerous biologic processes, including platelet aggregation, inflammation, hemorrhage, vasoconstriction and vasodilation, blood pressure, and tumorigenesis (116). Greater concentrations of n-3 fatty acids EPA and DHA are able to replace AA in membrane phospholipids. Aronson et al. found that short-term dietary intervention in men with prostate cancer leads to a significant increase in the n-3/n-6 fatty acid ratios in plasma and adipose tissue (117). The n-6 AA derived- eicosanoid is more mitogenic and more potent than n-3 PUFA derived- eicosanoids. In addition, eicosanoids derived from n-3 fatty acids have fewer inflammatory properties than those derived from n-6 fatty acids

(112, 118). The substrate utilized by COXs is critically important for prostaglandin effects on cell function (119). Supplementation of the diet with n-3 fish oils has been shown to decrease production of TNF, IL-1, IL-6 by mononuclear cell (120). As such, changes in the ratio of dietary n-3/n-6 fatty acids could have the potential to impact the generation of

Figure 2.5 The arachidonic acid cascade (121).

inflammatory eicosanoids and to contribute to the inflammatory component associated

with chronic diseases such as prostate cancer and bladder cancer.

One additional function of fatty acids is to regulate expression of genes involved in metabolism, as well as cell growth and proliferation. Both n-3 and n-6 fatty acids have been shown to regulate the activity of several transcription factors, including PPARs,

LXR, NFκB, c/EBPβ, and Ets-1 (122). However, whether fatty acids interact with these genes by directly binding or indirectly, such as through regulation of COX-2 and prostaglandin pathways, is still unknown.

Proposed mechanisms by which individual fatty acids control specific cell functions and gene expression allows us to envision how the type and quantity of ingested dietary fat ingested may contribute to development of prostate and bladder cancer. They also offer insights for identifying novel preventative and therapeutic strategies for such cancers.

20 2.3 Cancer chemoprevention

Cancer chemoprevention refers to the use of natural and/or synthetic substances to reduce the risk of developing cancer or lessen the likelihood that cancer will recur. Many cancers have a latency period of 10-20 years, which provides sufficient time for preventative strategies. Cancer chemoprevention is commonly divided into three categories, i.e., primary, secondary, and tertiary. Primary prevention involves interventions designed to help at-risk individuals delay or prevent the development of a target disease (123). Secondary prevention involves strategies developed to provide treatment of premalignant lesions with the goal of prevention progression to frank cancer.

Tertiary prevention involves the treatments of patients to prevent further functional loss and the development of secondary primary cancers. In a classic view, carcinogenesis involves the accumulation of genetic and epigenetic alterations in a multi-step process characterized by the occurrence of initiation, promotion, and progression (124) (Figure

2.6). Cancer chemoprevention can interfere with all three stages through modifying carcinogen activation, modulating DNA repair processes, scavenging reactive oxygen species, altering expression of genes involved in cell growth and cell death, inhibiting tumor angiogenesis and invasions, and regulating inflammatory processes (124).

Figure 2.6 Multistage carcinogenesis: processes and prevention strategies(124).

22 2.3.1 Prostate cancer chemoprevention

The discovery of prostate-specific antigen (PSA), a protein produced by prostate tissue, has significantly improved the early detection of prostate cancer (125). Advances have been made in both the diagnosis and treatment of prostate cancer, yet it is still the second leading cause of cancer mortality in men in the US. The features of prostate cancer—high prevalence and long latency suggest an opportunity for chemoprevention

(57).

At present, compounds that have been considered for chemoprevention of prostate cancer include lycopene, beta-carotene, vitamin E, selenium, vitamin D, soy isoflavones,

5-alpha-reductase inhibitors (finasteride and dutasteride ), and NSAIDs in both clinical and preclinical studies. In particular, several large-scale, randomized, population-based

studies have suggested a benefit of vitamin E or selenium in the prevention of prostate

cancer. One large-scale randomized placebo-controlled trail, the Alpha-Tocopherol Beta-

Carotene Cancer Prevention Trial (ATBC), found a statistically significant 32% reduction

in prostate cancer incidence and a 41% lower mortality in those receiving alpha-

tocopherol upon secondary analysis (126). The strongest evidence for a protective effect

of selenium for prostate cancer comes from the Nutritional Prevention of Cancer Trial, a

randomized placebo-controlled study of oral selenized yeast in patients with non-

melanoma skin cancer. In this study, the incidence of prostate cancer was reduced in the

selenium group by two thirds compared to the placebo after 4.5 years of follow-up, and

the incidence was further reduced with longer follow-up (126). The findings from these

two trials along with other clinical and pre-clinical evidence promoted the use of selenium and vitamin E in the large-scale phase III, randomized Selenium and Vitamin E

23 Cancer Prevention Trial (SELECT) involving 35, 534 men to test their efficacy in the prevention of prostate cancer (127, 128). Unlike trials with selenium and vitamin E reported so far in which prostate cancer was a secondary end point, clinical diagnosis of prostate cancer is the primary end point of SELECT (125). However, the final results will not be available until 2013.

Since the observation that castration was effective in reducing the symptoms of metastatic cancer in the 1940s by Huggins and Hodges (129), the role of testosterone and dihydrotestosterone (DHT) in prostate carcinogenesis has been well established.

Testosterone is converted to the active form DHT by the membrane-bound enzyme, steroid 5-alpha reductase. Therefore, the 5-alpha reductase inhibitors have been extensively studied for chemoprevention of prostate cancer. The Prostate Cancer

Prevention Trial (PCPT) that involved 18, 882 men was the largest population based, randomized phase III clinical trial to evaluate the efficacy of finasteride, the type 2 isoenzyme of 5-alpha reductase, in the prevention of prostate cancer (130). There was a

24.8 % reduction of prostate cancer incidence in the finasteride group compared to the placebo group, however, a large number of higher-graded cancers were noted among men randomized to finasteride (104, 131). Although enthusiasm for the widespread use of finsteride has been tempered, future studies are still warranted to definitively examine the chemopreventive activity of 5-alpha reductase inhibitors in prostate carcinogenesis. The ongoing The Reduction by Dutasteride (another 5-alpha reductase inhibitor) of Prostate

Cancer Events (REDUCE) trial may help in clarifying this association (131).

24 2.3. 2 Bladder cancer chemoprevention

Although several advances have also been made in the treatment of superficial

bladder cancer, recurrence rate can be as high as 70% (67), and approximately 10% to

30% of cases will progress to invasive cancer and potentially lethal (132). In addition,

bladder cancer is an expensive cancer to survey and treat due to the need for frequent

interval cystourethroscopy, urine cytology and radiological evaluations (133). Therefore,

strategies for preventing bladder cancer are needed to reduce both the development and

recurrence of this disease. Bladder cancer is an attractive target for prevention efforts in

part because renal excretion of certain agents enables prolonged exposure to high

concentrations of drugs or metabolites in the urine (134). At present, Bacillus Calmett

Guerin (BCG) is considered the most effective chemopreventative and chemotherapeutic

agent against recurrence of superficial bladder cancer (123, 135-137). However, BCG has

been associated with high rates of side effects and development of resistance after

prolonged usage (123, 138).

Chemoprevention of bladder cancer through dietary intervention has been

proposed based upon many studies. Test foods and compounds have included intakes of

fruits and vegetable (139, 140), alpha-carotene and beta-carotene (139), Vitamin A (141),

Vitamin C (141, 142) and pyridoxine (Vitamin B6) (141, 143). However, results from

these studies are inconsistent and demonstrate conflicting findings (144). Vitamin A and

its analogues (retinoids) have been extensively studied for their chemopreventive activity

against bladder cancer (133) and demonstrated promising results. However, large-scale

trials evaluating bladder cancer prevention are rare (134). Prospective randomized trials with a large sample size, longer follow-up, and an extended duration of treatment are

25 needed to clarify the association between micronutrients and bladder cancer protection

(132, 134).

2.3. 3 The use of NSAIDs for prostate and bladder cancer chemoprevention

The COXs inhibitors, NSAIDs, have been studied for their chemopreventive effects again prostate and bladder cancers. In general, clinical trails are very limited for

NSAIDs, which may be due to the risk of side effects related to NSAIDs. Evidence from both epidemiologic and pre-clinical studies demonstrates conflicting results regarding the role of COX-2 in prostate cancer carcinogenesis, but demonstrates a much stronger association of COX-2 with bladder carcinogenesis. A more detailed review on COX-2 expression and evidence regarding NSAIDs and cancer chemoprevention in both the prostate and bladder cancer will be discussed in the later part of this review. The use of

COX-2 selective inhibitors has recently been associated with an increased risk for cardiovascular heart disease (145). It is therefore essential to carefully examine the biologic basis for targeting COX-2 for the prevention of prostate and bladder cancers.

In summary, prostate and bladder cancers are good targets for chemoprevention.

Although some agents have demonstrated promising effects, new chemopreventive agents against these two diseases need to be developed and validated. Before identifying any effective agents for chemoprevention, the molecular biology of these two diseases should be understood.

26 2.4 Molecular pathways in human prostate and bladder cancer

2.4.1 Hallmarks of cancer

The process by which normal cells become progressively transformed to malignancy requires the acquisition and accumulation of mutations which arise as a consequence of damage to the genome. It has become increasingly apparent that the six

essential pathways underling the molecular mechanisms of tumorigenesis described by

Hanahan and Weinberg are altered during carcinogenesis. These include 1) self-

sufficiency in growth signals, 2) insensitivity to growth-inhibitory signals, 3) evasion of apoptosis, 4) limitless replication, 5) sustained angiogenesis, and 6) tissue invasion and metastasis (146) (Figure 2.7). Genetic instability is a critical factor and which plays a key

role in driving the above six essential pathways. These six “Hallmarks” are believed to

be common to most or all human tumors, albeit through different mechanistic strategies

(146). An improved understanding of the genetic and molecular changes during prostate

and bladder cancer development and progression is helping us to identify appropriate

targets for chemoprevention.

Figure 2.7 The hallmarks of cancer- six essential pathways underling the molecular mechanisms of tumorigenesis (146).

28 2.4.2 Molecular pathway in prostate cancer

Prostate cancer contains many somatic genomic alterations, including point mutations, deletions, amplifications, chromosomal rearrangements, polymorphisms, and

changes in DNA methylation (147). However, unlike carcinomas such as colon and

pancreas where specific oncogenes such as k-ras or tumor suppressor genes such as p53 are mutated at a very high frequency, gene mutations reported thus far in prostate cancer appear quite heterogeneous. In addition, genetic alterations appear to accumulate with prostate cancer progression. Genes altered during prostate carcinogenesis include phosphatase and tensin homologue (PTEN), BRCA2, NKX3.1, androgen receptor (AR), telomerase, kruppel-like factor 6 (KLF6), P27, alpha-Methlacul-CoA racemase

(AMACR), and Rb (147). Epigenetic events including DNA methylation of many genes have been identified and include ras association domain family protein 1, isoform A

(RASSF1A), GSTP1, and COX-2 (63). The genetic instability leading to mutation in a diverse array of genes have been associated with prostate cancer carcinogenesis.

Many of the molecular changes described above have been identified in numerous studies, and appeared to be potential targets for prostate cancer chemoprevention (148).

Alterations in several signaling pathways have been identified to be associated with the promotion of prostate cancer growth. High levels of epidermal growth factor receptor

(EGFR), insulin-like growth factor receptor type-1 (IGF-1R), and transforming growth factor α (TGFα) have been found in prostate cancer and result in the increased

proliferation of prostate cancer cells. The signal transduction and activators of transcription (STATs) signaling, the beta-catenin signaling and the Toll-like receptor

(TLR) signaling have also been found to be activated and overexpressed in human

29 prostate cancers and all promote cell growth and survival. Many molecules involved in

cell cycle regulation including CDKs, INK4 (CDK inhibitor), Rb, E2F, and telomerase

were deregulated in prostate cancer. Cell survival and apoptosis related molecules

including NF-Kb, TNF-α, Bcl-2 and inhibitor of apoptosis protein (IAP) have been

identified in prostate cancer. Vascular endothelial growth factor (VEGF) plays an

important role during angiogenesis and promotes tumor growth. Overexpression of

VEGF and the matrix metalloproeinases (MMPs) that are involved in metastasis and

invasion have also been identified in human prostate cancer.

2.4.3 Molecular pathway in bladder cancer

The most common genetic alterations in low grade papillary transitional cell

carcinoma (TCC) of bladder cancer are the loss of heterozygosity of part of all of chromosome 9 and mutations in the fibroblast growth factor receptor 3 (FGFR3). As described earlier (Figure 2.2 and 2.3), the pathway to development of invasive TCC seems to start with dysplasia, progresses to carcinoma in situ (CIS), and is followed by invasion of the lamina propria (72). The most frequent genetic alteration in dysplasia and

CIS is mutation of the tumor suppressor gene p53, followed by loss of heterozygosity of chromosome 9. The dyregulation of tumor suppressor genes pRb and p53 has been reported to be associated with bladder cancer cell proliferation, resistance to apoptosis, cell cycle progression and invasion (149, 150). Increased genomic instability and aberrant methylation of cytosine residues in DNA has been shown to be correlated with increased tumor invasion and progression. Figure 2.8 shows the model for bladder cancer progression involving genetic and molecular changes.

Figure 2.8 Model for bladder cancer progression showing the molecular pathways of tumorigenesis (72).

31 Similar to prostate cancer, many molecular changes in the six described pathways have been identified and represent potential targets for bladder cancer chemoprevention

(72, 149). The first important group of molecules are growth factors and their receptors.

Numerous studies have demonstrated that members of the epidermal growth factor family are overexpressed in bladder carcinoma , including EGF (151-153), TGFα (152-154), epiregluin (155, 156), and others. EGFR is also associated with the stage and grade of bladder cancer development and progression (150). Pathologic biomarkers of proliferation, such as Ki67 and proliferating cell nuclear antigen (PCNA), are increased with bladder cancer progression (157-159). In contrast, signaling molecules that inhibit growth, such as TGFβ (160, 161), CDK inhibitors P15 and P16 (153) are downregulated in TCC. Insulin growth factor 1 (IGF-1) plays a role in proliferation and survival, which has been reported to be upregulated in patients of bladder cancer (149, 162).

Overexpression of VEGF is significantly associated with early recurrence, progression to high stage and grade of urinary bladder cancer (151, 163-165). In contrast, the anti- angiogenic molecule, thrombospondin-1 has been reported to be downregulated during the development of bladder cancer (166, 167). In addition, numerous studies have demonstrated that COX-2 is overexpressed in human TCC of urinary bladder, and this may affect many of the above pathways (4-7, 168).

32 2.5 Cyclooxygenase genes and enzymes

2.5.1 Isozymes of cyclooxygenase

Cyclooxygenase, also known as prostaglandin H synthase (PGHS) or prostaglandin endoperoxide synthase, is the enzyme that catalyzes the rate-limiting step in prostaglandin synthesis by converting arachidonic acid into prostaglandin H2 (121).

PGH2 is then converted to a series of different PGs dependent on the presence of the cell- specific PG synthase enzymes (47, 169). Two isoforms of cyclooxygenase have been identified, i.e., COX-1 and COX-2. Cyclooxygenase and its enzymatic activity was first identified in 1976 by two different groups (121). The gene structure of COX-1 was identified in 1988 (170-172), and subsequently COX-2 (115, 173, 174). The human gene encoding the COX-1 enzyme is located on chromosome 9 (9q32-0q33.3), contains 11 exons and is 40kb; its mRNA is approximately 2.8kb (175). The gene encoding COX-2 is located on chromosomal 1 (1q25.2-25.3), containing 10 exons and encompasses 7.5 kb with a 4.5 kb transcript (1). Despite the difference in genomic structure and transcript size,

COX-1 possesses approximately 60% amino acid identity with COX-2 (115). Both COX enzymes have about 600 amino acids with the calculated molecular weight of the nascent polypeptide 68 kDa and the mature protein 70-80 kDa as a result of glycosylation (176).

More recently, a variant of COX-1 named as COX-3, has been identified in the brain of dog (177).

33 2.5.2 Cellular localization of COX isozymes

The structures of the four domains of COX-1 and COX-2 predict that both

enzymes are located in the lumen of the nuclear envelope and endoplasmic reticulum.

The amino-terminal signal peptides on the nascent COX-1 and COX-2 polypeptides direct COX enzymes into the lumen of the endoplasmic reticulum (115). Both COX-1 and COX-2 form dimers via hydrophobic interactions, hydrogen bonding, and salt

bridges between the dimerization domains of each monomer. Three disulfide bonds links

the dimerization domains together, and a fourth disulfide bond links the dimerization

domain with the globular catalytic domain. The presence of disulfide bonds, which require an oxidizing environment, is consistent with the location of COXs inside the lumen of the nuclear envelope and ER which have redox states that are significantly more oxidized than cytosol. COX isozymes also contain a tandem series of four amphipathic helices which create a hydrophobic surface that penetrates into the upper portion of the luminal side of the hydrophobic core of the lipid bilayer of the lumen of the ER and nuclear envelope. The catalytic domain of the COX isozmes which contains two distinct enzymatic active sites (e.g. peroxidase active site and cyclooxygenase active site) and

processes the KDEL sequence that acts as a signal for retention of proteins in the ER

(115). Figure 2.9 shows the crystallographic structures of ovine COX-1 and murine

COX-2. Several laboratories determined that COX-1 and COX-2 traffic within the

nucleus following a variety of stimuli (121). Morita et al. suggest that COX-1 is

predominantly localized to the ER, while COX-2 is localized to both ER and nuclear

membrane (178).

Figure 2.9 Crystallographic structures of ovine COX-1 (left) and murine COX-2 (right) homodimers. The crystallographic structure of ovine COX-1 was taken from Protein Data Bank file 1PRH and the murine COX-2 structure was from file COX5 of the same source. Functional domains: 1) membrane binding domain (yellow); 2) dimerization domain (green); catalytic domain (blue) heme (red). The open cleft of the peroxidase active site is observable at the top of each monomer (121).

35 2.5.3 Regulation and tissue expression of COX isozymes

2.5.3.1 Tissue expression

Despite their structural similarities, the regulation and expression pattern of COX-

1 and COX-2 isomers are different (179). Although there are some exceptions, COX-1 is generally considered as constitutively expressed in many tissues, whereas COX-2 is an inducible isoform whose expression is stimulated by growth factors, hormone, cytokines

and tumor promoters (1, 121). COX-2 is up-regulated in many epithelial cancers and its prostaglandin products (PGs) are proposed to increase proliferation, enhance angiogenesis, inhibit apoptosis, and induce invasion in several tissues (1, 121). RT-PCR analysis revealed that the COX-1 and COX-2 mRNA were expressed in all human tissues examined including lung, uterus, testis, brain, pancreas, kidney, liver, thymus, prostate, mammary gland, stomach and small intestine (180). In this study, the highest levels of

COX mRNA were detected in the prostate with approximately equal levels of COX-1 and

COX-2 transcripts present. High levels of COX-2 also were observed whereas COX-1 mRNA levels were about 2-fold lower in lung. An intermediate level of expression of both COX-1 and COX-2 mRNA was observed in the mammary gland, stomach, small intestine, and uterus. The lowest levels of COX-1 and COX-2 mRNA were observed in the testis, pancreas, kidney, liver, thymus, and brain (180). Although protein of COX-2 is considered to be absent in most tissues under normal conditions, COX-2 protein is highly expressed in normal prostate (180), kidney and brain (181). Extremely high levels of PGs are found in semen as a product of prostate and seminal vesicles. The constitutive expression of COX-2 in these tissues is likely to be important in maintaining normal homeostasis and function (181, 182).

36 2.5.3.2 Regulation of COX expression

Many factors have been shown to upregulate COX-2 expression at the

transcriptional and post-transcriptional levels, including activator protein 1 (AP-1),

nuclear factor interleukin-6 (NF-IL6), nuclear factor κB (NF-κB), nuclear factor of

activated T cells (NFAT) and polyomavirus enhancer activator 3 (PEA3)(183) (Figure

2.10). In contrast, few negative effectors have been reported. Wild-type p53 has been shown to inhibit COX-2 transcription by competing with TATA-binding proteins for binding to COX-2 gene (184). Another interesting observation is that the CpG island upstream of the COX-2 gene is methylated in greater than 85% of primary and metastatic prostate cancers and in all the 7 tested prostate cancer cells lines, but not in normal tissues (185). Although COX-2 is frequently overexpressed in colorectal cancer, evidence suggests that COX-2 expression is reduced in colorectal cancer with a defective mismatch repair system (186). This phenomenon is commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC). Interestingly, the lack of tumor COX-2 expression in HNPCC is strongly associated with COX-2 promotor hypermethylation.

Although traditionally assumed that COX-1 is constitutively expressed and involved in the maintenance of tissue homeostasis, this view has been challenged by studies with COX-1 knockout mice. Disruption of the COX-1 gene is as effective as

Figure 2.10 Regulation of cyclooxygenase 2 (COX-2) in cancer (183).

38 2.6 COX-2 and Cancer

Early evidence for a potential role of COX-2 in human carcinogenesis was

reported in 1994, when the mRNA for COX-2 was found to be markedly elevated in

colorectal carcinomas (192). Subsequently, numerous studies have established a positive link between COX-2 overexpression and tumorigenesis of several human cancers. Table

2.1 lists the premalignant and malignant tissues that have been identified to have COX-2 overexpression. Evidence from genetic studies suggests that overexpression of COX-2 is sufficient to enhance tumorigenesis in animal models and inhibition of the COX-2 pathway results in a reduction in tumor incidence and progression (1, 2). For example, transgenic mice overexpressing COX-2 developed mammary gland hyperplasia, dysplasia and metastatic tumors (193). COX-2 overexpression also was able to induced hyperplasia and dysplasia in skin (194) and bladder (3) in transgenic mouse models. In contrast, knocking out COX-2 significantly reduced the development of intestinal tumors

(188) and skin papillomas (187) in mouse models.

Several mechanisms have been proposed for the role of COX-2 in tumorigenesis.

COX-2 may promote angiogenesis by increasing the production of angiogenic factors, such as VEGF, PDGF, bFGF, and TGFβ ((195-198). Other mechanisms include the inhibition of apoptosis, increased invasiveness/motility, and modulation of inflammation and immune responses (199). Figure 2.11 summarizes the possible mechanisms of COX-

2 in tumorigenesis.

Table 2.1 Cyclooxygenase 2 is overexpressed in several premalignant and malignant conditions (183).

Figure 2.11 Possible mechanisms of COX-2-derived PGE2 contributions to tumor development. In epithelial tumors of the mammary gland, COX-2-derived PGE2 may stimulate proangiogenic factors such as vascular endothelial growth factor, which promotes tumor-associated angiogenesis. Solid malignancies are made up of multiple types of cells that produce signals that work in both a paracrine and autocrine manner as depicted (199).

41 2.6.1 COX-2 and prostate cancer

Although there is a strong correlation between COX-2 and the carcinogenesis of several cancers, available data regarding the expression of COX-2 in prostate cancer and its role in prostate carcinogenesis have not been consistent. In humans, the use of

NSAIDs has been associated with a decreased risk of developing prostate cancer.

Retrospective case-control and prospective cohort epidemiologic analyses examining the risk of prostate cancer relative to NSAID use have resulted in mixed findings. While results from some studies indicate no protective effect of NSAIDS (15-19), there are several reports of a significant reduction in prostate cancer risk attributable to frequent

NSAID use (20-23).

Initial reports from several research groups indicated that COX-2 was also overexpressed in human prostate cancers (24-29). However, there are a number of discrepancies in these studies. These include differences in the cell types found to express

COX-2 within prostate tissue, expression in normal or benign tissues, and the relationship of level of expression to grade or stage of prostate cancer (31, 200). To address this issue,

Zha and coworkers conducted a very comprehensive and well-controlled analysis of

COX-2 expression in PC tissues (31). Tissues from a total of 144 PC patients were analyzed, far more than in any similar study. In contrast to previous results, the authors found only scattered expression of COX-2 in less than 1% of cells, and no difference between cancer and normal cells as determined by immunohistochemistry. The investigators found an increased COX-2 expression only in areas of inflammation, which was in agreement with Wang et al. (30), that COX-2 upregulation is only associated with chronic inflammation in benign prostate hyperplasia. Zha et al. further performed

42 immunoblotting and qRT-PCR analyses on a subset of frozen tissues and found that mean

mRNA levels of COX-2 were higher in normal prostate tissues compared to malignant

prostate tissues (31). Similarly, , Shappell et al. (27) found that COX-2 expression was

more often reduced in prostate tumors compared to benign tissues by using well-

controlled immunohistochemistry and in situ hybridization analysis. The observed reduction was significant in Gleason score 5 and 6 tumors (27). Previous studies in

general suggest that the expression of COX-2 is either low or non-detectable in the

normal prostate tissue. However, this notion is challenged by observation that prostate

expresses the highest level of both COX-2 and COX-1 among the various tissues

examined (180). A recent study by Wagner et al. found that both normal and the benign

prostate tissues have either focal or rare COX-2 staining, whereas the adenocarcinoma

tissues were uniformly negative for COX-2 expression (201).

There have been relatively few animal studies that have tested the level of COX-2

expression and the effect of NSAIDs on prostate carcinogenesis. Two groups of

investigators recently reported that the COX-2 selective inhibitor celecoxib decreased

prostate tumorigenesis in the TRAMP transgenic model (34, 35). Celecoxib, however, has been shown to have multiple COX-2 independent activities that could account for its effect on tumorigenesis in this model (36, 37). Until now, investigations into the role of

COX-2 in prostate cancer were largely hindered by the lack of prostate specific COX-2

overexpression and knockout mouse models.

In contrast, numerous in vitro studies have examined the expression and role of

COX-2 in prostate normal or cancer cell lines. The majority of the in vitro studies examined the effects of NSAIDs on prostate cancer inhibition. Only studies that

43 determined the biological role of COX inhibitors and the expression of COX in cells are reviewed. In general, these studies found that the expression of COX-2 was either very

low or non-detectable in prostate cancer cell lines PC-3, DU145, LNCaP and TSU (31,

182, 201-203). In addition, the growth of PC-3 and DU145 was resistant to the treatment

of COX-2 selective inhibitor (201). Interestingly, Subbarayan et al. demonstrated that in

contrast to the low mRNA and protein levels of COX-2 in prostate cancer cells, the basal

level of COX-2 expression was high in the normal PrEC cells (182). Another study found

that COX-2 protein expression was high in the normal PrEC and the benign BPH-1 cells,

but low in LNCaP and PC-3 cells (202).

Collectively, the above data suggest that the expression of COX-2 may actually

be reduced during late stages of prostate carcinogenesis.

2.6.2 COX-2 and bladder cancer

In contrast to prostate cancer, numerous studies have demonstrated that COX-2 is

overexpressed in TCC of human urinary bladder (4-7). The degree of COX-2 expression

is significantly correlated with the tumor grade and depth of invasion (T stage) of TCC

(8-10). Increased COX-2 expression has also been reported in rat and canine models of

bladder cancer (204, 205). Previous study by Klein et al. have shown that forced

expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause

mouse urinary bladder transitional cell hyperplasia (TCH) in an age-dependent manner

(3). They further observed that TCH was strongly associated with increased Ki67 staining,

induced inflammation, lymphocytes and macrophage infiltration in COX-2

overexpression transgenic (K5.COX-2) mouse bladders. Additionally, TCC was observed

44 in approximately 10% of the K5.COX-2 transgenic mice with increased vascular

proliferation and VEGF expression (3).The level of prostaglandin E2 was significantly

increased in the bladder of BK5.COX-2 mice, as has been reported to be elevated in patients with bladder cancer (4, 206).

COX inhibitors have been shown to induce remission of chemically induced bladder tumors in rodents (207, 208), naturally occurring invasive TCC in dogs (209,

210), and bladder carcinoma in an orthotopic mouse model (211). Smakman et al. found that selective COX-2 inhibitor, NS-398, abrogated PGE2 secretion and inhibited

proliferation of human bladder cancer T24 cells (211). They further observed that in vivo

administration of NS-398 reduced the outgrowth of experimental orthotopic T24 bladder

carcinoma. A substantial number of studies demonstrated that NSAIDs inhibited growth, induced apoptosis, and arrested cell cycling of bladder cancer cells in vitro (11-14, 212).

Despite the strong evidence of the association between COX-2 and bladder cancer

from numerous clinicopathologic studies and the consistent results from in vivo and in

vitro studies, the epidemiological and clinical data on NSAIDs and bladder cancer are very limited. At present, only one case control study which involved 1514 bladder cancer cases and an equal number of controls has been reported. In this study, Castelao et al.

reported that NSAIDs users had almost 20% decreased risk for urinary bladder cancer

(213). Both phase I and phase II clinical trials using NSAIDs for bladder cancer

chemoprevention are underway at present. Dr. Jaye Viner of the NCI Division of Cancer

Prevention has stated, “NSAIDs are arguably the most promising chemopreventive

agents for epithelial cancers, such as bladder cancer”. More prevention trials that

targeting COX/PGs pathway are expected to be carried out in the near future.

45 These studies suggest that there is an association between COX-2, prostaglandin

synthesis and the development and progression of urinary bladder cancer. However, the

exact role and mechanisms of COX-2 overexpression during bladder carcinogenesis have not been well defined.

2.7 Family of PGs an their role in cancer

2.7.1 Family of PGs

Prostaglandins (PGs) are the most abundant lipid-derived eicosanoids generated by sequential metabolism of AA by the COX enzymes and PG synthase enzymes. Upon released from membrane by PLA2, AA is converted to an unstable PG endoperoxide

intermediate PGG2 and then PGH2 in reactions catalyzed by the rate-limiting enzyme

COX (47). PGH2 is then converted to a series of different PGs that is dependent on the presence of the cell-specific PG synthase enzymes (47, 169). At present, five PGs have been identified, including PGE2, PGD2, PGF2α, PGI2, and tromboxane A2 (169) (Figure

2.5). Among these PGs, PGE2 is the most common and ubiquitously produced

metabolite and acts locally in an autocrine and paracrine manner to elicit a wide range of

physiological responses (44). PGE2 was first extracted from semen, prostate, and seminal

vesicles by Goldblatt and von Euler in the 1930s and were shown to lower blood pressure

and cause smooth muscle contraction (121). It is now well established that PGE2 is important in control of gastric secretion, renal function, smooth muscle contraction and relaxation (depending on the tissue), and immunity (214). In addition, PGE2 has been

implicated in a broad array of diseases including cancer, inflammation, cardiovascular disease and hypertension (44). PGE2 may contribute to tumor progression by inducing

46 cell proliferation, angiogenesis, invasion and metastasis (2, 3). Also, PGE2 has been reported to participate in these complex processes by stimulating vascular endothelial

growth factor (VEGF) secretion, cell migration and matrix metalloproteinase-2 (MMP-2) expression and activation (34, 215).

2.7.2 PGE2 and prostate cancer

Human prostate epithelial cells synthesize PGs, particularly PGE2. However, the exact role of PGE2 in prostate carcinogenesis remains unknown. Although several

studies have assumed that PGE2 levels are increased in prostate cancer tissues and in vitro

cultured prostate cancer cells, the level of PGE2 was not determined in any of these studies (24, 216-218). Collectively, these studies suggested that PGE2 could mediate

signaling pathways involved in increased cell proliferation, promotion of angiogenesis,

and inhibition of apoptosis in prostate cancer cells. A potential mechanism for the effects

of PGE2 on angiogenesis is through the induction of VEGF which has been shown to be

upregulated by COX-2 activity and PGE2 treatment in PC-3 ML prostate cancer cells

(219).

An in vitro study demonstrated that PGE2 increased prostate PC-3 cell growth and

the mRNA level of COX-2 (218). The investigators further found that flurbiprofen

inhibited PGE2-induced cell proliferation, and COX-2 up-regulation in PC-3 cells.

Another in vitro study reported by Liu et al. demonstrated that PGE2 significantly

increased hypoxia-inducible factor-1α (HIF-1α) expression, a transcriptional activator of

VEGF, particularly in the nucleus (220). They also demonstrated that two selective

COX-2 inhibitors, meloxicam and NS398, were both able to decrease HIF-1α levels and

47 nuclear localization. However, two investigators who actually measured PGE2 levels in prostate found that PGE2 production is low or non-detectable in prostate benign and

tumor tissues (27), and in PC-3 and DU145 cells (201).

Taken together, the exact role and direct molecular mechanisms involved in the

tumor stimulating effects of PGE2 in the prostate remain largely unknown. Given the fact

that both COX expression and activity might diminish during prostate carcinogenesis, it

is essential to elucidate the contribution of the upstream of PG pathway (the COX

expression) to prostate carcinogenesis. Moreover, COX inhibitors have been criticized for

causing gastrointestinal side effects and other potential risks, which is a major obstacle

for large-scale application to the prevention of prostate cancer. Therefore, targeting

down-stream signaling pathways of PGE2 may be an attractive new strategy for

prevention and therapy of prostate cancer.

2.7.3 PGE2 and bladder cancer

Elevated COX-2 expression and PGE2 has been reported in patients with urinary

tract infections and with bladder cancer when compared with age matched controls (168).

A study from Badawi et al. found that higher COX-2 expression and PGE2 synthesis were

associated with patients with bladder cancer who are smokers compared with nonsmokers

(206). These results suggest that elevated COX-2 expression and PGE2 production may

be the mechanisms by which cigarette smoking and infection influence the development

of human urinary bladder cancer. Shi et al. determined that both the mRNA level of

COX-2 and the concentration of PGE2 were significantly increased in the TCC of Wistar

rats induced by terephthalic acid (TPA) (221). Increased secretion of PGE2 has been

48 observed in the bladder cancer cell lines including TSGH8301, TCC 8702, RT4,

TCC8701, TCC9101, HT1376 and T24 compared to normal urothelial cells (222).

Although compelling evidence suggests that PGE2 secretion is increased in bladder cancer, studies directly examining the role of PGE2 in bladder carcinogenesis are lacking.

Additional studies are needed to better understand the role of COX-2 and PGE2 in

bladder carcinogenesis. As for prostate cancer, targeting down-stream signaling pathways

of PGE2 may represent novel strategies for prevention and therapy of bladder cancer.

2.8 Mechanism of PGE2 action

2.8.1 PG receptors

The physiological effects of PGs are mediated primarily by G-protein-coupled

receptors, a family of rhodopsin-like seven transmembrane spanning receptors (44).

There are at least nine known PG receptor forms found in human (169). PG receptors are

designated by the letter “P” and a prefix of “D”, “E”, “F”, “I” and “T” according to their

preference for PGD2, PGE2, PGF2α, PGI2, and thromboxane A2 respectively (121). Four

of the receptor subtypes bind PGE2 (EP1-EP4), two bind PGD2 (DP1 and DP2), and FP,

IP, and TP binds to PGF2α, PGI2, and tromboxane A2 respectively (169) (Figure 2.12).

The four PGE2 receptors are encoded by different genes.

Figure 2.11 Prostaglandin synthesis and actions (169).

50 EP1 is coupled to phospholipase C, generating two second messengers inositol

triphosphate, which is involved in the liberation of intracellular calcium (Ca2+), and

diacylglycerol, an activator of protein kinase C (220, 223). EP2 and EP4 are coupled to

adenylated cyclase and generate cAMP that activates the protein kinase A signaling

pathways. The EP3 receptor is known to have multiple splice variants in humans (at least nine). The signaling of EP3 is more complex, with a wide range of action from inhibiting cAMP to increasing Ca2+ and inositol triphosphate (169). The exact physiologic roles PG

receptors play in normal and pathologic settings are determined by multiple factors

including cellular contex, receptor expression profile, ligand affinity, and different

coupling to signal transduction pathways (44).

