Suppression of Carcinogenesis and Tumor Progression by an Energy Restriction-Mimetic Agent in Murine Models of Prostate Cancer

SUPPRESSION OF CARCINOGENESIS AND TUMOR PROGRESSION BY AN ENERGY RESTRICTION-MIMETIC AGENT IN MURINE MODELS OF PROSTATE CANCER

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Lisa Danielle Berman-Booty, V.M.D

Graduate Program in Veterinary Biosciences

The Ohio State University

2013

Dissertation Committee:

Professor Ching-Shih Chen, Advisor

Professor Robert Brueggemeier

Professor Steven Clinton

Professor Thomas Rosol

Copyrighted by

Lisa Danielle Berman-Booty

2013

ABSTRACT

Cancer cells preferentially utilize glycolysis to generate energy even in the presence of sufficient oxygen for oxidative phosphorylation. This shift in energy metabolism, termed the Warburg effect, is responsible for cancer cells’ high metabolic rate. Therapies that inhibit cancer cell energy metabolism have proved effective in vitro, and dietary caloric restriction is a valuable experimental chemotherapeutic strategy. While animal studies utilizing caloric restriction typically restrict the experimental group’s caloric intake by

20-40%, this degree of caloric restriction is not realistic for human cancer patients.

Therefore, agents that can induce a response similar to that of glucose restriction in vitro and caloric restriction in vivo are needed. These agents are termed energy-restriction mimetic agents (ERMAs) and include 2-deoxyglucose, resveratrol, and the thiazolidinedione derivatives OSU-CG12 and OSU-CG5. Here, we characterized OSU-

CG5’s mechanism of action in vitro and its chemotherapeutic and chemopreventive activities in vivo. Specifically, we evaluated OSU-CG5’s activity in three different human prostate cancer cell lines that range from androgen-dependent (LNCaP) to castration- resistant (LNCaP-abl and PC3). OSU-CG5 was cytotoxic and induced a response similar to that of glucose deprivation in these cell lines. Additionally, treatment with OSU-CG5 resulted in decreased expression of genes that promote the Warburg effect, namely, those

ii for glucose transporter 1 (GLUT1) and a number of metabolic enzymes. Regarding its in vivo chemotherapeutic activity, 100 mg/kg/day of OSU-CG5 administered via daily oral gavage suppressed the growth of LNCaP-abl xenograft tumors by 81%. Tumor growth suppression was associated with decreased tumor cell proliferation, as determined by proliferating cell nuclear antigen (PCNA) levels, and modulation of intratumoral biomarkers associated with cell survival, growth, and metabolism, including insulin like growth factor 1 (IGF-1) and its receptor IGF-1R, Myc, the androgen receptor (AR), and cyclins D1 and E. OSU-CG5’s activity was also investigated using transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Treatment of 6-week-old

TRAMP mice with 100 mg/kg/day of OSU-CG5 via oral gavage for 4 weeks significantly reduced absolute and relative urogenital tract weights, as well as the weights of the individual lobes of the prostate. Reductions in lobe weight were associated with decreased cell proliferation within prostatic intraepithelial neoplasia (PIN) lesions, as determined by immunostaining for Ki67 and western blotting for PCNA. OSU-CG5 also decreased the levels of the AR, phosphorylated Akt (p-Akt), and IGF-1R within the prostates of these mice. When 6-week-old, intact, male TRAMP mice were fed an AIN-

76A diet containing 1286 ppm of OSU-CG5 for approximately 18 weeks, prostate tumor development was not suppressed or significantly delayed. However, the tumors that developed in OSU-CG5-treated mice were 54.6% smaller by volume and 54.1% smaller by mass than the control mouse tumors. Decreased tumor size was associated with decreased tumor cell proliferation, as determined by Ki67 immunostaining, and reductions in the AR and p-Akt. Collectively, these studies demonstrate OSU-CG5’s

iii efficacy as a chemotherapeutic agent. OSU-CG5 mediated this effect by reducing tumor cell proliferation and modulating pro-growth and pro-survival biomarkers.

DEDICATION

This document is dedicated to my husband Jordan and parents Marvin and Ronna

ACKNOWLEDGMENTS

I thank my advisor Dr. Ching-Shih Chen for his advice, mentorship and unwavering support. I thank the members of my advisory committee for their guidance. I thank Dr. Samuel Kulp for his advice and help with experimental design, animal studies, and manuscript preparation. I appreciate all the help and encouragement provided by the other members of the Chen lab. In particular, I acknowledge Dr. Po-Chen Chu for his technical expertise and assistance and Dr. Dasheng Wang for synthesizing OSU-CG5.

I thank Dr. Jennifer Thomas-Ahner of the Clinton lab for her assistance with mouse necropsies and urogenital tract microdissection. I thank Drs. Brad Bolon and

Krista La Perle of the Comparative Pathology and Mouse Phenotyping Shared Resource

(CPMPSR) for their guidance and mentorship in mouse and toxicologic pathology. I appreciate Alan Fletchner, Anne Saulsbery, Bill Kimble, and Florinda Jaynes of the

CPMPSR for their help with tissue and slide processing and immunohistochemistry. I thank Jody Sneddon of the CPMPSR for performing serum chemistries and complete blood counts.

I acknowledge the National Institute of Health for my research support and funding. From June 2009 through April 2012, my research was supported by the T-32

Institutional National Research Service Award (NRSA) in Mouse Models of Human

Disease (Ruth L. Kirschstein award). From April 2012 through the present time, my research has been supported by a K01 SERCA.

Lastly, I thank my husband and parents for their love and support throughout this entire process.

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VITA

March 7, 1982 ...... Born—Brookline, MA, USA

2004 ...... Bachelors of Arts, Biology School of Arts and Sciences University of Pennsylvania Philadelphia, PA

2008 ...... Veterinariae Medicinae Doctoris School of Veterinary Medicine University of Pennsylvania Philadelphia, PA

2008 to present ...... Residency in Veterinary Anatomic Pathology Department of Veterinary Biosciences The Ohio State University Columbus, OH

2008 to present ...... Postdoctoral Fellow Department of Veterinary Biosciences The Ohio State University Columbus, OH

2012...... Diplomate, American College of Veterinary Pathologists (anatomic pathology)

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PUBLICATIONS

1. Berman-Booty LD, Chu PC, Thomas-Ahner JM, Bolon B, Wang D, Yang T, Clinton SK, Kulp SK, Chen CS. Suppression of prostate epithelial proliferation and pro-growth signaling in transgenic mice by a new energy-restriction mimetic agent. Cancer Prev Res in press (PMID: 23275006)

2.Omar HA, Berman-Booty L, Weng JR. Energy restriction: stepping stones towards cancer therapy. Future Oncol 2012; 8 (12): 1503-6

3.Berman-Booty LD, Garzel LM, Bergdall V, La Perle KM. A prostate fibromyxoid sarcoma with smooth muscle differentiation in a F344xBNF1 rat. Vet Pathol 2012; 49(4): 642-647

4.Berman-Booty LD, Sargeant AM, Rosol TJ, Rengel RC, Clinton SK, Chen CS, Kulp SK. A review of the existing grading schemes and a proposal for a modified grading scheme for prostatic lesions in TRAMP mice. Toxicol Pathol 2012; 40(1):5-17

5.Omar HA, Chou CC, Berman-Booty LD, Ma Y, Hung JH, Wang D, Kogure T, Patel T, Terracciano L, Muthusamy N, Byrd JC, Kulp SK, Chen CS. Antitumor effects of OSU- 2S, a non-immunosuppressive analogue of FTY720, in hepatocellular carcinoma. Hepatology 2011; 53(6):1943-58

6.Omar HA, Berman-Booty LD, Kulp SK, Chen CS. Energy restriction as an antitumor target. Future Oncol 2010; 6(11): 1675-9

7.Berman-Booty LD, Cui J, Horvath SJ, Premanandan C. Pathology in Practice. Tularemia. J Am Vet Med Assoc 2010; 237(2): 163-5

8.Zaph C, Troy AE, Taylor BC, Berman-Booty LD, Guild KJ, Du Y, Yost EA, Gruber AD, May MJ, Greten FR, Eckmann L, Karin M, Artis D. Epithelial-cell-intrinsic IKK- beta expression regulates intestinal immune homeostasis. Nature 2007; 446(7135): 552-6

9.Blais MC, Berman L, Oakley D, and Giger U. The canine DAL blood type: a red cell antigen lacking in some Dalmatians. J Vet Intern Med 2007; 21(2): 281-6.