Most PGE2 receptors are localized at the plasma membrane, although some are

found situated at the nuclear envelope. Bhattacharya et al. found that EP4 and a splice

variant of EP3 receptors are clearly visualized in the nuclear envelope of HEK 239 cells

stably overexpressing these receptors (224). However, the function of these receptors in

the nuclear envelope are not clear. Interestingly, nuclear peroxisome proliferators

activated receptors δ and γ (PPARδ/PPARγ) are receptors for PGJ2 and cyclopentenone

PGs, such as 15-d-PGJ2 (a metabolite of PGD2), respectively (47). PGE2 has been shown

to increase angiogenesis and inhibit apoptosis through the classical plasma membrane EP receptors (121). In contrast, accumulating evidence suggest that 15d-PGJ2 is able to induce apoptosis and inhibit angiogenesis upon activating PPARγ (121, 225). A recent study from Subbarayan et al. suggests that prostate cancer PC-3 cells express significantly more PPARγ than normal prostate epithelial cells (226). They further demonstrated that the PPARγ ligand 15-d-PGJ2 is able to induce cell death in PC-3 cells

51 expressing PPARγ. These findings suggest that the nuclear-acting PG (15-d-PGJ2) and

PPARγ may be involved in negative regulation of prostate cancer cell growth. However, the implication of these results and the molecular mechanisms involved need further investigation.

2.8.2 EP receptor and cancer

Several studies have demonstrated that EP receptors (individually or in combination) are implicated in promoting carcinogenesis in different types of tumors, including lung (45), ovarian (46), colorectal (47), colon (48), and breast (49). Spinella et al. found that EP2 and EP4 are involved in up-regulating VEGF, MMP and ovarian cell invasiveness upon endothelin-1 stimulation, a key mediator in ovarian tumorigenesis

(46) . A study from Miyata et al. suggests that only EP4 receptor is co-expressed with

COX-2 in transitional cell carcinoma of the upper urinary tract (227). Interestingly, Shoji et al. found that the mRNA level of both EP1 and EP2 receptors increased in colon cancer tissue, but not the EP4 receptor, which is constitively expressed in normal and cancer tissues (48). However, they also found that EP3 receptor mRNA level is significantly decreased in colon cancer tissue compared to normal tissue. They proposed that EP3 may play a key role in the suppression of colon cancer cell growth and its downregulation may enhance colon carcinogenesis at a later stage. Results from these studies suggest that EP receptors modulate tumorigenesis in a tissue-specific manner.

The use of genetically modified EP receptor knock out mouse models have greatly enhanced our understanding of their potential functions in tumorigenesis. A study from Yang et al. found that EP2(-/-) mice that had undergone isograft injection of MC26

52 or Lewis lung carcinoma cells exhibited significant reduction in tumor growth and prolonged lifespan as compared with the wild type mice (228) . Knockout of EP4

receptor, but not the EP2 receptor, decreased the formation of aberrant crypt foci in

animals treated with the colon carcinogen azoxymethane (229). A recent study from

Kawamori et al. demonstrated that the EP1(-/-) mice significantly reduced colon cancer

incidence and tumor volume, suggesting that EP1 receptor may also play an important

role in development and progression of colon cancer (230).

Despite many studies demonstrating that PGE2 and its downstream EP receptors

are important in tumorigenesis, the actual molecular targets of activated EP receptors

remain illusive. It has been demonstrated that PGE2 increases VEGF secretion via the

cAMP-protein kinase A (PKA) signaling pathway in a EP2- and EP4- dependent manner

(223). However, the relative contribution of EP2 verse EP4 receptors is not well defined.

In addition to increased VEGF secretion, EP2 receptor has also been demonstrated to inhibit glycogen synthase kinase-3 (GSK-3) by a PKA-dependent mechanism (223).

However, EP4 preferentially uses a phosphatidylinositol 3-kinase (PI3K)-dependent

pathway involving Akt/protein kinase B and activating MAPK/Erk signaling in HEK

cells (231, 232). Buchanan et al. found that PGE2 is able to induce migration and invasion

of colorectal carcinoma cells, which is partially due to the rapid transactivation and

phosphorylation of the epidermal growth factor receptor (EGFR) and Akt activation

(233). Although the investigators did not implicate EP receptor in their study, these

results suggest that EP receptors may be involved in EGFR activation and one of its

downstream signaling events, the Akt pathway. Fukuda and co-workers found that PGE2

induce the up-regulation of VEGF mRNA level in HCT116 human colon carcinoma cells

53 (234). They further demonstrated that EP1 is the key receptor contributing to VEGF up-

regulation through mediating multiple signaling pathways, including ERK, Akt and HIF-

1. These results suggest that EP receptors are able to target a variety of molecules

involved in several signaling pathways, including cell proliferation, apoptosis, invasion

and angiogenesis. These findings also suggest that there are important differences in the

signaling potential of the EP receptors, which should be the primary consideration for

developing new cancer chemopreventive strategies through targeting EP receptors and

their down-stream effectors. The development and use of selective EP receptor agonists

and antagonists have greatly facilitated our understanding about the role and mechanisms

for COX-2 and PGE2 in human carcinogenesis. Table 2.2 lists the current developed EP

receptor agonist and antagonists.

Unfortunately, the study of the expression of EP receptors in both prostate and

bladder tissues and their direct role in tumorigenesis has been limited. So far, there are

only two in vitro studies that determined the expression of EP receptors and molecular

pathways in prostate cancer cells. Chen et al. found that treatment of PC-3 cells with

arachidonic acid remarkably increased PGE2 secretion, which leads to the induction of c-

fos in these cells (52). They further determined the expression of the four EP receptors in

PC-3 cells together with two other prostate cancer cell lines (DU145, LNCaP) and a human normal prostate isolate (PrEC) by RT-PCR. Results show that only EP2 and EP4 are expressed in the examined prostate cells, but not EP1 and EP3 receptors. Liu et al. reported that EP2, EP3 and EP4 are expressed in prostate cancer PC ML cells, and EP2 and EP4 receptors are involved in PGE2-induced HIF-1α in these cells (42). Similarly,

there is only one study showing that the expression of EP4 receptor was upregulated in

54 patients with TCC (227). Taken together, results from these studies are insufficient to

make any definitive conclusions regarding the role PGE2 and its EP receptors play in

prostate and bladder carcinogenesis.

In summary, prostate and bladder cancers are significant health problems for middle-aged and elderly population of the United States. Therefore, prostate and bladder cancer chemoprevention through drugs, such as NSAIDs, and dietary intervention, is an

important issue. However, before we could use any agents to prevent these two diseases,

the molecular targets and their modes of action need to be validated. Although there is a

strong association between COX-2/PGE2 synthesis pathway and bladder cancer

carcinogenesis, the molecular mechanisms and gene regulation upon COX-2

overexpression in bladder cancer have not been defined. In contrast, whether targeting

COX-2/PGE2 pathway would be an effective chemopreventive strategy for prostate

cancer needs to be critically examined. Recently, COX inhibitors have been criticized for

causing gastrointestinal side effects and other potential risks, such as heart disease.

Therefore, inhibition of downstream effectors of PGE2, such as EP receptors, has the

potential to be efficacies for prevention and therapy of prostate and bladder cancer.

However, both the expression profile of EP receptors and their role in PGE2-elicited carcinogenic effects need to be determined in these two cancers.

PG Agonist Company Antagonist Company Natural Receptor Ligand EP1 ONO-DI-004 Ono Pharm. Jap. SC 19220 Cayman PGE 2 ONO-8713 Ono Pharm. Jap.

EP2 Butaprost, free acid Cayman None PGE2 CAY10399 Cayman ONO-AE-259-01 Ono Pharm. Jap.

EP3 Sulprostone Cayman ONO-AE3-240Ono Pharm. Jap.PGE 2 ONO-AE-248 Ono Pharm. Jap. EP4 ONO-4819 Ono Pharm. Jap. AH23848B Glaxo, UK PGE 2 ONO-AE-329 Ono Pharm. Jap.

Table 2.2 Prostaglandin receptor agonists and antagonists.

CHAPTER 3

PROSTAGLANDIN E2 INDUCES VESCULAR ENDOTHELIAL GROWTH FACTORS SECRETION IN PROSTATE CACNER CELLS THROUGH EP2 RECEPTOR MEDIATED PATHWAY

Xingya Wang1 and Russell D. Klein1,2,3

1The Ohio State University, Interdisciplinary Ph.D. Program in Nutrition (OSUN) 2the Ohio State University Comprehensive Cancer Center, Molecular Carcinogenesis and Chemoprevention Program 3Department of Human Nutrition, 325 Campbell Hall, 1787 Neil Avenue, Columbus, OH 43210

This work was supported by the National Cancer Institute grant K07 CA091865 (RDK).

57 3.1 ABSTRACT

Prostaglandin E2 (PGE2) has been shown to induce expression of vascular

endothelial growth factor (VEGF) and other signaling molecules in several cancers. PGE2

elicits its functions through four G-protein coupled membrane receptors (EP1-4). In this

study, we investigated the role of EP receptors in PGE2-induced molecular events in prostate cancer cells. qRT-PCR analysis revealed that PC-3 cells express a substantially higher level of EP2 and moderately higher EP4 than DU145 and LNCaP cells. LNCaP cells had virtually no detectable EP2 mRNA. EP1 and EP3 mRNAs were not detected in these cells. Treatment of prostate cancer cells with PGE2 (1 nM -10 µM) increased both

VEGF secretion and cAMP production. Levels of induction in PC-3 cells were greater

than in DU145 and LNCaP cells. The selective EP2 agonist CAY10399 also significantly

increased VEGF secretion and cAMP production in PC-3 cells, but not in DU145 and

LNCaP cells. Moreover, PGE2 and CAY10399 increased MAPK/Erk and Akt

phosphorylation in PC-3 and DU145 cells, but not in LNCaP cells. However, neither the

MAPK/Erk inhibitor U0126 nor the PI3K/Akt inhibitor LY294002 abolished PGE2- induced VEGF secretion in PC-3 cells. We further demonstrated that the adenylate cyclase activator forskolin and the cAMP anologue 8-bromo-cAMP mimicked the effects of PGE2 on VEGF secretion in PC-3 cells. Meanwhile, the adenylate cyclase inhibitor

2’5’-dideoxyadenosine, at concentrations that inhibited PGE2-induced cAMP,

significantly blocked PGE2-induced VEGF secretion in PC-3 cells. We conclude that

PGE2- induced VEGF secretion in prostate cancer cells is mediated through EP2, and possibly EP4, -dependent cAMP signaling pathways.

58 3.2 INTRODUCTION

Prostaglandins (PGs) are produced from fatty acids released from membrane lipids by the rate-limiting cyclooxygenases (COX-1 and COX-2) and have been implicated in a broad array of diseases including cancer. At present, five PGs have been identified, including PGE2, PGD2, PGF2α, PGI2, and thromboxane A2 (169). PGE2 is the most common and ubiquitously produced PG, which acts locally in an autocrine and over short distances in a paracrine manner to elicit a wide range of physiological functions, including controlling of gastric secretion, renal function and other processes. Extremely high levels of PGs are found in semen as a product of prostate and seminal vesicles (235).

PGE2 is the most abundant PG in prostate. Although the exact physiological function of

PGE2 in prostate is still unknown, it is likely to be important in maintaining normal homeostasis and function of the prostate (182).

It is well established that PGE2 may contribute to tumorigenesis via induction of cell proliferation, angiogenesis, invasion and metastasis in several cancers, such as colon, lung, breast, skin, and bladder (1-3). In particular, PGE2 has been shown to induce the production of VEGF, which is a key mediator of angiogenesis in these cancers (38-43,

236). In these studies, either elevated PGE2 levels have been found in cancer compared to normal conditions or direct role of PGE2 in tumorigenesis of these cancers have been determined. On the contrast, there is no definitive evidence suggest that PGE2 plays a key role in prostate carcinogenesis at present. Despite the lack of direct evidence, it has been proposed that the association between PGE2 and prostate carcinogenesis is supported by results from the use of COX inhibitors in epidemiological (21, 23), in vivo (34, 35), and

59 in vitro studies (218, 220). The majority of these studies have focused on COX inhibitors in regulating prostate carcinogenesis. Available data suggest that PGE2 may mediate

signaling pathways that promote cell proliferation (52, 219), invasion (217), and

angiogenesis (42) in prostate cancer cells. However, the direct role and exact molecular

mechanisms involved in the carcinogenic effects of PGE2 in prostate cancer development

and progression have received limited attention. Moreover, COX inhibitors are criticized

for causing unwanted side effects, which is a major obstacle for widespread use in the

prevention of prostate cancer. Therefore, targeting down-stream signaling pathways of

PGE2, if it plays a role in prostate carcinogenesis, may represent an attractive new

strategy for prostate cancer prevention and therapy.

The physiological effects of PGE2 are mediated by four G protein coupled

membrane receptors (EP1, EP2, EP3 and EP4), which are encoded by different genes

(44). EP1 is coupled to phospholipase C to generate the second messengers inositol

trisphosphate that is involved in the liberation of intracellular calcium (Ca2+) and diacylglycerol, an activator of protein kinase C (PKC) (47). EP2 and EP4 are coupled to adenylate cyclase and generate cAMP that activates the protein kinase A (PKA) signaling pathways (223). The signaling of EP3 is more complex, with a wide range of actions from inhibition of cAMP to increases in Ca2+ and inositol trisphosphate (169). This is due

to the presence of multiple splice variants of EP3 receptor. The exact physiologic roles

PG receptors play in normal and pathologic settings are determined by multiple factors

including cellular type, receptor expression profile, ligand affinity, and coupling to

different signal transduction pathways (44). EP receptors have been implicated in

promoting carcinogenesis in different types of tumors, including lung (45), ovarian (46),

60 colorectal (47, 48), breast (49), and skin (50, 51). The use of genetically modified EP

receptor knock-out mouse models has greatly enhanced our understanding of their

potential functions in tumorigenesis. Despite many studies demonstrating that PGE2 and its downstream EP receptors are important in tumorigenesis, the molecular targets of activated EP receptors remain illusive. Accumulating evidence demonstrates that EP receptors (individual or in combination) are able to target a variety of signaling molecules in many cancer models. These include VEGF upregulation by PKA-dependent (223) and hypoxia-inducible factor-α (HIF-1α)-dependent (234) pathways, epidermal growth factor receptor (EGFR) (233), c-fos (52), PI3K/Akt (231), Erk (232), and beta-catenin (237).

Due to the complexity of PGE2 and its EP receptors induced signaling system, their

effects need to be determined in specific cell types.

Unfortunately, the study of the expression of EP receptors in prostate tissue and

their direct role in prostate carcinogenesis is limited. To our knowledge, there are only

two in vitro studies that are concerned with the expression of EP receptors and their

potential role in prostate cancer cells (42, 52). Chen et al. found that EP2 and EP4 are

predominantly expressed in three prostate cancer cells, PC-3, DU145 and LNCaP (52).

They further demonstrated that both EP2 and EP4 receptors were involved in PGE2-

induced c-fos mRNA expression through PKA signaling pathways in PC-3 cells. Another

study reported that EP2, EP3 and EP4 are expressed in prostate cancer PC-3 ML cells,

but only EP2 and EP4 are involved in PGE2-induced HIF-1α in these cells (42). However,

results from these two in vitro studies are insufficient for making definitive conclusions

about the roles of PGE2 and its EP receptors in prostate carcinogenesis.

61 In the present study, we investigated the expression profile of the EP receptors

and their role in PGE2-induced signaling events in several prostate cancer cell lines. We

found that EP2 and EP4, but not EP1 or EP3 receptors, are predominantly expressed in

human prostate epithelial cells. We further demonstrated that PC-3 cells express a

substantially higher level of EP2 and a moderately higher level of EP4 as compared to

DU145 and LNCaP cells. We report that PGE2 induces VEGF protein secretion in

prostate cancer cells, which is primarily mediated by the EP2/cAMP-dependent signaling

pathway, but not through MAPK/Erk and Akt signaling pathways. In addition, the

activation of both MAPK/Erk and Akt are also correlated with the expression level of the

EP2 receptor in prostate cancer cells. However, the role of EP4 could not be ruled out at

present. Our study provides evidence that the EP2 receptor is primarily responsible for

PGE2-induced molecular events in prostate cancer cells. Assuming PGE2 plays a role in

prostate carcinogenesis, the EP2 receptor may serve as a potentially useful target for

prostate cancer chemoprevention and therapy.

3.3 MATERIALS AND METHODS

3.3.1 Materials

PGE2 and EP2 receptor agonist CAY10399, indomethacin and polycolonal EP1,

EP2, EP3 and EP4 antibodies were purchased from Cayman Chemical (Ann Arbor, MI).

Forskolin, 8-bromo-cAMP and 2’5’-dideoxyadenosine were purchased from Calbiochem

(San Diego, CA). MAPK/Erk kinase inhibitor U0126 and PI3K/Akt inhibitor LY294002

were from Cell Signaling (Beverly, MA). The Quantikine human VEGF ELISA kit was purchased from R&D Systems (Minneapolis, MN). The Direct Cyclic AMP enzyme

62 immunoassay (EIA) kit was purchased from Assay Design (Ann Arbor, MI). Phospho- and total Erk, Akt (ser 473) polyclonal antibodies, and anti-Rabbit HRP-linked IgG were purchased from Cell Signaling (Beverly, MA). TRIzol reagent was purchased from

Invitrogen (Carlsbad, CA). RNeasy Mini RNA extraction kit was purchased from Qiagen

(Valencia, CA). The iScript cDNA synthesis kit was purchased from Bio-Rad (Hercules,

CA). Taqman® Gene Expression Assay kits for all primers: EP1 (Hs00168752-m1), EP2

(Hs00168754-m1), EP3 (Hs00168755-m1), EP4 (Hs00168761-m1), VEGF

(Hs00173626-m1) and GAPDH (Hs99999905-m1), as well as Taqman® Universal PCR

Mastermix were purchased from Applied Biosystems (Foster City, CA). BCA protein

Assay kit and RadioImmuno Precipitation Assay (RIPA) cell lysis buffer were purchased from Pierce (Rockford, IL).

3.3.2 Cell culture

Human prostate cancer cell lines PC-3, DU145 and LNCaP were purchased from the American Type Culture Collection. PC-3 and DU145 cells were cultured in

Dulbecco’s modified Eagle’s medium (GIBCO) containing 10% fetal bovine serum

(FBS). LNCaP cells were cultured in RPMI-1640 medium (GIBCO) containing 10% FBS.

The PrEC normal human prostate epithelial cells were purchased from Clonetics (San

Diego, CA), and incubated in the PrECM medium (Clonetics). All cells were maintained in a humidified atmosphere with 5% CO2 at 37ºC. For the experiments, cells were seeded at 2 x 105 cells/well in six-well plates, and incubated 24 h in serum containing medium.

Cells were then serum-starved overnight followed by treatment with different reagents.

63 3.3.3 RNA extraction and Real-Time PCR (qRT-PCR)

For EP receptor expression assay, the prostate cancer cells (PC-3, DU145, LNCaP)

and the primary PrEC cells were seeded in 10 cm dishes and incubated in their respective

medium. When cells reached 70% confluence, they were rinsed with phosphate buffer

saline (PBS) twice and RNA isolation was performed. Total RNA from cells was isolated

by TRIzol reagent and further purified by RNeasy Mini kit as described by Qiagen. Both

the quantity and quality of total RNA were analyzed by the Agilent Bioanalyzer 2100

system. Total RNA (1 µg) was reverse transcribed with iScript cDNA synthesis kit. qRT-

PCR was performed to determine the expression of EP receptors using a 1:10 dilution of

cDNA with Taqman gene expression primers and ABI mastermix according to the

manufacturer’s instructions on an iCycler IQ Real-Time PCR detection system (Bio-Rad).

GAPDH was used as the reference gene for all samples.

3.3.4 VEGF measurement by ELISA

Cells were seeded at 2 x 105 cells/well in 6-well plates and allowed to attach for

24 h. Medium was removed, cells were washed twice with PBS, and cultures were

incubated in serum-free medium for 16 h prior to treatment. Cells were treated with 1 nM

-10 µM PGE2 (and/or CAY10399) in serum-free medium containing 1% bovine serum

albumin for 24 h. PGE2 and CAY10399 were dissolved in absolute ethanol. To examine

VEGF induction by the cAMP analogue 8-bromo-cAMP and forskolin, PC-3 cells were treated with 500 nM to 50 µM 8-bromo-cAMP and 100 nM to 10 µM foskolin for 24 h.

Both forskolin and 8-bromo-cAMP were solubilized in DMSO. For inhibitor studies, cells were pretreated with the respective inhibitors at indicated working concentrations

64 for 1 h in serum-free medium prior to adding PGE2. U0126, LY294002, and 2’5’-

dideoxyadenosine were solubilized in DMSO. For all experiments, the amounts of

solvent were normalized among the treatments and controls. After treatment, medium

was collected to measure the concentration of secreted VEGF165 subtype by Quantikine

human VEGF ELISA kit (R&D Systems). Cells were washed with PBS and lysed with

RIPA lysis buffer. Protein concentrations in the medium and cell lysates were determined by the bicinchoninic acid method (BCA) according to the manufacturer’s instruction

(Pierce).

3.3.5 Cyclic AMP measurement

Cells were seeded in 6-well plates as described above. For dose-response

experiments, the same doses of PGE2 and CAY10399 for VEGF assay were used in

serum free medium and cells were collected after 30 min. For the time course

experiments, cells were treated with 0, 1 nM or 10 µM PGE2 for 0, 15, 30 min, and 1, 2, 4,

8 and 24 h. After treatment, cells were washed once with PBS before lysing with 0.5 ml

0.1 M HCl and Triton X-100 (0.1%) for 15 min. Cell lysates were subsequently collected and subjected to cAMP EIA assay according to the manufacturer’s instruction (Assay

Design). Protein concentrations in the cell lysates were determined by the BCA assay according to the manufacturer’s instruction (Pierce).

3.3.6 Immunoblotting

Cells were seeded in 6-well plates as described above. After appropriate treatment, growth medium was removed, cells were washed twice with PBS, and 60 µl of 1x NEB

65 buffer (62.5 mM Tris-Base (pH6.8), 2 % SDS, 10 % Glycerol, 50 mM DTT, 0.1%

bromophenol blue) was added to each well. Cells lysates were collected, denatured for 5

min at 100ºC, and centrifuged at 13,000 rpm for 5 min at 4ºC. Approximately 20 µl of

each respective supernant was loaded onto a 10% SDS-polyacrylamide gel and

electrophoresed at 170 V for about 1 h. Separated proteins were transferred onto a PVDF

membrane at 100 V for 30 min on ice. After transfer, membranes were blocked with 5% non-fat dry milk in 1x TBST (50 mM Tris pH7.5, 150 mM NaCl, 0.1% Tween-20) at room temperature for 1 h. Phosphorylated and total Erk1/2 or Akt (serine 473) were determined by western blot using the indicated antibodies according to the manufacturer’s instructions (Cell Signaling). EP receptor western blotting was carried out according to manufacturer’s instructions (Cayman Chemical) using antibody dilutions of

1:333 (EP1), 1:1000 (EP2), 1:500 (EP3), 1:750 (EP4) and 1:2000 (anti-Rb HRP) in 5% non-fat dry milk/TBS with 0.1% Tween-20.

3.3.7 Statistical Analysis

All the experiments were repeated at least three times, and data are presented as means + standard deviation (SD). Comparisons among multiple groups were performed

by one-way analysis of variance (ANOVA) with Tukey’s post-hoc comparisons. A p-

value of <0.05 was considered statistically significant. All analyses were performed using

SPSS 13.0 software (Chicago, IL).

66 3.4 RESULTS

3.4.1 EP2 and EP4 receptors are predominantly expressed in prostate cells

The expression of all the four EP receptors in prostate cells was examined by qRT-PCR and immunoblotting analysis. The mRNA of both the EP2 and EP4 receptors, but not EP1 or EP3 receptors, were detected in prostate cells. PC-3 cells express a substantially higher level of EP2 than DU145 and PrEC cells (Figure 3.1 A). However,

EP2 mRNA was not detected in LNCaP cells. PC-3 cells also express higher level of EP4 mRNA than DU145, LNCaP and PrEC cells (Figure 3.1 B). We also determined the expression of the four EP receptors in four other prostate cell lines, viz., RWPE-1,

RWPE-2, PWR-1E and 22RV1. Again, EP2 and EP4 receptor mRNA were present in these cell lines, whereas EP1 and EP3 were not detected. However, results from immunoblotting did not correlate with results from qRT-PCR (Figure A.1-see appendix).

Further experiments demonstrated that the sensitivity and specificity of EP receptor antibodies from Cayman Chemical and Alpha Diagnostic (San Antonio, TX) were unreliable. For example, we found that EP4 binds to unknown antigens in the serum- containing medium (Figure A.2). Results from qRT-PCR suggest that PGE2-elicited

physiological functions in prostate cells are primarily mediated by EP2 and EP4 receptors.

3.4.2 VEGF secretion upon PGE2 treatment is correlated with the presence of EP2

receptor

To determine the effects of PGE2 on angiogenic pathways in prostate cells, we

examined whether treatment with PGE2 altered expression of VEGF in three prostate

cancer cell lines (PC-3, DU145 and LNCaP). The basal level of PGE2 in PC-3, DU145

2500 A EP2

2000

1500

1000

500 expression mRNA Relative

0 PC-3 DU145 LNCaP PrEC

B 100 EP4

Relative mRNA expression

PC-3 DU145 LNCaP PrEC

Figure 3.1 Relative mRNA expression of EP2 and EP4 receptors in PC-3, DU145, LNCaP and PrEC cells determined by qRT-PCR. A, PC-3 cells express a substantially higher level of EP2 receptor mRNA than DU145 and PrEC cells. No EP2 mRNA was detected in LNCaP cells. B, PC-3 cells express a higher level of EP4 receptor mRNA than DU145, LNCaP and PrEC cells. GAPDH was used as the reference gene for all samples. Experiments were repeated three times in triplicate. Results are represented as average expression relative to lowest expressing sample; error bars indicate the 95% CI. 68 and LNCaP cell culture medium before treatment were 19.5 pmol/L, 16.6 pmol/L and

22.4 pmol/L, respectively. Due to the low basal levels of PGE2 in these cells compared to

the concentrations we used in this study, the COX inhibitor indomethacin was not added

before our treatment with PGE2 in subsequent experiments. There was a significant

increase of VEGF protein secretion compared to controls in all three cell lines after 24 h

incubation with concentrations of PGE2 ranging from 1 nM to 10 µM in serum-free

medium (Figure 3.2 A). The response of PC-3 cells to each dose of PGE2 significantly

exceeded that of DU145 and LNCaP cells. Although the relative increase in VEGF

secretion by PGE2 in PC-3 cells was greatest, the basal level of VEGF secretion was

much lower on average (45 pg/ml) as compared to DU145 and LNCaP cells (e.g. 1650

pg/ml and 825 pg/ml on average, respectively). VEGF mRNA was not altered by PGE2 treatment in the three prostate cancer cells. To determine whether the EP2 receptor was critical for PGE2-induced VEGF secretion in prostate cancer cells, PC-3 cells were

treated with an EP2 selective agonist, CAY10399, using the same concentrations as for

PGE2 treatment. Stimulation of the EP2 receptor by CAY10399 significantly increased

VEGF secretion in PC-3 cells, although the extent of stimulation was less than that for cells treated with equivalent concentration of PGE2 (Figure 3.2 B). However, there were

no additive or synergistic effects of combined PGE2 and CAY10399 on VEGF protein

secretion in PC-3 cells (data not shown). Treatment with CAY10399 slightly, but not

significantly, increased VEGF secretion in DU145 and LNCaP cells (data not shown). In

addition, VEGF secretion was not significantly increased in the non-cancerous prostate

cell line RWPE-1 and its transformed tumorigenic RWPE-2 counterpart cells upon PGE2 treatment (data not shown). For all the above experiments, there were no observed

69 A LNCaP

250.00 d’’

) ) l

l DU145

o o r

r PC-3 t

t 225.00 n

n c’’ o

o c’’ c

c s

s 200.00 c’’ v

e e s s

175.00 a

a b’’ e

e b’ r r c

c b’ b’ c n

n 150.00

i b’

f b’ b b b o

125.00 a

% %

( (

F 100.00 a a’a’’

G G E

V V 75.00

Con 1 nM 10 nM 100 nM 1 µM 10 µM

Treatment (PGE ) B 2

d’ 110.00 Cay d’ PGE

2 )

) 100.00 c’ m

m c’ d

u u

i i d

d 90.00 e

e b’ c m

f 80.00

o o

l b

m m /

/ 70.00

g g p

( (

60.00 a F

F a a

G G E E a’

50.00 V V 40.00

30.00

Con 1 nM 10 nM 100 nM 1 µM 10 µM Treatment

Figure 3.2 PGE2 and the EP2 receptor agonist CAY10399 induce VEGF protein secretion in human prostate cancer cells. A, Percent increase of VEGF secretion with PGE2 treatment (0 to 10 µM) in PC-3, DU145 and LNCaP cells. Except for LNCaP cells treated with 1 nM PGE2, all treatments significantly increased VEGF secretion compared to control (p <0.05, n=4). B, The EP2 receptor selective agonist CAY10399 increases VEGF secretion to a lesser extent than equivalent concentrations of PGE2 (1 nM to 10 µM) in PC-3 cells (p <0.05 at concentrations above 100 nM). VEGF was determined by ELISA. All experiments were repeated at least three times in duplicate. Data are represented as mean ± SD. A p-value of <0.05 is considered as significant. 70 differences in cell proliferation and protein concentrations (in either the cell lysates or

incubation medium) associated with PGE2 treatment after 24 h in serum free medium.

Collectively, these data suggest that PGE2 induces VEGF secretion in prostate cancer cell

lines posttranscriptionally by a process mediated primarily by EP2 and possibly EP4

receptors.

3.4.3 PGE2-induced cAMP production is correlated with the expression level of EP2 receptor

Since EP2 and EP4 receptors are coupled to an increase in cAMP, the effect of

PGE2 on intracellular cAMP regulation was examined. As shown in Figure 3A,

incubation with concentrations of PGE2 above 10 nM for 30 min significantly increased

cAMP concentration in a dose-dependent manner in PC-3 cells. Similarly, CAY10399

(above 100 nM) significantly, but to a much lesser extent than PGE2, increased cAMP

concentration in PC-3 cells (Figure 3.3A). PGE2 treatment also increased cAMP

concentration in DU145 and LNCaP cells, although the extent of the increase was much

less than that in the PC-3 cells (Figure 3.3B). LNCaP cells which only express low levels of EP4 had the least induction of cAMP upon PGE2 treatment. These data suggest that the

EP2 receptor is the most important receptor for bulk stimulation of intracellular cAMP production in prostate cancer cells. We also determined the rate of increase in cAMP levels upon stimulation with low (1 nM) and high (10 µM) concentrations of PGE2 in PC-

3 cells (Figure 3.4). Both concentrations of PGE2 increased cAMP concentration within

15 min and maximum concentrations were reached by 1 or 2 h. The increase of cAMP

concentration by either 1 nM or 10 µM of PGE2 remained significantly elevated

)

A ) PGE2

s s e

e 600.00 CAY d t

a a s

s PC-3 y

l l

l 500.00 l

e e

c c

o o

400.00 l

m m

/ / l

l o

o 300.00 c m

p p (

( P

P 200.00 M

A A c c d' b 100.00 ’

c' a,a’ a,a’ a,a’ b' 0.00 ’ Con 1nM 10nM 100nM 1µM 10µM Treatment

) ) ) )

s s s

B s

e e e e

t t t

t DU145 d

a a a

s s s

s LNCaP

y y y y

l l l l

e e e

c c c

c 6.00

f f f f

o o o

l l l l

m m m m

/ / / /

l l l

o o o

o 4.00

m m m

m c

p p p p

( ( ( (

P P P

M M M

M b

A A A

A 2.00

c c c c b b c’ a b’ b’ b’ b’ 0.00 a’

Con 1nM 10nM 100nM 1µM 10µM Treatment

Figure 3.3 PGE2 and the EP2 receptor selective agonist CAY10399 increases cAMP concentrations in PC-3, DU145 and LNCaP cells. A, Treatment of PC-3 cells with PGE2 and CAY10399 for 30 min dose-dependently increases intracellular cAMP production in PC-3 cells (p <0.05, n=3). B, PGE2 treatment increases cAMP production in DU145 and LNCaP cells, although the extent of the induction was much less than that for the PC-3 cells (p <0.05, n=3).

72 )

A ) No PGE2 s

s d

e e t

t 1nM

a 12.00 s

y y

l l

l 10.00 e

e c

c c

o o

8.00 l

m m

/ / l

l b,c a,b b o

o 6.00 m

m b p

p b

( (

a,b P

P 4.00 M

M a a a a A

A a a c c 2.00

0min 15min 30min 1h 2h 4h 8h Time

B No PGE2

1400.00 d )

) 10 µM

s s e

e t

t 1200.00

a a s

s y

y c

l l

l l l

l 1000.00 c e

c c

f o

o 800.00

m m

/ / l

o o 600.00

pm pm (

( e

400.00 P

M M A

A c c 200.00 f a,a a a a a a a

0min 15min 30min 1h 2h 4h 8h

Time

Figure 3.4 PGE2 at 1 nM (A) and 10 µM (B) induces cAMP production in PC-3 cells in a time-dependent manner. PC-3 cells were treated with either 1 nM or 10 µM PGE2 in serum-free medium and cell lysates were collected at indicated times to quantify cAMP concentrations by enzyme immunoassay (EIA). All experiments were repeated three times in duplicate. Data are represented as mean ± SD. A p-value of <0.05 is considered as significant. 73 compared to controls even after 24 h incubation. These results suggest that PGE2-elicited down-stream signaling events are constitutively activated in PC-3 cells.

3.4.4 EP2 and possibly EP4 receptors mediate PGE2-induced activation of ERK1/2

and Akt

To determine the effects of PGE2 on Erk and Akt activation and the involvement

of EP receptors, prostate cancer cells (PC-3, DU145 and LNCaP) were treated with either

PGE2 or CAY10399 at the indicated doses and times. To eliminate the influence of

growth factors on Erk phosphorylation, cells were serum-starved overnight before

treatments. After serum starvation, the basal levels of phospho-Erk were nearly

undetectable in PC-3 and LNCaP cells. However, phosphorylation of Erk remained high

in serum-starved DU145 cells. The phosphorylation of Erk in PC-3 cells was

significantly upregulated within 15 min by either 10 µM PGE2 or CAY10399 (Figure 3.5

A). The maximum stimulation was reached at 15 min or 30 min by treating with PGE2 or

CAY10399, respectively, and Erk phosphorylation declined to basal level after 2 h.

Figure 3.5 B shows the dose-response of Erk activation in PC-3 cells upon treatment with concentrations of PGE2 or CAY10399 ranging from 1 nM to 10 µM for 15 min.

Treatment of PC-3 cells with PGE2 induced the phosphorylation of Erk in an inverse dose-dependent manner, whereas activation elicited by CAY10399 was dose independent

(Figure 3.5 B). The dose- and time-dependent patterns of Erk phosphorylation by either

PGE2 or CAY10399 in DU145 cells were similar to that observed in PC-3 cells (Figure

A.3 A and B). In contrast, Erk activation in LNCaP cells, which only express relatively

low levels of EP4 receptor mRNA, was less robust in response to PGE2

A 10 µ M PGE 2 (min) 0 15 30 60 120 240

p-Erk PC -3 Erk

10 µ M CAY10399 (min) 0 15 30 60 120 240

p-Erk

PC -3 Erk

μ B PGE 2 ( M) 00 0.001 0.001 0.01 0.01 0.10.1 1 10

p -Erk PC - 3 Erk

CAY10399 (µM) 0 0.001 0.001 0.01 0.01 0.10.1 1 1 10 10

p-Erk PC - 3 Erk

Figure 3.5 A, PGE2 and CAY10399 induce phosphorylation of Erk in time-dependent manner in PC-3 cells. Cells were treated with either 10 µM PGE2 or CAY10399 for 15 to 240 min. Total and phosphorylated Erk in cell lysates were analyzed by immunoblotting. B, PGE2 induces phosphorylation of Erk in an inverse dose-dependent manner in PC-3 cells. Cells were treated for 15 min with concentrations of PGE2 and CAY10399 ranging from 1 nM to 10 µM followed by immunoblotting.

75 treatment. A noticeable increase of phospho-Erk in LNCaP cells was only observed after

extended exposure of the film in the dark room.

After serum starvation, the basal level of phospho-Akt was very low in DU145 cells, moderately low in PC-3 cells, but high in LNCaP cells. Phospho-Akt was activated by 10 µM PGE2 within 15 min in PC-3 cells, within 60 min in DU145 cells, but was not

further increased in LNCaP cells (Figure 3.6). The time-dependent pattern of Akt

phosphorylation elicited by CAY10399 in DU145 cells was similar to that observed in

PGE2-treated cells (Figure A.3 C). Akt phosphorylation by PGE2 remained elevated in

DU145 cells after 12 h treatment (Figure A.3 C). Similarly, the effects of concentrations of PGE2 or CAY10399 on Akt phosphorylation in PC-3 and DU145 cells were similar to

Erk activation in these cells. Figure 3.7 summarizes and compares the dose-response of

Erk and Akt phosphorylation in all the three cell lines upon treatment with PGE2 or

CAY10399 (1 nM or 10 µM). Collectively, these results suggest that PGE2-induced Erk

and Akt phosphorylation in prostate cancer cells is mediated primarily by the EP2

receptor.

3.4.5 PGE2-induced VEGF secretion is not dependent on Erk and Akt signaling

pathways

Based on the above results, we decided to focus on PC-3 cells to determine the

mechanisms involved in the increase of VEGF protein secretion upon PGE2 treatment.

We first examined the role of Erk and Akt signaling pathways in PGE2-induced VEGF

secretion in prostate cancer cells by treating PC-3 cells with either the MAPK/Erk

10 µM PGE 2 (min) 0 15 30 60 120 240

p-Akt PC -3 Akt

10 µM PGE 2 (min) 0 15 30 60 120 240

p-Akt DU145 Akt

10 µM PGE 2 (min) 0 15 30 60 120 240

p-Akt LNCaP

Akt

Figure 3.6 PGE2 induces phosphorylation of Akt only in EP2- expressing PC-3 and DU145 cells. Cells were treated with either 10 µM PGE2 or CAY10399 for 15 to 240 min. Cell lysates were analyzed by immunoblotting for total and phosphorylated Akt.

A PC- 3 DU145 LNCaP 0 0.001 10 0 0.001 10 0 0.001 10 (µM)

p-Erk

PGE2 Erk

p-Erk CAY Erk

B PC-3 DU145 LNCaP 0 0.001 10 0 0.001 10 0 0.001 10 (µM)

p-Akt PGE 2 Akt

p-Akt CAY Akt

Figure 3.7 Comparison of the effects of PGE2 and CAY10399 at 1 nM or 10 µM on Erk and Akt phosphorylation in PC-3, DU145 and LNCaP cells. Cells were treated with either PGE2 or CAY10399 in serum free medium for 15 min and 60 min to determine the activation of Erk (A) and Akt (B), respectively. All experiments were repeated at least three times, and only one representative blot is shown for each experiments.

78 inhibitor U0126 or the PI3K/Akt inhibitor LY294002. Pretreatment of PC-3 cells with

U0126 (≥ 1 µM) or LY294002 (≥ 0.625 µM) for 1 h significantly blocked 10 µM PGE2- induced phosphorylation of Erk and Akt, respectively (Figure 3.8 A). Based on these data, 1 µM U0126 and 0.625 µM LY290024 were used for subsequent inhibitor experiments. However, inhibition of either MAPK or PI3K/Akt signaling did not attenuate PGE2-induced VEGF secretion in PC-3 cells (Figure 3.8 B). Interestingly,

U0126 at 1 µM alone increases VEGF secretion, and U0126 in combination with 10 µM

PGE2 significantly increased VEGF secretion for unknown reasons. These data suggest that both MAPK/Erk and Akt signaling pathways are not involved in PGE2-induced

VEGF secretion in prostate cancer cells.

3.4.6 PGE2-induced VEGF secretion is dependent on cAMP mediated signaling

events

To further determine the mechanism involved in PGE2-induced VEGF secretion in PC-3 cells, the effects of the cAMP analogue 8-bromo-cAMP and the adenylate cyclase activator forskolin on VEGF secretion were examined. As shown in Figure 3.9 A

and B, forskolin and 8-bromo-cAMP significantly increased VEGF secretion in PC-3

cells. Next we investigated the effects of the adenylate cyclase inhibitor 2’5’-

dideoxyadenosine on PGE2-induced VEGF secretion in PC-3 cells. Treatment of PC-3 cells with 2’5’-dideoxyadenosine at 500 µM and 5 mM alone for 1.5 h did not alter basal levels of cAMP in PC-3 cells (Figure 3.10 A). However, pretreatment of these cells with

500 µM and 5 mM 2’5’-dideoxyadenosine for 1 h followed by 10 µM PGE2 stimulation

for an additional 30 min significantly blocked 10 µM PGE2-induced increase in cAMP by

79 96% and 98%, respectively (Figure 3.10 A). Similarly, 2’5’-dideoxyadenosine alone had

no effects on basal level of VEGF secretion in PC-3 cells after 24 h incubation (Figure

3.10 B). However, 500 µM and 5 mM 2’5’-dideoxyadenosine significantly inhibited 10

µM PGE2-induced VEGF secretion from PC-3 cells by 46% and 68%, respectively

(Figure 3.10 B). We also determined that treatment of PC-3 cells with 50 µM 2’5’- dideoxyadenosine completely blocked 1 nM PGE2-induced VEGF protein secretion

(Figure A.4). These results suggest that PGE2-induced VEGF secretion in prostate cancer cells is mediated through cAMP-dependent signaling pathways.

80 A p-Erk

Erk PGE2 (10 µM) - + + + + + U0126 ( µ M) - - 0.1 1 10 100

p- Akt Akt

PGE2 (10 µM) - + + + + + LY294002 (µM) --0.625 1.25 2.5 5

100.00 c

) ) m

m u

u 80.00 b

i i d

d e

e b m

f f

o o

60.00

l l

m m /

g g

p p (

(

a F F 40.00

a G

G a

E E V V 20.00

Con PGE2 U0126 LY U0126/P LY/P Treatment

Figure 3.8 The MAPK/Erk and Akt signaling pathways are not involved in PGE2- induced VEGF protein secretion in PC-3 cells. A, MAPK/Erk and Akt pathway specific inhibitors block PGE2-induced activation of Erk and Akt, respectively. PC-3 cells were pretreated with U0126 (0.1 to 100 µM) for 1 h followed by stimulation with 10 µM PGE2 for an additional 15 min. For Akt analysis, PC-3 cells were pretreated with LY294002 (0.625 to 5 µM) for 1 h followed by 10 µM PGE2 stimulation for an additional 1 h. Cell lysates were analyzed for total and phospho-Erk and Akt by immunoblotting. The experiment was performed three times, and one representative blot is shown. B, Inhibition of MAPK/Erk and Akt signaling pathways failed to block PGE2-induced VEGF secretion in PC-3 cells. PC-3 cells were pretreated with either 1 µM U0126 or 0.625 µM LY294002 for 1 h followed by treatment with 10 µM PGE2 for an additional 24 h. Cell culture media were collected and analyzed for VEGF protein concentration by ELISA. U0, U0126; LY, LY294004; P, PGE2. All experiments were repeated at least three times in duplicate. Data are means ± SD. Bars with different letters are significantly different from each other with a p-value <0.05. 81 A

70.00 b )

) b

m m u

u b i

i 60.00

d d e

e d

m m f f

f o

o 50.00

m m /

g g (p

(p 40.00

c F

G G E

E V V 30.00 a

20.00

Con PGE2 100nM F 1µM F 10µM F 100µM F Treatment B 90.00 b

)

) 80.00 m

u u i

d d e

e 70.00

m m

of of

60.00 l

l m

m d g/

g/ 50.00

p p ( ( c

c c F F

F G

G 40.00 E

E V V a 30.00

20.00

Con PGE2 500nM b 5µM b 50µM b 500µM b Treatment

Figure 3.9 A, VEGF protein secretion is significantly increased by the adenylate cyclase activator forskolin in a dose-dependent manner in PC-3 cells (p <0.05). PC-3 cells were treated with either 10 µM PGE2 or various concentrations of forskolin (0.1 to 10 µM) for 24 h in serum-free medium. F, Forskolin. B, VEGF protein secretion is significantly increased by the cAMP analogue 8-bromo-cAMP (p <0.05). PC-3 cells were treated with either 10 µM PGE2 or various concentrations of 8-bromo-cAMP (0.5 to 50 µM) for 24 h in serum-free medium. B, 8-bromo-cAMP. 82 )

A ) s

s b

e e t

a a s

s 500.00

y y

l l

l l l

e e

c c

400.00

f f

o o

m m

/ / l

l 300.00

o o m

p p

( (

P 200.00 M

A A c c 100.00 c d a a a Con PGE2 0.5mM I 5mM I .5mM I/P 5mM I/P Treatment

B b

60.00

) ) m

u u

i i d

d 50.00 c e

e m

m c

f f f o

l 40.00

m m

/ / g

g a

(p (p

a a F

F 30.00 G

E E V V 20.00

Con PGE2 0.5mM I 5mM I .5mM I/P 5mM I/P Treatment

Figure 3.10 A, The adenylate cyclase inhibitor 2’5’-dideoxyadenosine significantly blocks PGE2-induced cAMP production (p <0.05). PC-3 cells were pretreated with 500 µM and 5 mM 2’5’-dideoxyadenosine for 1 h followed by 10 µM PGE2 stimulation for an additional 30 min. B, Inhibition of cAMP production significantly blocks PGE2- induced secretion of VEGF. PC-3 cells were pretreated with either 500 µM or 5 mM 2’5’-dideoxyadenosine for 1 h followed by 10 µM PGE2 treatment for an additional 24 h in serum-free medium. I, 2’5’-dideoxyadenosine; P, PGE2. All experiments were repeated at least three times in duplicate. Data are means ± SD. Bars with different letters are significantly different from each other with a p-value <0.05. 83 3.5 DISCUSSION

Numerous studies have demonstrated that PGE2 and its EP receptors are implicated in promoting carcinogenesis in different types of cancer. PGE2 binds to the

four EP receptors that are coupled to different G proteins and induce a variety of intracellular signaling cascades. Thus, the predominance of a particular EP receptor type

may determine the mechanisms of PGE2-mediated action in a particular cell type. In the

present study, we report that EP2 and EP4 mRNA are the predominant receptors

expressed in prostate epithelial cells. We further demonstrate that EP2 is expressed in

PC-3 cells at a significantly higher level than in DU145 cells. In addition, EP4 expression

is moderately higher in PC-3 cells compared to DU145 cells. LNCaP cells express the lowest EP4 and EP2 was non-detectable. Consistent with our results, previous studies by

Chen et al. (52) and Liu et al. (42) found that only EP2 and EP4 mRNA were expressed

in prostate cancer cells. However, neither of these studies used qRT-PCR to quantitate

the expression of the EP receptors in prostate cells. Our study is novel in that we

determined the contribution of each EP receptor to PGE2-elicited signaling events by

comparing the three prostate cancer cell lines (PC-3, DU145 and LNCaP) in the

subsequent studies.

VEGF is considered to be one of the most important angiogenic factors in tumor

angiogenesis (238). There are at least four subtypes of VEGF that are generated by

alternative splicing found in human, i.e. VEGF121, VEGF165, VEGF189, and VEGF206

(239). Among these subtypes, VEGF165 is the most commonly studied form and is considered to be the strongest signal transducer of all the subtypes (238). Here we report

84 that PGE2 significantly induced VEGF165 protein secretion in PC-3 cells and to a lesser

extent in DU145 and LNCaP cells, which correlates with the expression level of EP2 and

EP4 receptors in these cells. Both EP2 and EP4 are linked to the generation of cAMP as a

second messenger. Indeed, in the present study, we found PGE2 induced a substantially

high level of intracellular cAMP in PC-3, but much lower level in DU145 and LNCaP

cells. Although EP2 and EP4 are both coupled to Gs proteins and generate cAMP, their

activation has markedly distinct impacts on cAMP levels. It was reported that 1 µM PGE2 stimulated cAMP formation about 71 fold in EP2 expressing cells, but only about 10 fold in EP4 expressing cells (232, 240). However, this difference is not due to the distinct ligand affinity for PGE2, since EP4 actually has higher affinity than EP2 for PGE2 (240).

EP4, but not EP2, undergoes rapid agonist-induced desensitization and internalization

(241, 242). Thus, the differential effects of EP2 and EP4 on intracellular cAMP levels in prostate cancer cells appeared to be a result from differences in PGE2-mediated receptor

desensitization and internalization. Consistent with our results, Chang et al. (39) found

that PGE2 significantly increased VEGF secretion through cAMP-dependent pathway in

mammary tumor cells highly expressing EP2, but not in EP4 expressing cells.

CAY10399, the EP2 selective agonist, has been reported to have both a lower

affinity than PGE2 for EP2 receptor (Ki: 92nM for CAY10399 vs. Ki: 38nM for PGE2) and a lower ability to increase cAMP levels at the same concentrations as PGE2 (EC50:

43nM for CAY10399 vs. EC50: 2.1nM for PGE2) (243). This may explain why

CAY10399 induced increases in VEGF and cAMP in PC-3 cells was less robust

compared with PGE2 in our study. The observation that CAY10399 in combination with

PGE2 did not enhance the effects of PGE2 on VEGF secretion suggests that either these

85 two compounds competitively bind to EP2 or EP2 receptor is fully saturated at the

concentrations tested. It is interesting that the similar magnitude of increase in cAMP

concentration induced by 10µM CAY10399 and 100nM PGE2 also is associated with

similar levels of VEGF secreted into medium (Figure 3.2). The observation that

CAY10399 significantly increases VEGF secretion and cAMP concentration in PC3 cells,

but not in DU145 and LNCaP cells (data not shown), suggests that PGE2-mediated VEGF

protein secretion is predominantly mediated by EP2 receptor in prostate cancer cells.

However, the observation that VEGF secretion is also slightly but significantly induced by PGE2 (>10 nM) in LNCaP cells that only express EP4 suggests that a role for EP4 cannot be excluded.

Considerable evidence demonstrates that PGE2 is rapidly converted to its inactive

metabolite15-keto-PGE2 and, subsequently, to 13,14-dihydro-15-keto-PGE2 by the

enzyme 5-hydroxy prostaglandin dehydrogenase (15-PGDH) (242, 244, 245). Recently, a

two-step model of PGE2 metabolic clearance that consisted of sequential PGE2 uptake

mediated by a PGE2 transporter followed by cytoplasmic inactivation by 15-PGDH was

reported (245). In this study we determined that although PGE2 may be inactivated

rapidly after addition to cultures, the down-stream signaling molecule cAMP remains

elevated in prostate cancer cells for up to 24 hours compared to non-treatment controls.

The possible significance of this chronic elevation of cAMP on the expression of other

genes needs further examination.

In addition to inducing VEGF secretion, PGE2 has been reported to activate a

variety of other signaling molecules by binding to specific EP receptors coupled to a

variety of downstream signaling pathways. Here we report that PGE2 and CAY10399

86 significantly increased Erk and Akt phosphorylation in EP2 expressing cells (PC-3 and

DU145), but not in EP2-null LNCaP cells. Our results suggest that EP2 plays an

important role in PGE2-induced activation of the Erk and Akt signaling pathways in

prostate cancer cells. We observed a very slight increase of Erk phosphorylation in

LNCaP cells after an extended exposure time (data not shown), suggesting that EP4

might play a minor role in PGE2-induced Erk activation in these cells. Studies by others suggested that EP4 is preferentially coupled to Akt and induced Erk activation in a

phosphatidylinositol 3-kinase (PI3K)-dependent pathway in HEK cells (232, 246).

Mendez and Lapointe also found that PGE2 induces rapid Erk phosphorylation which is

mediated through EP4-dependent EGFR transactivation in cardiac myocytes (247).

Krysan et al. (248) reported that PGE2-induced Erk activation is mediated by EP1-

dependent protein kinase C, but not EGFR signaling pathways in non-small lung cancer

cells. We observed that PGE2 induced activation of Erk and Akt is an inverse dose- dependent manner, which is contrary to the dose-response of PGE2 for cAMP

production. This observation suggests that cAMP is not likely to be involved in PGE2

induced Erk and Akt activation in prostate cancer cells. Consistent with our results,

Mendez and Lapointe reported that PGE2-induced Erk activation occurred through

cAMP-independent signaling process (247). However, another study found that cAMP is

involved in PGE2-induced transactivation of the EGFR and Erk via EP2-mediated

pathways (249). Collectively, these findings suggest that there are important differences

in the signaling potential of the EP receptors which are cell-type dependent and such

differences need to be considered for developing new cancer chemopreventative

strategies that target EP receptors and their down-stream effectors.

87 Although PGE2 induces the activation of Erk and Akt activation in PC-3 cells,

neither of these two signal pathways seem to be involved in PGE2-induced secretion of

VEGF in these cells based on our inhibitor studies. The observation that PGE2-induced

Erk and Akt activation occurs in an inverse dose-response manner with VEGF secretion

further suggests that neither Erk nor Akt is likely to be involved in PGE2-induced VEGF

secretion in prostate cancer cells. Results from Chen et al. (52) and our laboratory suggest

that the human primary epithelial PrEC cells express both EP2 and EP4 receptors. The

mechanism of the lose of EP2 mRNA expression in LNCaP cells is unknown at present.

LNCaP cells responded to PGE2 stimulation the least for regulating signaling events such as cAMP generation, VEGF upregulation, Erk and Akt activation under experimental conditions. These observations suggest that LNCaP cells may not be an appropriate model for studying the role of PGE2 and its EP receptor-mediated signal pathways during

the carcinogenesis of human prostate cancer, if PGE2 plays a role.

In our study, both the adenylate cyclase activator forskolin and the cAMP analogue 8-bromo-cAMP significantly induced VEGF secretion in PC-3 cells. We further

demonstrated that the adenylate cyclase inhibitor 2’5’-dideoxyadenosine completely and

partially blocked 1 nM and 10 µM PGE2-induced VEGF secretion in PC-3 cells,

respectively. These results suggest that PGE2-induced VEGF secretion is mediated at

least partially through cAMP-dependent signaling pathways. Upon generating cAMP by

EP2/EP4 coupled Gs protein, cAMP activates protein kinase A and results in the phosphorylation of the cAMP response element binding protein (CREB), a transcription

factor that regulate genes whose promoters contain cAMP response element (CRE) (246).

In our study, the increase of VEGF protein secretion upon PGE2 stimulation is not

88 regulated transcriptionally. In addition, we determined that the PKA inhibitor H89 failed

to block PGE2-induced VEGF secretion by PC-3 cells (data not shown), suggesting that

cAMP-mediated PKA activation and the downstream signaling events may not be

involved in PGE2-induced VEGF secretion in these cells. More recently, the exchange

protein directly activated by cAMP (Epac) has been found to be another important cAMP

effector molecule (250). In future studies, the mechanisms involved in posttranscriptional

regulation of VEGF and cAMP-mediated signaling cascades other than PKA (such as

Epac) that are responsible for VEGF secretion in prostate cancer cells need to be further

addressed.

In conclusion (as shown in Figure 3. 11), our study demonstrates that PGE2- induces VEGF secretion in prostate cancer cells through EP2 and possibly EP4 mediated cAMP-dependent pathways. EP2 is also the major EP receptor that is responsible for

PGE2- induced activation of Erk and Akt in prostate cancer cells. However, a role for

EP4 in regulating these processes cannot be ruled out at present. PC-3 cells which express higher levels of both EP2 and EP4 receptors than DU145 and LNCaP cells may serve as a potentially useful and relevant model for the study of the role of PGE2 in prostate carcinogenesis, if PGE2 plays a role. Because COX inhibitors may not be useful

as chemoprevention agents due to various toxicities associated with long term use, EP

receptors, located downstream of PG synthesis in the COX pathway may represent

attractive alternative targets for chemoprevention and treatment of prostate cancer, if this pathway is important in prostate carcinogenesis.

PGE2 (Autocrine/Paracrine) EP2 inhibitor

EP4 EP2 CAY10399

cAMP P P Erk ? Akt ? VEGF ?

genes involved in prostate tumorigenesis

Angiogenesis Vascular Remodeling prostate tumorigenesis

Figure 3.11 Working model of the major signaling pathways elicited by PGE2 in human prostate cancer cells. PGE2 synthesized by prostate epithelial cells acts locally in an autocrine, and over short distances, in a paracrine manner by binding to EP2 and EP4 receptors located on prostate epithelial and endothelial cells. EP2 is the primary receptor responsible for the bulk activation of cAMP which mediates VEGF protein secretion in prostate cancer cells. EP2 is also the primary receptor that mediates PGE2-induced phosphorylation of Erk and Akt in prostate cancer cells. CAY10399, the EP2 selective agonist, mimics PGE2-induced signaling pathways in prostate cancer cells. EP4 may play a role in PGE2-induced VEGF secretion and Erk phosphorylation in prostate cancer cells. However, the downstream genes regulated by PGE2 that may contribute to prostate tumorigenesis have not been well defined. The development of new agents that target EP2 receptor may exhibit chemopreventive and chemotherapeutic effects during prostate carcinogenesis.

CHAPTER 4

PROSTATE TUMORIGENESIS IN TRAMP MICE IS NOT INHIBITED BY NSAIDS OR GENETIC DISRUPTION OF CYCLOOXYGENASE-2 EXPRESSION

Xingya Wang1, Jennifer K.L. Colby3, Peiying Yang4, Susan M. Fischer3,, Robert A. Newman4, Russell D. Klein1,2,5

1The Ohio State University, Interdisciplinary Ph.D. Program in Nutrition (OSUN) 2the Ohio State University Comprehensive Cancer Center, Molecular Carcinogenesis and Chemoprevention Program

3The University of Texas M.D. Anderson Cancer Center Science Park-Research Division P.O. Box 389, Park Rd. 1C, Smithville, TX 78957

4The University of Texas M.D. Anderson Cancer Center Department of Experimental Therapeutics 8000 El Rio, Houston, TX 77054

5The Ohio State University, Department of Human Nutrition 325 Campbell Hall, 1787 Neil Avenue, Columbus, OH 43210

This work was supported by the National Cancer Institute Grant CA091865 (RDK) and CA107588 (SMF).

91 4.1 ABSTRACT

Overexpression of cyclooxgenase-2 (COX-2) and prostaglandin E2 (PGE2) have been demonstrated to play a significant role in the tumorigenesis of colon, lung, breast, bladder, and skin cancer. However, inconsistent and controversial reports on the expression and activity of COX-2 in prostate cancer raised the question of whether COX-

2 plays a pivotal role in prostate carcinogenesis. To address this question we examined the effects of COX-2 inhibition on prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Three wk old TRAMP mice were fed control, celecoxib-, or indomethacin-supplemented diets for 27 weeks. A TRAMP/COX-

2 knockout mouse model was also generated to determine the effects of the loss of the

COX-2 gene on prostate tumorigenesis in TRAMP mice. These studies demonstrated that neither non-steroidal anti-inflammatory drugs (NSAIDs) nor genetic disruption of COX-2 was inhibitory in terms of tumor and metastases incidence, lobe weight or types of pathological lesions. However, the endogenous levels of PGE2 were significantly reduced by both approaches in TRAMP prostate tissue compared to control. A careful analysis of wild-type and TRAMP prostate tissues was undertaken for the expression of COX-1 and

COX-2 using immunoblotting, qRT-PCR and immunohistochemistry approaches in

TRAMP dorsal prostate tissue from mice 10 and 16 wk old, as well as tumor from 30 wk old mice. We found that the expression of both COX-1 and COX-2 dramatically decreased during TRAMP prostate carcinogenesis. In contrast to previous reports, we did not detect overexpression of COX-2 by immunohistochemistry in any prostate tissues from wild-type mice or TRAMP mice. Using the probascin promoter, a COX-2 92 overexpressing mouse was also generated but failed to show any pathology in any of the

prostate lobes. Collectively, our results suggest that COX-2 may not play a tumorigenic

role during prostate carcinogenesis in the TRAMP model.

3.2 INTRODUCTION

COX-2 expression is elevated in a number of cancer types, and inhibition of

COX-2 by NSAIDs has been tested as effective chemoprevention strategy for these

cancers, including colon, breast, lung, bladder, and skin. However, studies on the role of

COX-2 in prostate carcinogenesis have resulted in mixed findings.

The first discrepancy from available data is whether COX-2 is overexpressed in

prostate cancer. Since the first report that COX-2 was overexpressed in human prostate

adenocarcinoma compared to the benign prostate tissues by Gupta et al. in 2000 (251),

many other investigators also addressed the similar question. In general, results from

these studies are inconsistent and controversial. While some studies suggest that COX-2

was overexpressed in human prostate cancers (24-26, 28, 29), other studies indicate that

COX-2 was either minimally expressed or even decreased in prostate cancers (27, 30, 31).

The second discrepancy is whether PGE2 secretion is also increased in human

prostate cancers and whether PGE2 plays a direct role in prostate carcinogenesis. The

primary mechanism by which NSAIDs are thought to prevent cancer is through inhibition

of the synthesis of PGE2. Unfortunately, studies that directly measure the level of PGE2 in prostate cancers are very limited. Although several studies have assumed that PGE2

levels are increased in prostate cancer tissues and in vitro cultured prostate cancer cells,

93 none of these studies actually determined the level of PGE2 (24, 217, 218, 252). However,

two investigators who actually measured PGE2 levels in prostate found that PGE2 production is low or non-detectable in prostate benign and tumor tissues (27), and in prostate cancer PC-3 and DU145 cells (201). In our above in vitro study, we also determined that the basal concentrations of PGE2 in prostate cancer cell lines (PC-3,

DU145, and LNCaP) were very low. Although stimulation with high concentration of

exogenous PGE2 was able to induce VEGF secretion in these cancer cells, PGE2 had no effects on promoting proliferation in these cells. We also determined that both COX-2 and COX-1 proteins were highly expressed in non-tumorigenic prostate cells, but not detectable in all the cancer cell lines that determined by immunoblotting (Figure A.10).

The third discrepancy is whether NSAIDs are able to effectively reduce the risk to develop prostate cancer and to inhibit the progression of this disease. Similarly, several retrospective case-control and prospective cohort epidemiologic analyses examining the risk of prostate cancer relative to NSAID use have resulted in mixed findings. While results from some studies indicate no protective effect of NSAIDs (15-19), there are several reports of a significant reduction in prostate cancer risk attributable to frequent

NSAID use (20-23). Among studies that suggest a protective effect of NSAIDs against prostate cancer, aspirin but not other NSAIDs was primarily responsible for the decreased prostate cancer risk (21, 253, 254).

However, evidence from chemoprevention clinical trials using NSAIDs to prevent prostate cancer progression has been largely lacking, which is mainly due to the concerns of the cardiovascular toxicity related with the use of selective COX-2 inhibitors (127,

255). At present, there are only two clinical trials that were conducted by the same

94 investigators to examine the effects of celecoxib on the PSA level in patients with

recurrent prostate cancers after definitive radiation therapy or radical prostatectomy (256,

257). Although results from these two trials suggest that celecoxib may affect serum PSA

levels in these patients, these studies were limited by small number of patients, short

follow-up duration and a single end point (PSA level only).

There have been relatively few animal studies that have tested the level of COX-2

expression and the effect of NSAIDs on prostate carcinogenesis. The TRAMP model for

prostate cancer is now well established and demonstrates several characteristics that

make it advantageous for chemoprevention studies (32, 33). The pathobiology of the

TRAMP model has recently been extensively reviewed (33). Two groups of investigators

recently reported that the COX-2 selective inhibitor celecoxib decreased prostate

tumorigenesis in the TRAMP transgenic model (34, 35). Celecoxib, however, has been

shown to have multiple non-COX-2 mediated activities that could account for its effect

on tumorigenesis in this model (36, 37).

Taken together, the exact role and direct molecular mechanisms involved in the

tumorigenesic effects of PGE2 in prostate cancer are still largely unknown. The potential

for non-COX mediated effects of NSAIDs, combined with the lack of expression of

COX-2 in human prostate cancers (27, 30, 31), led us to question whether COX-2

inhibition was actually responsible for the reported chemopreventive activity of celecoxib

in the TRAMP model. In addition, investigations into the role of COX-2 in prostate

cancer were largely hindered by the lack of prostate specific COX-2 overexpression and

knockout mouse models. To address this question we compared the effect of a non-

selective COX inhibitor (indomethacin), a COX-2 selective inhibitor (celecoxib), and

95 genetic disruption of COX-2 gene expression on prostate carcinogenesis in the TRAMP

model. Interestingly, we found that the expression and activity of both COX-1 and COX-

2 are dramatically decreased during TRAMP prostate carcinogenesis, and that inhibition

of COX activity by either NSAID treatment or by disruption of the COX-2 gene is largely ineffective in preventing prostate cancer in the TRAMP model. We recently generated a transgenic mouse model (Pb.COX-2) with the COX-2 gene under the control of the probasin promoter, which allows for COX-2 overexpression only in prostate tissue.

In support of our findings in the TRAMP model, overexpression of COX-2 in mouse prostate tissue had no pro-tumorigenic effects on prostates of Pb.COX-2 mice.

To our knowledge, we are the first laboratory to examine a direct role of COX-2

in prostate carcinogenesis by using genetically modified mouse models that either

overexpress or lack expression of COX-2 in the prostate. Our results suggest that COX-2

does not play a tumorigenic role in prostate carcinogenesis in the TRAMP model. The

discrepancy of current knowledge on COX-2 in human prostate carcinogenesis needs to

be further elucidated in future studies.

4.3 MATERIALS AND METHODS

4.3.1 Materials

PGE2, polyclonal anti-COX-2 (murine), polyclonal anti-COX-1 (murine), COX-1

(murine) blocking peptide, and goat anti-rabbit IgG horseradish peroxidase (HRP) were

purchased from Cayman Chemical (Ann Arbor, MI). Polyclonal anti-rabbit PGHS-2 was

purchased from Oxford Biomedical Research (Oxford, MI). Biotin conjugated mouse

96 anti-COX-2 monoclonal antibody was from BD (Franklin Lakes, NJ). Goat polyclonal

anti-COX-2 (c-20), goat polyclonal anti-COX-1 (M-20) and donkey-anti-goat IgG HRP

were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal

anti-beta-actin was from Abcam (Cambridge, MA). TRIzol reagent and goat serum were

purchased from Invitrogen (Carlsbad, CA). RNeasy Mini RNA extraction kit was

purchased from Qiagen (Valencia, CA). The iScript cDNA synthesis kit was purchased

from Bio-Rad (Hercules, CA). Taqman® Gene Expression Assay kits for all primers

(COX-1, COX-2 and 18s), and Taqman® Universal PCR Mastermix were purchased

from Applied Biosystems (Foster City, CA). Dako Envision Plus kit (K0411) was

purchased from DakoCytomation (Denmark).

4.3.2 Genetically-engineered Mice

C57BL/6J TRAMP mice were originally obtained as a kind gift from Dr. Norman

Greenberg (32) and were maintained as a hemizygote colony. TRAMP [C57xFVB] F1

mice were generated by crossing C57BL/6J TRAMP hemizygotes with wild-type FVB/n

mice. Male COX-2 knockout (KO) mice (B6;129P2-Ptgs2tm1Smi) were obtained from the

Taconic (Hudson, NY) Emerging Models Program by kind permission of Dr. Robert

Langenbach (258). Male COX-2(-/-) mice were crossed with female C57BL/6J mice to generate male and female COX-2(+/-) mice. COX-2(+/-) mice from this cross were then

mated to generate male COX-2(-/-) and COX-2(+/+) mice. Male COX-2(-/-) were then

crossed with female hemizygous C57BL/6J TRAMP mice to generate female mice

heterozygous for COX-2 and hemizygous for the TRAMP transgene (C+/-:T+/- ). To

produce mice for the tumor study female C+/-:T+/- mice were crossed with either male

97 COX-2 -/- or COX-2 +/+ mice in order to generate hemizygous TRAMP males wild-type

(C+/+:T+/- ) or deficient (C-/-:T+/- ) for COX-2. The Pb.COX-2 transgenic mice were on

the FVB background, which overexpress COX-2 under AAR2 probasin promoter

(pAAR2Pb) control. We generated several lines of Pb.COX-2 transgenic mice, and only

the lines with positive expression of COX-2 in prostate lobes were bred for subsequent

studies. All genotyping was done by PCR on DNA isolated from tail snips according to

established protocols for the TRAMP transgene (32), the Ptgs2tm1Smi mutation (258), and

pAAR2Pb.COX-2 that was developed in our laboratory. All mice were maintained in

HEPA-filtered cages according to Institutional Laboratory Animal Care and Use

Committee (ILACUC) approved protocols at the Science Park-Research Division,

University of Texas, MD Anderson Cancer Center, and at the Ohio State University.

4.3.3 TRAMP Diet Study

Diets were formulated and produced by Research Diets (New Brunswick, NJ) and were based on the AIN-76A semi-purified rodent diet. Celecoxib (LKT Laboratories, St.

Paul, MN) at either 500 ppm or 1500 ppm and indomethacin (Sigma, St. Louis, MO) at 4 ppm were added to the diets prior to pelleting. The selected doses of celecoxib and indomethacin were well tolerated by TRAMP mice as reported in a previous study (34).

TRAMP mice at 3 wk of age were randomly distributed by weight into experimental and control groups (n=30 per group). The TRAMP mice were fed either AIN-76A control diet or COX-inhibitor supplemented AIN-76A diets until the animals were 30 wk old.

Throughout the experiment the animals had access to drinking water and diets ad libitum.

98 Fresh diets were provided and food intakes were recorded every other day. Body weights were measured weekly.

4.3.4 Tumor Studies

All transgenic mice were weighed and palpated for tumors weekly starting at 12 wk of age. Mice were necropsied when palpable tumors reached 2 cm in diameter, or mice reached 30 wk of age. Mice that died prior to 30 wk from causes unrelated to prostate tumor growth were censored from the analyses. Early necropsies were performed

on mice with palpable tumors greater than 2 cm in diameter regardless of age. At

necropsy the dorsal (DP), lateral (LP), ventral (VP), and anterior (AP) lobes of the

prostate were microdissected into individual lobes whenever possible. Weights of all

prostatic lobes, regional lymph nodes (LN) and tumors were recorded. The techniques

and protocols for histological dissection of the prostate lobes were in concordance with

the 2001 Bar Harbor Pathology Workshop (259). Mice were examined for any grossly

observable metastatic tumors. The prostate lobes, LN, tumor tissues, and sections of the

lung and liver were fixed in 10% buffered formalin and then paraffin-embedded for

histopathological and immunohistochemical examination or were flash-frozen in liquid

nitrogen for determination of PGE2.

4.3.5 Histopathology

The histopathology of embedded tissues from all mice was assessed by scoring 4-

micron thick sections stained with hematoxylin and eosin (H&E). All sections were

coded, blinded and randomized prior to independent analysis by RDK and XW. Prostatic

99 lobes, including DP, LP, VP, and AP, were assessed according to published criteria that

have been well established for the TRAMP model (33). Only lobes that were fully

dissectible were included in the analysis. All glands in a given section were classified as

either normal, prostatic intraepithelial neoplasia (PIN), well-differentiated carcinoma

(WD), moderately-differentiated carcinoma (MD), or poorly-differentiated carcinoma

(PD). The phylloides-like lesions (Phyl) described by Kaplan-Lefko et al. (33) were also

examined and recorded. An additional classification of Atrophy as described by Shappell et al (259) was also included in the analysis of the mouse prostate tissues. The numbers of glandular ducts exhibiting each grade were determined for each section, as well as the total number of glandular ducts present. For each pair of lobes, the percent of the tissue that was normal, PIN, WD, MD, PD, Phyl, and Atrophy was determined. These data was used to score each pair of prostatic lobes for the percentage of each pathological grade present and these scores were averaged for all mice in a group. LN, lungs, and liver were assessed for metastases by scoring H&E sections for the presence or absence of apparent metastatic lesions.