FIELDS OF STUDY

Major Field: Veterinary Biosciences

TABLE OF CONTENTS

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... viiii

List of Tables ...... xiv

List of Figures ...... xvi

Chapter 1: A review of the existing grading schemes and a proposal for a modified grading scheme for prostatic lesions in TRAMP mice ...... 1

Abstract ...... 1

Introduction ...... 2

Review of available grading schemes for TRAMP mice ...... 5

Rationale for a refined grading scheme...... 12

The refined grading scheme ...... 13

Utilization of the grading scheme ...... 18

Materials and Methods ...... 19

Results ...... 20

Discussion ...... 21

References ...... 27

Chapter 2: The Novel Energy Restriction-Mimetic Agent OSU-CG5 Down-Regulates

Expression of Genes That Promote the Warburg Effect and Suppresses the Growth of

Castration-Resistant Xenograft Tumors ...... 43

Abstract ...... 43

Introduction ...... 44

Materials and Methods ...... 48

Results ...... 54

Discussion ...... 61

References ...... 67

Chapter 3: Suppression of Prostate Epithelial Proliferation and Intraprostatic Pro-Growth

Signaling in Transgenic Mice by a New Energy Restriction-Mimetic Agent ...... 82

Abstract ...... 82

Introduction ...... 83

Materials and Methods ...... 85

Results ...... 92

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Discussion ...... 97

References ...... 103

Chapter 4: The Novel Energy-Restriction Mimetic Agent OSU-CG5 Reduces Prostate

Cancer Severity in a Transgenic Mouse Model of Prostate Cancer ...... 114

Abstract ...... 114

Introduction ...... 115

Materials and Methods ...... 118

Results ...... 125

Discussion ...... 132

References ...... 140

References ...... 156

Appendix A: Supplemental Data Tables from Chapter 3………………………………176

Appendix B: Supplemental Discussion of the Microarray Data from Chapter 3 ...... 191

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LIST OF TABLES

Table 1.1 Determination of the adjusted lesion score from the lesion grade and distribution ...... 39

Table 1.2 The incidences (%) of the prostate lesions identified as most severe in each lobe of the prostates of TRAMP mice...... 40

Table 1.3 The incidences (%) of the prostate lesions identified as most common in each lobe of the prostates of TRAMP mice...... 41

Table 1.4 Summary of published and proposed refined grading schemes for the prostate glands of TRAMP mice………………………………………………...... 42

Table 2.1 A subset of metabolic genes involved in the regulation of glycolysis, lactate synthesis, and fatty acid metabolism...... 79

Table 2.2 Serum biochemical and hematologic values of athymic nude mice treated with

100 mg/kg/day of OSU-CG5 for 59 days………………………………………………..81

Table 3.1 Evidence that OSU-CG5 caused no systemic toxicity in young adult mice after

4 weeks of treatment ...... 113

Table 4.1 1286 ppm of OSU-CG5 administered via an AIN-76A diet did not significantly reduce the average weights of the UGT or individual lobes of the prostate...... 150

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Table 4.2 The average SALS and scores for the most severe and most common lesions in individual lobes of prostates of TRAMP mice that received either a control AIN-76A diet or an experimental AIN-76A diet with 1286 ppm of OSU-CG5 for 18 weeks………...151

Table 4.3 The number and frequency of the most severe lesion in each lobe of the prostate of TRAMP mice that received either a control AIN-76A diet or an experimental diet containing 1286 ppm of OSU-CG5 for 18 weeks…………………………………152

Table 4.4 Absolute and relative organ weights of the TRAMP mice that received either the control AIN-76A diet or the AIN-76A with 1286 ppm of OSU-CG5 for 18 weeks……………………………………………………………………………………153

Table 4.5 Administration of OSU-CG5 in an experimental diet for 18 weeks did not result in any detectable abnormalities in serum chemistries or complete blood counts..154

Table A.1 SALS and average scores for the most severe and most common lesions in individual lobes of the prostates in vehicle-treated versus OSU-CG5 treated TRAMP mice……………………………………………………………………………………..176

Table A.2 Top 5 affected gene networks generated by IPA in OSU-CG5 treated TRAMP mice…...... 177

Table A.3 Select upregulated genes in the dorsal and lateral prostates of TRAMP mice treated with OSU-CG5 (N=3)………………………………………………………...... 178

Table A.4 Select downregulated genes in the dorsal and lateral prostates of TRAMP mice treated with OSU-CG5 (N=3)………………………………………………………...... 181

Table A.5 Genes shown to be modulated by caloric restriction that are up- or down- regulated in prostates of TRAMP mice treated with OSU-CG5 (N=3)………………..184

LIST OF FIGURES

Figure 1.1 Representative images of low grade PIN (Lesion grade 1) in TRAMP prostate

...... 31

Figure 1.2 Representative images of moderate grade PIN (Lesion grade 2) in TRAMP prostate...... 32

Figure 1.3 Representative images of high grade PIN (Lesion grade 3) in TRAMP prostate...... 33

Figure 1.4 Representative images of phyllodes-like tumor (Lesion grade 4) in TRAMP prostate...... 35

Figure 1.5 Representative images of well differentiated adenocarcinoma (Lesion grade 5) in TRAMP prostate...... 36

Figure 1.6. Representative images of moderately differentiated adenocarcinoma (Lesion grade 6) and poorly differentiated carcinoma (Lesion grade 7) in the lateral lobe of

TRAMP prostate ...... 38

Figure 2.1 Evidence that OSU-CG5 reduced prostate cancer cell viability and modulated biomarker expression ...... 70

Figure 2.2 Treatment of LNCaP, LNCaP-abl, and PC3 cells with 5 μM of OSU-CG5 downregulated the expression of many genes that promote the Warburg effect...... 72

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Figure 2.3 Treatment of athymic nude mice baring LNCaP-abl tumors with 100 mg/kg/day of OSU-CG5 resulted in statistically significant tumor growth suppression.. 74

Figure 2.4 Treatment of LNCaP-abl xenograft tumors with 100 mg/kg/day of OSU-CG5 resulted in reductions in intratumoral biomarker expression ...... 76

Figure 2.5 OSU-CG5 treatment of LNCaP-abl xenograft tumors resulted in statistically significant downregulation of a number of genes involves in glycolysis and fatty acid metabolism ...... 78

Figure 3.1. Evidence that OSU-CG5 reduces energy production...... 106

Figure 3.2 OSU-CG5 reduced urogenital tract and prostate lobe weights in TRAMP mice

...... 107

Figure 3.3 OSU-CG5 reduced prostate epithelial proliferation in TRAMP mice...... 108

Figure 3.4 Gene network in the combined dorsal and lateral lobes of prostate of TRAMP mice treated for 4-weeks with 100mg/kg/day of OSU-CG5 created using Ingenuity pathway analysis software...... 110

Figure 3.5 Western blot analysis of the effects of OSU-CG5 on the combined dorsal and lateral lobes of the prostate of 10 week old TRAMP mice ...... 112

Figure 4.1 OSU-CG5 reduced the viability of TRAMP-C2 cells after either 48 or 72 hours of treatment...... 143

Figure 4.2 1286 ppm OSU-CG5 did not significantly reduce the absolute or relative urogenital tract weights or alter TRAMP mouse survival ...... 144

Figure 4.3 1286 ppm of OSU-CG5 in an AIN-76A diet resulted in statistically significant reductions in prostate tumor volume and prostate tumor weight………………………145

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Figure 4.4 1286 ppm of OSU-CG5 in an AIN-76A diet reduced tumor cell proliferation in prostate tumors from TRAMP mice but did not significantly increase the apoptotic index…………………………………………………………………………………….146

Figure 4.5 Western blot analysis of the effects of dietary administration of OSU-CG5 on

Akt-Ser-473 and GSK3β phosphorylation and AR and IGF-1R expression in the prostate tumors of TRAMP mice………………………………………………………………...148

Figure 4.6 Dietary intake of OSU-CG5 and mouse body weights over the course of the study……………………………………………………………………………………149

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CHAPTER 1: A REVIEW OF THE EXISTING GRADING SCHEMES AND A

PROPOSAL FOR A MODIFIED GRADING SCHEME FOR PROSTATIC LESIONS

IN TRAMP MICE

ABSTRACT

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model is well established and offers several advantages for the study of chemopreventive agents, including its well defined course of disease progression and high incidence of poorly differentiated carcinomas within a relatively short length of time. However, there is no consensus on the grading of prostatic lesions in these mice. In particular, agreement is lacking on the criteria for differentiating prostatic intraepithelial neoplasia (PIN) from well differentiated adenocarcinoma, specifically as it relates to evidence of invasion. This differentiation is critical for evaluating the effects of putative chemopreventive agents on progression to neoplasia. Moreover, only one of the published grading schemes assigns numerical grades to prostatic lesions which facilitate statistical analysis. Here, we review five currently available grading schemes and propose a refined scheme that provides a useful definition of invasion for the differentiation of PIN from well differentiated adenocarcinoma, and includes a numerical scoring system that accounts for both the most severe and most common histopathological lesions in each of the lobes of the prostate

and their distributions. We expect that researchers will find this refined grading scheme to be useful for chemoprevention studies in TRAMP mice.

INTRODUCTION

In the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, the

SV40 large and small T antigens are expressed under the androgen-dependent control of the rat probasin promoter in the prostatic epithelium. Expression of the transgene results in inhibition of p53 and Rb tumor suppressor activities (Gingrich et al., 1999, Gingrich et al., 1996, Greenberg et al., 1994, Greenberg et al., 1995, Greenberg, 1996). Two lines of

TRAMP mice exist, the C57BL/6 parent line and the first generation offspring of the parent line crossed to FvB mice (C57BL/6 TRAMP x FvB). In the literature, both lines are referred to as TRAMP mice. The two lines exhibit similar progression of prostate lesions, with the lesions of C57BL/6 TRAMP x FvB mice progressing at a slightly more rapid rate (Gingrich et al., 1999).

Male TRAMP mice reproducibly develop poorly differentiated prostate carcinomas. The prostates of these mice progress through different preneoplastic and neoplastic lesions similar to what occurs in man (Kaplan-Lefko et al., 2003, Gingrich et al., 1999, Greenberg et al., 1995, Gingrich et al., 1996). Between six and twelve-weeks of age, the prostatic epithelial cells develop varying degrees of hyperplasia or prostatic intraepithelial neoplasia (PIN). By approximately 18-weeks of age, mice develop well differentiated prostatic adenocarcinomas. Between the ages of 24 to 30 weeks, almost

one-hundred percent of the TRAMP mice will have poorly differentiated carcinomas with metastasis to the iliac lymph nodes and lungs most commonly (Kaplan-Lefko et al., 2003,

Gingrich et al., 1996, Gingrich et al., 1999, Hurwitz et al., 2001, Greenberg, 1996).

Because of tumor size and metastatic burden, C57BL/6 TRAMP x FvB mice rarely live beyond 33 weeks of age, while C57BL/6 TRAMP mice are frequently able to live for up to 36-40 weeks, with occasional mice surviving up to 52 weeks of age (Gingrich et al.,

1999).

In addition to progressing through the various preneoplastic and neoplastic stages similar to those observed in men, tumorigenesis in the TRAMP model has other similarities to human prostate carcinogenesis. As in men, prostate cancer of TRAMP mice progresses from an initial androgen-dependent stage to a castration-resistant stage.

Castration at 12 weeks of age results in approximately 80% of C57BL/6 TRAMP x FvB mice developing hormone-insensitive, poorly differentiated carcinomas by 24 weeks of age, which metastasize at a higher frequency than those in intact mice (Gingrich et al.,

1997, Kaplan-Lefko et al., 2003). Androgen receptor immunostaining is variable, and may even be absent, in moderately differentiated or poorly differentiated carcinomas

(Kaplan-Lefko et al., 2003). Similar to immunohistochemical changes observed in human prostatic carcinomas, decreased cytokeratin 8 and E-cadherin immunostaining are observed in poorly differentiated carcinomas of TRAMP mice, compared to PIN and well differentiated adenocarcinomas. The changes in tumor cell immunoreactivity suggest that an epithelial-to-mesenchymal transition is occurring with loss of tumor differentiation

(Kaplan-Lefko et al., 2003).

Despite the similarities in the progression of prostate lesions in men and TRAMP mice, controversy exists regarding the applicability of the TRAMP model for the study of human prostate tumorigenesis. The main point of contention is the existence of the neuroendocrine phenotype. Poorly differentiated carcinomas in the TRAMP model frequently exhibit a neuroendocrine phenotype with positive immunostaining for neuroendocrine markers, such as synaptophysin (Chiaverotti et al., 2008, Kaplan-Lefko et al., 2003). There are two opinions regarding the origin of these neuroendocrine cells.

Since preneoplastic PIN lesions and well differentiated adenocarcinomas rarely express neuroendocrine markers, it is speculated that the poorly differentiated carcinomas arise from cells undergoing epithelial-to-neuroendocrine transition with loss of differentiation

(Kaplan-Lefko et al., 2003), analogous to what occurs in men (Bonkhoff, 2001). The second opinion is that the poorly differentiated carcinomas arise from a neuroendocrine stem cell with no relation to the other lesions occurring in the prostates (Chiaverotti et al.,

2008). In part because we have observed prostates in which poorly differentiated carcinomas coexist with preexisting PIN or well differentiated adenocarcinoma, we support the first hypothesis, namely, that tumor cells that express neuroendocrine markers have undergone epithelial-to-neuroendocrine transition. In addition, since approximately

10% of human prostate carcinomas have a neuroendocrine phenotype in association with increased aggressive behavior (Bonkhoff, 2001), we do not believe that the neuroendocrine phenotype in poorly differentiated carcinomas of TRAMP mice obviates its use as a model for prostate tumorigenesis in man. Rather, we believe that the TRAMP

model with its progression from preneoplastic lesions to highly aggressive neoplasia has an advantage over other transgenic mouse models of prostate cancer.

The TRAMP model has proven useful to investigate the ability of drugs to prevent preneoplastic lesions (PIN) from progressing to aggressive neoplasia.

Interventions that have successfully slowed the progression of lesions in TRAMP mice and reduced the incidence of poorly differentiated carcinomas include the histone deacetylase inhibitor OSU-HDAC42 (a.k.a. AR42, Arno Therapeutics, Parsippany,

NJ)(Sargeant et al., 2008), the non-steroidal anti-inflammatory drug E-7869 (R- flurbiprofen)(Wechter et al., 2000), and 20% dietary restriction (Suttie et al., 2003). An efficient and inclusive grading scheme for the evaluation of prostate lesions in TRAMP mice is an important component of such chemoprevention studies. Unlike human prostate pathology, in which low-grade and high-grade PIN are used to describe the preneoplastic lesions and the Gleason system is used to grade prostate cancer (Kumar et al., 2005), multiple grading schemes for the lesions in the prostates of TRAMP mice exist (Gingrich et al., 1999, Kaplan-Lefko et al., 2003, Shappell et al., 2004, Suttie et al., 2003,

Chiaverotti et al., 2008, Hurwitz et al., 2001).

REVIEW OF AVAILABLE GRADING SCHEMES FOR TRAMP MICE

The first comprehensive grading scheme for the C57BL/6 TRAMP and C57BL/6

TRAMP x FvB mice was described by Gingrich, et al in 1999 (Gingrich et al., 1999). In this scheme, lesions were described as low grade PIN, high grade PIN, well differentiated adenocarcinoma, moderately differentiated adenocarcinoma, or poorly differentiated

adenocarcinoma. Low grade PIN was defined based on the appearance of elongated, instead of round, nuclei with condensed chromatin. The designation of high grade PIN was made based on nuclear morphology and cell changes, including variation in nuclear shape, condensed chromatin, and mitotic figures. Instead of being oriented in a single layer, prostate epithelial cells stratify and form cribriform and papillary structures. Well differentiated adenocarcinoma was diagnosed when epithelial cells invaded into the stroma surrounding glands, or, more frequently, when epithelial cells with rounded nuclei were present within the hypertrophied and hyperplastic fibromuscular stroma surrounding the glands. The rounded nuclei were reported to be frequently immunohistochemically positive for the T antigen, suggesting that these rounded nuclei belong to invading transformed epithelial cells. The diagnosis of moderately differentiated adenocarcinoma was made based on the neoplasm containing irregularly shaped glands, while poorly differentiated adenocarcinoma was diagnosed if no glandular elements remained and the tumor was composed of solid sheets of cells (Gingrich et al., 1999).

Since the introduction of the grading scheme by Gingrich, et al (Gingrich et al.,

1999), other investigators have refined the classification of lesions of TRAMP mice and proposed modified grading schemes. Four of these modified schemes are reviewed here.