4.3.6 Immunohistochemistry

Paraffin-embedded specimens were cut into 4 micron thick sections, deparaffinized, rehydrated, antigen retrieved in citrate buffer (pH 6.0), and blocked for endogenous peroxidase with H2O2 (Dako k0411 kit). The specimens were incubated in

5% goat serum for 10 min to block non-specific binding. Primary polyclonal antibodies

against COX-1 and COX-2 (both from Cayman Chemical) were used at a dilution of

1:500 and applied to sections for 1 h. The specimens were subsequently washed in

100 phosphate-buffered saline (PBS) and treated with secondary antibody (Dako k0411 kit) for 30 min. To visualize the antibody, 3,3’-diaminobenzidine tetrahydrochloride (DAB,

Dako k0411 kit) was incubated for 7 min. All immunohistochemical stainings for COX-1 and COX-2 were performed in an OptiMax Automated Cell Staining System (BioGenex,

San Ramon, CA). Finally, the slides were dehydrated and mounted. Vas-deferens tissue from wild-type mice was used as positive control for COX-1 and COX-2, while vas- deferens from a COX-2 knockout mouse served as a negative control for COX-2. There was no negative control for COX-1, however, a COX-1 blocking peptide was used as control for the specificity of COX-1 staining.

4.3.7 Immunoblotting

Total proteins were collected from frozen tissue by standard methods.

Approximately 40 µg protein was electrophoresed on a 10% SDS-polyacrylamide gel followed by standard immunoblotting procedures. Four commercial sources of the primary antibodies against COX-2 were analyzed at the following dilutions: 1:500 (Santa

Cruz), 1:750 (Cayman), 1:250 (BD) and 1:500 (Oxford). Two sources of the COX-1 antibodies were analyzed at dilutions of 1:500 for the Santa Cruz antibody and 1:1000 for the Cayman Chemical antibody. The secondary antibody was goat anti-rabbit IgG HRP for COX-1 (Cayman) and COX-2 (Cayman, BD and Oxford). The donkey anti-goat IgG

HRP was used for COX-1 and COX-2 antibodies from Santa Cruz. Beta-actin was used as a loading control. Cell lysates from a wild type mouse vas-deferens was used as positive control for COX-1 and COX-2 immunoblotting.

101 4.3.8 RNA Extraction and Real-Time PCR (qRT-PCR)

Total RNA was isolated by TRIzol reagent and further purified by RNeasy Mini

kit as described by Qiagen. Both the quantity and quality of total RNA were analyzed by the Agilent Bioanalyzer 2100 system. Total RNA was reverse transcribed with an iScript

cDNA synthesis kit. qRT-PCR was performed to determine the expression of COX-1 and

COX-2 using a 1:10 dilution of cDNA with Taqman gene expression primers and ABI

mastermix according to the manufacturer’s instructions on an iCycler IQ qRT-PCR

detection system (Bio-Rad). 18s RNA was used as the reference gene for all samples.

4.3.9 Quantitation of PGE2

PGE2 was measured on DP and PD tumors using an LC/MS/MS system as described by Kempen et al. (260). For endogenous measurements snap-frozen tissues

were homogenized followed by extraction with hexane: ethyl acetate (1:1) and total

protein was determined on the homogenate. LC/MS/MS analyses were performed using a

Quattro Ultima tandem mass spectrometer (Micromass, Beverly, MA) equipped with an

Agilent HP 1100 binary pump HPLC inlet. PGE2 was separated from other eicosanoids

using a YMC ODS-AQ 2.0 × 100-mm column with a solvent system of 10 mM

ammonium acetate/methanol and a flow rate of 0.3 ml/min. PGE2 was detected using

electrospray negative ionization and multiple reaction monitoring (MRM) and the result

was expressed as nanograms of PGE2 per mg protein.

102 4.3.10 Statistical Analysis

Data are presented as means + standard deviation (SD). Inter-group comparisons of prostate cancer incidence were made using Fisher’s exact test (two sided). Survival curves were compared using Kaplan-Meier analysis, and the difference between groups was estimated using the log-ranked test. Comparisons among multiple groups were performed by one-way analysis of variance (ANOVA). A p-value of < 0.05 was considered statistically significant. All analyses were performed using SPSS 13.0 software (Chicago, IL).

4.4 RESULTS

4.4.1 Cyclooxygenase expression and activity in TRAMP prostates

We first determined the level of expression and activity of COX enzymes in the prostate tissue of wild-type (WT) and TRAMP transgenic mice. As shown in the immunoblot in Figure 4.1 A, both COX-1 and COX-2 proteins are expressed in DP tissue from WT mice as determined by commercial antibodies from Santa Cruz

Biotechnology. Interestingly, the expression of both COX proteins was dramatically reduced in TRAMP DP tissue as early as 10 wk of age. COX protein expression remained at a reduced level at 16 wk of age and was further reduced in tissue from PD tumors. The commercial antibody against COX-2 from Cayman Chemical detected the expression of

COX-2 in both the WT and TRAMP prostate tissue, although there was high background binding. The COX-2 antibody from BD only detected COX-2 expression in the positive control, while the COX-2 antibody from Oxford Biomedical Research failed to produce any signal. The Cayman Chemical polyclonal COX-1 antibody worked as well as the

103

B 10.00 * COX-1 10.00 COX-2 *

1.00 1.00

0.10 0.10

0.01 0.01

Relative expression mRNA 10 16 Relative expression mRNA 10 16 0.00 WT WK WK PD 0.00 WT WK WK PD

Figure 4.1 Expression of cyclooxygenase enzymes in dorsal prostate (DP) tissue is reduced in TRAMP mice compared to wild-type (WT) mice. A, Western blot analysis of COX-1 and COX-2 protein levels in DP tissue from WT (10 wk), TRAMP DP (10 and 16 wk), and from histological confirmed poorly-differentiated tumor tissue (PD). Mouse vas deferens tissue was used as positive control for COX (+). Tissue lysates from two different mice were analyzed for each tissue type and images are representative of two independent experiments. B, Quantitative RT-PCR of COX-1 and COX-2 mRNA levels in WT (10 wk) and TRAMP DP tissues (10 and 16 wk), and PD tumor. Each bar represents the mean COX-1 or COX-2 mRNA expression (error bars are 95% confidence intervals) of five animals relative to a common calibrator sample generated by pooling the dorsal, ventral, and lateral prostates of a single wild-type mouse. 18s RNA levels were used to normalize the expression of individual samples. Data points with an asterisk are significantly different from other data points in the same panel (p < 0.05).

104 COX-1 antibody from Santa Cruz. To further confirm the results obtained by immunoblot

analysis, we performed real-time RT-PCR to quantify relative expression levels of COX

mRNA in WT and TRAMP prostate tissues (Figure 4.1 B). The relative expression

levels of COX mRNA in DP and PD tissues correlated extremely well with protein levels.

TRAMP DP tissue expressed significantly less COX mRNA at 10 wk and 16 wk of age

and expression was further decreased in PD tumor tissue. Since most studies reporting

COX-2 over-expression were determined by immunohistochemistry methods (24-26, 28,

29, 261), we also undertook a careful analysis of COX-1 and COX-2 expressions in WT

and TRAMP prostate tissues using validated methods. The COX-2 immunohistochemical

staining protocol was optimized to consistently produce strong staining of positive

control tissue (vas deferens) from WT mouse or prostate lobes from the Pb.COX-2 mouse,

while not staining negative control vas deferens from COX-2 knockout mouse (Figure

4.2). We did not, however, detect COX-2 protein expression in prostate tissues from WT

or TRAMP mice at any stage of pathology (Figure 4.2). These results suggest that COX-

2 antibody detected COX-2 staining in the positive control tissues but was not sensitive enough to detect the relatively low expression in prostate tissues. We also found that commercially available COX-2 antibodies produce strong non-specific epithelial and stromal staining in both the TRAMP and COX-2 null prostate tissues if immunohistochemical conditions are not carefully controlled. Strong COX-1 protein expression was initially observed in both WT and TRAMP prostate tissues under conditions lacking a negative control. However, use of the COX-1 blocking peptide demonstrated that COX-1 staining in prostate tissue is non-specific. We found two

105

Figure 4.2 COX-2 immunohistochemical staining of serial sections from TRAMP dorsal prostate (DP) tissue with prostatic intraepithelial neoplasia (PIN), moderately- differentiated carcinoma (MD), and poorly differentiated (PD) tumors. Vas deferens (Vas-D) from WT mouse served as positive control, while vas deferens from a COX-2 knockout mouse served as negative control.

106 additional bands migrating at ~ 110 and ~ 40 kDa in immunoblots, suggesting cross

reactivity of this antibody with non-COX-1 antigens.

To further determine the activity of COX enzymes in the TRAMP mice, we

measured the endogenous PGE2 level in DP from 10 wk old WT and TRAMP mice using

LC/MS/MS. In the WT mice, DP had the highest level of PGE2 as compared to other

lobes (Figure A.5). In correlation with the expression of COX enzymes, PGE2 levels

were significantly lower in DP from TRAMP mice compared to WT littermates (Figure

4.3). PGE2 levels remained significantly lower in TRAMP PD tumors than in DP from

WT mice (Figure 4.9). Collectively, the above results suggest that both the expression

and the activity of COX enzymes are significantly decreased in the TRAMP prostate

tissue compared to the WT littermates.

4.4.2 Tumorigenesis and histopathological evaluation of TRAMP mice

We characterized the histopathology of TRAMP mice (chow diet-fed) by collecting prostate tissues at different ages (6 to 35 wk). The results correlated well with the pathological changes previously described (33). Briefly, histological PIN and early carcinoma was present in 100% of TRAMP prostates by 8 wk of age, which progresses to

PD with distant site metastasis by 16 to 32 wk of age. Metastatic sites included the pelvic lymph nodes, liver and lung. In very rare cases, metastases were found in the kidney and adrenal glands. The WD and MD were observed at all ages between 8 to 32 wk old. We scored prostatic glandular ducts and metastases in TRAMP prostate tissues for the tumor studies according to the histopathologic criteria described previously (33). The

107

Figure 4.3 Prostaglandin E2 (PGE2) levels in dorsal prostate (DP) tissue are reduced in 10 wk old TRAMP mice compared to wild-type (WT) mice. Tissues were analyzed for PGE2 by LC/MS/MS. Bars represent the mean from at least five mice (error bars are 95% confidence intervals). * p < 0.05.

108 representative H&E stained sections of DP (Figure 4.4 A), LP (Figure 4.4 B), VP

(Figure 4.4 C), and AP (Figure 4.4 D) tissues from TRAMP mice and sections of

TRAMP lung (Figure 4.4 E) and liver (Figure 4.4 F) tissue with micro-metastases are shown.

4.4.3 Effects of celecoxib and indomethacin on the incidence of poorly-differentiated tumors and metastases in TRAMP mice

To determine the effects of cyclooxygenase inhibition on prostate tumorigenesis, we fed TRAMP mice either control diet (AIN-76A) or diets containing celecoxib at 500

ppm and 1500 ppm, or indomethacin at 4 ppm. All animals (30 per group) were fed diets

starting at weaning (3 wk old) and continued for 27 weeks unless mice were necropsied

or died prior to 30 wk of age. Mice that died prior to 30 wk of age without a PD

carcinoma or that had primary tumors of non-prostatic origin were excluded from the

analysis. There were no significant differences in body weights or food intake for animals

that received either control or experimental diets (data not shown). As summarized in

Table 4.1, feeding TRAMP mice NSAID diets did not reduce the incidence of tumor and

metastases in LN, liver and lung compared to TRAMP mice fed the control diet. By 30

wk, approximately 60 % of TRAMP mice developed palpable tumors (PD) in control-

(15/26), 500 ppm celecoxib- (18/28), and 1500 ppm celecoxib- (15/25) fed groups.

Although there was a slight reduction in the incidence of PD tumors (12/26) and LN

metastases (13/26) in mice fed 4 ppm indomethacin, this difference was not significant

statistically. There were no significant differences in the size and weight of the PD

109

Figure 4.4 Histopathologic criteria used to score prostatic glandular ducts and metastases in tissues from TRAMP tumor studies. Representative H&E stained sections of dorsal (A), lateral (B), ventral (C), and anterior (D) prostate tissue from TRAMP mice as well as sections of TRAMP lung (E) and liver (F) tissue with micro-metastases are shown. For sections from each lobe glandular ducts with histological features of normal ducts (N), prostatic intraepithelial neoplasia (PIN), well-differentiated carcinoma (WD), and moderately-differentiated carcinoma (MD) are indicated if present. Micro-metastases in the lung and the liver are indicated by arrows. Length of the black bar present in each panel is 100 microns. 110

Lymph nodes Lung Treatment n* PD tumors Mets Mets Liver Mets Control 26 58% 77% 42% 69% 500 ppm 28 64% 71% 50% 53% Celecoxib 1500 ppm 25 60% 68% 68% 80% Celecoxib 4 ppm 26 46% 50% 46% 65% Indomethacin

Table 4.1 Incidence of poorly-differentiated (PD) carcinomas and metastases (Mets) in TRAMP mice fed diets containing cyclooxygenase inhibitors. *Mice that died prior to 30 weeks of age without a poorly-differentiated carcinoma or had primary tumors of non- prostatic origin were excluded from the analysis.

111 tumors between groups. Tumor-free survival analysis revealed that NSAID treatment did not prolong the time to development of palpable tumor of TRAMP mice (Figure 4.5).

4.4.4 Effects of NSAIDs on prostate histopathology and PGE2 levels in TRAMP mice

To further evaluate the effects of celecoxib and indomethacin on the development and progression of prostate adenocarcinoma in TRAMP mice, we analyzed the pathologic lesions in the glandular ducts of each individual pair of prostatic lobes from the control and experimental mice. We first analyzed the wet weight of each individual lobe by using lobes that were only fully dissectible upon necropsy, e.g., DP (n = 12 to 16), LP (n = 8 to

12), VP (n= 10 to 13), and AP (n = 13 to 16). Although the average wet weights of AP,

LP and DP were lower in the celecoxib-fed groups than in the control and indomethacin treated groups, the differences were not statistically significant (Figure 4.6). However, there was a significant reduction in wet weight of VP from mice fed 1500 ppm of celecoxib (Figure 4.6).

As described above, the pathologic lesions in the glandular ducts of individual prostatic lobes were assessed using published criteria (33). Only lobes clearly identifiable with microscopic analysis were included. Therefore, we analyzed total lobes of DP (n=19 to 26), LP (n=14 to 17), VP (n= 14 to 17) and AP (n=17 to 24). As shown in

Figure 4.7, treatment of TRAMP mice with either celecoxib or indomethacin did not significantly alter the distribution of pathologic lesions in the glandular ducts of prostatic lobes. Compared to other lobes, DP had the most severe lesions in the glandular ducts in

112

Figure 4.5 Kaplan-Meier survival analysis for the incidence of palpable tumors. Treatment of TRAMP mice with cyclooxygenase inhibitors does not significantly alter prostate carcinogenesis. Three wk old TRAMP mice were treated with an AIN-76A semi- purified diet, or the control diet supplemented with 500 ppm celecoxib, 1500 ppm celecoxib, or 4 ppm indomethacin for 27 weeks. Mice that did not complete the entire 27 week treatment period due to causes other than a palpable tumor are indicated as being censored. 113

a a

a,b b

C 500 1500 4 ppm C 500 1500 4 ppm

Cele Cele Indo Cele Cele Indo

Figure 4.6 Treatment of TRAMP mice with cyclooxygenase inhibitors does not significantly alter prostate wet weights of prostate lobes. Three wk old TRAMP mice were fed either AIN-76A semi-purified diet (Control, C) or the control diet supplemented with 500 ppm celecoxib (500 Cele), 1500 ppm celecoxib (1500 Cele), or 4 ppm indomethacin (Indo) for 27 weeks. Wet weights of anterior (AP, n = 13 to 16), dorsal (DP, n = 12 to 16), lateral (LP, n = 8 to 12), and ventral (VP, n = 10 to 13) prostatic lobes dissected from mice treated with the indicated diets. Only lobes that were fully dissectible were included in the analysis. Bars represent means, error bars are 95% confidence intervals (CI), and means within a panel with different letters significantly differ from each other (p < 0.05).

114

C 5 0 0 1 5 0 0 4 p p m vvvvvv C e le C e le In d o

continued Figure 4.7 Treatment of TRAMP mice with cyclooxygenase inhibitors does not significantly alter prostate carcinogenesis. Three wk old TRAMP mice were fed AIN-

115 Figure 4.7 continued

76A semi-purified diet (Control, C) or the control diet supplemented with 500 ppm celecoxib (500 Cele), 1500 ppm celecoxib (1500 Cele), or 4 ppm indomethacin (Indo) for 27 weeks. Incidence of pathologic lesions in the glandular ducts of prostatic lobes from TRAMP mice fed the indicated diets. The percent of normal (Norm) ducts and ducts containing prostatic intraepithelial neoplasia (PIN), well-differentiated carcinoma (WD), moderately-differentiated carcinoma (MD), phylloides-like lesions (Phyl), atrophic lesions (Atrophy), and poorly-differentiated carcinoma (PD) was determined for each lobe. Only clearly identifiable lobes were included in the analysis; AP (n = 17 to 24), DP (n = 19 to 28), LP (n = 14 to 17), VP (n = 14 to 17). Bars represent means and error bars are 95% CI.

116 that they have higher percentages of WD and MD, but lower percentages of normal and

PIN. Interestingly, in correlation with the effects of 1500 ppm celecoxib on the wet weight of VP, celecoxib at 1500 ppm slightly, but not significantly, increased the percentage of PIN and decreased the percentage of WD in VP compared to the TRAMP mice fed control diet. However, these effects could be due to the non-COX effects of celecoxib.

To verify the effectiveness of our feeding regimen on TRAMP prostate tissue, 10 wk old TRAMP mice (n = 5 per group) were fed either AIN-76A diet (control) or the experimental diets (500 ppm, 1500 ppm celecoxib, and 4 ppm indomethacin) for two weeks. We found that celecoxib at 500 ppm has essentially no effects on PGE2 levels in

DP tissue compared to the DP of control mice (Figure 4.8). However, PGE2 concentration in the DP tissue was significantly reduced with 1500 ppm celecoxib.

Indomethacin had the greatest significant inhibitory effect on the concentration of PGE2 in the DP tissue of TRAMP mice compared to other groups (Figure 4.8). The above data suggest that NSAIDs effectively reduced the activity of the COX enzymes in TRAMP mice, but had very minimal, if any, effect on the inhibition of prostate tumorigenesis in this animal model.

117

C 500 1500 4 ppm

vvvv v Cele Cele Indo

Figure 4.8 PGE2 levels in dorsal prostate (DP) tissue from 10 wk old TRAMP mice fed AIN-76A semi-purified diet (control), or control diet containing 500 ppm celecoxib, 1500 ppm celecoxib, or 4 ppm indomethacin for two weeks. Tissues were analyzed for PGE2 by LC/MS/MS. Bars represent the mean obtained from at least five mice (error bars are 95% confidence intervals). Bars with different letters significantly differ from each other (p < 0.05).

118 4.4.5 Effects of genetic disruption of COX-2 on prostate tumorigenesis in TRAMP mice

We next examined whether disruption of the COX-2 gene in the TRAMP mice could inhibit prostate tumorigenesis by crossing TRAMP transgenic mice with COX-2

KO mice. We generated the TRAMP (+/-) x COX-2 (+/+) as the COX-2 WT (n = 33), and TRAMP (+/-) x COX-2 (-/-) as the COX-2 KO mice (n = 35). Figure 4.9 shows that the endogenous PGE2 level was significantly lower in the DP tissue of COX-2 KO mice compared to the COX-2 WT mice. PGE2 concentrations were further reduced in the PD

of TRAMP mice with either COX-2 WT or COX-2 KO (Figure 4.9). Similar to the treatment with celecoxib and indomethacin, there were no significant effects of the loss of COX-2 gene expression on the incidence of PD tumors, LN or liver metastases in

TRAMP mice by 30 wk of age (Table 4.2). We observed a significant increase in the percentage of lung metastases in TRAMP x COX-2 KO mice compared to the control littermates, which might be due to the intrinsic variation in the TRAMP model. There were no significant differences in the size and weight of the PD tumors between groups.

Tumor-free survival analysis revealed that loss of the COX-2 gene slightly, but not significantly, prolonged the time to development of palpable tumor in the TRAMP (+/-) x

COX-2 (-/-) mice compared to the COX-2 positive littermates (Figure 4.10). The lower overall incidence of PD tumors in these mice (as compared to the NSAID study) is likely due to background strain difference as these mice were not crossed with FVB mice.

119

c c

Figure 4.9 PGE2 levels are significantly reduced in 10 wk old dorsal prostate (DP) tissues of the COX-2 knockout TRAMP mice (COX-2 KO) compared to COX-2 WT TRAMP mice (COX-2 WT). PGE2 levels are further reduced in the 30 wk old poorly- differentiated tumors (PD) of both the COX-2 WT and the COX-2 KO mice compared to 10 wk old DP. Tissues were analyzed by LC/MS/MS and bars represent the mean obtained from at least five mice (error bars are 95% confidence intervals). Bars with different letters significantly differ from each other (p < 0.05).

120

Lymph nodes Lung Genotype n* PD tumors Mets Mets Liver Mets COX-2 WT 30 43% 60% 70% 83%

COX-2 KO 28 36% 54% 93% 82%

Table 4.2 Incidence of poorly-differentiated carcinomas (PD) and metastases (Mets) in TRAMP mice either wild-type (WT) or homozygous mutant (KO) for the COX-2 gene. *Mice that died prior to 30 weeks of age without a poorly-differentiated (PD) tumor or had primary tumors of non-prostatic origin were excluded from the analysis. †Significantly different from COX-2 wild-type (p = 0.026).

121

Figure 4.10 Kaplan-Meier survival analysis for the incidence of palpable tumors. Genetic disruption of COX-2 expression dose not prolong the development of poorly- differentiated (PD) tumors. TRAMP mice either wild-type (COX-2 WT, n = 33) or homozygous mutant (COX-2 KO, n = 35) for the COX-2 gene were followed until 30 weeks of age at which time all surviving animals were necropsied. Mice that did not complete the entire 27 week experimental period due to causes other than a palpable tumor are indicated as being censored.

122 As shown in Figure 4.11, disruption of the COX-2 gene in the TRAMP mice decreased the wet weight of LP in a significant manner, but not the weights of other lobes.

We further analyzed the effects of loss of COX-2 on the pathologic lesions in the glandular ducts of individual prostatic lobes. We found that genetic disruption of COX-2 expression only very modestly reduced prostate tumorigenesis in the LP and VP lobes of

TRAMP mice (Figure 4.12). Taken together, these data suggest that COX-2 deficiency was not sufficient to inhibit tumorigenesis in the TRAMP model.

4.4.6 Effects of COX-2 overexpression on prostate tumorigenesis

To provide further evidence for a role for COX-2 in prostate tumorigenesis, we generated transgenic mice with COX-2 under the control of a probasin promoter

(Pb.COX-2), allowing prostate-specific expression of COX-2. We originally developed

more than 10 founder lines, and only two founder lines had positive COX-2 expression as

determined by immunohistochemistry and immunoblot. Analysis using

immunohistochemistry demonstrated that COX-2 is predominantly expressed in VP,

followed by LP and DP (Figure A.6). The expression of COX-2 in AP was at similar

levels as in WT control. The synthesis of PGE2 was also significantly increased in the

three lobes from the Pb. COX-2 mice than from the WT littermates. PGE2 levels in DP,

LP and VP were 31, 37 and 28 ng/mg protein in the Pb.COX-2 mice, respectively, while

the concentrations of PGE2 were 15, 2 and 1 ng/mg protein in DP, LP and VP in the WT

littermates, respectively (Figure A.7). Consistent with what we observed from the

NSAID study, overexpression of COX-2 in the Pb.COX-2 mice failed to induce any of the histopathology seen in the TRAMP model. Interestingly, we found lymphocyte

123

WT KO WT KO WT KO WT KO

COX-2 Genotype

Figure 4.11 Wet weights of prostate lobes in COX-2 knockout TRAMP (KO) compared to COX-2 WT TRAMP (WT) mice. TRAMP mice either wild-type (n = 33) or homozygous mutant (n = 35) for the COX-2 gene were followed until 30 weeks of age at which time all surviving animals were necropsied. Wet weights of anterior (AP, n = 23 WT, 19 KO), dorsal (DP, n = 23 WT, 22 KO), lateral (LP, n = 18 WT, 16 KO), and ventral (VP, n = 18 WT, 18 KO) prostatic lobes dissected from mice with the indicated genotypes were determined. Only lobes that were fully dissectible were included in the analysis. Bars represent means, error bars are 95% confidence intervals (CI), and KO means within a lobe with an asterisk significantly differ from WT (p < 0.05).

124

continued Figure 4.12 Incidence of pathologic lesions in the glandular ducts of prostatic lobes from TRAMP mice with the indicated genotypes (COX-2 WT and COX-2 KO). The

125 Figure 4.12 continued percent of Normal (Norm) ducts and ducts containing prostatic intraepithelial neoplasia (PIN), well-differentiated carcinoma (WD), moderately-differentiated carcinoma (MD), phylloides-like lesions (Phyl), and poorly-differentiated carcinoma (PD) was determined for each lobe. Only clearly identifiable lobes were included in the analysis; the number of lobes assessed for each genotype was the same as that for the wet weight analysis. Bars represent means and error bars are 95% CI. Bars with an asterisk significantly differ from other bars in the same panel (p < 0.05).

126 infiltration into the VP and the appearance of hyperplasia in a very small percentage of the mice in one of the Pb.COX-2 founder lines (Figure A.8).

4.5 DISSCUSSION

Prostate cancer (PC) is a major public health problem in the US which results in significant morbidity and mortality each year. New strategies that target molecular changes in prostate carcinogenesis are needed for both the prevention and treatment of this disease. There is substantial evidence for the upregulation of COX-2 in a number of cancers and its pro-tumorigenic activity in these cancers (1). COX-2 selective inhibitors have been intensely studied for their cancer chemopreventive potential. However, available data regarding the expression of COX-2 in prostate cancer and its role in prostate carcinogenesis have been inconclusive. Recently, the use of COX-2 selective inhibitors has been associated with an increased risk for cardiovascular heart disease (145,

255). The lack of definitive evidence showing a benefit of COX-2 inhibition in PC calls for a more critical examination of the role for COX-2 in prostate carcinogenesis before considering interventions to inhibit COX-2 in PC (201).

In this study, we addressed this question using both pharmacologic and genetic approaches. We first examined the effect of NSAID treatment on prostate tumorigenesis in the TRAMP model. We found that neither celecoxib nor indomethacin was able to inhibit the incidence of tumors or metastases or the progression of pathological lesions.

Our results contradict two earlier studies in TRAMP mice in which celecoxib reduced the incidence of PIN, decreased tumor incidence and blocked metastasis (34, 35). However, t

127 there are marked differences in experiments between our study and those studies. We used the C57/BL6J TRAMP transgenics that were crossed with WT FVB mice, while the

TRAMP mice used in the above two published studies were on a pure C57BL/6 background. Background strain has been reported to affect the prostate tumor phenotype in the TRAMP model (262). In addition, due to the stochastic development of PD in the

TRAMP model between 16 to 32 wk of age and the occasional idiopathic loss of mice from the study, at least 30 mice per treatment group are needed to provide sufficient power to detect statistical significance between experimental groups. In agreement with our findings, Zeng et al. (263) found that the selective COX-2 inhibitor nimesulide failed to inhibit prostate tumorigenesis in probasin/SV40 T antigen transgenic rats, a model in which carcinogenesis is genetically initiated in a same manner as in the TRAMP model.

Until now, only a few animal studies have been carried out to test the effect of NSAIDs on prostate carcinogenesis. Recently, COX-2 selective inhibitors have been reported to have effects that are independent of COX-2 activity that could account for their effects on prostate tumorigenesis (36). Taken together, experiments using pharmacologic approaches do not appear to provide strong evidence that COX-2 plays a pro-tumorigenic role in prostate carcinogenesis.

We next used genetic approaches in which the COX-2 gene was either knocked out or overexpressed in the mouse prostate to definitively determine the role of COX-2 in prostate carcinogenesis. In support of the results from the NSAID study, deletion of

COX-2 in the TRAMP mice had essentially no effect on prostate tumorigenesis, although there was a slight inhibitory effect on the severity of the lesions in the LP and VP.

Similarly, we did not observe any pathological lesions in the prostates of the Pb.COX-2

128 mice. In contrast, genetic studies using either transgenic or knockout technology have firmly established a link between COX-2 and tumorigenesis in cancers of colon, mammary, skin and bladder (1). To our knowledge, we are the first laboratory to examine the role of COX-2 in prostate carcinogenesis using genetic approaches. Our results indicate that COX-2 may not play a critical role in prostate carcinogenesis. However, in one of the Pb.COX-2 founder lines we found lymphocyte infiltration into the VP and the appearance of hyperplasia in a small percentage of the mice (Figure A.8). Given the recent evidence that COX-2 may be overexpressed only in areas of inflammation in PC tissues (30, 31), a role for COX-2 in either the initiation or promotion of this disease by eliciting an inflammatory response cannot be ruled out.

Since our results contradict many previously published studies, we performed a careful analysis of the expression and activity of the COXs in the WT and TRAMP mouse prostate tissues. Unexpectedly, both the expression and activity of COX-1 and

COX-2 were substantially decreased in the TRAMP prostates compared to the WT littermates. The expression and activity of the COXs were also progressively reduced as the PC progressed. To show that this phenomenon was not unique to the TRAMP model, we examined the expression of COX mRNAs in the AP and DP of 10 wk old prostate specific phosphatase and tensin homolog deleted on chromosome 10 (PTEN) knockout mice (34, 36). Unlike TRAMP mice in which carcinogenesis is driven by the SV40 viral oncogene, prostate carcinogenesis in prostate-specific PTEN knockout mouse is driven by the deregulation of downstream pathways of PTEN (262, 264), in which AP has the most severe phenotype. Similar to our observation in TRAMP mice, COX mRNA levels were significantly reduced in both the AP and DP of conditional PTEN knockout mice

129 compared to the WT littermates (Figure A.9). Taken together, the loss of COX

expression and its activity during prostate carcinogenesis concurs well with results from

our tumor studies and further suggests that COX-2 is unlikely to play a critical role in

prostate carcinogenesis.

A review of the literature reveals possible dissimilarities between studies on

COX-2 expression in PC, including differences in the cell types found to express COX-2,

expression levels in normal vs. benign tissue, and the relationship of level of expression

to grade or stage of PC (1, 200). Some studies only compared the expression of COX-2 in

the benign versus the malignant tissue but ignored measurement in normal tissue. Most studies used non-quantitative methods (26, 28, 29, 34). Additionally, the majority of the studies that used immunohistochemistry to determine COX-2 expression had neither positive nor negative controls. To address this issue, we carefully optimized immunohistochemical protocols to consistently produce strong staining of positive control tissue, while not staining negative control tissue. Under this condition, we found that the commercial COX-2 antibody could only detect highly expressed COX-2 in

positive control tissue, but was not sufficiently sensitive enough to detect the relatively

low expression of COX-2 in WT and TRAMP prostate tissues. To address the same issue,

Zha and coworkers (31) conducted a very comprehensive and well-controlled analysis of

COX-2 expression in human PC tissues. Tissues from a total of 144 PC patients were

analyzed, far more than in any similar study. In contrast to previous results, these

investigators found only scattered expression of COX-2 in less than 1% of cells, and no

difference between cancer and normal cells as determined by immunohistochemistry.

They observed increased COX-2 expression only in areas of inflammation, which was in

130 agreement with Wang et al. (30) who reported that COX-2 upregulation is only associated with chronic inflammation in benign prostate hyperplasia. Zha et al. (31) further performed immunoblotting and qRT-PCR analyses on a subset of frozen tissues.

Interestingly, in agreement with our findings, mean mRNA levels of COX-2 were considerablely reduced in malignant prostate as compared to normal tissue, suggesting a down-regulation of COX-2 in PC. Similarly, by using well-controlled immunohistochemistry and in situ hybridization analysis, Shappell et al. (27) found that

COX-2 expression was more often reduced in prostate tumor compared to benign tissue, and this reduction was significant for Gleason score 5 and 6 tumors. However, the authors did not examine the level of COX-2 expression in normal prostate tissue.

Although Gupta et al. (34) found a progressive increase in COX-2 expression in the

TRAMP prostate tissue from 8 to 24 wk old, COX-2 expression was lost after 24 wk of age. Thus, there are a substantial number of reports on human and murine tissues that show COX-2 expression diminishes during progression of the lesion.

Although the concept that COX-2 diminishes with the tumorigenesis of PC has not been well acknowledged, results from a substantial number of in vitro studies actually support this concept. Either very low or non-detectable expression of COX-2 in prostate cancer cells PC-3, DU145, LNCaP and TSU has been reported by several investigators

(31, 182, 201-203). In addition, growth of PC-3 and DU145 have been found to be resistant to the treatment with a COX-2 selective inhibitor (201). Interestingly,

Subbarayan et al. demonstrated that in contrast to the low mRNA and protein levels of

COX-2 in prostate cancer cells, the basal level of COX-2 expression was high in the normal PrEC cells (182). Another study found that COX-2 protein expression was high in

131 the normal PrEC and the benign BPH-1 cells, but low in LNCaP and PC-3 cells (202).

We also determined the expression of COX-1 and COX-2 in seven prostate cell lines by immunoblotting. In agreement with results from our in vivo study and the above studies, we found that COX-1 and COX-2 were not detectable in four prostate cancer cell lines, i.e., PC-3, DU145, LNCaP and 22RV1. However, high level of COX-1 and COX-2 expression were observed in the non-tumorigenic prostate PWR-1E, RWPE-1 and its tumorigenic form RWPE-2 cells (Figure A.10).

Although it has been generally assumed that COX-1 is constitutively expressed and involved in the maintenance of tissue homeostasis, this view has been challenged by studies with COX-1 knockout mice. Disruption of the COX-1 gene is as effective as

COX-2 gene disruption in imparting resistance to chemically induced skin tumors (187) and intestinal polyps in Min mice (188). Furthermore, COX-1 expression has been shown to be elevated in renal, cervical, and ovarian cancers and to regulate angiogenic growth factors (189-191). Our study also suggests that the expression of COX-1 was progressively lost during prostate tumorigenesis in the TRAMP model. Given the fact that indomethacin failed to inhibit prostate tumorigenesis in TRAMP mice, COX-1 may also not play a role in the development and progression of this disease.

Contrary to the conventional wisdom about COX-1, COX-2 is considered to be absent in most tissue under normal conditions, but rapidly induced by growth factors or cytokines and promotes tumorigenesis in several tissues. However, unlike most epithelial tissues where COX-2 is not expressed under normal conditions, COX-2 is highly expressed in normal prostate (180). Extremely high levels of PGs are found in semen as a product of prostate and seminal vesicles (235). The constitutive expression of COX-2 is

132 likely to be important in maintaining normal homeostasis and function of the prostate

(182). Collectively, strong evidence suggests that the expression of COX-2 and maybe

COX-1 (which needs more evidence) is either decreased or lost during prostate

carcinogenesis. The loss of COX-1 and COX-2 may be characteristic of the molecular

changes during prostate carcinogenesis.

Although the mechanisms underlying the marked decrease of COX mRNA and

protein levels during prostate tumorigenesis have yet to be determined, there are a

number of possible mechanisms may explain. The down-regulation of COX enzymes

could be a result of negative-feed back regulation due to decreased substrate availability

due to competition with LOX for arachidonic acid. In vitro evidence that COX-2 mRNA

can be upregulated by COX-2 stimulators in prostate cancer cells that only minimally

express COX-2 suggests that COX-2 gene was not impaired in prostate cancer cells (31,

182). Although many factors have been shown to upregulate COX-2 expression at the

transcriptional and post-transcriptional levels, few inhibitory effectors have been reported.

Wild-type p53 has been shown to inhibit COX-2 transcription by competing with TATA-

binding proteins for binding to COX-2 gene (184). Although the SV40 large T antigen

interacts with p53, p53 activity is actually increased in the TRAMP mice (32). This

observation is also supported by the observation of Perez-Stable et al. that p53 was upregulated in a prostate transgenic mouse line driven by SV 40 T antigen (265).

Upregulation of the WT p53 in human PC has also been reported. The downregulation of

COX-2 in prostate carcinogenesis may result from suppression by p53. Another interesting observation is that the CpG island upstream of the COX-2 gene is methylated in greater than 85% of primary and metastatic prostate cancers and in all the seven tested

133 prostate cancer cell lines, but not in normal tissues (185). Although COX-2 is frequently overexpressed in colorectal cancer, evidence suggests that COX-2 expression is reduced in colorectal cancer with a defective mismatch repair system (186), a phenomenon commonly associated with hereditary nonpolyposis colorectal cancer (HNPCC).

Interestingly, the lack of tumor COX-2 expression in HNPCC is strongly associated with

COX-2 promoter hypermethylation. This evidence suggests that the loss of COX-2 expression may due to the hypermethylation of COX-2 in several tumors, including prostate. However, the epigenetic mechanism for COX-1 down-regulation in cancer has not been examined. Regardless of the mechanism, it is not likely that loss of COX expression contributes to the tumorigenesis of PC. Further studies are needed to address this question and the basis for this molecular change.

Upregulation of COX-2 results in increased synthesis of PGE2. This product is the key mediator of the COX-2 pathway that activates downstream signaling and thus contributes to tumorigenesis by inducing cell proliferation, angiogenesis, invasion and metastasis (3). The exact role of PGE2 in prostate carcinogenesis, however, is currently unknown. Although several studies have assumed that PGE2 levels are increased in prostate cancer tissues and in vitro cultured prostate cancer cells, PGE2 levels were not measured in most studies. We found that PGE2 levels were significantly reduced in

TRAMP prostate tissue compared to wild type littermates. In support of our findings, two investigators found that PGE2 production is low or non-detectable in human benign prostates and prostate tumor tissues (27), and in PC-3 and DU145 cells (201). However, there are no reports on PGE2 levels in normal human or mouse prostate tissue.

134 A substantial amount of evidence suggests that the level of arachidonic acid, precursor of PGE2, is decreased in prostate malignant tissues compared with non-cancer

or benign tissues in human (252, 266-270) and rat (252). It was initially assumed that

decreased arachidonic acid in prostate cancer is a result of increased metabolism of

arachidonic acid from producing prostaglandins or leukotrienes (267). However, given

the evidence that the expression of COX and level of PGE2 are significantly decreased in

prostate cancer, it is not likely that the increased metabolism of arachidonic acid is to

produce prostaglandins. Lipoxygenase (LOX) metabolizes arachidonic acid to various

hydroxyl-eicosatetranoic acids (HETE) which have cancer-promoting effects by several

mechanisms (268). Indeed, recent evidence suggests that expression of different LOX

isomers and their metabolites were increased in the urine and tissue from prostate benign

or cancer patients (27, 270, 271). Interestingly, Kelavkar et al. found that the majority of

arachidonic acid metabolites are 12-HETE and 15-HETE in TRAMP prostate with

increased 12/15-LOX activity, an ortholog of human 15-LOX-1, compared to prostate in

WT mice (272). The same investigators further demonstrated that conditional expression

of human 15-LOX-1 induces PIN in transgenic mouse model (273). However, the

synthesis of prostaglandins in these studies was low or non-detectable. We also found

that 12-HETE production was significantly increased in the TRAMP DP compared to the

WT DP (Figure A.11). However, COX metabolites other than PGE2, including PGA2,

PGD2, and 15-d-PGJ2, were also decreased in the TRAMP DP (Figure A.11).

Collectively, these results suggest that LOX isomers and their metabolites originated

from arachidonic acid present in high fat diets may play a significant role in prostate

carcinogenesis.

135 In conclusion, we have demonstrated that neither pharmacological inhibition nor genetic modification of COX-2 had an effect on prostate carcinogenesis. The lack of a pro-tumorigenic role for COX-1 and COX-2 in prostate carcinogenesis is further supported by our observation that both the expression and activity of COX-1 and COX-2 are progressively lost during prostate carcinogenesis. This is the first study to definitively establish the lack of a positive link between COX-2 and the progression of prostate cancer. Given the epidemiological evidence that NSAID use may be protective against

PC (20-23) and the evidence that COX-2 may be associated with inflammation processes in PC, a role for COX-2 in the initiation of prostate cancer cannot yet be ruled out.

Further work will be required to determine the significance of the loss of COX genes in prostate carcinogenesis, and the role of COX-2, if there is any, in the early stage of prostate cancer development.

136

CHAPTER 5

OVEREXPRESSION OF CYCLOOXYGENASE-2 (COX-2) IN THE MOUSE URINARY BLADDER INDUCES THE EXPRESSION OF IMMUNE AND CELL PROLIFERATION RELATED GENES

Xingya Wang1, Jennifer K.L. Colby3, Robert Rengel2, Susan M. Fischer3,, Steve K. Clinton1,2, Russell D. Klein1,2,4

1The Ohio State University, Interdisciplinary Ph.D. Program in Nutrition (OSUN) 2the Ohio State University Comprehensive Cancer Center, Molecular Carcinogenesis and Chemoprevention Program

3The University of Texas M.D. Anderson Cancer Center Science Park-Research Division P.O. Box 389, Park Rd. 1C, Smithville, TX 78957

4Department of Human Nutrition, the Ohio State University 325 Campbell Hall, 1787 Neil Aveunue, Columbus, OH 43210

This study was supported by grants CA091865 (RDK) and CA100140 (SMF) from the National Cancer Institute.

137 5.1 ABSTRACT

Increased COX-2 expression has been reported in transitional cell carcinoma

(TCC) of the urinary bladder and has been positively correlated with grade and stage.

However, the exact role of COX-2/PGs play in bladder carcinogenesis remains unknown.

We have recently developed a transgenic mouse model which overexpresses COX-2 under the control of a bovine keratin 5 (BK5) promoter. BK5.COX2 mice had a high incidence of bladder transitional cell hyperplasia (TCH), but a relatively low incidence of bladder neoplasia. We have now conducted microarray gene analysis to determine the effects of COX-2 overexpression on the regulation of gene expression profile in urinary bladder. Total RNA was isolated from whole urinary bladders of 10 wk old BK5.COX2 transgenic mice and wild-type FVB/n mice. Gene array analysis was carried out using

Affymetrix 430 2.0 mouse gene chips. Analysis of gene array data by t-test (p<0.05) revealed 2-fold or more changes in 70 of the upregulated genes and 60 of the downregulated genes in bladders from transgenic compared to wild-type mice. Gene set analysis using Expression Analysis Systematic Explorer (EASE; NIH) software revealed that genes associated with Immune/Stress Response and Cell Cycle/Proliferation biologic processes were significantly overexpressed in the top 100 upregulated genes. Relevant downregulated genes included three TGF beta related genes (Tgfb2, Tgfb3, and Tgfbi), and the anti-angiogenic gene thrombospondin 2 (Thbs2). Expression analysis of the growth factors and TGF beta-related genes by qRT-PCR was carried out to validate these findings. The growth factor, epiregulin, was the most highly induced gene among those validated by qRT-PCR in the transgenic bladders compared to the wild-type bladders,

138 with 3.7 fold and 5.3 fold increase at 10 and 30 wk of age, respectively. Increased staining of Ki67, the proliferation biomarker, was observed in TCH of BK5.COX2 mouse bladder compared to wild type mouse bladder tissues. We then found that PGE2 at concentration in BK5.COX-2 transgenic mouse bladders significantly induced the expression of epiregulin mRNA in bladders from wild-type FVB mice ex vivo, suggesting a direct regulatory effect of PGE2 on epiregulin expression. We further determined that treating bladder cancer UMUC-3 cells with recombinant hEpiregulin increased both cell proliferation and Erk phosphorylation. The results of gene expression analysis indicate that the initial response of the mouse urinary bladder to elevated COX-2 expression includes mounting an inflammatory response and inducing cell proliferation. Epiregulin, the growth factor that associated with cell proliferation, may serve as a novel biomarker and target for future prevention and treatment of bladder cancer.

5.2 INTRODUCTION

Bladder cancer is the fourth most common type of cancer in men and the ninth most common type in women in the US. The morbidity of bladder cancer is increasing due to high rate of recurrence. It is widely accepted that specific molecular targets in bladder carcinogenesis must be identified to decrease both the incidence and mortality of this disease. There is strong evidence that COX-2 is overexpressed in transitional cell carcinoma (TCC) of human urinary bladder (4-7). The degree of COX-2 expression is significantly correlated with the tumor grade and depth of invasion (T stage) of TCC (8-

10). Increased COX-2 expression has also been reported in rat and canine models of

139 bladder cancer (204, 205). A case control study involving 1514 incident bladder cancer

cases and an equal number of controls reported that use of NSAIDs decreased the risk for

urinary bladder cancer by almost 20% (213). NSAIDs have been shown to inhibit the

growth of bladder cancer cells in vitro (11-14, 212). In addition, COX inhibitors also

induced remission of chemically induced bladder tumors in rodents (207, 208), naturally

occurring invasive TCC in dogs (209, 210), and bladder carcinoma in an orthotopic

mouse model (211).

Although the above evidence suggests that there is a strong association between

COX-2/prostaglandin synthesis and the development and progression of urinary bladder cancer, the exact role and mechanisms of COX-2 overexpression during bladder carcinogenesis have not been well defined. Overexpression of COX-2 has also been associated with the development of other types of cancer, including lung, colon, mammary, and skin. Evidence from genetic and pharmacologic studies suggest that

COX-2 and prostaglandins have tumorigenic effects in these cancers by increasing cell proliferation, enhancing angiogenesis, promoting invasion, and inhibiting apoptosis (1, 3).

However, the exact molecular pathways that are regulated by COX-2 overexpression are largely unknown. At present, there has been no study to determine the effects of COX-2 overexpression on gene profiles during tumorigenesis in any type of cancers.

The use of genetically modified mouse models has greatly enhanced our understanding of the potential function of certain genes, such as COX-2, in tumorigenesis.

A previous study by Klein et al. has shown that forced expression of COX-2 under the

control of a keratin 5 promoter is sufficient to cause mouse (NMRI background) urinary

bladder transitional cell hyperplasia (TCH) in an age-dependent manner (3). They further

140 observed that TCH was strongly associated with increased Ki67 staining, and induced

lymphocytes and macrophage infiltration in COX-2 overexpression transgenic K5.COX-

2 mouse bladders. We have now developed a transgenic mouse model (BK5.COX2) in

which overexpression of COX-2 is driven by bovine keratin 5 (BK5) promoter on FVB

background. In this model, a similar but more severe phenotype developed in the urinary

bladders compared to the K5.COX-2 mouse model. The level of prostaglandin E2 was

significantly increased in the bladder of BK5.COX-2 mice, as has been reported to be

elevated in patients with bladder cancer (4, 206). Here, we conducted Affymetrix

microarray analysis to determine the effects of COX-2 overexpression on genetic and molecular changes during the early stage of bladder carcinogenesis. We found that genes involved in the Cell Cycle/Proliferation biologic processes and Immune/Stress Response are significantly upregulated. Relevant genes that are downregulated include the TGF beta-related genes and the anti-angiogenic gene thrombospondin 2. We further determined that the growth factor, epiregulin, was the most highly induced gene among those validated by qRT-PCR.

Our study is innovative in that we developed a unique mouse model that provided us an opportunity to study the role of COX-2 and PGs during bladder carcinogenesis. The results clearly demonstrated that COX-2 overexpression and PGE2 overproduction play a

tumorigenic effect in bladder cancer through regulating growth/proliferation,

extracellular remodeling, and immune responses related genes. This is the first study that

determined the regulation of gene expression profile by COX-2 during early stages of

carcinogenesis of any type of cancer.

141 5.3 MATERIALS AND METHODS

5.3.1 Materials

PGE2, PGE2 EIA kit, polyclonal murine COX-1 and COX-2 antibodies, and goat anti-rabbit IgG horseradish peroxidase (HRP) were purchased from Cayman Chemical

(Ann Arbor, MI). Phospho- and total- Erk polyclonal antibodies, EGFR, erbB4, anti-

Rabbit HRP-linked IgG, and HRP-conjugated Anti-Biotin antibody were purchased from

Cell Signaling (Beverly, MA). Polyclonal beta actin antibody was obtained from abCAM

(Cambridge, MA). Recombinant hEpiregulin was purchased from R&D Systems

(Minneapolis, MN). TRIzol reagent and goat serum were purchased from Invitrogen

(Carlsbad, CA). RNeasy Mini RNA extraction kit was purchased from Qiagen (Valencia,

CA). The iScript cDNA synthesis kit was purchased from Bio-Rad (Hercules, CA).

Taqman® Gene Expression Assay kits for all primers (COX-1, COX-2, Ereg, IGF-1,

IGFBP6, TGFBi, TGFB2, TGFB3, GAPDH, and 18s), and Taqman® Universal PCR

Mastermix were purchased from Applied Biosystems (Foster City, CA). Dako Envision

Plus kit (K0411) was purchased from DakoCytomation (Denmark). BCA protein Assay kit and RadioImmuno Precipitation Assay (RIPA) cell lysis buffer were purchased from

Pierce (Rockford, IL). The 3-[4,5-2-yl]-2-, 5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO).

5.3.2 Genetically-engineered mice

The BK5.COX2 transgenic mice were generated on the FVB background by pronuclear injection of a bovine keratin 5 (BK5) promoter construct containing the

142 coding region of the mouse COX-2 gene, as well as a rabbit beta-globin intron and an

SV40 poly-A tail (Figure A.12). Nine founder lines were initially generated and one line that expressed high level of COX-2 and also produced offspring was used from subsequent studies (line #7). In order to maintain the line, BK5.COX2 transgenic males were bred to FVB/n wild type females. Because fertility of female transgenics was greatly reduced, female offspring only were used for experimental analysis. It was noticed in early attempts at breeding the mice that many females were undergoing premature parturition as a result of high PGE2 levels derived from placenta of transgenic

pups. In order to avoid loss of entire litters, premature delivery was prevented by feeding

the dams low levels of 4 ppm indomethacin-supplemented AIN-76 A diet (Research

Diets, St.Paul, MN) several days before delivery. Hemizygous offspring were used for all

subsequent analyses. Both male and female transgenic mice were used for histopathology.

However, only female transgenic mice were used for microarray and other biologic

studies due to the higher rate of reproduction than the male transgenic mice. Initial

genotyping was done by PCR using genomic DNA extracted from tail snips. Primers

used to recognize the COX-2 transgene are as follows: 5’-TCA-AAG-ACA-CTC-AGG-

TAG-AG-3’(forward); 5’-CTT-GAG-TTT-GAA-GTG-GTA-AC-3’(reverse). Since

BK5.COX2 mice were hairless due to defects in hair follicles, transgenic mice were identified by a sparse hair coat without performing genotyping assay. All mice were maintained in HEPA-filtered cages according to Institutional Laboratory Animal Care and Use Committee (ILACUC) approved protocols at the Science Park-Research

Division, University of Texas, MD Anderson Cancer Center, and at the Ohio State

University.

143 5.3.3 Tissue preparation and histopathology analysis

The bladder tissues were collected at different ages (10, 20, 30 and 40 wk old).

The urinary bladders were excised and cut in half and fixed in 10% buffered formalin.

The fixed urinary bladders were then positioned with the cut faces oriented in the same

direction for paraffin embedding. Four-micron sections were stained with hematoxylin

and eosin (H&E) by standard protocols. All bladder sections from different age of mice

(about 15 mice/age group) were evaluated according to established published criteria

(274, 275).

5.3.4 Immunohistochemistry

Paraffin-embedded specimens were cut into 4 micron thick sections,

deparaffinized, rehydrated, antigen retrieved in citrate buffer (pH 6.0), and blocked for

endogenous peroxidase with H2O2 (Dako k0411 kit). The specimens were incubated in

5% goat serum for 10 min to block non-specific binding. Primary polyclonal antibody

against COX-2 were used at a dilution of 1:1000 and applied to sections for 1 h. The

specimens were subsequently washed in phosphate-buffered saline (PBS) and treated

with secondary antibody (Dako k0411 kit) for 30 min. To visualize the antibody, 3,3’-

diaminobenzidine tetrahydrochloride (DAB, Dako k0411 kit) was incubated for 5 min.

Finally, the slides were dehydrated and mounted. The mKi67 staining was performed by the pathology core facility of the CCC at OSU.

144 5.3.5 Immunoblotting

Total proteins were collected from frozen bladder tissues of BK5.COX2 mice or

cultured cells by standard methods. Approximately 40 µg protein (from tissue) or 25 µg protein (from cell culture) was electrophoresed on a 10% SDS-polyacrylamide gel followed by standard immunoblotting procedures. The primary antibodies against COX-1 and COX-2 were analyzed at dilution 1:1000. Beta-actin was used as a loading control.

Cell lysates from a wild-type mouse vas-deferens was used as positive control for COX-1 and COX-2 immunoblotting. Phosphorylated and total Erk1/2 were determined by western blot according to the manufacturer’s instructions (Cell Signaling).

5.3.6 RNA extraction and Real-Time PCR (qRT-PCR)

Total RNA was isolated by TRIzol reagent and further purified by RNeasy Mini kit as described by Qiagen. Both the quantity and quality of total RNA were analyzed by the Agilent Bioanalyzer 2100 system at the core facility of the OSU Comprehensive

Cancer Center. Total RNA was reverse transcribed with an iScript cDNA synthesis kit.

qRT-PCR was performed to determine the expression of all the examined genes using a

1:10 dilution of cDNA with Taqman gene expression primers and ABI mastermix

according to the manufacturer’s instructions on an iCycler IQ qRT-PCR detection system

(Bio-Rad). GAPDH and 18s RNA were used as the reference gene for all samples.

5.3.7 Quantitation of PGE2

PGE2 levels in the bladder tissues of the BK5.COX2 and wild-type mice were

measured using a PGE2 enzyme-linked-immunosorbent (EIA) kit from Cayman Chemical

145 (Ann Arbor, MI). Briefly, bladder tissues were snap frozen and pulverized in liquid

nitrogen, then purified with methanol on a C-18 column. The eluate was collected and

further processed according to the manufacturer’s instructions (Cayman Chemical).

Levels of PGE2 were determined using the multiple linear regression program and expressed as ng/mg of tissue. Experiments were performed in duplicate and 5 mice from

each genotype were used in individual experiment.

5.3.8 Affymetrix microarray and EASE Analysis

Bladder tissues were excised from 10 wk old wild-type and BK5. COX2

transgenic mice (5 mice/group). Total RNA were extracted and purified as described

above. The Affymetrix analysis was performed by the Affymetrix Core Facility at the

Columbus Children’s Hospital Medical Center. One Mouse 430 2.0 GeneChip

(Affymetrix Inc., Santa Clara, CA) was used for each of 10 samples. The mouse genome

430 2.0 array is a high-density oligonuleotide array comprised of over 45,101 probe sets

representing over 40,000 mouse genes. The database for the oligonucleotide in the Mouse

430 2.0 gene chip is available at

http://www.affymetrix.com/support/technical/libraryfielsmain.affx. Briefly, 10µg of total

RNA was used to synthesize double-stranded cDNA. In vitro transcription was performed

to produce biotin-labeled cRNA. The biotinylated cRNA was cleaned with an RNeasy

Mini Kit (Qiagen), fragmented to 50-200 nucleotides, and hybridized to Affymetrix 430

2.0 arrays. Affymetrix MicroArray Suite was used to scan and quantitate the Genechips.

Data were collected for subsequent analysis.

146 5.3.9 Microarray data and EASE Analysis

Genes differentially expressed in BK5.COX2 mice versus wild-type mice were

identified at a p value < 0.05 and fold change ( > 2) by two-way analysis of variance.

Intensity data were collected from each chip and the top 100 up- and top 100 down-

regulated probesets with p <0.05 were then analyzed using Expression Analysis

Systematic Explorer (EASE; NIH) (276). EASE analysis allows one to discern particular

gene classes associated with biologic processes and molecular functions. The results were

segregated into Biological Process, Cellular Component and Molecular Function

ontologies. Within each of these groups, themes are sorted according to EASE-score. The

probability of over-representation is called the EASE score, which is the upper boundary

of the distribution of Jackknife Fisher exact probabilities as determined by EASE. The

probesets having EASE scores of less than 0.05 were considered to be significantly over-

represented.

5.3.10 Cell culture

Human bladder cancer cell line UMUC-3 was purchased from the American Type

Culture Collection. UMUC-3 cells were cultured in RPMI-1640 medium (GIBCO) containing 10% FBS. Cells were maintained in a humidified atmosphere with 5% CO2 at

37ºC. For the experiments, cells were seeded at 2 x 105 cells/well in six-well plates, and

incubated 24 h in serum containing medium. Cells were then serum-starved overnight

followed by treatment with hEpiregulin.

147 5.3.11 Cell Proliferation Assay

UMUC-3 bladder cancer cells were plated in 96-well microtiter plates at an initial

density of 2 x 104 cells per well. Cells were treated with hEpiregulin (0-1 ng/ml) and incubated for 48 h. After incubation, cell proliferation was determined by MTT assay according to manufacturer’s instruction (Sigma, MO).

5.3.12 Organ culture experiment

A total of 12 wild-type FVB mice at 10 wk of age were ordered from Taconic

(Germantown, NY). Upon arrival, mice were maintained in HEPA-filtered cages overnight with access to diet and water. At necropsy, bladder tissues from all the mice

were excised and cut in half. A total of 24 half bladders from the 12 FVB mice were

emerged into 24 individual wells in a 96-well plate containing RMPI medium with 10µM

PGE2. Tissues were incubated (37ºC, 5% CO2) for 0, 1h, 2h, 4h, 6h and 8 h. Four samples (4 wells) were collected at each individual time point. Tissues were snap frozen

and total mRNA was extracted as described above. The effects of PGE2 on epiregulin

mRNA expression in the FVB mice bladders were determined using qRT-PCR as

described above.

5.3.13 Statistical Analysis

Data are presented as means + standard deviation (SD). Comparisons among

multiple groups were performed by one-way analysis of variance (ANOVA) with

Tukey’s post-hoc comparisons. A p-value of <0.05 was considered statistically

significant. All analyses were performed using SPSS 13.0 software (Chicago, IL).

148 5.4 RESULTS

5.4.1 The expression and activity of transgene in BK5.COX2 mice urinary bladder

COX-2 expression was detected in bladder tissues of the BK5.COX2 mice as early as 3 wk of age. At 10 wk, increased COX-2 expression was present in the basal epithelial cells with diffusion into superficial epithelial cells of transgenic urinary bladders, whereas no staining was observed in wild-type epithelial cells (Figure 5.1 A).

COX2 expression remained high as BK5.COX2 mice aged, whereas there was no change in wild-type mice (data not shown). Results form immunoblot analysis also demonstrate

that COX-2 is highly expressed in bladder tissue of BK5.COX2 mice, but not detectable

in the wild-type mice (Figure 5.1 B). The level of COX-2 expression varies considerably

within transgenic mice at the same age. Interestingly, COX-1 expression was reduced in

the urinary bladder of the BK5.COX2 mice compared to the wild-type control (Figure

5.1 B). The relative expression levels of COX mRNA in bladder tissues from transgenic

and wild-type mice correlated extremely well with protein levels as determined by

immunoblotting. COX-2 mRNA expression increased about 400 fold in the bladder tissue

of transgenic mice compared to the control mice, while level of COX-1 mRNA was

reduced by 50% in the transgenic mice compared to the wild-type mice (Figure 5.1 C).

We also determined the level of PGE2 in the transgenic mice bladder as a result of COX-

2 overexpression in these mice. As shown in Figure 5.1 D, the level of PGE2

significantly increased about 100 fold in the BK5.COX2 mouse bladder tissue compared

to the PGE2 level in age-matched wild-type bladders.

149

H&E COX-2

100x 100x

continued

Figure 5.1 Expression of cyclooxygenase enzymes and PGE2 synthesis in bladder tissues of 10 wk old BK5.COX2 (Tg) mice compared to wild-type (Wt) mice. A. COX-2 immunohistochemical staining in Tg and Wt mice bladders. B. Western blot analysis of COX-1 and COX-2 protein levels in Tg and Wt mice bladders. Cell lysates of two individual mice from both Wt (1-2) and Tg (3-4) were used. Mouse vas deferens tissue was used as positive control for COX (+). Beta-actin was used as loading control. C. Quantitative RT-PCR of COX-1 and COX-2 mRNA levels in Tg and Wt mice bladder tissues. Each bar represents the mean COX-1 or COX-2 mRNA expression (error bars are 95% confidence intervals) of five animals relative to a common calibrator sample generated by pooling the 3 bladders from wild-type mice. GAPDH mRNA levels were used to normalize the expression of individual samples. Bar with an asterisk is significantly different from other bar in the same panel (p < 0.05). D. PGE2 levels in Tg and Wt bladder tissues. Tissues were analyzed for PGE2 by EIA kit. Bars represent the mean from 4 mice, and error bars are 95% confidence intervals. Bar with an asterisk is significantly different from other bar in the same panel (p < 0.05).

150 Figure 5.1 continued Wt Tg (+) B 1 2 3 4 80kDa 72kDa COX-2 60kDa

42kDa ß-actin

80kDa 70kDa COX-1 60kDa

42kDa ß-actin

COX-2 C * COX-1

800.00

n n

o o

i i

s s

s s re

re re

re 1.00 p

p 600.00

p p

x x x

e e

A A A

A N

N *

N N R

R 400.00 R

m m

0.50 e

e e

v v

i i

t t

t t a

a 200.00

a a

l l

e e e

e R R R R 0.00 Wt Tg Wt Tg Genotype Genotype

D *

10 (ng/mg of tissue ) 2 5

PGE

(ng/mg ofPGE2 tissue) 0 Wt Tg Genotype Genotype

151 5.4.2 Histopathological evaluation of BK5.COX2 mice

A total of 73 urinary bladders from 10- to 40-wk-old BK5.COX2 transgenic and wild-type FVB mice were examined for pathologic lesions. Histopathologic analysis revealed that 100% incidence of transitional cell hyperplasia in 10 wk old BK5.COX2 transgenic mice (Table 5.1). Only simple (~ 47%) and papillary (~53%) hyperplasia were observed in 10 wk old transgenic mice. Simple TCH is characterized by an increase in the number of epithelial cell layers compared with control animals, which only have 2-3 layers of transitional cells (Figure 5.2, panel a-c). Papillary TCH is characterized by an increase in the number of epithelial cell layers, papillary extensions into the bladder lumen, and some times thickened lamina propria (Figure 5.2, panel d-f). No TCH was observed in any of the urinary bladders from wild-type mice. As mice aged to over 20 wk old, the pathologic lesions are very heterogeneous. We found 29 out of a total of 35 transgenic mice developed TCH from 20 to 40 wk of age and 6 developed transitional cell neoplasia (Table 5.1). Besides simple (~22%) and papillary (~37%) TCH, we also observed nodular hyperplasia characterized by the downward proliferation into the lamina propria and nests of epithelial cells (Figure 5.3 panel a, b) in mice over 20 wk old. The lamina propria in the transgenic mice was significantly thickened in almost all the transgenic mice as indicated in Figure 5.3 panel a (*). There was a marked increasing lymphocyte infiltration nto lamina propria and more often into muscle (Figure 5.3 panel a, c). The degree of lamina propria thickening appeared to be proportional to the severity of epithelial proliferation. Carcinoma in situ (CIS, 6% ) and possible transitional cell carcinoma (TCC, 6%) were observed in a small percentage of the transgenic mice

(Figure 5.3 panel d.e). We also observed transitional cell papilloma in about 6% of

152

Genotype Age (wk) Transitional Cell Hyperplasia* Transitional Cell Neoplasia* simple papillary nodular CIS papilloma possible TCC Wild type 10 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) Wild type > 20 1/13 (7) 0/13 (0) 0/13 (0) 0/13 (0) 0/13 (0) 0/13 (0)

BK5.COX2 10 7/15 (47) 8/15 (53) 0/10 (0) 0/10 (0) 0/10 (0) 0/10 (0) BK5.COX2 > 20 8/35 (22) 13/35 (37) 8/35 (22) 2/35 (6) 2/35 (6) 2/35 (6) *number of positive/number of total (%)

Table 5.1 Histopathologic analysis of the phenotype of urinary bladders in BK5.COX2 transgenic mice and wild-type mice at different ages.

153

10 wk Wt 10 wk Tg

a d

40x 40x e b

200x 200x c f

400x 400x

Figure 5.2 Transitional Cell hyperplasia (TCH) in 10 wk old BK5.COX2 transgenic mouse urinary bladder. Bladders from wild-type (Wt, a-c) and BK5.COX2 (Tg, d-f) mice were stained with hematoxylin and eosin. Note the thin epithelium (black arrow) in Wt bladders in panel a, b and c. Papillary TCH denoted by papillary extensions into the bladder lumen (d - f)) and thickened hyperplastic epilthelium (black arrow in panel d and f).

154 Over 20 wk old BK5.COX2

a e

40x 40x b f

100x 40x c g

200x 40x d h

40x 40x

Figure 5.3 Figure 2. Histopathology of mouse urinary bladders from older (20-30 wk) BK5.COX2 mice. Note the thickened lamina propria (* in a) and increased TCH, very often, the nodular hyperplasia (white arrow in a) that characterized by the downward proliferation into the lamina propria and nests of epithelial cells (a-b). Note the nests of darkly strained inflammatory cells in panel a (black arrow) and panel c at higher magnification. Panel d is an example of carcinoma in situ (CIS, black arrow) in a small percentage of the transgenic mice. Possible transitional cell carcinoma (TCC, panel e) and transitional cell papilloma (f) were observed in a small percentage of the transgenic mice. Both lamina propria invasion (g, black arrow) and muscle invasion (h, black arrow) were observed in sections from TCC.

155 the total transgenic mice examined (Figure 5.3 panel f). Increased vascular vessels were

observed in stromal and lamina propria in many sections from the BK5.COX2 transgenic

mice (data not shown). A total of 15 wild-type mice that over 20 wk old was examined as

controls and only one mouse developed simple hyperplasia which may due to an

incidental lesion (Table 5.1).

5.4.3 Gene regulation profile in BK5.COX2 mice and EASE analysis

The effects of COX-2 overexpression on gene expression profile during bladder carcinogenesis were determined by microarray on 10 wk old BK5.COX2 transgenic and wild-type mice. We chose this age of mice so that we could test the early effects of increased COX-2 expression and elevated prostaglandin levels on gene expression before the induction of substantial TCH has occurred. Analysis of gene array data by t-test

(p<0.05) revealed 2270 genes were significantly (p<0.05) upregulated and 1740 genes were significantly downregulated in transgenic compared to wild-type bladders. However, only 70 of the upregulated genes and 60 of the downregulated genes had expression changed by 2-fold or more in transgenic compared to wild-type bladders. The expression of COX-2 was increased 11 fold in the transgenic mice compared to the wild-type mice.

Gene set analysis using EASE software revealed that genes associated with

Immune/Stress Response, Cell Cycle/Proliferation and Cell Adhesion biologic processes were significantly (EASE score < 0.05) overrepresented in the top 100 upregulated genes as listed in Table 5.2. Examples of upregulated genes in Immune/Stress response biologic processes include immunoglobin genes (Igj, Igh-4, IgVJ558), tumor suppressor

156 Upregulated Genes by Biological Process Affymetrix ID Gene Symbol Gene Name Fold Increase Cell Proliferation/Cell cycle 1419431_AT Ereg epiregulin 2.48 1419519_AT Igf1 insulin-like growth factor 1 2.08 1426817_AT Mki67 antigen identified by monoclonal 2.68 antibody Ki 67 1454694_A_AT Top2a topoisomerase (DNA) II alpha 2.42 1416251_AT Mcm6 minichromosome maintenance deficient 6 1.96 1417314_AT H2-Bf histocompatibility 2, complement 1.56 component factor B 1423774_A_AT Prc1 protein regulator of cytokinesis 1 2.36 1434437_X_AT Rrm2 ribonucleotide reductase M2 1.84 1439436_X_AT Incenp inner centromere protein 1.66 1448314_AT Cdc2a cell division cycle 2 homolog A 1.96 1416802_A_AT Cdca5 cell division cycle associated 5 1.78 Immune-Stress Response 1417318_AT Dbccr1 deleted in bladder cancer chromosome 1.94 region candidate 1 1417314_AT H2-Bf histocompatibility 2, complement 1.56 component factor B 1418021_AT C4 complement component 4 (within H-2S) 4.08 1419684_AT Ccl8 chemokine (C-C motif) ligand 8 2.74 1424305_AT Igj immunoglobulin joining chain 2.6 1425247_A_AT Igh-4 immunoglobulin heavy chain 4 (serum IgG1) 4.54 1425763_X_AT Igh-VJ558 immunoglobulin heavy chain (J558 family) 3.64 1425985_S_AT Masp1 mannan-binding lectin serine protease 1 1.88 1428306_AT Ddit4 DNA-damage-inducible transcript 4 1.72 1448550_AT Lbp lipopolysaccharide binding protein 2.66 1448872_AT Reg3g regenerating islet-derived 3 gamma 4.38 Cell Adhesion 1448872_AT Reg3g regenerating islet-derived 3 gamma 4.38 1422444_AT Itga6 integrin alpha 6 1.96 1452595_At Adamts4 a disintegrin-like and metalloprotease 3.7 with thrombospondin type 1 motif, 4 1451440_AT Chodl chondrolectin 1.92

1421694_a_at Cspg2 chondroitin sulfate proteoglycan 2 3.32 1448600_S_AT Vav3 vav 3 oncogene 2.46

Table 5.2 Expression Analysis Systematic Explorer (EASE) analysis of top 100 upregulated genes that associated with Biologic Process ontology.

157 genes (Dbccr1) and complement cascade genes (H2-Bf and C4). Upregulated Cell

Cycle/Proliferation biologic process genes include growth factors (Ereg and Igf1) and cell cycle genes (Mki67, Cdc2a, Top2a, and Prc1). EASE analysis also revealed that genes associated with Immune Regulatory, Hydrolase/Peptidase Activity, and

Oxidoreductase Activity molecular function were significantly overrepresented in the top

100 upregulated genes as listed in Table 5.3. These findings correspond well with the phenotype observed in the bladders of BK5.COX2 mice as described above. Analysis of the top 100 downregulated genes by EASE failed to identify genes associated with any ontologies. However, relevant downregulated genes included three TGF beta related genes (Tgfb2, Tgfb3, Tgfbi), the anti-angiogenic gene thrombospondin 2 (Thbs2), and the insulin-like growth factor binding protein 6 (gfbp6). Examples of downregulated genes are listed in Table 5.4.