The grading scheme proposed by Kaplan-Lefko, et al in 2003 (Kaplan-Lefko et al., 2003) for C57BL/6 TRAMP x FvB mice classified the prostatic lesions in TRAMP mice as

PIN, well differentiated carcinoma, moderately differentiated carcinoma, poorly differentiated carcinoma, or phyllodes-like lesions. The single grade of PIN described by the authors was characterized by cells exhibiting stratification and papillary projections,

cribriform patterns, and tufts. Nuclei were reported to be elongated with hyperchromatic chromatin, and cells had increased mitotic and apoptotic indices. As PIN progressed to well differentiated carcinoma, invasion was frequently noted. In the well differentiated carcinomas, there were increased small glandular structures with desmoplasia. The cells had round nuclei, with less hyperchromasia compared to nuclei of PIN-containing glands, and mitotic and apoptotic indices were increased. Moderately differentiated carcinoma was distinguished from well differentiated carcinoma by the replacement of the neoplastic glands by solid sheets of cells, with remnants of glandular structures. In the poorly differentiated carcinomas, the tumors were composed of solid sheets of cells with high nuclear to cytoplasmic ratios, anisokaryosis, and marked pleomorphism. Phyllodes- like lesions were composed of hypercellular stroma that was tightly packed or loose and edematous, and exhibited epithelial changes compatible to those of PIN or carcinoma.

The phylloides-like lesions occurred at a higher frequency in C57BL/6 TRAMP mice than in C57BL/6 TRAMP x FvB mice (Kaplan-Lefko et al., 2003).

In 2003, Suttie, et al (Suttie et al., 2003) proposed a grading scheme for the prostates of C57BL/6 TRAMP mice that recommended evaluating each lobe of the prostate and assigning a grade from one to six, based on the most severe lesion within the lobe. Grades one through three represented varying degrees of hyperplasia, grades four and five denoted adenomas, and grade six indicated adenocarcinoma. The distribution of the lesion within each individual lobe was estimated and described as either focal (fewer than two lesions), multifocal (3 or more lesions with less than 30% of the lobe involved), or diffuse (greater than 30% of the lobe involved). The distribution and lesion grade were

then combined to calculate a “distribution-adjusted lesion grade” ranging from 0-18 that could be used for statistical analysis. Prostate glands with grade 1 hyperplasia were defined as those lined by hyperbasophilic epithelial cells with frequent crowding, but no stratification. Glands with grade 2 hyperplasia were differentiated from grade 1 by occasional stratification of epithelial cells and the formation of papilla or cribriform structures. In grade 3 hyperplasia, papilla and cribriform structures that extended into the lumen of the gland were observed. Grade 3 hyperplasia may have concurrent hyperplasia of the smooth muscle surrounding the dorsal and anterior lobes of the prostate. Grade 4 adenomas were characterized by glands filled with proliferating epithelium. Hyperplasia of the smooth muscle surrounding the anterior and dorsal lobes may be more severe in grade 4 adenomas compared to grade 3 hyperplasia. The grade 5 adenoma was distinguished from the grade 4 adenoma based on enlargement of the gland. Finally, the grade 6 adenocarcinoma exhibited poorly differentiated epithelial cells with local invasion through the capsule and/or distant metastasis. Invasion was defined as islands of less well differentiated epithelial cells within the smooth muscle stroma that were not associated with the epithelium lining the gland. The authors emphasized the need to differentiate invasive epithelial cells from herniation of dysplastic epithelial cells associated with the grade 4 and 5 adenomas (Suttie et al., 2003).

In 2004, the Bar Harbor Meeting of the Mouse Models of Human Cancer

Consortium Prostate Pathology Committee released a consensus report that reviewed and classified the pathologic changes in different transgenic mouse models of prostate cancer, including TRAMP mice (Shappell et al., 2004). The pathologic changes that were

described in the prostates of C57BL/6 TRAMP and C57BL/6 TRAMP x FvB mice included epithelial hyperplasia, combined epithelial and stromal hyperplasia, mouse PIN

(mPIN), microinvasive carcinoma, and invasive carcinoma. Invasive carcinoma included well, moderately, and poorly differentiated adenocarcinomas, as well as neuroendocrine carcinomas. It was noted that the combined epithelial and stromal hyperplasia described in this report may resemble phyllodes tumor of the human breast when the stroma was exuberant and edematous. Hyperplasia was classified as either focal or diffuse based on whether 50% or more of the glands was involved. In both epithelial hyperplasia and mPIN, the cells may exhibit nuclear atypia (such as hyperchromasia, anisokaryosis, and increased mitotic index) and often stratify and form tufts, papillary or polypoid projections, and/or cribriform patterns. mPIN was differentiated from hyperplasia based on the opinion that mPIN begins focally and progresses with regards to the proportion of each gland involved, number of glands affected, and the severity of the observed atypia, whereas hyperplasia is non-progressive and may involve the whole gland simultaneously.

Moreover, mPIN was not classified as either low or high grade, because the histologic features of mPIN that are associated with progression to invasion had not been defined.

Microinvasive carcinoma was indicated by the penetration of the basement membrane by carcinoma cells in a gland with mPIN. Invasive carcinoma was characterized by desmoplasia associated with the epithelial cells that invaded into the smooth muscle surrounding the glands, “adequate” distance separating the epithelial cells within the smooth muscle from those lining the gland, or evidence that the basement membrane was breached using immunohistochemistry, special stains, or electron microscopy. Invasive

was distinguished from microinvasive carcinoma based on the degree of invasion into the surrounding stroma or tissues, as well as evidence of metastasis. Moreover, the importance of differentiating invasive epithelial cells from herniated epithelial cells was emphasized. Invasive carcinomas were categorized as well, moderately and poorly differentiated adenocarcinomas and neuroendocrine carcinomas. The degree of differentiation was distinguished by the extent of gland formation by the neoplastic cells, with lesser differentiation associated with the replacement of neoplastic glands by solid sheets of cells. Neuroendocrine carcinomas were defined as invasive carcinomas that exhibited histologic, immunohistochemical, and ultrastructural features of neuroendocrine differentiation. Specifically, these lesions would be composed of solid sheets of cells with rosettes or cribriform patterns and the absence of glands. The cells should stain for at least one immunohistochemical marker of neuroendocrine differentiation, such as synaptophysin or chromogranin A. Dense, neuroendocrine type, secretory granules should be visible by electron microscopy (Shappell et al., 2004).

The lesions described in the grading scheme presented by Chiaverotti, et al in

2008 (Chiaverotti et al., 2008) included mild, moderate, and severe atypical hyperplasia of Tag (with Tag denoting that the mice express the SV40 T antigen-containing transgene), papillary adenoma, adenocarcinoma (well, moderately, and poorly differentiated), and neuroendocrine carcinoma in C57BL/6 TRAMP and C57BL/6

TRAMP x FvB mice. Atypical hyperplasia of Tag was the term used instead of PIN because these authors concluded that the proliferative lesions in the TRAMP mice did not conform to their definition of PIN. These authors differentiate PIN, a lesion where only

the epithelium is affected, from atypical hyperplasia of Tag, in which both the epithelium and stroma are involved. Low grade PIN was described as epithelium forming “plaque- like” lesions one to two cells thick and high grade PIN as epithelium forming cribriform structures, as well as plaques that may be multiple cell layers thick. In addition, it was the opinion of the authors that the proliferative lesions in TRAMP mice did not progress to invasion, so the lesions (that others have defined as PIN) were named atypical hyperplasia of Tag. Mild atypical hyperplasia of Tag was described as epithelial crowding with cells having increased nuclear to cytoplasmic ratios and loss of cell polarity. The focal areas of cell crowding progressed to affect the whole gland diffusely with concurrent smooth muscle hyperplasia. In moderate atypical hyperplasia of Tag, the epithelial proliferation and dysplasia were greater with herniation, but not invasion, of hyperplastic cells into the smooth muscle surrounding the glands. Herniation of dysplastic epithelial cells was distinguished from invasion by the absence of a stromal reaction (replacement of the stromal smooth muscle component by reactive fibroblasts and myofibroblasts). In severe atypical hyperplasia of Tag, the proliferative epithelium formed cribriform structures. Papillary adenomas were defined as benign proliferations of epithelium and associated stroma that protruded as polypoid masses into the lumen and were similar to phyllodes-like lesions described by others. Neuroendocrine carcinomas were composed of solid sheets of highly anaplastic cells that were synaptophysin-positive and occasionally weakly positive for the androgen receptor. The authors defined well differentiated adenocarcinomas as neoplasms with apparent epithelial origins and distinct glandular morphology, and those less well differentiated as having a more solid

morphology, but, importantly, as being negative for synaptophysin. Moreover, invasion

(as determined by a stromal reaction) or metastasis were considered features of adenocarcinomas. The authors reported that no adenocarcinomas occurred in the TRAMP mice, and they speculated that the observed neuroendocrine carcinomas arose from distinct neuroendocrine stem cells within the prostate, and not from progression of atypical hyperplasia of Tag (Chiaverotti et al., 2008).

RATIONALE FOR A REFINED GRADING SCHEME

It may be argued that the differences among grading schemes are not important for determining whether a drug succeeds in preventing neoplasia in TRAMP mice.

However, we suggest that there are significant differences among the available schemes that necessitate the development of a concise and inclusive grading scheme. Moreover, we believe that refinements can be made in distinguishing lesions that can enhance assessments of chemopreventive interventions in this model. Specifically, our rationales for creating a refined grading scheme for prostatic lesions in TRAMP mice are three-fold.

First, we sought to establish criteria for distinguishing among low, moderate, and high grade PIN. Such distinctions would make it possible to determine if a drug alters the severity of PIN, which may signify that it slows the progression of lesions in the TRAMP model, an important endpoint in a chemoprevention study. Second, we wanted to define invasion to permit differentiation of herniated dysplastic epithelial cells (high grade PIN) from epithelial cells that have invaded into the underlying stroma (adenocarcinoma).

Differentiating high grade PIN, a preneoplastic lesion, from well differentiated

adenocarcinoma, a neoplastic lesion, is critical for determining if an intervention has successfully prevented progression to neoplasia in the TRAMP model. Criteria previously described for differentiating invasive cells from herniated cells include the presence of epithelial cells within the smooth muscle surrounding the gland (Kaplan-

Lefko et al., 2003), a stromal reaction (Chiaverotti et al., 2008, Shappell et al., 2004), and the distance between cells outside of the gland and those within the acinus (Suttie et al.,

2003, Shappell et al., 2004). We propose that classifying invasion based on the distance between the free epithelial cells within the smooth muscle surrounding the acinus and the epithelial cells lining the acinus is inconsistent, since this distance will vary from section to section of gland examined. Third, we sought to incorporate a numerical grading system that allows simultaneous assessment of lesion severity and extent of the lobe involvement. An ideal grading scheme should account for not only lesion type, but also its distribution, since the extent of lobe involvement is a measure of lesion progression. A lesion that involves more of a gland is assumed to have progressed further and, therefore, is interpreted as more severe. Combining the type of lesion with an indication of its distribution to generate a numerical value will facilitate the assessment of lesion severity and permit statistical evaluation. Such an approach has been proposed by Suttie, et al

(Suttie et al., 2003).

THE REFINED GRADING SCHEME

Each of the four lobes (dorsal, ventral, lateral, anterior) of the prostates of

C57BL/6 TRAMP x FvB mice is assessed individually and assigned two grades each

ranging from 0 to 7. The first grade represents the most severe lesion within that lobe with normal prostate as the least severe (grade 0) and poorly differentiated carcinoma as the most severe lesion (grade 7). The second grade, using the same scale, identifies the most common lesion in the lobe, which is defined as the lesion with an extent of lobe involvement that is greater than or equal to that of the most severe lesion. By assessing both the most severe lesion and most common lesion, we believe that a more complete picture of disease status can be obtained. Additionally, by accounting for both of these lesions, the refined grading scheme begins to approximate the Gleason system used to grade prostate cancer in men (Kumar et al., 2005).

The lesion grades for the refined scheme are as follows. Grade 0 is normal prostate. Grades 1, 2, and 3 represent low, moderate, and high grade PIN, respectively.

Although different terms have been used to describe the preneoplastic lesions in the prostates of TRAMP mice, such as hyperplasia (Suttie et al., 2003), atypical hyperplasia of Tag mice (Chiaverotti et al., 2008), and PIN (Kaplan-Lefko et al., 2003), we prefer the term PIN because it implies progression to adenocarcinoma. Grade 4 includes phyllodes- like lesions. Grades 5 and 6 represent well and moderately differentiated adenocarcinomas, respectively. Grade 7 is poorly differentiated carcinoma which may have neuroendocrine features.

After the most severe and most common lesions within a lobe are identified, the distributions of each of these lesions within the individual lobe are determined and described as focal, multifocal, or diffuse. Focal indicates there are fewer than three foci within the lobe. Lesions are described as multifocal if there are three or more foci within

the lobe, with less than fifty percent of the lobe containing the lesion of interest. Finally, lesions are diffuse if greater than fifty percent of the lobe is affected.

Adjusting the lesion grades to include an indication of distribution provides two adjusted scores, one for the most severe lesion and the other for the most common, ranging from 0 (normal) to 21 (diffuse, poorly differentiated carcinoma) (Table 1). These adjusted scores are then added to obtain a sum that reflects the most severe lesion and its distribution and the most common lesion and its distribution (sum of the adjusted lesion scores). If the most severe lesion is also the most common lesion, then the adjusted score for the most severe lesion is simply doubled to obtain the sum of the adjusted lesion scores. If the majority of the prostate is normal, then the sum is simply the adjusted lesion score for most severe lesion plus zero. This refined grading scheme incorporates both the most severe and most common lesions, both of which contribute to the pathology present within a lobe. A detailed description of the seven grades follows.

Grade 0: Normal: Prostate glands are lined by a monolayer of cuboidal to columnar epithelial cells with basally oriented nuclei. There may be mild to moderate in-folding of epithelial cells in the dorsal and anterior prostate lobes, and rare in-folding in the ventral and lateral prostate lobes.

Grade 1: Low grade PIN: There is crowding and occasional stratification of prostate epithelial cells with an increased nuclear to cytoplasmic ratio (Figure 1A). There may be rare short papillary projections of hyperplastic epithelium into the glandular lumen in the lateral and ventral lobes (Figure 1B). Cytoplasmic and nuclear atypia are minimal.

Grade 2: Moderate grade PIN: Similar to low grade PIN, but there is more frequent prostate epithelial cell stratification. Hyperplastic cells may be arranged as short to tall papillary projections protruding into glandular lumen (Figures 2A and 2B); rarely, the hyperplastic epithelial cells form a cribriform pattern. There may be mild hyperplasia of smooth muscle surrounding the glands. Occasional herniation of hyperplastic epithelial cells into the underlying smooth muscle may be observed.

Grade 3: High grade PIN: Like low and moderate grade PIN, there is cell crowding, cell stratification, and an increased nuclear to cytoplasmic ratio. Hyperplastic cells project into the lumen as papillary projections or form a cribriform pattern (Figure 3A). Mitotic figures may be frequent. There is frequent herniation of epithelial cells into hyperplastic smooth muscle surrounding the glands (Figure 3B and C). Herniated dysplastic glands have no stromal reaction. Proliferating epithelium may fill and/or expand the lumen of the gland. The hyperplastic epithelium filling the lumen may consist of small hyperplastic acini without associated stroma (Figure 3D). Alternatively, the epithelium expanding the lumen may form a discrete mass that is continuous with the proliferative epithelium lining the lumen of the gland (Figure 3E and 3F). The proliferating mass of epithelium may contain associated tightly packed stroma. In this refined grading scheme such lesions are defined as high grade PIN, and not adenomas. There may be mild to moderate hyperplasia of the smooth muscle surrounding the individual glands with high grade PIN.

In the ventral and lateral lobes, there may be minimal to mild proliferations of fibroblasts intervening between and surrounding the glands (Figure 3D). There is no evidence of invasion or metastasis.

Grade 4: Phyllodes-like tumor: These lesions are most commonly observed in the dorsal or anterior lobes. They consist of papillary projections of loose stroma with loosely arranged stellate mesenchymal cells (Figure 4A). The epithelium overlying the stroma of the phyllodes-like tumor is cuboidal to columnar, with minimal to no nuclear or cytoplasmic atypia and rare mitotic figures (Figure 4B). The main component of a phyllodes-like tumor is stroma. Previous descriptions of these tumors by others suggest that these tumors are not malignant (Tani et al., 2005) and likely represent a variant of an adenoma.

Grade 5: Well differentiated adenocarcinoma: The lumen of the affected gland contains a mass of epithelial cells forming well differentiated acini or tubule-like structures. The epithelial cells frequently have nuclear and cytoplasmic atypia and a high mitotic index.

The acini may be separated by a mild amount of tightly packed stroma. Well differentiated adenocarcinomas are differentiated from high grade PIN by the presence of invasion, which is evident as invasion of epithelial cells into the underlying smooth muscle, with cells crossing the basement membrane and replacement of the normal smooth muscle surrounding the gland by reactive fibroblasts and myoepithelial cells in the area of invasion (Figures 5A, B, and C). This stromal reaction may be exuberant and encompass adjacent glands or may be more focused around the lone invading epithelial cells. It must be noted that there may be a minimal to mild amount of fibrous connective tissue with fibroblasts and myoepithelial cells in the interglandular regions of the ventral and lateral lobes with high grade PIN. However, if cells do not penetrate through the basement membrane in these areas, this is considered high grade PIN and not invasion.