5.4.4 qRT-PCR and immunohistochemistry confirmation of microarray findings

We next validated some the genes by qRT-PCR and immunohistochemistry. qRT-

PCR analysis confirmed that epiregulin and IGF-1 mRNAs were significantly upregulated, while IGFBP6, TGFbi, TGFb2, and TGFb3 mRNAs were significantly downregulated in 10 wk old transgenic mice bladders compared to the wild-type control

(Figure 5.4). We further examined the expression of these genes in 30 wk old transgenic and wild-type mice. Interestingly, the mRNA levels of the three members of TGF beta related genes remained significantly reduced in transgenic compared to wild-type bladders (Figure 5.5). However, the mRNA levels of IGF-1 and IGFBP6 in BK5.COX2

158 Upregulated Genes by Molecular Function Affymetrix ID Gene Symbol Gene Name Fold Increase Immune Regulatory 1417318_AT Dbccr1 deleted in bladder cancer chromosome 1.94 region candidate 1 1417314_AT H2-Bf histocompatibility 2, complement 1.56 component factor B 1418021_AT C4 complement component 4 (within H-2S) 4.08 1424305_AT Igj immunoglobulin joining chain 2.6 1425247_A_AT Igh-4 immunoglobulin heavy chain 4 (serum IgG1) 4.54 1425763_X_AT Igh-VJ558 immunoglobulin heavy chain (J558 family) 3.64 1428306_AT Ddit4 DNA-damage-inducible transcript 4 1.72 Hydrolase/Peptidase Activity 1416251_AT Mcm6 minichromosome maintenance deficient 6 1.96 1421921_AT Serpina3m serine (or cysteine) proteinase inhibitor, 2.34 clade A, member 3M 1422760_AT Padi4 peptidyl arginine deiminase, type IV 1.94 1426302_AT Tmprss4 transmembrane protease, serine 4 1.8 1448136_AT Enpp2 ectonucleotide pyrophosphatase/ 2.04 phosphodiesterase 2 1417314_AT H2-Bf histocompatibility 2, complement 1.56 component factor B 1418937_AT Dio2 deiodinase, iodothyronine, type II 2.32 1419476_AT Adamdec1 ADAM-like, decysin 1 3.1 1419323_AT Padi1 peptidyl arginine deiminase, type I 2.08 1422868_S_AT Gda guanine deaminase 1.72 1425985_S_AT Masp1 mannan-binding lectin serine protease 1 1.88 1451537_AT Chi3l1 chitinase 3-like 1 4.16 Oxidoreductase Activity 1418264_AT Solt SoxLZ/Sox6 leucine zipper binding 1.56 protein in testis 1418509_AT Cbr2 carbonyl reductase 2 1.76 1418937_AT Dio2 deiodinase, iodothyronine, type II 2.32 1419618_AT Bbox1 butyrobetaine (gamma), 2-oxoglutarate 2.9 dioxygenase 1 1434437_X_AT Rrm2 ribonucleotide reductase M2 1.84 1448792_A_AT Cyp2f2 cytochrome P450, family 2, subfamily f, 7.9 polypeptide 2

Table 5.3 Expression Analysis Systematic Explorer (EASE) analysis of top 100 upregulated genes that associated with Molecular Function ontology.

159 Down-regulated Genes Affymetrix ID Gene Symbol Gene Name Fold Decrease 1417455_AT Tgfb3 transforming growth factor, beta 3 1.88 1415871_At Tgfbi transforming growth factor, beta induced 1.34 1438303_At Tgfb2 transforming growth factor, beta 2 1.2 1417933_AT gfbp6 insulin-like growth factor binding protein 6 1.58 1422571_AT Thbs2 thrombospondin 2 2.06 1422324_A_AT Pthlh parathyroid hormone-like peptide 2.46 1460230_AT Syn2 synapsin II 2.44 1435459_AT Fmo2 flavin containing monooxygenase 2 2.38 1448162_AT Vcam1 vascular cell adhesion molecule 1 2.08 1449979_A_AT Spock3 sparc/osteonectin, cwcv and kazal-like 2.08 domains proteoglycan 3 1451527_AT Pcolce2 procollagen C-endopeptidase enhancer 2 1.98 1425644_AT Lepr leptin receptor 1.94 1449015_AT Retnla resistin like alpha 1.88 1452514_A_AT kit kit oncogene 1.86 1422561_AT Adamts5 a disintegrin-like and metalloprotease (reprolysin type) 1.82 with thrombospondin type 1 motif, 5 (aggrecanase-2) 1419492_S_AT Defb1 defensin beta 1 1.78 1420796_AT Ahrr aryl-hydrocarbon receptor repressor 1.78 1427139_AT Adamts10 a disintegrin-like and metalloprotease (reprolysin type) 1.78 with thrombospondin type 1 motif, 10 1437671_X_AT Prss23 protease, serine, 23 1.74 1419473_A_AT Cck cholecystokinin 1.68 1421006_AT Col4a6 procollagen, type IV, alpha 6 1.66 1426622_A_AT Qpct glutaminyl-peptide cyclotransferase (glutaminyl cyclase) 1.66 1418486_AT Vnn1 vanin 1 1.64 1456084_X_AT Fmod fibromodulin 1.64 1416168_AT Serpinf1 serine (or cysteine) proteinase inhibitor, clade F, member 1 1.58 1420512_AT Dkk2 dickkopf homolog 2 (Xenopus laevis) 1.56

Table 5.4 Expression Analysis Systematic Explorer (EASE) analysis of top 100 downregulated genes.

160 bladders were not significantly different from the levels in wild-type bladders (Figure

5.5). Among these genes, epiregulin is the most significantly increased gene in the

BK5.COX2 transgenic mice bladders. The mRNA level of epiregulin increased by 3.7 fold in 10 wk old transgenic mice bladders and 5.3 fold in 30 wk old transgenic mice bladders compared to age-matched wild-type bladders.

A subset of tissues sections from 10 wk old BK5.COX2 and wild-type mice bladders were analyzed for expression of the proliferation marker Ki67 by immunohistochemistry. We found very few cells were stained positive for mKi67 in the wild-type mice bladders (Figure 5.6 panel a-c). However, the number of epithelial cells positive for Ki67 is significantly increased in bladders from BK5.COX2 transgenic mice

(Figure 5.6 panel d-f).

5.4.5 Epiregulin mRNA induction by PGE2

As shown in Figure 5.1 D, the average concentration of PGE2 in bladder tissues of

BK5.COX2 mice was about 17ng/mg of tissue. This concentration (10µM after

conversion) was used to treat bladder tissues from FVB wild-type mice to determined

whether epiregulin mRNA level could be induced by PGE2 at this concentration under an

ex-vivo organ culture condition. We found that 10µM PGE2 induced epiregulin mRNA

expression in a time-dependent manner in bladders from 10 wk old wild-type mice

(Figure 5.7). The maximum level of epiregulin mRNA was reached at 4 h. The increase

of epiregulin mRNA remained significantly elevated compared to controls after 8 h

incubation. These results suggest that PGE2 has a direct role in the induction of epiregulin mRNA expression. However, the regulation of epiregulin at protein level by PGE2 was

161

Gene validation in 10 wk old BK5.COX2

Epiregulin IGF-1 IGFBP6

5.00 n

n 2.50

n n o

i i

o o i

10.00 i

n n n n

s s

o o o

o 4.00

i i i i s s e

s s s s e

2.00 e

r r

s s s s

p p

e e e e

x x

r r r r

x x e

7.50 e

p p p p e

P<0.05 e 3.00

x x x x A

A 1.50

A A

e e e e

N N

A A A A

R R

N N N N m

5.00 m

m 2.00

R R R R

P<0.01 1.00 e

e P< 0.05

e e

v v

m m m m

i i

v v

i i

t t

e e e e

t t a

v v v v

l l

a a

i i i i l

2.50 l

e e t t t

t 0.50 1.00

e e

a a a a

R R

l l l l R

e e e e

R R R R 0.00 Wt Tg Wt Tg Wt Tg Genotype Genotype Genotype

TGFbi TGFb2 TGFb3

2.50

n n n n

o o o

2.00 o

o o o o

i i i i

i i i

i 2.00

s s s s

s s s

s 2.00

s s s s

e e e e

r r r r

r r r

p p p p

p p p

p 1.50

x x x x

x x x

x 1.50

e e e e

e e e

e P<0.05 1.50

A A A A

A A A

A P< 0.05 N N N N

N N N N

R R R R

R R R R 1.00 P<0.01 1.00

1.00

m m m m

e e e e

v v v

v v v v

i i i i

t t t t

a a a a

l l l

0.50 0.50 l

l l l l

l l l

l 0.50

e e e

e e e e

R R R R

R R R R 0.00 0.00 Wt Tg Wt Tg Wt Tg Genotype Genotype Genotype

Figure 5.4 qRT-PCR validation of six genes of interest that were significantly changed in 10 wk old BK5.COX2 transgenic mice (Tg) compared to wild-type (Wt) mice by Gene Array analysis. Note that epiregulin is the most significantly upregulated gene (increased in BK5.COX2 by 3.7 fold, p<0.01). Each bar represents the mean mRNA expression of indicated gene (error bars are 95% confidence intervals) of five animals relative to a common calibrator sample generated by pooling 3 bladders from wild-type mice. GAPDH mRNA levels were used to normalize the expression of individual samples. A p value < 0.05 was considered as significantly different from each other.

162

Gene validation in 30 wk old BK5.COX2

n n n n

n n n

Epiregulin n IGF--1 IGFBP6

o o o

8.00 o

o o o o

i i i

i i i i i

n n n n

s s s s

o o o o

i i i i

s s s

s 1.50

s s s

8.00 s

s s s s

e e e e

e e e

r r r r

s s s s

r r r r

e e e e

p p p

P=0.37 p

p p p p

r r r r

x x x x

x x x

x 6.00

p p p

e e e

e P=0.14

e e e e

x x x x

e e e

e 6.00

A A A A

N N N

N 1.00

N N N N

A A A A

R R R R

N N N N

R R R R

R R R

m m m

4.00 m

m m m m

m m m

m 4.00

e e e e

v v v v

e e e e

v v v v

i i i

i i i i

v v v v

t t t t

i i i i

t t t t

a a a a

l l l

l 0.50

l l l l

a a a a

l l l l

e e e

e 2.00

e e e

e P<0.01

R R R 2.00 R

R R R R

0.00 Wt Tg Wt Tg Wt Tg Genotype

Genotype Genotype

TGFbi n TGFb2 TGFb3 o

2.00 o

i i

n n n

n 2.50

n n n n

s s

o o o o

i i i i

i i i i s

s s s s

e e

s s s s

r r

s s s s

e e e

e 1.50

e e e e p

p 2.00

r r r

r r r r

1.50 r

x x

p p p p

e e

x x x x

x x x

P<0.05 x

e e e e

e e e

P<0.05 A A

1.50

A A A A

N N

N N N N 1.00

N N N

N R

1.00 R P<0.05

R R R R

m m

m m m m

1.00

e e

e e e e

v v

i i

v v v v

i i i i

t t

i i i i

t t t

0.50 0.50 t

a a

a a a a

l l

a a a a

l l l l

l l l

l 0.50

e e

e e e e

e e e

R R R R

R R

R R R R 0.00 0.00 Wt Tg Wt Tg Wt Tg Genotype Genotype Genotype

Figure 5.5 Expression of the six validated genes in 30 wk old BK5.COX2 (Tg) transgenic mice and age matched wild-type (Wt) mice by qRT-PCR analysis. Note that epiregulin is again the most significantly upregulated gene in the 30 wk old animals (increased in BK5.COX2 by 5.3 fold, p<0.01). Each bar represents the mean mRNA expression of indicated gene (error bars are 95% confidence intervals) of five animals relative to a common calibrator sample generated by pooling 3 bladders from wild-type mice. GAPDH mRNA levels were used to normalize the expression of individual samples. A p value < 0.05 was considered as significantly different from each other.

163

Ki67 staining

a b c

100x 200x 400x d e f

100x 200x 400x

Figure 5.6 Immunohistochemistry analysis of the proliferation marker mKi67 10 wk old BK5.COX2 (Tg) transgenic mice and wild-type (Wt) mice bladders. Note that the number of epithelial cells positive for mKi67 is significantly increased in bladders from Tg mice (d-f) . Whereas, very few cells were stained positive for mKi67 in the wild-type bladder epithelial cells (a-c, arrow in panel c).

164

140

120 c

c 100

b 40 b Relative mRNA expression mRNA Relative 20 a a 0 0h 1h 2h 4h 6h 8h

Time

Figure 5.7 PGE2 at 10µM induces epiregulin mRNA expression in a time-dependent manner in bladder tissues from 10 wk old FVB wild-type mice. Bladder tissues were excised from mice, cut in half, and then incubated in RPMI medium with 10µM PGE2 for indicated time. Levels of epiregulin mRNA were determined by qRT-PCR. Bars represent means, error bars are 95% confidence intervals (CI), and means with different letters significantly differ from each other (p<0.05, n=4).

165 not detected at this time due to the non-specificity and insensitivity of the commercial

available antibody against epiregulin (Santa Cruz).

5.4.6 Effects of Epiregulin on proliferation and Erk activation in UMCC-3 cells

We next screened five bladder cells line (RT4, HT1376, J82, T24, and UMUC-3)

for the effects of recombinant hEpiregulin on cell proliferation in these cells. We found

that hEpiregulin has essentially no effects on proliferation in RT4, HT1376, J82, and T24

cells (data not shown). These cells have both high level of COX-2 expression and PGE2 concentration. However, UMUC-3 cells which have very low COX-2 expression and

PGE2 production, significantly responded to epiregulin treatment. The recombinant

hEpiregulin significantly increased UMUC-3 cell proliferation at 48 h at concentrations

ranging from 10pg/ml to 1 ng/ml compared to untreated controls (Figure 5.8 A). In

addition, hEpiregulin induced phosphorylation of Erk in a dose- and time-dependent

manner in UMUC-3 cells (Figure 5.8 B, C). The phosphorylation of Erk in UMUC-3

cells was significantly upregulated within 15 min by 10 ng/ml hEpiregulin. The

maximum stimulation was reached at 15 min and Erk phosphorylation declined but

remained high after 4 h (Figure 5.8 C).

166 A 180 c 160 b,c 140 a a,b 120 a

100 80

Cell proliferation ( % of increase ) increase of ( % proliferation Cell 20 Con 1 pg/ml 10 pg/ml 100 pg/ml 1 ng/ml

Concentration of hEpiregulin

p-Erk

t-Erk

0 1 10 100 1000 10000 (pg/ml) C Concentration of hEpiregulin

p-Erk

t-Erk

0 15 30 60 120 240 (min) Time

Figure 5.8 Recombinant hEpiregulin induces proliferation and Erk phosphorylation in bladder cancer UMUC-3 cells. A. hEpiregulin increases cell proliferation in UMUC-3 cells. Cells were treated with hEpiregulin (0, 1pg/ml, 10pg/ml, 100pg/ml and 1ng/ml) for 24 hr, cell proliferation was determined by MTT assay. Bars represent means, error bars are 95% confidence intervals (CI), and means with different letters significantly differ from each other (p<0.05, n=3). B. hEpiregulin induces phosphorylation of Erk in a dose- dependent manner in UMUC-3 cells. Cells were treated for 30 min with concentrations of hEpiregulin ranging from 1 pg/ml to 10000 pg/ml followed by immunoblotting. C. hEpiregulin at 10ng/ml induces phosphorylation of Erk in time-dependent manner in UMUC-3 cells. Cells were treated with 10 ng/ml hEpiregulin for 15 to 240 min followed by immunoblotting.

167 4.5 DISSCUSSION

COX-2 overexpression and increased PGE2 synthesis have been associated with bladder tumorigenesis by numerous studies (3). We have developed a transgenic mouse model which over expresses COX-2 under the control of a bovine keratin 5 (BK5) promoter. We found that BK5. COX2 mice developed high incidence of urinary bladder

TCH and transitional cell neoplasia in a small percentage of the transgenic mice. To determine the molecular pathways elicited by COX-2 overexpression during the early events of bladder carcinogenesis, we conducted DNA microarray to determine the effects of COX-2 and prostaglandin synthesis on regulating gene profile in the urinary bladder of

10 wk old BK5.COX2 mice before the onset of substantial TCH. We found that genes involved in the Cell Cycle/Proliferation and Immune/Stress response biologic processes were significantly upregulated. These findings correspond well with the previously described phenotype observed in the bladders of BK5.COX2 mice.

Mohammed et al. (5) were the first to characterize the expression of COX-2 in normal human urothelium, TCC, and CIS in bladder cancer patients. The investigators observed that COX-2 was not expressed in all normal bladder urothelium examined, although COX-2 was overexpressed in 83% of TCC specimens and 75% of CIS specimens (5). Since then, a substantial number of studies has confirmed these findings, and have correlated the intensity of COX-2 overexpression to tumor grade and stage of this disease (6-10). Evidence for a functional role of COX-2 in bladder cancer is primarily from preclinical studies including genetic and pharmacologic studies. Results from these studies suggest that COX-2 and PGE2 play tumorigenic effects in a number of

168 cancers through increasing proliferation, enhancing angiogenesis, promoting invasion, and inhibiting apoptosis (3). However, the exact molecular mechanisms involved have not been well studied. DNA microarray allowed us to identify early genetic and molecular changes that lead to superficial and invasive bladder cancer drove by COX-2 overexpression. Many molecular changes have been identified during human bladder carcinogenesis (72, 150). Interestingly, we found that certain genes that are deregulated in the BK5.COX2 urinary mouse bladder model also have been reported by others, such as upregulation of epiregulin, IGF-1 and ki67, and downregulation of members of TGF- beta and thrombospondin families (149, 150). Therefore, the BK5.COX2 model certainly appears to be relevant to human urinary bladder cancer, thereby allowing us to study mechanisms of tumorigenic effects of COX-2/PGs and their downstream pathways.

The development of TCH in the bladder of BK5.COX2 mice was associated with a dramatic increase in proliferating cells in the basal cell layers of the urothelium. Results from gene array analysis supported our original hypothesis that prostaglandin synthesis via COX-2 overexpression induces cell proliferation through induction of growth factors.

Both epiregulin and IGF-1 were induced more than 2 fold greater in the bladders of 10 wk old BK5.COX2 transgenic mice compared to bladders from wild-type mice. However, there was no such marked difference in IGF-1 mRNA between transgenic and wild-type mice at 30 wk of age. This suggests that IGF-1 may only play a carcinogenic role upon

COX-2 overexpression in the early stage of tumor development in bladder.

Epiregulin is a novel member of the EGF family. It was the most significantly upregulated gene among genes validated by qRT-PCR and remained significantly high in bladders of 30 wk old transgenic mice. This suggests that epiregulin may serve as a key

169 modulator of cell proliferation as a result of COX-2 overexpression in urinary bladders.

Epiregulin binds to ErbB1 (EGFR) and Erbb4 and is a potent mitogen for rat primary

hepatocytes (69). A study by Zhu et al. found that epiregulin is highly expressed in

pancreatic cancer cells, and recombinant human epiregulin was able to induce the growth

of pancreatic cancer cells in a dose-dependent manner (277). Epiregulin binds to various

combinations of erbB receptors with low affinity, but transmits a more potent mitogenic

signal than EGF (137). Nicholson et al. found that epiregulin is one of the four

biomarkers that are consistently and progressively upregulated in a mouse lung metastatic

bladder model (155). The mRNA level of epiregulin was also increased in HCV28

bladder cancer cells upon the stimulation with the DNA damage agent VP16 (278).

Thogersen et al. reported that epiregulin is the most significantly increased EGF like

ligand in human T2-T4 tumors as compared with Ta tumors (154). Together with our

finding that epiregulin was highly induced in BK5.COX2 transgenic bladders, these

studies suggest that epiregulin may be a potential important biomarker of bladder cancer.

We further found that PGE2 at a concentration equivalent to that in the bladder tissue of

BK5.COX2 mice increased epiregulin mRNA in bladders from FVB mice ex-vivo. This

suggests that PGE2 may directly induce the transcription of this gene. We further

determined that epiregulin significantly induced cell proliferation and MAPK/Erk

phosphorylation in bladder cancer UMUC-3 cells, but not other bladder cell lines

examined. UMUC-3 cells are differ from the other cell lines (RT4, HT1376, J82, and T24)

in that it has non-detectable expression of COX-2 and low levels of PGE2 (11, 279).

However, ERGF and ErbB4 were not activated in UMUC-3 cells upon treatment with

170 epiregulin. The different response of UMUC-3 cells to epiregulin will require further

investigation.

Although EASE analysis failed to group the top 100 downregulated genes into

any gene ontologies, we found that the three members of TGFβ family, i.e., Tgfb2, Tgfb3,

and Tgfbi, were significantly downregulated in the bladder of transgenic mice at 10 and

30 wk of age. The TGFβ family is multifuntional cytokines responsible for cell proliferation, differentiation, extracellular matrix synthesis/degradation, and mediation of

apoptosis (280). Reduced expression of TGFβ-1 is significantly associated with high- grade of bladder tumors (160, 161, 281). Loss of expression of TGFβ receptors is significantly associated with higher tumor grade and stage, presence of lymph node metastases, progression and reduced survival (138, 159, 160, 281). Conditional deletion of TGFβ Receptor type II is associated with thickened lamina propria and myofibroblast proliferation in Tgfbr2fspko mice bladders (282). One significant observation in the phenotype that developed in the bladder tissue of BK5.COX2 mice is that TCH was strongly associated with thickness of stroma and laminar propria. The downregulation of

TGFβ-related genes and together with other genes associated with Cell Adhesion and

Hydrolase/Peptidase Activity identified by EASE analysis may play a key role in the

remolding of extracellular matrix compartment of urinary bladder during carcinogenesis

driven by COX-2 overexpression.

Another observation in the urinary bladder of BK5.COX2 mice is that a dramatic

increase in vascularization was observed in the lamina propria and stroma.

Neovascularization depends on an increase in proangiogenic factors relative to

antiangiogenic factor. In the K5.COX-2 mouse model, Klein et al. (3) demonstrated that

171 the proangiogenic factor VEGF was clearly elevated in the urinary bladder of transgenic

mice. However, the gene array study did not identify significant dysregulation in genes

associated with angiogenesis, except for the downregulation of the anti-angiogenic gene

thrombospondin 2. We previously reported that PGE2 induces VEGF secretion in prostate

cancer cells by posttranscriptional regulation (283). Immunohistochemical analysis of the

expression of angigenic factors, such as VEGF in sections of BK5.COX2 bladders merits

further investigation.

In the K5.COX-2 mouse model, inflammation was proposed as the causative

factor in the development of TCH in these mice upon COX-2 overexpression (3). We

also observed significant lymphocyte infiltration in the lamina propria and more often in

the muscle area. EASE analysis of the gene array data revealed that a substantial number

of genes involved in Immune-Stress Response in biological process and Immune

Regulatory in molecular function were significantly upregulated. Although the exact

function of these immune response related genes is not clear, it is evident that the initial

response of the mouse urinary bladder to elevated COX-2 expression includes mounting an inflammatory response. A better understanding of the mechanisms associated with the

observed inflammatory response is expected to provide insights about the etiology of

bladder cancer.

The physiological effects of PGE2 are mediated by four G protein coupled

membrane receptors, i.e., EP1, EP2, EP3 and EP4. EP receptors have been implicated in

promoting carcinogenesis in different types of tumors, including lung (45), ovarian (46),

colorectal (47, 48), breast (49), and skin (50, 51). Miyata et al. suggests that only EP4

receptor is co-expressed with COX-2 in TCC of the upper urinary tract (227).

172 Interestingly, gene array analysis found that EP4 is that only EP receptor that was significantly elevated in the BK5.COX2 transgenic mouse bladders, although it was only induced by 70% compared to the wild-type controls. Because COX-2 selective inhibitors may not be useful as chemoprevention agents due to potential side effects associated with long term use, EP receptors, located downstream of PG synthesis in the COX pathway may represent attractive alternative targets for chemoprevention and treatment of bladder cancer. However, further studies are needed to determine the role of EP receptors

(potentially the EP4 receptor) and mechanisms involved in bladder carcinogenesis.

Although bladder TCH developed in mice as young as 10 wk of age in 100% of the transgenic mice, TCC developed only in 6 % of the transgenic mice. The total percentage of the transitional cell neoplasia is about 18 %. These observations suggest that additional mutations are required for progression form TCH to TCC. The alteration and mutation of tumor suppressor gene p53 has been shown to be significantly associated with bladder cancer tumorigenesis (149, 150). However, crossing the BK5.COX2 mice with p53-/- mice did not enhance the severity of lesions in the urinary bladder of the double crossed transgenic mice (data not shown). The ErbB family, including EGFR,

ErbB-2, ErbB-3 and ErbB-4, has been strongly associated with the stage and grade of bladder cancer development and progression (151, 284). Overexpression of EGFR in urothelium has been demonstrated to induce hyperplasia and promotes bladder tumor growth in transgenic mice (285). Crosstalk between COX-2 and EGFR has been established to be associated with carcinogenesis by many studies (286). In future study, we may cross EGFR overexpressing mouse with BK5.COX2 transgenic mouse to determine if the incidence of TCC would be increased. It will also be interesting to learn

173 whether knocking out EGFR in BK5.COX2 mice prevents the development of TCH.

Netherless, the BK5.COX2 transgenic mouse model may serve as a potential useful pre-

clinical model for superficial bladder cancer.

In summary, our study provides evidence that COX-2 overexpression and PGE2

overproduction play a tumorigenic effect in bladder cancer through regulating

growth/proliferation, extracellular remodeling, and immune responses related genes. The

biomarkers of COX-2 overexpression may be useful targets for future prevention studies

focusing on bladder cancer. Our study also provides rational for future clinical trials to

test the ability of COX-2-selective inhibitor alone or in combination with EGFR inhibitors to prevent the development and progression of bladder cancer.

174

EPILOQUE

Prostate and bladder cancers are significant health problems. Identification of new

molecular targets may provide effective strategies to reduce the development and

progression of these two diseases. The research presented above was to determine

whether COX-2/PGE2/EP signaling pathway was valid targets for the prevention and

treatment of prostate and bladder cancers. The overall conclusion of my dissertation was that COX-2 may not play a tumorigenic role in prostate cancer progression, but has a pivotal role in bladder carcinogenesis though regulating the expression of specific patterns of genes.

The first study was conduct to examine the expression profile and the role of EP receptors in PGE2-mediated signaling cascades in prostate cancer cells. It was found that

EP2 is the predominantly expressed receptor in prostate cancer cell lines, and it also

played a key role in mediating PGE2-induced VEGF secretion and activation of Erk and

Akt in these cells. However, this kind of work has not been fully addressed in bladder

cancer cell lines. We have determined that all the EP receptors may express in both

bladder cancer cell lines and mouse bladder tissues. It would be interesting to determine

whether PGE2 could induce the similar signal cascades as we observed in prostate cell

lines, and to determined which EP receptors are involved in these biologic effects. Due to

the unwanted side effects associated with the use of COX inhibitors, the EP receptors

175 may be novel targets for the prevention and treatment of bladder cancer. Therefore, future studies are needed to determine the contribution of each individual EP receptor in bladder carcinogenesis using genetically modified mouse models.

One of the most significant findings from our second study was that the expression and activity of COX-1 and COX-2 are progressively lost during prostate carcinogenesis and tumor progression. Given the recent evidence that COX-2 may be overexpressed only in areas of inflammation in prostate cancer tissues, a role for COX-2 in either the initiation or promotion of this disease by eliciting an inflammatory response need to be investigated in future. In addition, the mechanisms underlying the marked decrease of COX mRNA and protein levels during prostate tumorigenesis need to be explored. Whether epigenetic regulation, such as gene methylation, plays a role in COX downregulation would be more interesting to study. Although it may not be likely that loss of COX expression contributes to the tumorigenesis of prostate cancer, further work will be required to determine the significance of this phenomenon.

Results from our third study demonstrated that COX-2 overexpression regulates the expression of genes involved in Immune/Stress, Proliferation/Cell Cycle and

Extracellular Matrix remodeling in the bladder of BK5.COX2 transgenic mice. However, the exact function of these immune response related genes and many genes associated with other ontologies in bladder carcinogenesis in this model are not clear. It would be very difficult to study each individual gene. A comparison of the molecular changes during bladder carcinogenesis between human bladder cancer and BK5.COX2 mouse model in future studies are needed to further elucidate the etiology and biomarkers of human bladder cancer. We found that the growth factor, epiregulin, may play a key role

176 in bladder carcinogenesis upon elevated PGE2 production. However, at present, little is known about the localization of epiregulin and which erbB receptors and down-stream signaling cascades are responsible for epiregulin-elicited biologic effects in bladder carcinogenesis. Further studies are needed to address these questions. Upon we get these information, we could target specific erbB receptors as individual or in combination with

COX-2 /PGE2/EP inhibition to prevent the development and progression of bladder carcinogenesis in pre-clinical and clinical studies. As we know, dietary fatty acids play an important role in the modulation of COX/PG synthesis and would therefore regulate bladder carcinogenesis. Whether dietary intervention through fish oil could inhibit bladder carcinogenesis in BK5.COX2 mouse model warrants evaluation in future studies.

177

REFERENCES

1. Zha, S., Yegnasubramanian, V., Nelson, W. G., Isaacs, W. B., and De Marzo, A. M. Cyclooxygenases in cancer: progress and perspective. Cancer Lett, 215: 1-20, 2004.

2. Cao, Y. and Prescott, S. M. Many actions of cyclooxygenase-2 in cellular dynamics and in cancer. J Cell Physiol, 190: 279-286, 2002.

3. Klein, R. D., Van Pelt, C. S., Sabichi, A. L., Dela Cerda, J., Fischer, S. M., Furstenberger, G., and Muller-Decker, K. Transitional cell hyperplasia and carcinomas in urinary bladders of transgenic mice with keratin 5 promoter-driven cyclooxygenase-2 overexpression. Cancer Res, 65: 1808-1813, 2005.

4. Hara, S., Kondo, Y., Matsuzawa, I., Hashimoto, Y., Kimura, G., Akimoto, M., and Imura, N. Expression of cycloxygenase-2 in human bladder and renal cell carcinoma. Adv Exp Med Biol, 507: 123-126, 2002.

5. Mohammed, S. I., Knapp, D. W., Bostwick, D. G., Foster, R. S., Khan, K. N., Masferrer, J. L., Woerner, B. M., Snyder, P. W., and Koki, A. T. Expression of cyclooxygenase-2 (COX-2) in human invasive transitional cell carcinoma (TCC) of the urinary bladder. Cancer Res, 59: 5647-5650, 1999.

6. Shirahama, T. Cyclooxygenase-2 expression is up-regulated in transitional cell carcinoma and its preneoplastic lesions in the human urinary bladder. Clin Cancer Res, 6: 2424-2430, 2000.

7. Sweeney, C. J., Marshall, M. S., Barnard, D. S., Heilman, D. K., Billings, S. D., Cheng, L., Marshall, S. J., and Yip-Schneider, M. T. Cyclo-oxygenase-2 expression in primary cancers of the lung and bladder compared to normal adjacent tissue. Cancer Detect Prev, 26: 238-244, 2002.

8. Gurocak, S., Sozen, S., Erdem, O., Ozkan, S., Kordan, Y., Alkibay, T., Akyol, G., and Bozkirli, I. Relation between cyclooxygenase-2 expression and clinicopathologic parameters with patient prognosis in transitional cell carcinoma of the bladder. Urol Int, 76: 51-56, 2006.

178 9. Wadhwa, P., Goswami, A. K., Joshi, K., and Sharma, S. K. Cyclooxygenase-2 expression increases with the stage and grade in transitional cell carcinoma of the urinary bladder. Int Urol Nephrol, 37: 47-53, 2005.

10. Wulfing, C., Eltze, E., von Struensee, D., Wulfing, P., Hertle, L., and Piechota, H. Cyclooxygenase-2 expression in bladder cancer: correlation with poor outcome after chemotherapy. Eur Urol, 45: 46-52, 2004.

11. Gee, J., Lee, I. L., Jendiroba, D., Fischer, S. M., Grossman, H. B., and Sabichi, A. L. Selective cyclooxygenase-2 inhibitors inhibit growth and induce apoptosis of bladder cancer. Oncol Rep, 15: 471-477, 2006.

12. Mizutani, Y., Kamoi, K., Ukimura, O., Kawauchi, A., and Miki, T. Synergistic cytotoxicity and apoptosis of JTE-522, a selective cyclooxygenase-2 inhibitor, and 5-fluorouracil against bladder cancer. J Urol, 168: 2650-2654, 2002.

13. Mizutani, Y., Nakanishi, H., Li, Y. N., Sato, N., Kawauchi, A., and Miki, T. Enhanced sensitivity of bladder cancer cells to cisplatin mediated cytotoxicity and apoptosis in vitro and in vivo by the selective cyclooxygenase-2 inhibitor JTE- 522. J Urol, 172: 1474-1479, 2004.

14. Mohseni, H., Zaslau, S., McFadden, D., Riggs, D. R., Jackson, B. J., and Kandzari, S. COX-2 inhibition demonstrates potent anti-proliferative effects on bladder cancer in vitro. J Surg Res, 119: 138-142, 2004.

15. Irani, J., Ravery, V., Pariente, J. L., Chartier-Kastler, E., Lechevallier, E., Soulie, M., Chautard, D., Coloby, P., Fontaine, E., Bladou, F., Desgrandchamps, F., and Haillot, O. Effect of nonsteroidal anti-inflammatory agents and finasteride on prostate cancer risk. J Urol, 168: 1985-1988, 2002.

16. Langman, M. J., Cheng, K. K., Gilman, E. A., and Lancashire, R. J. Effect of anti- inflammatory drugs on overall risk of common cancer: case-control study in general practice research database. BMJ, 320: 1642-1646, 2000.

17. Leitzmann, M. F., Stampfer, M. J., Ma, J., Chan, J. M., Colditz, G. A., Willett, W. C., and Giovannucci, E. Aspirin use in relation to risk of prostate cancer. Cancer Epidemiol Biomarkers Prev, 11: 1108-1111, 2002.

18. Norrish, A. E., Jackson, R. T., and McRae, C. U. Non-steroidal anti-inflammatory drugs and prostate cancer progression. Int J Cancer, 77: 511-515, 1998.

19. Paganini-Hill, A., Chao, A., Ross, R. K., and Henderson, B. E. Aspirin use and chronic diseases: a cohort study of the elderly. BMJ, 299: 1247-1250, 1989.

179 20. Garcia Rodriguez, L. A. and Gonzalez-Perez, A. Inverse association between nonsteroidal anti-inflammatory drugs and prostate cancer. Cancer Epidemiol Biomarkers Prev, 13: 649-653, 2004.

21. Habel, L. A., Zhao, W., and Stanford, J. L. Daily aspirin use and prostate cancer risk in a large, multiracial cohort in the US. Cancer Causes Control, 13: 427-434, 2002.

22. Nelson, J. E. and Harris, R. E. Inverse association of prostate cancer and nonsteroidal anti-inflammatory drugs (NSAIDs): results of a case-control study. Oncol Rep, 7: 169-170, 2000.

23. Roberts, R. O., Jacobson, D. J., Girman, C. J., Rhodes, T., Lieber, M. M., and Jacobsen, S. J. A population-based study of daily nonsteroidal anti-inflammatory drug use and prostate cancer. Mayo Clin Proc, 77: 219-225, 2002.

24. Kirschenbaum, A., Klausner, A. P., Lee, R., Unger, P., Yao, S., Liu, X. H., and Levine, A. C. Expression of cyclooxygenase-1 and cyclooxygenase-2 in the human prostate. Urology, 56: 671-676, 2000.

25. Lee, L. M., Pan, C. C., Cheng, C. J., Chi, C. W., and Liu, T. Y. Expression of cyclooxygenase-2 in prostate adenocarcinoma and benign prostatic hyperplasia. Anticancer Res, 21: 1291-1294, 2001.

26. Madaan, S., Abel, P. D., Chaudhary, K. S., Hewitt, R., Stott, M. A., Stamp, G. W., and Lalani, E. N. Cytoplasmic induction and over-expression of cyclooxygenase- 2 in human prostate cancer: implications for prevention and treatment. BJU Int, 86: 736-741, 2000.