Grade 6: Moderately differentiated adenocarcinoma: The lumen of the affected gland contains a mass of epithelial cells, some of which form acini or tubules (Figure 6A). The epithelial cells frequently have nuclear and cytoplasmic atypia and a high mitotic index.

The epithelial cells may be separated by a mild amount of tightly packed stroma. There is invasion of epithelial cells into the underlying smooth muscle, with cells crossing the basement membrane and the replacement of the normal smooth muscle by reactive fibroblasts.

Grade 7: Poorly differentiated carcinoma (neuroendocrine-type): The tumors consist of solid sheets of polygonal to elongated cells with high mitotic indices and frequent nuclear and cytoplasmic atypia (Figure 6B). Although the neoplastic cells do not form tubules or acini, they may entrap normal appearing glands.

UTILIZATION OF THE GRADING SCHEME

To demonstrate the utility of the refined grading scheme, we report the average sum of adjusted lesion scores for the lobes of the prostates of 42 intact C57BL/6 TRAMP x FvB mice between the ages of 18 and 24 weeks. Previous use of TRAMP mice in our laboratory have shown the advantage of using a 24-week endpoint for a chemoprevention study (Sargeant et al., 2008). Before the age of 18 weeks, few TRAMP mice will have developed carcinomas (Gingrich et al., 1999, Gingrich et al., 1996), thus a study having an end point prior to this age will be limited in its ability to determine whether an agent can prevent prostate tumorigenesis. In fact, our laboratory has shown that only 40% of intact C57BL/6 TRAMP x FvB mice develop malignant tumors by 18 weeks of age

(Sargeant et al., 2007). Conducting a chemopreventive study past 24-weeks of age is not recommended, since after that age, most control mice will have developed poorly differentiated prostate carcinomas (Gingrich, 1996 et al., Gingrich et al., 1999), necessitating humane euthanasia and thereby limiting the number of mice available for study.

MATERIALS AND METHODS

TRAMP mice (C57BL/6 TRAMP x FvB) were bred and housed as previously reported (Sargeant et al., 2007) and the presence of the transgene was confirmed by PCR.

Mice received a standard rodent diet and water ad libitum. Mice were euthanized between

18 and 24 weeks of age by carbon dioxide asphyxiation. All procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use

Committee of The Ohio State University. At necropsy, the four lobes of each prostate were collected, microdissected, isolated, and placed in 10% formalin and processed as previously described (Sargeant et al., 2007). Hematoxylin and eosin-stained sections of prostates were examined using light microscopy. Lesions were described and the sum of the adjusted lesion scores of each lobe were calculated for each mouse using the grading scheme described above. The lesion grades are presented as the mean (± standard deviation [SD]) of the sums of the adjusted lesion scores of each lobe for all 42 mice.

RESULTS

The individual lobes of the prostates from 42 intact male TRAMP mice were examined histologically, the most severe and most common lesions in each lobe were determined, graded, and adjusted lesion scores were calculated. The results are summarized in Tables 2 and 3 which show the lesion types identified as most severe and most common and their incidences in each lobe of the prostate. As shown in Table 2, poorly differentiated carcinoma was the most severe lesion observed in all four prostatic lobes. In the ventral, lateral and dorsal lobes, poorly differentiated carcinoma was the most severe lesion in the majority of mice with incidences of 66.7%, 64.3%, and 52.4%, respectively. In contrast, in the anterior lobe, high grade PIN was the lesion most frequently observed to be the most severe (52.4% of mice) with poorly differentiated carcinoma being the second most frequent lesion (38.1%). A similar pattern was observed for the most common lesions (Table 3). In the ventral, lateral and dorsal lobes, poorly differentiated carcinoma was the most common lesion with incidences of 64.3%,

64.3% and 52.4%, respectively. In the anterior prostate, however, normal prostatic morphology was observed to be the most common histologic feature in the majority of mice (59.5%).

This pattern of lesion severity and distribution among the prostatic lobes of

TRAMP mice was reflected in the average sums of the adjusted lesion scores. For the ventral, lateral and dorsal lobes, the average sums of the adjusted lesion scores were 32.1

± 14.7, 34.0 ± 11.2, and 30.9 ± 12.1 (mean ± SD), respectively, while that of the anterior lobe was 20.6 ± 17.2.

DISCUSSION

Based on our examination of the prostate glands of 42 intact TRAMP mice between the ages of 18 and 24-weeks, poorly differentiated carcinoma was the most severe lesion observed in all four prostatic lobes. However, only in the ventral, lateral, and dorsal lobes was poorly differentiated carcinoma the lesion most frequently identified as most severe and most common (Tables 2 and 3). In the anterior lobe, although poorly differentiated carcinoma was also present, high grade PIN was the lesion observed to be the most severe lesion in the largest proportion of mice (52.4%). In addition, in the anterior lobe, the most common “lesion” in the majority of mice was no lesion at all; i.e. normal prostate histology. These results are consistent with the previously reported descriptions of tumorigenic progression in TRAMP mice (Kaplan-Lefko et al., 2003,

Gingrich et al., 1996, Gingrich et al., 1999, Hurwitz et al., 2001).

Among the other published grading schemes that we reviewed (Table 4), that proposed by Suttie, et al (Suttie et al., 2003) is the most similar to the refined scheme described here. The Suttie scheme recommends evaluating each lobe of the prostate and assigning, based on the most severe lesion within the lobe, a grade from one to six. The grades one through three represent varying degrees of hyperplasia, grades four and five represent adenomas, and grade six represents adenocarcinomas. The distribution of the lesion within the lobe is then estimated as focal, multifocal, or diffuse, and is combined with the lesion grade to give a “distribution-adjusted lesion grade,” that ranges from 0-18.

The versatility of a grading scheme that combines lesion severity and distribution into a single score is commendable. However, to characterize the lesions within a lobe and

obtain a score that is indicative of the most significant lesions, i.e. not only the most severe, but also the most common, we assigned numerical values for both the severity and distribution of both the most severe and most common lesions to generate a score referred to above as the sum of the adjusted lesion scores. Another important distinction between the Suttie scheme and our refined scheme is how invasion is defined. Suttie et al define invasion as islands of epithelial cells within the smooth muscle stroma that are less well differentiated and not closely associated with the epithelium lining the gland.

We prefer a definition of invasion that does not rely on an estimation of the distance between epithelial cells in the smooth muscle and the glandular epithelium, since this distance may vary between tissue sections with plane of cut. Thus, we identify invasion based on the presence of stromal reaction associated with epithelial cells present within the smooth muscle capsule. Also, the refined grading scheme assigns only one grade for adenomas, instead of two. The histologic descriptions of adenomas in the Suttie scheme are similar to our definition of high grade PIN. Finally, the refined scheme segregates carcinomas into three distinct groups (well, moderately and poorly differentiated) since a treatment that shifts prostate phenotype from poorly differentiated carcinoma to well differentiated adenocarcinoma could be clinically important.

The grading scheme described by Gingrich, et al (Gingrich et al., 1999) defined low grade PIN, high grade PIN, well differentiated adenocarcinoma, moderately differentiated adenocarcinoma, and poorly differentiated adenocarcinoma as the lesions present in the prostates of TRAMP mice. The differences between the two grades of PIN described in the Gingrich scheme and the three grades of PIN in the refined grading

scheme are subtle. However, we propose that there is a distinct intermediate grade of PIN between low and high grade PIN. This could be helpful in defining the effects of chemopreventive agents that may target the PIN stage of prostate cancer progression. The

Gingrich scheme defined invasion as epithelial cells invading into the stroma surrounding glands, or as rounded nuclei within hypertrophied and hyperplastic fibromuscular stroma surrounding glands. The rounded nuclei were reported to be immunohistochemically positive for the T antigen, suggesting that they belonged to transformed epithelial cells

(Gingrich et al., 1999). The presence of nuclei that are immunopositive for the T antigen is a novel and interesting criterion for invasion. However, without demonstration of T antigen immunoreactivity, we assume these nuclei represent hyperplastic smooth muscle cells that frequently surround glands with moderate and high grade PIN lesions. For simplicity and consistency, we propose a scheme that does not rely on immunohistochemistry to identify invasion.

The grading scheme proposed by Kaplan-Lefko, et al (Kaplan-Lefko et al., 2003) classified the lesions in the prostate glands of TRAMP mice as either PIN, well differentiated carcinoma, moderately differentiated carcinoma, poorly differentiated carcinoma, or phylloides-like lesions. Although this scheme provides a simple and straightforward way to describe the lesions, in our view, the variability in the cytological and architectural changes that occur in PIN necessitated the division of PIN into three categories to emphasize the progression of hyperplasia and dysplasia that occurs in the

PIN lesions. Additionally, the Kaplan-Lefko scheme does not address the issue of differentiating herniation of dysplastic cells from invasion. Photomicrographs

accompanying the description of this scheme imply that herniation is equivalent to invasion.

In the descriptions of the prostatic lesions of TRAMP mice in the Consensus

Report from the Bar Harbor Meeting of the Mouse Models of Human Cancer Consortium

Prostate Pathology Committee (Shappell et al., 2004), the authors list hyperplasia (both epithelial alone, as well as combined epithelial and stromal), adenomas and papillomas, mPIN (mouse PIN), microinvasive carcinoma, and invasive carcinoma (which includes well differentiated, moderately differentiated, and poorly differentiated neuroendocrine carcinomas). Among the grading schemes reviewed, this is the only one that differentiated hyperplasia from mPIN. Since PIN progresses to adenocarcinoma in

TRAMP mice, we felt it unnecessary to describe a separate lesion of hyperplasia. While our definition of PIN is consistent with the authors’ definition of mPIN, the refined scheme identifies different categories of PIN. The Consensus Report defined invasion based on the presence of desmoplasia associated with epithelial cells that have invaded into the smooth muscle surrounding the glands, “adequate” distance separating the invasive epithelial cells from the gland, or unequivocal evidence that the basement membrane was penetrated by epithelial cells confirmed by immunohistochemistry, special stains, or electron microscopy. We agree that penetration of the basement membrane and a stromal reaction associated with the epithelial cells within the smooth muscle support a diagnosis of invasion as we have delineated in our scheme, but we prefer to not use the distance between epithelial cells within the smooth muscle stroma and the gland as a criterion for invasion, since this may vary between sections.

The grading scheme used by Chiaverotti, et al (Chiaverotti et al., 2008) described mild, moderate, and severe atypical hyperplasia of Tag, papillary adenoma, adenocarcinoma (well, moderately, and poorly differentiated), and neuroendocrine carcinoma as the lesions observed in TRAMP mice. Although the terminology differs, the description of atypical hyperplasia of Tag mice is similar to our description of PIN.

Chiaverotti et al suggest that the term PIN should not be used to describe the lesions in

TRAMP mice, since PIN implies epithelial hyperplasia and dysplasia, with no stromal involvement. We prefer to use the term PIN, because we and others believe that it is descriptive of the natural progression of these lesions. Nonetheless, we acknowledge that the PIN we describe in the prostates of TRAMP mice is different from that observed in human prostates. As for defining invasion, the Chiaverotti scheme, like our refined scheme, differentiates invasion from herniation of dysplastic epithelial based on the presence of a stromal reaction.

We have compared our new, refined grading scheme with selected examples of other reported schemes in TRAMP mice. The refined grading scheme was designed to be especially useful for chemoprevention studies. The proposed advantages of the refined grading scheme include distinctions among low, moderate, and high grade PIN, which will enable studies to determine if a treatment alters the severity of PIN and slows the progression of preneoplastic lesions in the TRAMP model. In addition, the refined grading scheme provides a useful definition of invasion and permits differentiation of herniated dysplastic epithelial cells from epithelial cells that have invaded into the underlying stroma. Differentiating high grade PIN from well differentiated carcinoma

with invasion is imperative for determining if a treatment prevents progression to neoplasia in the TRAMP model. Finally, the refined grading scheme assigns numerical values to lesion types and their distributions and combines the adjusted scores that represent the most severe lesion and the most common lesion and their respective distributions. By taking into consideration the most severe and most common lesions and their distributions, the refined scheme allows for the progression of lesions within a lobe to be fully assessed.

Although controversy still exists regarding the assessment, classification, and progression of the lesions that occur in the prostates of TRAMP mice, we believe that the discussions and debates that arise from it are essential for better understanding and use of this model. We anticipate that the readers will find our review of available grading schemes informative, and that further discussion will be stimulated. We believe that our refined grading scheme is a valuable addition to the grading schemes currently available to investigators. We hope that other investigators will accept our scheme’s utility for fully evaluating the prostate lesions of TRAMP mice.

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Figure 1.1

Representative images of low grade PIN (Lesion grade 1) in TRAMP prostate. (A) Note the focal cell stratification, increased nuclear to cytoplasmic ratio, and crowding of the epithelial cells (anterior lobe, H&E, magnification 200X). (B) Few, short papillary proliferations of hyperplastic epithelium project into the lumen (ventral lobe, H&E,

200X).

Figure 1.2

Representative images of moderate grade PIN (Lesion grade 2) in TRAMP prostate. Note the more prominent papillary projections of hyperplastic epithelial cells that project into the lumen in the dorsal (A, H&E, 200X) and ventral lobes (B, H&E, magnification

100X).

Figure 1.3: Representative images of high grade PIN (Lesion grade 3) in TRAMP prostate. (A) The epithelium forms a cribriform pattern filling the lumen of the gland

(star). Note that the epithelium adjacent to the area of the cribriform pattern, as well as the epithelium of adjacent glands form tall papillary projections extending into the lumens (dorsal lobe, H&E, 100X). (B) There is frequent herniation of clusters of dysplastic epithelial cells (arrows) into the underlying smooth muscle capsule (dorsal lobe, H&E, 80X).(C) Higher magnification of the image in panel B clearly shows herniation of epithelial cells into underlying smooth muscle (arrows) without any evidence of stromal reaction (dorsal lobe, H&E, 200X). (D) Acini of hyperplastic epithelial cells fill the lumen of the gland and minimal proliferation of fibroblasts between adjacent glands is evident (ventral lobe, H&E, 100X). (E) Note the mass of proliferating epithelial cells and tightly compacted stroma protruding into the lumen.

There is no distinction between the proliferating epithelium and the adjacent epithelium lining the gland (dorsal lobe, H&E, 100X). (F) Higher magnification of the image in panel E shows the smooth muscle capsule surrounding the gland is intact and there is no evidence of invasion. The stroma between acini of the proliferating dysplastic epithelium is tightly compacted and a minor component of the lesion (H&E, 200X).

Figure 1.3

Figure 1.4

Representative images of phyllodes-like tumor (Lesion grade 4) in TRAMP prostate. (A)

Phyllodes-like tumor in the anterior lobe of the prostate (H&E, 20X). (B) Higher magnification of the image in panel A shows that the phyllodes-like tumor consists of papillary projections of loose stroma with loosely arranged stellate mesenchymal cells and epithelial cells with minimal or no nuclear or cytoplasmic atypia and rare mitotic figures (H&E, 80X).

Figure 1.5: Representative images of well differentiated adenocarcinoma (Lesion grade

5) in TRAMP prostate. (A) Well differentiated adenocarcinoma forming a mass of epithelial cells and acini that expands the lumen of a gland from the dorsal lobe of the prostate. Note that this gland is no longer surrounded by a complete or distinct smooth muscle capsule (H&E, 40X). (B) Higher magnification of the image in panel A shows invasion of epithelial cells through the basement membrane and into the adjacent stroma

(arrow). A stromal reaction in response to invasion is evident as reactive fibroblasts and myoepithelial cells surrounding the invading cells (H&E, 400X). (C) Note the exuberant stromal reaction and replacement of the smooth muscle capsule by reactive fibroblasts in this focus of well differentiated adenocarcinoma from the lateral prostate (H&E, 200X).

Figure 1.5

Figure 1.6

Representative images of moderately differentiated adenocarcinoma (Lesion grade 6) and poorly differentiated carcinoma (Lesion grade 7) in the lateral lobe of TRAMP prostate.

(A) Moderately differentiated adenocarcinoma: Note the small acinar-like structures formed by cells exhibiting varying degrees of nuclear and cytoplasmic atypia (H&E,

100X). (B) Note the solid sheets of polygonal to elongated cells, a high mitotic index, frequent nuclear and cytoplasmic atypia, and the lack of glands (H&E, 400X).