27. Shappell, S. B., Manning, S., Boeglin, W. E., Guan, Y. F., Roberts, R. L., Davis, L., Olson, S. J., Jack, G. S., Coffey, C. S., Wheeler, T. M., Breyer, M. D., and Brash, A. R. Alterations in lipoxygenase and cyclooxygenase-2 catalytic activity and mRNA expression in prostate carcinoma. Neoplasia, 3: 287-303, 2001.

28. Uotila, P., Valve, E., Martikainen, P., Nevalainen, M., Nurmi, M., and Harkonen, P. Increased expression of cyclooxygenase-2 and nitric oxide synthase-2 in human prostate cancer. Urol Res, 29: 23-28, 2001.

29. Yoshimura, R., Sano, H., Masuda, C., Kawamura, M., Tsubouchi, Y., Chargui, J., Yoshimura, N., Hla, T., and Wada, S. Expression of cyclooxygenase-2 in prostate carcinoma. Cancer, 89: 589-596, 2000.

30. Wang, W., Bergh, A., and Damber, J. E. Cyclooxygenase-2 expression correlates with local chronic inflammation and tumor neovascularization in human prostate cancer. Clin Cancer Res, 11: 3250-3256, 2005.

180 31. Zha, S., Gage, W. R., Sauvageot, J., Saria, E. A., Putzi, M. J., Ewing, C. M., Faith, D. A., Nelson, W. G., De Marzo, A. M., and Isaacs, W. B. Cyclooxygenase-2 is up-regulated in proliferative inflammatory atrophy of the prostate, but not in prostate carcinoma. Cancer Res, 61: 8617-8623, 2001.

32. Greenberg, N. M., DeMayo, F., Finegold, M. J., Medina, D., Tilley, W. D., Aspinall, J. O., Cunha, G. R., Donjacour, A. A., Matusik, R. J., and Rosen, J. M. Prostate cancer in a transgenic mouse. Proc Natl Acad Sci USA, 92: 3439-3443, 1995.

33. Kaplan-Lefko, P. J., Chen, T. M., Ittmann, M. M., Barrios, R. J., Ayala, G. E., Huss, W. J., Maddison, L. A., Foster, B. A., and Greenberg, N. M. Pathobiology of autochthonous prostate cancer in a pre-clinical transgenic mouse model. Prostate, 55: 219-237, 2003.

34. Gupta, S., Adhami, V. M., Subbarayan, M., MacLennan, G. T., Lewin, J. S., Hafeli, U. O., Fu, P., and Mukhtar, H. Suppression of prostate carcinogenesis by dietary supplementation of celecoxib in transgenic adenocarcinoma of the mouse prostate model. Cancer Res, 64: 3334-3343, 2004.

35. Narayanan, B. A., Narayanan, N. K., Pittman, B., and Reddy, B. S. Regression of mouse prostatic intraepithelial neoplasia by nonsteroidal anti-inflammatory drugs in the transgenic adenocarcinoma mouse prostate model. Clin Cancer Res, 10: 7727-7737, 2004.

36. Kulp, S. K., Yang, Y. T., Hung, C. C., Chen, K. F., Lai, J. P., Tseng, P. H., Fowble, J. W., Ward, P. J., and Chen, C. S. 3-phosphoinositide-dependent protein kinase-1/Akt signaling represents a major cyclooxygenase-2-independent target for celecoxib in prostate cancer cells. Cancer Res, 64: 1444-1451, 2004.

37. Williams, C. S., Watson, A. J., Sheng, H., Helou, R., Shao, J., and DuBois, R. N. Celecoxib prevents tumor growth in vivo without toxicity to normal gut: lack of correlation between in vitro and in vivo models. Cancer Res, 60: 6045-6051, 2000.

38. Bradbury, D., Clarke, D., Seedhouse, C., Corbett, L., Stocks, J., and Knox, A. Vascular endothelial growth factor induction by prostaglandin E2 in human airway smooth muscle cells is mediated by E prostanoid EP2/EP4 receptors and SP-1 transcription factor binding sites. J Biol Chem, 280: 29993-30000, 2005.

39. Chang, S. H., Liu, C. H., Wu, M. T., and Hla, T. Regulation of vascular endothelial cell growth factor expression in mouse mammary tumor cells by the EP2 subtype of the prostaglandin E2 receptor. Prostaglandins Other Lipid Mediat, 76: 48-58, 2005.

181 40. Ding, Y. B., Shi, R. H., Tong, J. D., Li, X. Y., Zhang, G. X., Xiao, W. M., Yang, J. G., Bao, Y., Wu, J., Yan, Z. G., and Wang, X. H. PGE2 up-regulates vascular endothelial growth factor expression in MKN28 gastric cancer cells via epidermal growth factor receptor signaling system. Exp Oncol, 27: 108-113, 2005.

41. Li, Y. Q., Tao, K. S., Ren, N., and Wang, Y. H. Effect of c-fos antisense probe on prostaglandin E2-induced upregulation of vascular endothelial growth factor mRNA in human liver cancer cells. World J Gastroenterol, 11: 4427-4430, 2005.

42. Liu, X. H., Kirschenbaum, A., Lu, M., Yao, S., Dosoretz, A., Holland, J. F., and Levine, A. C. Prostaglandin E2 induces hypoxia-inducible factor-1alpha stabilization and nuclear localization in a human prostate cancer cell line. J Biol Chem, 277: 50081-50086, 2002.

43. Zhu, G., Saed, G. M., Deppe, G., Diamond, M. P., and Munkarah, A. R. Hypoxia up-regulates the effects of prostaglandin E2 on tumor angiogenesis in ovarian cancer cells. Gynecol Oncol, 94: 422-426, 2004.

44. Hata, A. N. and Breyer, R. M. Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol Ther, 103: 147-166, 2004.

45. Fang, K. M., Shu, W. H., Chang, H. C., Wang, J. J., and Mak, O. T. Study of prostaglandin receptors in mitochondria on apoptosis of human lung carcinoma cell line A549. Biochem Soc Trans, 32: 1078-1080, 2004.

46. Spinella, F., Rosano, L., Di Castro, V., Natali, P. G., and Bagnato, A. Endothelin- 1-induced prostaglandin E2-EP2, EP4 signaling regulates vascular endothelial growth factor production and ovarian carcinoma cell invasion. J Biol Chem, 279: 46700-46705, 2004.

47. Hull, M. A., Ko, S. C., and Hawcroft, G. Prostaglandin EP receptors: targets for treatment and prevention of colorectal cancer? Mol Cancer Ther, 3: 1031-1039, 2004.

48. Shoji, Y., Takahashi, M., Kitamura, T., Watanabe, K., Kawamori, T., Maruyama, T., Sugimoto, Y., Negishi, M., Narumiya, S., Sugimura, T., and Wakabayashi, K. Downregulation of prostaglandin E receptor subtype EP3 during colon cancer development. Gut, 53: 1151-1158, 2004.

49. Timoshenko, A. V., Xu, G., Chakrabarti, S., Lala, P. K., and Chakraborty, C. Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells. Exp Cell Res, 289: 265-274, 2003.

182 50. Sung, Y. M., He, G., and Fischer, S. M. Lack of expression of the EP2 but not EP3 receptor for prostaglandin E2 results in suppression of skin tumor development. Cancer Res, 65: 9304-9311, 2005.

51. Tober, K. L., Wilgus, T. A., Kusewitt, D. F., Thomas-Ahner, J. M., Maruyama, T., and Oberyszyn, T. M. Importance of the EP(1) receptor in cutaneous UVB- induced inflammation and tumor development. J Invest Dermatol, 126: 205-211, 2006.

52. Chen, Y. and Hughes-Fulford, M. Prostaglandin E2 and the protein kinase A pathway mediate arachidonic acid induction of c-fos in human prostate cancer cells. Br J Cancer, 82: 2000-2006, 2000.

53. Cancer facts and figures, 2007.

54. Nupponen, N. and Visakorpi, T. Molecular biology of progression of prostate cancer. Eur Urol, 35: 351-354, 1999.

55. Giovannucci, E. and Clinton, S. K. Tomatoes, lycopene, and prostate cancer. Proc Soc Exp Biol Med, 218: 129-139, 1998.

56. Agarwal, R. Cell signaling and regulators of cell cycle as molecular targets for prostate cancer prevention by dietary agents. Biochem Pharmacol, 60: 1051-1059, 2000.

57. Kelloff, G. J., Lieberman, R., Steele, V. E., Boone, C. W., Lubet, R. A., Kopelovitch, L., Malone, W. A., Crowell, J. A., and Sigman, C. C. Chemoprevention of prostate cancer: concepts and strategies. Eur Urol, 35: 342- 350, 1999.

58. Dennis, L. K., Lynch, C. F., and Torner, J. C. Epidemiologic association between prostatitis and prostate cancer. Urology, 60: 78-83, 2002.

59. Nelson, W. G., De Marzo, A. M., DeWeese, T. L., and Isaacs, W. B. The role of inflammation in the pathogenesis of prostate cancer. J Urol, 172: S6-11; discussion S11-12, 2004.

60. Nelson, W. G., DeWeese, T. L., and DeMarzo, A. M. The diet, prostate inflammation, and the development of prostate cancer. Cancer Metastasis Rev, 21: 3-16, 2002.

61. De Marzo, A. M., Platz, E. A., Sutcliffe, S., Xu, J., Gronberg, H., Drake, C. G., Nakai, Y., Isaacs, W. B., and Nelson, W. G. Inflammation in prostate carcinogenesis. Nat Rev Cancer, 7: 256-269, 2007.

183 62. Bostwick, D. G., Cooner, W. H., Denis, L., Jones, G. W., Scardino, P. T., and Murphy, G. P. The association of benign prostatic hyperplasia and cancer of the prostate. Cancer, 70: 291-301, 1992.

63. Gonzalgo, M. L. and Isaacs, W. B. Molecular pathways to prostate cancer. J Urol, 170: 2444-2452, 2003.

64. Prout, G. R., Jr., Wesley, M. N., Greenberg, R. S., Chen, V. W., Brown, C. C., Miller, A. W., Weinstein, R. S., Robboy, S. J., Haynes, M. A., Blacklow, R. S., and Edwards, B. K. Bladder cancer: race differences in extent of disease at diagnosis. Cancer, 89: 1349-1358, 2000.

65. Marsit, C. J., Karagas, M. R., Danaee, H., Liu, M., Andrew, A., Schned, A., Nelson, H. H., and Kelsey, K. T. Carcinogen exposure and gene promoter hypermethylation in bladder cancer. Carcinogenesis, 27: 112-116, 2006.

66. Pelucchi, C., Bosetti, C., Negri, E., Malvezzi, M., and La Vecchia, C. Mechanisms of disease: The epidemiology of bladder cancer. Nat Clin Pract Urol, 3: 327-340, 2006.

67. Gu, J., Barton Grossman, H., Dinney, C. P., and Wu, X. The pharmacogenetic impact of inflammatory genes on bladder cancer recurrence. Pharmacogenomics, 6: 575-584, 2005.

68. Leibovici, D., Grossman, H. B., Dinney, C. P., Millikan, R. E., Lerner, S., Wang, Y., Gu, J., Dong, Q., and Wu, X. Polymorphisms in inflammation genes and bladder cancer: from initiation to recurrence, progression, and survival. J Clin Oncol, 23: 5746-5756, 2005.

69. Colquhoun, A. J. and Mellon, J. K. Epidermal growth factor receptor and bladder cancer. Postgrad Med J, 78: 584-589, 2002.

70. Liou, L. S. Urothelial cancer biomarkers for detection and surveillance. Urology, 67: 25-33, 2006.

71. Dinney, C. P., McConkey, D. J., Millikan, R. E., Wu, X., Bar-Eli, M., Adam, L., Kamat, A. M., Siefker-Radtke, A. O., Tuziak, T., Sabichi, A. L., Grossman, H. B., Benedict, W. F., and Czerniak, B. Focus on bladder cancer. Cancer Cell, 6: 111- 116, 2004.

72. Wolff, E. M., Liang, G., and Jones, P. A. Mechanisms of Disease: genetic and epigenetic alterations that drive bladder cancer. Nat Clin Pract Urol, 2: 502-510, 2005.

184 73. Doll, R. and Peto, R. The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst, 66: 1191-1308, 1981.

74. Johnson, I. T. Cancers of the gut and Western ills. Science, 307: 1839, 2005.

75. Davis, C. D. Nutritional interactions: credentialing of molecular targets for cancer prevention. Exp Biol Med, 232: 176-183, 2007.

76. Michels, K. B. The role of nutrition in cancer development and prevention. Int J Cancer, 114: 163-165, 2005.

77. Suzuki, H., Toyota, M., Sato, H., Sonoda, T., Sakauchi, F., and Mori, M. Roles and causes of abnormal DNA methylation in gastrointestinal cancers. Asian Pac J Cancer Prev, 7: 177-185, 2006.

78. Adlercreutz, H. Phytoestrogens: epidemiology and a possible role in cancer protection. Environ Health Perspect, 103 Suppl 7: 103-112, 1995.

79. Parker, S. L., Tong, T., Bolden, S., and Wingo, P. A. Cancer statistics, 1997. CA Cancer J Clin, 47: 5-27, 1997.

80. Setchell, K. D., Borriello, S. P., Hulme, P., Kirk, D. N., and Axelson, M. Nonsteroidal estrogens of dietary origin: possible roles in hormone-dependent disease. Am J Clin Nutr, 40: 569-578, 1984.

81. Jemal, A., Thomas, A., Murray, T., and Thun, M. Cancer statistics, 2002. CA Cancer J Clin, 52: 23-47, 2002.

82. Haenszel, W. and Kurihara, M. Studies of Japanese migrants. I. Mortality from cancer and other diseases among Japanese in the United States. J Natl Cancer Inst, 40: 43-68, 1968.

83. Staszewski, J. and Haenszel, W. Cancer mortality among the Polish-born in the United States. J Natl Cancer Inst, 35: 291-297, 1965.

84. Shimizu, T., Ohta, Y., Ozawa, H., Matsushima, H., and Takeda, K. Papaverine combined with prostaglandin E2 synergistically induces neuron-like morphological changes and decrease of malignancy in human prostatic cancer LNCaP cells. Anticancer Res, 20: 761-767, 2000.

85. Chung, L. W. Implications of stromal-epithelial interaction in human prostate cancer growth, progression and differentiation. Semin Cancer Biol, 4: 183-192, 1993.

185 86. Breslow, N., Chan, C. W., Dhom, G., Drury, R. A., Franks, L. M., Gellei, B., Lee, Y. S., Lundberg, S., Sparke, B., Sternby, N. H., and Tulinius, H. Latent carcinoma of prostate at autopsy in seven areas. The International Agency for Research on Cancer, Lyons, France. Int J Cancer, 20: 680-688, 1977.

87. Hebert, J. R., Hurley, T. G., Olendzki, B. C., Teas, J., Ma, Y., and Hampl, J. S. Nutritional and socioeconomic factors in relation to prostate cancer mortality: a cross-national study. J Natl Cancer Inst, 90: 1637-1647, 1998.

88. Rose, D. P. Effects of dietary fatty acids on breast and prostate cancers: evidence from in vitro experiments and animal studies. Am J Clin Nutr, 66: 1513S-1522S, 1997.

89. Steinmetz, K. A. and Potter, J. D. Vegetables, fruit, and cancer. II. Mechanisms. Cancer Causes Control, 2: 427-442, 1991.

90. Tymchuk, C. N., Barnard, R. J., Heber, D., and Aronson, W. J. Evidence of an inhibitory effect of diet and exercise on prostate cancer cell growth. J Urol, 166: 1185-1189, 2001.

91. Kolonel, L. N. Nutrition and prostate cancer. Cancer Causes Control, 7: 83-44, 1996.

92. Cui, L., Takahashi, S., Tada, M., Kato, K., Yamada, Y., Kohri, K., and Shirai, T. Immunohistochemical detection of carcinogen-DNA adducts in normal human prostate tissues transplanted into the subcutis of athymic nude mice: results with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 3,2'-dimethyl-4- aminobiphenyl (DMAB) and relation to cytochrome P450s and N- acetyltransferase activity. Jpn J Cancer Res, 91: 52-58, 2000.

93. Bogen, K. T. and Keating, G. A. U.S. dietary exposures to heterocyclic amines. J Expo Anal Environ Epidemiol, 11: 155-168, 2001.

94. Clinton, S. K. and Giovannucci, E. Diet, nutrition, and prostate cancer. Annu Rev Nutr, 18: 413-440, 1998.

95. Cohen, J. H., Kristal, A. R., and Stanford, J. L. Fruit and vegetable intakes and prostate cancer risk. J Natl Cancer Inst, 92: 61-68, 2000.

96. Kolonel, L. N., Hankin, J. H., Whittemore, A. S., Wu, A. H., Gallagher, R. P., Wilkens, L. R., John, E. M., Howe, G. R., Dreon, D. M., West, D. W., and Paffenbarger, R. S., Jr. Vegetables, fruits, legumes and prostate cancer: a multiethnic case-control study. Cancer Epidemiol Biomarkers Prev, 9: 795-804, 2000.

186 97. Denis, L., Morton, M. S., and Griffiths, K. Diet and its preventive role in prostatic disease. Eur Urol, 35: 377-387, 1999.

98. Steinmaus, C. M., Nunez, S., and Smith, A. H. Diet and bladder cancer: a meta- analysis of six dietary variables. Am J Epidemiol, 151: 693-702, 2000.

99. Riboli, E., Gonzalez, C. A., Lopez-Abente, G., Errezola, M., Izarzugaza, I., Escolar, A., Nebot, M., Hemon, B., and Agudo, A. Diet and bladder cancer in Spain: a multi-centre case-control study. Int J Cancer, 49: 214-219, 1991.

100. Bruemmer, B., White, E., Vaughan, T. L., and Cheney, C. L. Nutrient intake in relation to bladder cancer among middle-aged men and women. Am J Epidemiol, 144: 485-495, 1996.

101. Michaud, D. S., Holick, C. N., Giovannucci, E., and Stampfer, M. J. Meat intake and bladder cancer risk in 2 prospective cohort studies. Am J Clin Nutr, 84: 1177- 1183, 2006.

102. Nagano, J., Kono, S., Preston, D. L., Moriwaki, H., Sharp, G. B., Koyama, K., and Mabuchi, K. Bladder-cancer incidence in relation to vegetable and fruit consumption: a prospective study of atomic-bomb survivors. Int J Cancer, 86: 132-138, 2000.

103. Vainio, H. and Weiderpass, E. Fruit and vegetables in cancer prevention. Nutr Cancer, 54: 111-142, 2006.

104. Fleshner, N., Bagnell, P. S., Klotz, L., and Venkateswaran, V. Dietary fat and prostate cancer. J Urol, 171: S19-24, 2004.

105. Laaksonen, D. E., Laukkanen, J. A., Niskanen, L., Nyyssonen, K., Rissanen, T. H., Voutilainen, S., Pukkala, E., Hakkarainen, A., and Salonen, J. T. Serum linoleic and total polyunsaturated fatty acids in relation to prostate and other cancers: a population-based cohort study. Int J Cancer, 111: 444-450, 2004.

106. Link, L. B., Thompson, S. M., Bosland, M. C., and Lumey, L. H. Adherence to a low-fat diet in men with prostate cancer. Urology, 64: 970-975, 2004.

107. Ngo, T. H., Barnard, R. J., Anton, T., Tran, C., Elashoff, D., Heber, D., Freedland, S. J., and Aronson, W. J. Effect of isocaloric low-fat diet on prostate cancer xenograft progression to androgen independence. Cancer Res, 64: 1252-1254, 2004.

108. Porter, M. P. and Stanford, J. L. Obesity and the risk of prostate cancer. Prostate, 62: 316-321, 2005.

187 109. Astorg, P. Dietary N-6 and N-3 polyunsaturated fatty acids and prostate cancer risk: a review of epidemiological and experimental evidence. Cancer Causes Control, 15: 367-386, 2004.

110. Augustsson, K., Michaud, D. S., Rimm, E. B., Leitzmann, M. F., Stampfer, M. J., Willett, W. C., and Giovannucci, E. A prospective study of intake of fish and marine fatty acids and prostate cancer. Cancer Epidemiol Biomarkers Prev, 12: 64-67, 2003.

111. Leitzmann, M. F., Stampfer, M. J., Michaud, D. S., Augustsson, K., Colditz, G. C., Willett, W. C., and Giovannucci, E. L. Dietary intake of n-3 and n-6 fatty acids and the risk of prostate cancer. Am J Clin Nutr, 80: 204-216, 2004.

112. Pischon, T., Hankinson, S. E., Hotamisligil, G. S., Rifai, N., Willett, W. C., and Rimm, E. B. Habitual dietary intake of n-3 and n-6 fatty acids in relation to inflammatory markers among US men and women. Circulation, 108: 155-160, 2003.

113. Terry, P. D., Rohan, T. E., and Wolk, A. Intakes of fish and marine fatty acids and the risks of cancers of the breast and prostate and of other hormone-related cancers: a review of the epidemiologic evidence. Am J Clin Nutr, 77: 532-543, 2003.

114. Woutersen, R. A., Appel, M. J., van Garderen-Hoetmer, A., and Wijnands, M. V. Dietary fat and carcinogenesis. Mutat Res, 443: 111-127, 1999.

115. Hla, T. and Neilson, K. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci U S A, 89: 7384-7388, 1992.

116. Smith, M. R., Manola, J., Kaufman, D. S., Oh, W. K., Bubley, G. J., and Kantoff, P. W. Celecoxib versus placebo for men with prostate cancer and a rising serum prostate-specific antigen after radical prostatectomy and/or radiation therapy. J Clin Oncol, 24: 2723-2728, 2006.

117. DuBois, R. N. Cyclooxygenase-2 selective inhibitors and prostate cancer: what is the clinical benefit? J Clin Oncol, 24: 2691-2693, 2006.

118. Aronson, W. J., Glaspy, J. A., Reddy, S. T., Reese, D., Heber, D., and Bagga, D. Modulation of omega-3/omega-6 polyunsaturated ratios with dietary fish oils in men with prostate cancer. Urology, 58: 283-288, 2001.

119. Banerjee, S., Bueso-Ramos, C., and Aggarwal, B. B. Suppression of 7,12- dimethylbenz(a)anthracene-induced mammary carcinogenesis in rats by resveratrol: role of nuclear factor-kappaB, cyclooxygenase 2, and matrix metalloprotease 9. Cancer Res, 62: 4945-4954, 2002.

188

120. Stoll, B. A. N-3 fatty acids and lipid peroxidation in breast cancer inhibition. Br J Nutr, 87: 193-198, 2002.

121. Simmons, D. L., Botting, R. M., and Hla, T. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev, 56: 387-437, 2004.

122. Jump, D. B. Fatty acid regulation of gene transcription. Crit Rev Clin Lab Sci, 41: 41-78, 2004.

123. Grossman, H. B. Chemoprevention of bladder cancer. Urology, 67: 19-22; discussion 23-14, 2006.

124. Hursting, S. D., Slaga, T. J., Fischer, S. M., DiGiovanni, J., and Phang, J. M. Mechanism-based cancer prevention approaches: targets, examples, and the use of transgenic mice. J Natl Cancer Inst, 91: 215-225, 1999.

125. Lowe, J. F. and Frazee, L. A. Update on prostate cancer chemoprevention. Pharmacotherapy, 26: 353-359, 2006.

126. Virtamo, J., Pietinen, P., Huttunen, J. K., Korhonen, P., Malila, N., Virtanen, M. J., Albanes, D., Taylor, P. R., and Albert, P. Incidence of cancer and mortality following alpha-tocopherol and beta-carotene supplementation: a postintervention follow-up. Jama, 290: 476-485, 2003.

127. Klein, E. A. Chemoprevention of prostate cancer. Annu Rev Med, 57: 49-63, 2006.

128. Klein, E. A., Lippman, S. M., Thompson, I. M., Goodman, P. J., Albanes, D., Taylor, P. R., and Coltman, C. The selenium and vitamin E cancer prevention trial. World J Urol, 21: 21-27, 2003.

129. Huggins, C. Endocrine substances in the treatment of cancers. J Am Med Assoc, 141: 750-754, 1949.

130. Thompson, I. M., Goodman, P. J., Tangen, C. M., Lucia, M. S., Miller, G. J., Ford, L. G., Lieber, M. M., Cespedes, R. D., Atkins, J. N., Lippman, S. M., Carlin, S. M., Ryan, A., Szczepanek, C. M., Crowley, J. J., and Coltman, C. A., Jr. The influence of finasteride on the development of prostate cancer. N Engl J Med, 349: 215-224, 2003.

131. Goetzl, M. A. and Holzbeierlein, J. M. Finasteride as a chemopreventive agent in prostate cancer: impact of the PCPT on urologic practice. Nat Clin Pract Urol, 3: 422-429, 2006.

189

132. Kamat, A. M. and Lamm, D. L. Chemoprevention of bladder cancer. Urol Clin North Am, 29: 157-168, 2002.

133. Busby, J. E. and Kamat, A. M. Chemoprevention for bladder cancer. J Urol, 176: 1914-1920, 2006.

134. Leppert, J. T., Shvarts, O., Kawaoka, K., Lieberman, R., Belldegrun, A. S., and Pantuck, A. J. Prevention of bladder cancer: a review. Eur Urol, 49: 226-234, 2006.

135. Harland, S. J. A second look at the pT1 G3 bladder tumour. Clin Oncol (R Coll Radiol), 17: 498-502, 2005.

136. Herr, H. W. Intravesical BCG: current results, natural history and implications for urothelial cancer prevention. J Cell Biochem Suppl, 16I: 112-119, 1992.

137. Shelly, M., Pinkas-Kramarski, R., Guarino, B. C., Waterman, H., Wang, L. M., Lyass, L., Alimandi, M., Kuo, A., Bacus, S. S., Pierce, J. H., Andrews, G. C., and Yarden, Y. Epiregulin is a potent pan-ErbB ligand that preferentially activates heterodimeric receptor complexes. J Biol Chem, 273: 10496-10505, 1998.

138. Kim, J. C. and Steinberg, G. D. The limits of bacillus Calmette-Guerin for carcinoma in situ of the bladder. J Urol, 165: 745-756, 2001.

139. Michaud, D. S., Pietinen, P., Taylor, P. R., Virtanen, M., Virtamo, J., and Albanes, D. Intakes of fruits and vegetables, carotenoids and vitamins A, E, C in relation to the risk of bladder cancer in the ATBC cohort study. Br J Cancer, 87: 960-965, 2002.

140. Radosavljevic, V., Jankovic, S., Marinkovic, J., and Dokic, M. Diet and bladder cancer: a case-control study. Int Urol Nephrol, 37: 283-289, 2005.

141. Lamm, D. L., Riggs, D. R., Shriver, J. S., vanGilder, P. F., Rach, J. F., and DeHaven, J. I. Megadose vitamins in bladder cancer: a double-blind clinical trial. J Urol, 151: 21-26, 1994.

142. Shibata, A., Paganini-Hill, A., Ross, R. K., and Henderson, B. E. Intake of vegetables, fruits, beta-carotene, vitamin C and vitamin supplements and cancer incidence among the elderly: a prospective study. Br J Cancer, 66: 673-679, 1992.

143. Newling, D. W., Robinson, M. R., Smith, P. H., Byar, D., Lockwood, R., Stevens, I., De Pauw, M., and Sylvester, R. Tryptophan metabolites, pyridoxine (vitamin B6) and their influence on the recurrence rate of superficial bladder cancer. Results of a prospective, randomised phase III study performed by the EORTC

190 GU Group. EORTC Genito-Urinary Tract Cancer Cooperative Group. Eur Urol, 27: 110-116, 1995.

144. Tsao, A. S., Kim, E. S., and Hong, W. K. Chemoprevention of cancer. CA Cancer J Clin, 54: 150-180, 2004.

145. Graham, D. J. COX-2 inhibitors, other NSAIDs, and cardiovascular risk: the seduction of common sense. Jama, 296: 1653-1656, 2006.

146. Hanahan, D. and Weinberg, R. A. The hallmarks of cancer. Cell, 100: 57-70, 2000.

147. De Marzo, A. M., DeWeese, T. L., Platz, E. A., Meeker, A. K., Nakayama, M., Epstein, J. I., Isaacs, W. B., and Nelson, W. G. Pathological and molecular mechanisms of prostate carcinogenesis: implications for diagnosis, detection, prevention, and treatment. J Cell Biochem, 91: 459-477, 2004.

148. Singh, R. P. and Agarwal, R. Mechanisms of action of novel agents for prostate cancer chemoprevention. Endocr Relat Cancer, 13: 751-778, 2006.

149. Bryan, R. T., Hussain, S. A., James, N. D., Jankowski, J. A., and Wallace, D. M. Molecular pathways in bladder cancer: part 1. BJU Int, 95: 485-490, 2005.

150. Bryan, R. T., Hussain, S. A., James, N. D., Jankowski, J. A., and Wallace, D. M. Molecular pathways in bladder cancer: part 2. BJU Int, 95: 491-496, 2005.

151. Bellmunt, J., Hussain, M., and Dinney, C. P. Novel approaches with targeted therapies in bladder cancer. Therapy of bladder cancer by blockade of the epidermal growth factor receptor family. Crit Rev Oncol Hematol, 46 Suppl: S85- 104, 2003.

152. Ruck, A. and Paulie, S. EGF, TGF alpha, AR and HB-EGF are autocrine growth factors for human bladder carcinoma cell lines. Anticancer Res, 18: 1447-1452, 1998.

153. Swellam, M., El-Aal, A. A., and AbuGabel, K. M. Deletions of p15 and p16 in schistosomal bladder cancer correlate with transforming growth factor-alpha expression. Clin Biochem, 37: 1098-1104, 2004.

154. Thogersen, V. B., Jorgensen, P. E., Sorensen, B. S., Bross, P., Orntoft, T., Wolf, H., and Nexo, E. Expression of transforming growth factor alpha and epidermal growth factor receptor in human bladder cancer. Scand J Clin Lab Invest, 59: 267-277, 1999.

191 155. Nicholson, B. E., Frierson, H. F., Conaway, M. R., Seraj, J. M., Harding, M. A., Hampton, G. M., and Theodorescu, D. Profiling the evolution of human metastatic bladder cancer. Cancer Res, 64: 7813-7821, 2004.

156. Thogersen, V. B., Sorensen, B. S., Poulsen, S. S., Orntoft, T. F., Wolf, H., and Nexo, E. A subclass of HER1 ligands are prognostic markers for survival in bladder cancer patients. Cancer Res, 61: 6227-6233, 2001.

157. Bol, M. G., Baak, J. P., van Diermen, B., Buhr-Wildhagen, S., Janssen, E. A., Kjellevold, K. H., Kruse, A. J., Mestad, O., and Ogreid, P. Proliferation markers and DNA content analysis in urinary bladder TaT1 urothelial cell carcinomas: identification of subgroups with low and high stage progression risks. J Clin Pathol, 56: 447-452, 2003.

158. Bozlu, M., Orhan, D., Baltaci, S., Yaman, O., Elhan, A. H., Tulunay, O., and Muftuoglu, Y. Z. The prognostic value of proliferating cell nuclear antigen, Ki-67 and nucleolar organizer region in transitional cell carcinoma of the bladder. Int Urol Nephrol, 33: 59-66, 2002.

159. Molinie, V., Longchampt, E., Ouazana, D., and Lebret, T. [Bladder tumors and molecular markers. Current status and perspectives]. Ann Pathol, 23: 306-331, 2003.

160. Coombs, L. M., Pigott, D. A., Eydmann, M. E., Proctor, A. J., and Knowles, M. A. Reduced expression of TGF beta is associated with advanced disease in transitional cell carcinoma. Br J Cancer, 67: 578-584, 1993.

161. Miyamoto, H., Kubota, Y., Shuin, T., Torigoe, S., Dobashi, Y., and Hosaka, M. Expression of transforming growth factor-beta 1 in human bladder cancer. Cancer, 75: 2565-2570, 1995.

162. Zhao, H., Grossman, H. B., Spitz, M. R., Lerner, S. P., Zhang, K., and Wu, X. Plasma levels of insulin-like growth factor-1 and binding protein-3, and their association with bladder cancer risk. J Urol, 169: 714-717, 2003.

163. Bernardini, S., Fauconnet, S., Chabannes, E., Henry, P. C., Adessi, G., and Bittard, H. Serum levels of vascular endothelial growth factor as a prognostic factor in bladder cancer. J Urol, 166: 1275-1279, 2001.

164. Crew, J. P., O'Brien, T., Bicknell, R., Fuggle, S., Cranston, D., and Harris, A. L. Urinary vascular endothelial growth factor and its correlation with bladder cancer recurrence rates. J Urol, 161: 799-804, 1999.

192 165. Crew, J. P., O'Brien, T., Bradburn, M., Fuggle, S., Bicknell, R., Cranston, D., and Harris, A. L. Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer. Cancer Res, 57: 5281-5285, 1997.

166. Campbell, S. C., Volpert, O. V., Ivanovich, M., and Bouck, N. P. Molecular mediators of angiogenesis in bladder cancer. Cancer Res, 58: 1298-1304, 1998.

167. Goddard, J. C., Sutton, C. D., Jones, J. L., O'Byrne, K. J., and Kockelbergh, R. C. Reduced thrombospondin-1 at presentation predicts disease progression in superficial bladder cancer. Eur Urol, 42: 464-468, 2002.

168. Wheeler, M. A., Hausladen, D. A., Yoon, J. H., and Weiss, R. M. Prostaglandin E2 production and cyclooxygenase-2 induction in human urinary tract infections and bladder cancer. J Urol, 168: 1568-1573, 2002.

169. Funk, C. D. Prostaglandins and leukotrienes: advances in eicosanoid biology. Science, 294: 1871-1875, 2001.

170. DeWitt, D. L. and Smith, W. L. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc Natl Acad Sci U S A, 85: 1412-1416, 1988.

171. Merlie, J. P., Fagan, D., Mudd, J., and Needleman, P. Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J Biol Chem, 263: 3550- 3553, 1988.

172. Yokoyama, C., Takai, T., and Tanabe, T. Primary structure of sheep prostaglandin endoperoxide synthase deduced from cDNA sequence. FEBS Lett, 231: 347-351, 1988.

173. Jones, D. A., Carlton, D. P., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. Molecular cloning of human prostaglandin endoperoxide synthase type II and demonstration of expression in response to cytokines. J Biol Chem, 268: 9049- 9054, 1993.

174. O'Banion, M. K., Sadowski, H. B., Winn, V., and Young, D. A. A serum- and glucocorticoid-regulated 4-kilobase mRNA encodes a cyclooxygenase-related protein. J Biol Chem, 266: 23261-23267, 1991.

175. Funk, C. D., Funk, L. B., Kennedy, M. E., Pong, A. S., and Fitzgerald, G. A. Human platelet/erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression, and gene chromosomal assignment. Faseb J, 5: 2304-2312, 1991.

193 176. Otto, J. C., DeWitt, D. L., and Smith, W. L. N-glycosylation of prostaglandin endoperoxide synthases-1 and -2 and their orientations in the endoplasmic reticulum. J Biol Chem, 268: 18234-18242, 1993.

177. Chandrasekharan, N. V., Dai, H., Roos, K. L., Evanson, N. K., Tomsik, J., Elton, T. S., and Simmons, D. L. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci U S A, 99: 13926-13931, 2002.

178. Morita, I., Schindler, M., Regier, M. K., Otto, J. C., Hori, T., DeWitt, D. L., and Smith, W. L. Different intracellular locations for prostaglandin endoperoxide H synthase-1 and -2. J Biol Chem, 270: 10902-10908, 1995.

179. Gilroy, D. W. and Colville-Nash, P. R. New insights into the role of COX 2 in inflammation. J Mol Med, 78: 121-129, 2000.

180. O'Neill, G. P. and Ford-Hutchinson, A. W. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues. FEBS Lett, 330: 156- 160, 1993.

181. Seibert, K., Zhang, Y., Leahy, K., Hauser, S., Masferrer, J., Perkins, W., Lee, L., and Isakson, P. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci U S A, 91: 12013-12017, 1994.

182. Subbarayan, V., Sabichi, A. L., Llansa, N., Lippman, S. M., and Menter, D. G. Differential expression of cyclooxygenase-2 and its regulation by tumor necrosis factor-alpha in normal and malignant prostate cells. Cancer Res, 61: 2720-2726, 2001.

183. Subbaramaiah, K. and Dannenberg, A. J. Cyclooxygenase 2: a molecular target for cancer prevention and treatment. Trends Pharmacol Sci, 24: 96-102, 2003.

184. Subbaramaiah, K., Altorki, N., Chung, W. J., Mestre, J. R., Sampat, A., and Dannenberg, A. J. Inhibition of cyclooxygenase-2 gene expression by p53. J Biol Chem, 274: 10911-10915, 1999.

185. Yegnasubramanian, S., Kowalski, J., Gonzalgo, M. L., Zahurak, M., Piantadosi, S., Walsh, P. C., Bova, G. S., De Marzo, A. M., Isaacs, W. B., and Nelson, W. G. Hypermethylation of CpG islands in primary and metastatic human prostate cancer. Cancer Res, 64: 1975-1986, 2004.

186. Castells, A., Paya, A., Alenda, C., Rodriguez-Moranta, F., Agrelo, R., Andreu, M., Pinol, V., Castellvi-Bel, S., Jover, R., Llor, X., Pons, E., Elizalde, J. I., Bessa, X., Alcedo, J., Salo, J., Medina, E., Naranjo, A., Esteller, M., and Pique, J. M.

194 Cyclooxygenase 2 expression in colorectal cancer with DNA mismatch repair deficiency. Clin Cancer Res, 12: 1686-1692, 2006.

187. Tiano, H. F., Loftin, C. D., Akunda, J., Lee, C. A., Spalding, J., Sessoms, A., Dunson, D. B., Rogan, E. G., Morham, S. G., Smart, R. C., and Langenbach, R. Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis. Cancer Res, 62: 3395-3401, 2002.

188. Chulada, P. C., Thompson, M. B., Mahler, J. F., Doyle, C. M., Gaul, B. W., Lee, C., Tiano, H. F., Morham, S. G., Smithies, O., and Langenbach, R. Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice. Cancer Res, 60: 4705-4708, 2000.

189. Gupta, R. A., Tejada, L. V., Tong, B. J., Das, S. K., Morrow, J. D., Dey, S. K., and DuBois, R. N. Cyclooxygenase-1 is overexpressed and promotes angiogenic growth factor production in ovarian cancer. Cancer Res, 63: 906-911, 2003.

190. Okamoto, T., Hara, A., and Hino, O. Down-regulation of cyclooxygenase-2 expression but up-regulation of cyclooxygenase-1 in renal carcinomas of the Eker (TSC2 gene mutant) rat model. Cancer Sci, 94: 22-25, 2003.

191. Sales, K. J., Katz, A. A., Howard, B., Soeters, R. P., Millar, R. P., and Jabbour, H. N. Cyclooxygenase-1 is up-regulated in cervical carcinomas: autocrine/paracrine regulation of cyclooxygenase-2, prostaglandin e receptors, and angiogenic factors by cyclooxygenase-1. Cancer Res, 62: 424-432, 2002.

192. Eberhart, C. E., Coffey, R. J., Radhika, A., Giardiello, F. M., Ferrenbach, S., and DuBois, R. N. Up-regulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas. Gastroenterology, 107: 1183-1188, 1994.

193. Liu, C. H., Chang, S. H., Narko, K., Trifan, O. C., Wu, M. T., Smith, E., Haudenschild, C., Lane, T. F., and Hla, T. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice. J Biol Chem, 276: 18563- 18569, 2001.

194. Neufang, G., Furstenberger, G., Heidt, M., Marks, F., and Muller-Decker, K. Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin. Proc Natl Acad Sci U S A, 98: 7629-7634, 2001.

195. Fosslien, E. Review: molecular pathology of cyclooxygenase-2 in cancer-induced angiogenesis. Ann Clin Lab Sci, 31: 325-348, 2001.

195 196. Joo, Y. E., Rew, J. S., Seo, Y. H., Choi, S. K., Kim, Y. J., Park, C. S., and Kim, S. J. Cyclooxygenase-2 overexpression correlates with vascular endothelial growth factor expression and tumor angiogenesis in gastric cancer. J Clin Gastroenterol, 37: 28-33, 2003.

197. Tatsuguchi, A., Matsui, K., Shinji, Y., Gudis, K., Tsukui, T., Kishida, T., Fukuda, Y., Sugisaki, Y., Tokunaga, A., Tajiri, T., and Sakamoto, C. Cyclooxygenase-2 expression correlates with angiogenesis and apoptosis in gastric cancer tissue. Hum Pathol, 35: 488-495, 2004.

198. Tsujii, M., Kawano, S., Tsuji, S., Sawaoka, H., Hori, M., and DuBois, R. N. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell, 93: 705-716, 1998.

199. Wang, D. and DuBois, R. N. Cyclooxygenase 2-derived prostaglandin E2 regulates the angiogenic switch. Proc Natl Acad Sci U S A, 101: 415-416, 2004.

200. Hussain, T., Gupta, S., and Mukhtar, H. Cyclooxygenase-2 and prostate carcinogenesis. Cancer Lett, 191: 125-135, 2003.

201. Wagner, M., Loos, J., Weksler, N., Gantner, M., Corless, C. L., Barry, J. M., Beer, T. M., and Garzotto, M. Resistance of prostate cancer cell lines to COX-2 inhibitor treatment. Biochem Biophys Res Commun, 332: 800-807, 2005.

202. Lim, J. T., Piazza, G. A., Han, E. K., Delohery, T. M., Li, H., Finn, T. S., Buttyan, R., Yamamoto, H., Sperl, G. J., Brendel, K., Gross, P. H., Pamukcu, R., and Weinstein, I. B. Sulindac derivatives inhibit growth and induce apoptosis in human prostate cancer cell lines. Biochem Pharmacol, 58: 1097-1107, 1999.

203. Patel, M. I., Subbaramaiah, K., Du, B., Chang, M., Yang, P., Newman, R. A., Cordon-Cardo, C., Thaler, H. T., and Dannenberg, A. J. Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism. Clin Cancer Res, 11: 1999-2007, 2005.

204. Khan, K. N., Knapp, D. W., Denicola, D. B., and Harris, R. K. Expression of cyclooxygenase-2 in transitional cell carcinoma of the urinary bladder in dogs. Am J Vet Res, 61: 478-481, 2000.

205. Kitayama, W., Denda, A., Okajima, E., Tsujiuchi, T., and Konishi, Y. Increased expression of cyclooxygenase-2 protein in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine. Carcinogenesis, 20: 2305-2310, 1999.

206. Badawi, A. F., Habib, S. L., Mohammed, M. A., Abadi, A. A., and Michael, M. S. Influence of cigarette smoking on prostaglandin synthesis and cyclooxygenase-2

196 gene expression in human urinary bladder cancer. Cancer Invest, 20: 651-656, 2002.

207. Grubbs, C. J., Lubet, R. A., Koki, A. T., Leahy, K. M., Masferrer, J. L., Steele, V. E., Kelloff, G. J., Hill, D. L., and Seibert, K. Celecoxib inhibits N-butyl-N-(4- hydroxybutyl)-nitrosamine-induced urinary bladder cancers in male B6D2F1 mice and female Fischer-344 rats. Cancer Res, 60: 5599-5602, 2000.

208. Okajima, E., Denda, A., Ozono, S., Takahama, M., Akai, H., Sasaki, Y., Kitayama, W., Wakabayashi, K., and Konishi, Y. Chemopreventive effects of nimesulide, a selective cyclooxygenase-2 inhibitor, on the development of rat urinary bladder carcinomas initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine. Cancer Res, 58: 3028-3031, 1998.

209. Knapp, D. W., Glickman, N. W., Widmer, W. R., DeNicola, D. B., Adams, L. G., Kuczek, T., Bonney, P. L., DeGortari, A. E., Han, C., and Glickman, L. T. Cisplatin versus cisplatin combined with piroxicam in a canine model of human invasive urinary bladder cancer. Cancer Chemother Pharmacol, 46: 221-226, 2000.

210. Mohammed, S. I., Bennett, P. F., Craig, B. A., Glickman, N. W., Mutsaers, A. J., Snyder, P. W., Widmer, W. R., DeGortari, A. E., Bonney, P. L., and Knapp, D. W. Effects of the cyclooxygenase inhibitor, piroxicam, on tumor response, apoptosis, and angiogenesis in a canine model of human invasive urinary bladder cancer. Cancer Res, 62: 356-358, 2002.

211. Smakman, N., Schaap, N., Snijckers, C. M., Borel Rinkes, I. H., and Kranenburg, O. NS-398, a selective cyclooxygenase-2 inhibitor, reduces experimental bladder carcinoma outgrowth by inhibiting tumor cell proliferation. Urology, 66: 434-440, 2005.

212. Farivar-Mohseni, H., Kandzari, S. J., Zaslau, S., Riggs, D. R., Jackson, B. J., and McFadden, D. W. Synergistic effects of Cox-1 and -2 inhibition on bladder and prostate cancer in vitro. Am J Surg, 188: 505-510, 2004.

213. Castelao, J. E., Yuan, J. M., Gago-Dominguez, M., Yu, M. C., and Ross, R. K. Non-steroidal anti-inflammatory drugs and bladder cancer prevention. Br J Cancer, 82: 1364-1369, 2000.

214. Southall, M. D. and Vasko, M. R. Prostaglandin receptor subtypes, EP3C and EP4, mediate the prostaglandin E2-induced cAMP production and sensitization of sensory neurons. J Biol Chem, 276: 16083-16091, 2001.

215. Dohadwala, M., Batra, R. K., Luo, J., Lin, Y., Krysan, K., Pold, M., Sharma, S., and Dubinett, S. M. Autocrine/paracrine prostaglandin E2 production by non-

197 small cell lung cancer cells regulates matrix metalloproteinase-2 and CD44 in cyclooxygenase-2-dependent invasion. J Biol Chem, 277: 50828-50833, 2002.

216. Faas, F. H., Dang, A. Q., Pollard, M., Hong, X. M., Fan, K., Luckert, P. H., and Schutz, M. Increased phospholipid fatty acid remodeling in human and rat prostatic adenocarcinoma tissues. J Urol, 156: 243-248, 1996.

217. Nithipatikom, K., Isbell, M. A., Lindholm, P. F., Kajdacsy-Balla, A., Kaul, S., and Campell, W. B. Requirement of cyclooxygenase-2 expression and prostaglandins for human prostate cancer cell invasion. Clin Exp Metastasis, 19: 593-601, 2002.

218. Tjandrawinata, R. R. and Hughes-Fulford, M. Up-regulation of cyclooxygenase-2 by product-prostaglandin E2. Adv Exp Med Biol, 407: 163-170, 1997.

219. Liu, X. H., Kirschenbaum, A., Lu, M., Yao, S., Klausner, A., Preston, C., Holland, J. F., and Levine, A. C. Prostaglandin E(2) stimulates prostatic intraepithelial neoplasia cell growth through activation of the interleukin-6/GP130/STAT-3 signaling pathway. Biochem Biophys Res Commun, 290: 249-255, 2002.

220. Liu, L. X., Lu, H., Luo, Y., Date, T., Belanger, A. J., Vincent, K. A., Akita, G. Y., Goldberg, M., Cheng, S. H., Gregory, R. J., and Jiang, C. Stabilization of vascular endothelial growth factor mRNA by hypoxia-inducible factor 1. Biochem Biophys Res Commun, 291: 908-914, 2002.

221. Shi, Y., Cui, L., Dai, G., Chen, J., Pan, H., Song, L., Cheng, S., and Wang, X. Elevated prostaglandin E2 level via cPLA2--COX-2--mPGES-1 pathway involved in bladder carcinogenesis induced by terephthalic acid-calculi in Wistar rats. Prostaglandins Leukot Essent Fatty Acids, 74: 309-315, 2006.

222. Yu, D. S., Chen, H. I., and Chang, S. Y. The expression of cyclooxygenase in transitional cell carcinoma cell lines: its correlation with tumor differentiation, invasiveness and prostanoids production. Eur Urol, 44: 491-494, 2003.

223. Arosh, J. A., Banu, S. K., Chapdelaine, P., Emond, V., Kim, J. J., MacLaren, L. A., and Fortier, M. A. Molecular cloning and characterization of bovine prostaglandin E2 receptors EP2 and EP4: expression and regulation in endometrium and myometrium during the estrous cycle and early pregnancy. Endocrinology, 144: 3076-3091, 2003.

224. Bhattacharya, M., Peri, K., Ribeiro-da-Silva, A., Almazan, G., Shichi, H., Hou, X., Varma, D. R., and Chemtob, S. Localization of functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. J Biol Chem, 274: 15719-15724, 1999.

198 225. Bishop-Bailey, D. and Wray, J. Peroxisome proliferator-activated receptors: a critical review on endogenous pathways for ligand generation. Prostaglandins Other Lipid Mediat, 71: 1-22, 2003.

226. Subbarayan, V., Sabichi, A. L., Kim, J., Llansa, N., Logothetis, C. J., Lippman, S. M., and Menter, D. G. Differential peroxisome proliferator-activated receptor- gamma isoform expression and agonist effects in normal and malignant prostate cells. Cancer Epidemiol Biomarkers Prev, 13: 1710-1716, 2004.

227. Miyata, Y., Kanda, S., Nomata, K., Eguchi, J., and Kanetake, H. Expression of cyclooxygenase-2 and EP4 receptor in transitional cell carcinoma of the upper urinary tract. J Urol, 173: 56-60, 2005.

228. Yang, L., Yamagata, N., Yadav, R., Brandon, S., Courtney, R. L., Morrow, J. D., Shyr, Y., Boothby, M., Joyce, S., Carbone, D. P., and Breyer, R. M. Cancer- associated immunodeficiency and dendritic cell abnormalities mediated by the prostaglandin EP2 receptor. J Clin Invest, 111: 727-735, 2003.

229. Mutoh, M., Watanabe, K., Kitamura, T., Shoji, Y., Takahashi, M., Kawamori, T., Tani, K., Kobayashi, M., Maruyama, T., Kobayashi, K., Ohuchida, S., Sugimoto, Y., Narumiya, S., Sugimura, T., and Wakabayashi, K. Involvement of prostaglandin E receptor subtype EP(4) in colon carcinogenesis. Cancer Res, 62: 28-32, 2002.

230. Kawamori, T., Kitamura, T., Watanabe, K., Uchiya, N., Maruyama, T., Narumiya, S., Sugimura, T., and Wakabayashi, K. Prostaglandin E receptor subtype EP(1) deficiency inhibits colon cancer development. Carcinogenesis, 26: 353-357, 2005.

231. Fujino, H., West, K. A., and Regan, J. W. Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. J Biol Chem, 277: 2614-2619, 2002.

232. Fujino, H., Xu, W., and Regan, J. W. Prostaglandin E2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. J Biol Chem, 278: 12151-12156, 2003.

233. Buchanan, F. G., Wang, D., Bargiacchi, F., and DuBois, R. N. Prostaglandin E2 regulates cell migration via the intracellular activation of the epidermal growth factor receptor. J Biol Chem, 278: 35451-35457, 2003.

234. Fukuda, R., Kelly, B., and Semenza, G. L. Vascular endothelial growth factor gene expression in colon cancer cells exposed to prostaglandin E2 is mediated by hypoxia-inducible factor 1. Cancer Res, 63: 2330-2334, 2003.

199 235. Bendvold, E., Svanborg, K., Bygdeman, M., and Noren, S. On the origin of prostaglandins in human seminal fluid. Int J Androl, 8: 37-43, 1985.

236. Abdel-Majid, R. M. and Marshall, J. S. Prostaglandin E2 induces degranulation- independent production of vascular endothelial growth factor by human mast cells. J Immunol, 172: 1227-1236, 2004.

237. Castellone, M. D., Teramoto, H., Williams, B. O., Druey, K. M., and Gutkind, J. S. Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta- catenin signaling axis. Science, 310: 1504-1510, 2005.

238. Shibuya, M. Structure and function of VEGF/VEGF-receptor system involved in angiogenesis. Cell Struct Funct, 26: 25-35, 2001.

239. Harada, S., Nagy, J. A., Sullivan, K. A., Thomas, K. A., Endo, N., Rodan, G. A., and Rodan, S. B. Induction of vascular endothelial growth factor expression by prostaglandin E2 and E1 in osteoblasts. J Clin Invest, 93: 2490-2496, 1994.

240. Regan, J. W. EP2 and EP4 prostanoid receptor signaling. Life Sci, 74: 143-153, 2003.

241. Desai, S., April, H., Nwaneshiudu, C., and Ashby, B. Comparison of agonist- induced internalization of the human EP2 and EP4 prostaglandin receptors: role of the carboxyl terminus in EP4 receptor sequestration. Mol Pharmacol, 58: 1279- 1286, 2000.

242. Nishigaki, N., Negishi, M., and Ichikawa, A. Two Gs-coupled prostaglandin E receptor subtypes, EP2 and EP4, differ in desensitization and sensitivity to the metabolic inactivation of the agonist. Mol Pharmacol, 50: 1031-1037, 1996.

243. Tani, K., Naganawa, A., Ishida, A., Egashira, H., Sagawa, K., Harada, H., Ogawa, M., Maruyama, T., Ohuchida, S., Nakai, H., Kondo, K., and Toda, M. Design and synthesis of a highly selective EP2-receptor agonist. Bioorg Med Chem Lett, 11: 2025-2028, 2001.

244. Eling, T. E., Hawkins, H. J., and Anderson, M. W. Structural requirements for, and the effects of chemicals on, the rat pulmonary inactivation of prostaglandins. Prostaglandins, 14: 51-60, 1977.

245. Nomura, T., Lu, R., Pucci, M. L., and Schuster, V. L. The two-step model of prostaglandin signal termination: in vitro reconstitution with the prostaglandin transporter and prostaglandin 15 dehydrogenase. Mol Pharmacol, 65: 973-978, 2004.

200 246. Fujino, H., Salvi, S., and Regan, J. W. Differential regulation of phosphorylation of the cAMP response element-binding protein after activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. Mol Pharmacol, 68: 251-259, 2005.

247. Mendez, M. and LaPointe, M. C. PGE2-induced hypertrophy of cardiac myocytes involves EP4 receptor-dependent activation of p42/44 MAPK and EGFR transactivation. Am J Physiol Heart Circ Physiol, 288: H2111-2117, 2005.

248. Krysan, K., Reckamp, K. L., Dalwadi, H., Sharma, S., Rozengurt, E., Dohadwala, M., and Dubinett, S. M. Prostaglandin E2 activates mitogen-activated protein kinase/Erk pathway signaling and cell proliferation in non-small cell lung cancer cells in an epidermal growth factor receptor-independent manner. Cancer Res, 65: 6275-6281, 2005.

249. Sales, K. J., Maudsley, S., and Jabbour, H. N. Elevated prostaglandin EP2 receptor in endometrial adenocarcinoma cells promotes vascular endothelial growth factor expression via cyclic 3',5'-adenosine monophosphate-mediated transactivation of the epidermal growth factor receptor and extracellular signal- regulated kinase 1/2 signaling pathways. Mol Endocrinol, 18: 1533-1545, 2004.

250. Holz, G. G., Kang, G., Harbeck, M., Roe, M. W., and Chepurny, O. G. Cell physiology of cAMP sensor Epac. J Physiol, 577: 5-15, 2006.

251. Gupta, S., Srivastava, M., Ahmad, N., Bostwick, D. G., and Mukhtar, H. Over- expression of cyclooxygenase-2 in human prostate adenocarcinoma. Prostate, 42: 73-78, 2000.

252. Faas, F. H., Dang, A. Q., White, J., Schaefer, R., and Johnson, D. Increased prostatic lysophosphatidylcholine acyltransferase activity in human prostate cancer: a marker for malignancy. J Urol, 165: 463-468, 2001.

253. Mahmud, S., Franco, E., and Aprikian, A. Prostate cancer and use of nonsteroidal anti-inflammatory drugs: systematic review and meta-analysis. Br J Cancer, 90: 93-99, 2004.

254. Perron, L., Bairati, I., Moore, L., and Meyer, F. Dosage, duration and timing of nonsteroidal antiinflammatory drug use and risk of prostate cancer. Int J Cancer, 106: 409-415, 2003.

255. Bresalier, R. S., Sandler, R. S., Quan, H., Bolognese, J. A., Oxenius, B., Horgan, K., Lines, C., Riddell, R., Morton, D., Lanas, A., Konstam, M. A., and Baron, J. A. Cardiovascular events associated with rofecoxib in a colorectal adenoma chemoprevention trial. N Engl J Med, 352: 1092-1102, 2005.

201 256. Pruthi, R. S., Derksen, J. E., and Moore, D. A pilot study of use of the cyclooxygenase-2 inhibitor celecoxib in recurrent prostate cancer after definitive radiation therapy or radical prostatectomy. BJU Int, 93: 275-278, 2004.

257. Pruthi, R. S., Derksen, J. E., Moore, D., Carson, C. C., Grigson, G., Watkins, C., and Wallen, E. Phase II trial of celecoxib in prostate-specific antigen recurrent prostate cancer after definitive radiation therapy or radical prostatectomy. Clin Cancer Res, 12: 2172-2177, 2006.

258. Morham, S. G., Langenbach, R., Loftin, C. D., Tiano, H. F., Vouloumanos, N., Jennette, J. C., Mahler, J. F., Kluckman, K. D., Ledford, A., Lee, C. A., and Smithies, O. Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse. Cell, 83: 473-482, 1995.

259. Shappell, S. B., Thomas, G. V., Roberts, R. L., Herbert, R., Ittmann, M. M., Rubin, M. A., Humphrey, P. A., Sundberg, J. P., Rozengurt, N., Barrios, R., Ward, J. M., and Cardiff, R. D. Prostate pathology of genetically engineered mice: definitions and classification. The consensus report from the Bar Harbor meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee. Cancer Res, 64: 2270-2305, 2004.

260. Kempen, E. C., Yang, P., Felix, E., Madden, T., and Newman, R. A. Simultaneous quantification of arachidonic acid metabolites in cultured tumor cells using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry. Anal Biochem, 297: 183-190, 2001.

261. Lee, J. H., Cheong, J., Park, Y. M., and Choi, Y. H. Down-regulation of cyclooxygenase-2 and telomerase activity by beta-lapachone in human prostate carcinoma cells. Pharmacol Res, 51: 553-560, 2005.

262. Klein, R. D. The use of genetically engineered mouse models of prostate cancer for nutrition and cancer chemoprevention research. Mutat Res, 576: 111-119, 2005.

263. Zeng, Y., Yokohira, M., Saoo, K., Takeuchi, H., Chen, Y., Yamakawa, K., Matsuda, Y., Kakehi, Y., and Imaida, K. Inhibition of prostate carcinogenesis in probasin/SV40 T antigen transgenic rats by raloxifene, an antiestrogen with anti- androgen action, but not nimesulide, a selective cyclooxygenase-2 inhibitor. Carcinogenesis, 26: 1109-1116, 2005.

264. Wang, S., Gao, J., Lei, Q., Rozengurt, N., Pritchard, C., Jiao, J., Thomas, G. V., Li, G., Roy-Burman, P., Nelson, P. S., Liu, X., and Wu, H. Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer Cell, 4: 209-221, 2003.

202 265. Perez-Stable, C., Altman, N. H., Mehta, P. P., Deftos, L. J., and Roos, B. A. Prostate cancer progression, metastasis, and gene expression in transgenic mice. Cancer Res, 57: 900-906, 1997.

266. Chaudry, A., McClinton, S., Moffat, L. E., and Wahle, K. W. Essential fatty acid distribution in the plasma and tissue phospholipids of patients with benign and malignant prostatic disease. Br J Cancer, 64: 1157-1160, 1991.

267. Faas, F. H., Dang, A. Q., White, J., Schaefer, R. F., and Johnson, D. E. Decreased prostatic arachidonic acid in human prostatic carcinoma. BJU Int, 92: 551-554, 2003.

268. Freeman, V. L., Meydani, M., Hur, K., and Flanigan, R. C. Inverse association between prostatic polyunsaturated fatty acid and risk of locally advanced prostate carcinoma. Cancer, 101: 2744-2754, 2004.

269. Mamalakis, G., Kafatos, A., Kalogeropoulos, N., Andrikopoulos, N., Daskalopulos, G., and Kranidis, A. Prostate cancer vs hyperplasia: relationships with prostatic and adipose tissue fatty acid composition. Prostaglandins Leukot Essent Fatty Acids, 66: 467-477, 2002.

270. Narayan, P. and Dahiya, R. Alterations in sphingomyelin and fatty acids in human benign prostatic hyperplasia and prostatic cancer. Biomed Biochim Acta, 50: 1099-1108, 1991.

271. Nithipatikom, K., Isbell, M. A., See, W. A., and Campbell, W. B. Elevated 12- and 20-hydroxyeicosatetraenoic acid in urine of patients with prostatic diseases. Cancer Lett, 233: 219-225, 2006.

272. Kelavkar, U. P., Glasgow, W., Olson, S. J., Foster, B. A., and Shappell, S. B. Overexpression of 12/15-lipoxygenase, an ortholog of human 15-lipoxygenase-1, in the prostate tumors of TRAMP mice. Neoplasia, 6: 821-830, 2004.

273. Kelavkar, U. P., Parwani, A. V., Shappell, S. B., and Martin, W. D. Conditional expression of human 15-lipoxygenase-1 in mouse prostate induces prostatic intraepithelial neoplasia: the FLiMP mouse model. Neoplasia, 8: 510-522, 2006.

274. Pathology of the Mouse, ed. R.R. Maranpot, 1st edition: 236-251, 1999.

275. Cohen, S. M. Urinary bladder carcinogenesis. Toxicol Pathol, 26: 121-127, 1998.

276. Hosack, D. A., Dennis, G., Jr., Sherman, B. T., Lane, H. C., and Lempicki, R. A. Identifying biological themes within lists of genes with EASE. Genome Biol, 4: R70, 2003.

203 277. Zhu, Z., Kleeff, J., Friess, H., Wang, L., Zimmermann, A., Yarden, Y., Buchler, M. W., and Korc, M. Epiregulin is Up-regulated in pancreatic cancer and stimulates pancreatic cancer cell growth. Biochem Biophys Res Commun, 273: 1019-1024, 2000.

278. Sorensen, B. S., Torring, N., Bor, M. V., and Nexo, E. The DNA damaging agent VP16 induces the expression of a subset of ligands from the EGF system in bladder cancer cells, whereas none of the four EGF receptors are induced. Mol Cell Biochem, 260: 129-135, 2004.

279. Mohammed, S. I., Dhawan, D., Abraham, S., Snyder, P. W., Waters, D. J., Craig, B. A., Lu, M., Wu, L., Zheng, R., Stewart, J., and Knapp, D. W. Cyclooxygenase inhibitors in urinary bladder cancer: in vitro and in vivo effects. Mol Cancer Ther, 5: 329-336, 2006.

280. Harradine, K. A. and Akhurst, R. J. Mutations of TGFbeta signaling molecules in human disease. Ann Med, 38: 403-414, 2006.

281. McGarvey, T. W., Tait, E., Tomaszewski, J. E., and Malkowicz, S. B. Expression of Transforming Growth Factor-beta Receptors and Related Cell-Cycle Components in Transitional-Cell Carcinoma of the Bladder. Mol Urol, 3: 371-380, 1999.

282. Sharif-Afshar, A. R., Donohoe, J. M., Pope, J. C. t., Adams, M. C., Brock, J. W., 3rd, and Bhowmick, N. A. Stromal hyperplasia in male bladders upon loss of transforming growth factor-beta signaling in fibroblasts. J Urol, 174: 1704-1707; discussion 1707, 2005.

283. Wang, X. and Klein, R. D. Prostaglandin E(2) induces vascular endothelial growth factor secretion in prostate cancer cells through EP2 receptor-mediated cAMP pathway. Mol Carcinog, 2007.

284. Oxford, G. and Theodorescu, D. The role of Ras superfamily proteins in bladder cancer progression. J Urol, 170: 1987-1993, 2003.

285. Cheng, J., Huang, H., Zhang, Z. T., Shapiro, E., Pellicer, A., Sun, T. T., and Wu, X. R. Overexpression of epidermal growth factor receptor in urothelium elicits urothelial hyperplasia and promotes bladder tumor growth. Cancer Res, 62: 4157- 4163, 2002.

286. Dannenberg, A. J., Lippman, S. M., Mann, J. R., Subbaramaiah, K., and DuBois, R. N. Cyclooxygenase-2 and epidermal growth factor receptor: pharmacologic targets for chemoprevention. J Clin Oncol, 23: 254-266, 2005.

204

APPENDIX A

(figures)

205

PWE-1E RWPE-1 RWPE-2 22RV1 PC-3 DU145 LNCaP Kidney

Figure A.1 EP2, EP3 and EP4 are expressed in different pattern in examined human prostate cell lines (PWR-1E, RWPE-1, RWPE-2, 22RV1, PC-3, DU145, and LNCaP). Cell lysates from the seven human prostate cell lines were run on 4%-15% SDS-PAGE criterion gel (Bio-Rad) in duplicates. The expression of EP2, EP3 and EP4 in these cell lines were analyzed by immnunoblotting. Lysate from mouse kidney was used as positive control. EP1 was not detectable in any of the cell lines and mouse kidney.

206

22RV1 PC-3 DU145 LNCaP

EP4

Serum + - + - + - + -

B PC-3 medium only

EP4

1 2 3 4

Figure A.2 Non-specificity of antibody against EP4 receptor that from Cayman Chemical. A, EP4 is only detectable in cell lysates that were not serum-starved. B, EP4 binds to unknown antigen in serum-containing medium. 1, cell lysates from serum starved PC-3 cells. 2, cell lysates from PC-3 cells in medium with serum. 3, cells lysates from 2 that added with additional 10% serum-containing medium. 4, serum-containing medium only (no cell lysates).

207 A

10µM PGE2 (minutes) 0 15 30 60 120 240

p-Erk DU145

10µM CAY10399 (minutes) 0 15 30 60 120 240

p-Erk DU145

B PGE2 (µM) 0 0.001 0.01 0.1 1 10

DU145 p-Erk CAY10399 (µM) 0 0.001 0.01 0.1 1 10

p-Erk DU145

C 10µM PGE (minutes) 2 0 15 30 60 120 240 360 480 720

p-Akt DU145

10µM CAY10399 (minutes)

0 15 30 60 120 240 360 480 720

p-Akt DU145

Figure A.3 PGE2- and CAY10399-induced Erk and Akt activation in DU145 cells. A, PGE2 and CAY10399 induce phosphorylation of Erk in time-dependent manner in DU145 cells. Cells were treated with either 10 µM PGE2 or CAY10399 for 15 to 240 min. Total and phosphorylated Erk in cell lysates were analyzed by immunoblotting. B, The dose-response of PGE2 and CAY10399 induced phosphorylation of Erk in DU145 cells. Cells were treated for 15 min with concentrations of PGE2 and CAY10399 ranging from 1 nM to 10 µM. C, Extended time course of phosphorylation of Akt in DU145 cells. Cells were treated with either 10 µM PGE2 or CAY10399 for 15 to 720 min (12h). 208

40 b 35 a,b 30 a a a 25 a 20

10 VEGF (pg/ml of medium) of VEGF (pg/ml 5

0 Con 1nM 5µM I 50µM I 5µM I/P 50µM I/P

PGE2 Treatment

Figure A.4 Adenylate cyclase inhibitor 2’5’-dideoxyadenosine significantly blocks 1nM PGE2-induced secretion of VEGF in PC-3 cells. Cells were pretreated with either 5µM or 50 µM 2’5’-dideoxyadenosine for 1 h followed by 1nM PGE2 treatment for an additional 24 h in serum-free medium. I, 2’5’-dideoxyadenosine; P, PGE2. Data are means ± SD. Bars with different letters are significantly different from each other with a p-value <0.05.

209

4 protein) (ng/mg PGE2 Mean 2

DP LP VP Prostate Lobe

Figure A.5 PGE2 levels in three prostate lobes of 10 wk old wild-type mice. Tissues were analyzed for PGE2 by LC/MS/MS. Bars represent the mean obtained from at least five mice (error bars are 95% confidence intervals). DP (dorsal prostate), LP (lateral prostate), and VP (ventral prostate).

210

Figure A.6 COX-2 immunohistochemical staining of the same section in the dorsal prostate (DP), lateral prostate (LP), and ventral prostate (VP) tissues of a 10 wk old Pb.COX-2 mouse.

211

Wild-type 45 Pb.COX2 40

35 30

25 20

15 10

Mean PGE2 (ng/mg protein) (ng/mg PGE2 Mean 5 0 DP LP VP

Prostate Lobe

Figure A.7 PGE2 levels in three prostate lobes of 10 wk old Pb.COX-2 and wild-type mice. Tissues were analyzed for PGE2 by LC/MS/MS. Bars represent the mean obtained from at least five mice (error bars are 95% confidence intervals). DP (dorsal prostate), LP (lateral prostate), and VP (ventral prostate).

212

Figure A.8 H&E stained section of ventral prostate (VP) from one mouse of a founder line of Pb.COX-2 mouse model. Please note lymphocyte infiltration (white arrow) and the appearance of hyperplasia (yellow arrow).

213 A: anterior prostate

4.00 8.00 COX-1 COX-2 3.50 7.00

3.00 6.00 2.50 5.00

2.00 4.00

1.50 3.00 1.00 * 2.00 mRNA relative expression mRNA expression mRNA relative 0.50 1.00 * 0.00 0.00 WT Tg WT Tg

Genotype Genotype B: dorsal prostate

1.80 0.80 COX-1 COX-2 1.60 0.70 1.40 0.60

1.20 ession 0.50 1.00 0.40 0.80 0.30 0.60 0.20 0.40 * 0.10 mRNA relative expression relative mRNA 0.20 expr relative mRNA * 0.00 0.00 WT Tg WT Tg

Genotype Genotype

Figure A.9 Eexpression of COX-1 and COX-2 mRNA are significantly reduced in both anterior prostate (AP) and dorsal prostate (DP) tissues of the 10 wk old conditional PTEN knock mice (Tg) compared to wild-type (WT) mice. Each bar represents the mean COX- 1 or COX-2 mRNA (error bars are 95% confidence intervals) of five animals relative to a common calibrator sample generated by pooling the dorsal, ventral, and lateral prostates of a single WT mouse. 18s RNA levels were used to normalize the expression of individual samples. * P < 0.05. A, anterior prostate; B, dorsal prostate.

214

PWE-1E RWPE-1 RWPE-2 22RV1 PC-3 DU145 LNCaP (+)*

COX-1 70kDa

COX-2 72kDa

Figure A.10 Western blot analysis of COX-1 and COX-2 protein levels in seven prostate cell lines (PWR-1E, RWPE-1, RWPE-2, 22RV1, PC-3, DU145, and LNCaP). Mouse vas deferens tissue was used as positive control for COX (+).

215

14.00 WT * TRAMP 12.00

10.00

8.00

6.00

4.00

protein) (ng/mg Concentration 2.00 * * 0.00 PGA2 PGD2 15-d-PGJ2 12-HETE

Figure A.11 Prostaglandin A2 (PGA2), PGD2, 15-d-PGJ2 and 12-HETE levels in dorsal prostate (DP) tissues of 10 wk old wild-type (WT) and TRAMP mice. Tissues were analyzed for eicosanoids by LC/MS/MS. Bars represent the mean of values obtained from at least five mice (error bars are 95% confidence intervals). * p < 0.05.

216

Bovine Keratin 5 Β-globin 5’ - Murine COX-2 cDNA SV40-poly A -3’ Promoter intron

Figure A.12 Transgene construct of BK5.COX2 mouse model.

217