Lesion Adjusted Grade Distribution Lesion Score 0 Diffuse 0 1 Focal 1 1 Multifocal 2 1 Diffuse 3 2 Focal 4 2 Multifocal 5 2 Diffuse 6 3 Focal 7 3 Multifocal 8 3 Diffuse 9 4 Focal 10 4 Multifocal 11 4 Diffuse 12 5 Focal 13 5 Multifocal 14 5 Diffuse 15 6 Focal 16 6 Multifocal 17 6 Diffuse 18 7 Focal 19 7 Multifocal 20 7 Diffuse 21 Table 1.1: Determination of the adjusted lesion score from the lesion grade and distribution.

Prostate Lobe Lesion Ventral Lateral Dorsal Anterior

Normal 0b 0b 0b 2.4

Low Grade PIN 4.8 0b 0b 4.8

Moderate Grade PIN 14.3 7.1 0b 0b

High Grade PIN 2.4 2.4 40.5 52.4

Phyllodes-like 0c 0c 2.4 2.4

Well differentiated 9.5 23.8 4.8 0c adenocarcinoma

Moderately differentiated 2.4 2.4 0c 0c adenocarcinoma

Poorly differentiated 66.7 64.3 52.4 38.1 carcinoma a Incidence expressed as a percentage of a total of 42 mice. b Lesions of this type were observed, but did not represent the most severe lesion in this lobe in any of the mice. c No lesions of this type were observed in this lobe.

Table 1.2: The incidences (%)a of the prostate lesions identified as most severe in each lobe of the prostates of TRAMP mice

Prostate Lobe Lesion Ventral Lateral Dorsal Anterior

Normal 4.8 0b 0b 59.5

Low Grade PIN 9.5 0b 0b 0b

Moderate Grade PIN 14.3 14.3 2.4 0b

High Grade PIN 2.4 19.0 45.2 2.4

Phyllodes-like 0c 0c 0b 0b

Well differentiated 2.4 2.4 0b 0c adenocarcinoma

Moderately differentiated 2.4 0b 0c 0c adenocarcinoma

Poorly differentiated 64.3 64.3 52.4 38.1 carcinoma a Incidence expressed as a percentage of a total of 42 mice. b Lesions of this type were observed, but did not represent the most common lesion in this lobe in any of the mice. c No lesions of this type were observed in this lobe.

Table 1.3: The incidences (%)a of the prostate lesions identified as most common in each lobe of the prostates of TRAMP mice

Number of Number of Grading different preneoplastic Terminology for Numerical Scheme lesions lesions preneoplastic lesions Definition of invasion score Gingrich et al., 5 2 PIN Epithelial cells within the stroma surrounding No 1999 glands; rounded nuclei present within the fibromuscular stroma surrounding glands Kaplan-Lefko et 5 1 PIN Epithelial cells within smooth muscle stroma; No al., 2003 increased number of small glandular structures with associated desmoplasia Suttie et al., 6 3 Hyperplasia Islands of epithelial cells within the smooth muscle Yes: 2003 stroma appear less well differentiated and are not distribution- closely associated with the outline of epithelium adjusted lining the gland lesion grade Bar Harbor 5 1 Epithelial hyperplasia, Penetration of the basement membrane by epithelial No

4 Meeting, 2004 combined epithelial and cells. Recognized based on presence of desmoplasia 2

stromal hyperplasia, mouse adjacent to the epithelial cells that have invaded PIN (mPIN) into the smooth muscle, “adequate” distance separating the cells within the smooth muscle from the epithelial cells lining the gland, or clear evidence of the basement membrane being penetrated (determined using immunohistochemistry, special stains, or electron microscopy) Chiaverotti et 6 3 Atypical hyperplasia of Tag Presence of stromal reaction to the invading No al., 2008 epithelial cells Berman-Booty 7 3 PIN Presence of epithelial cells in the underlying Yes: sum of et al., 2011 smooth muscle, with cells crossing the basement the adjusted membrane and replacement of the normal smooth lesion scores muscle surrounding gland by reactive fibroblasts and myoepithelial cells Table 1.4: Summary of published and proposed refined grading schemes for the prostate glands of TRAMP mice 42

CHAPTER 2: THE NOVEL ENERGY RESTRICTION-MIMETIC AGENT OSU-CG5

DOWN-REGULATES EXPRESSION OF GENES THAT PROMOTE THE

WARBURG EFFECT AND SUPPRESSES THE GROWTH OF CASTRATION-

RESISTANT XENOGRAFT TUMORS

ABSTRACT

Neoplastic cells often utilize glycolysis to generate energy, even in the presence of normal oxygen tensions. This shift in energy metabolism is known as the Warburg effect. Agents that mimic glucose restriction, such as the energy restriction-mimetic agent

(ERMA) 2-deoxyglucose (2-DG), reduce tumor cell proliferation in vitro and in multiple animal models. Our laboratory has developed a novel class of ERMAs derived from thiazolidinediones (TZDs) that exhibit higher potency relative to 2-DG in eliciting starvation-associated cellular responses and epigenetic gene activation in cancer cells.

We have previously described the mechanism of action of the parent TZD-derived-

ERMA OSU-CG12. In this paper we evaluated the in vitro mechanism of action of the

OSU-CG12 derivative OSU-CG5 in the human LNCaP, LNCaP-abl, and PC3 prostate cancer cell lines. Like OSU-CG12, OSU-CG5 elicited a starvation-associated response characterized by increased β-TrCP expression with resultant proteolysis of downstream targets including Sp1 and downregulation of the expression of

Sp1 target genes including the androgen receptor (AR). OSU-CG5 also suppressed biomarkers that promote the Warburg effect, namely, phosphorylated-Akt and Myc.

Additionally, OSU-CG5 modulated the expression of metabolic enzymes and glucose transporter 1 in these cell lines. In addition to its in vitro efficacy, 100 mg/kg/day of

OSU-CG5 suppressed LNCaP-abl xenograft tumor growth by 81% by reducing cell proliferation within the tumors, as determined by a decrease in proliferating cell nuclear antigen. The tumor suppressive dose of OSU-CG5 did not induce systemic or biochemical evidence of toxicity. Tumor growth suppression was associated with statistically significant down-regulation of a number of biomarkers associated with cell proliferation, survival, and energy metabolism, including the AR, insulin-like growth factor-1 (IGF-1), IGF-1 receptor, Myc, cyclin D1, and many metabolic enzymes that promote the Warburg effect. These results, coupled with the lack of systemic toxicity, indicate that OSU-CG5 has great potential as a chemotherapeutic agent for the treatment of castration-resistant prostate cancer.

INTRODUCTION

Utilization of glycolysis in the presence of normal oxygen tensions (aerobic glycolysis—the Warburg effect) as the primary means of energy generation is a fundamental way in which cancer cell energy metabolism differs from the metabolism of non-neoplastic cells (1-7). Briefly, this shift in energy metabolism is favored by multiple mechanisms including increased PI3K/Akt signaling, upregulation of Myc, and

44 stabilization of hypoxia inducible factor-1 (HIF-1). Signaling via the PI3K/Akt pro- survival pathway promotes aerobic glycolysis by increasing glucose transport into the cell via increased transcription of glucose transporter (GLUT) 1 and translocation of

GLUT4 to the plasma membrane (3-4). PI3K/Akt signaling also enhances the glycolytic rate by activating glycolytic enzymes including hexokinase 2 (HK2), which catalyzes the first enzymatic reaction in glycolysis, and phosphofructokinase 2 which synthesizes the molecule that activates phosphofructokinase 1 (PFK-1) (3-4). Increased expression of the transcription factor Myc promotes the Warburg effect by upregulating the expression of many metabolic enzymes including HK2, enolase (4, 8), and the muscle isozyme of PFK-

1 (8). Additionally, Myc increases the expression of GLUT1 (8) and lactate dehydrogenase A (LDH-A), the enzyme the converts pyruvate to lactate, the end product of aerobic glycolysis (4, 9). HIF-1 is a heterodimeric transcription factor with an α subunit that is normally only stabilized under hypoxic conditions. Activated HIF-1 increases the glycolytic rate by inducing the transcription of many metabolic enzymes, including HK2 (3), LDH-A (3, 9), the liver isoform of phosphofructokinase-1 (PFK-1L), aldolase A (ALDOA), pyruvate kinase muscle isoform 2 (PKM2), and enolase (9). HIF-1 also increases glucose transport into the cell by increasing the transcription of GLUT1(3).

Interestingly, given the similarities between the genes upregulated by Myc and HIF-1, it has been suggested that HIF-1 is responsible for activating that subset of genes under low oxygen tensions, while Myc activates them under normal oxygen tensions (8).

Additionally, signaling via PI3K/Akt has been implicated in HIF-1 stabilization at normal oxygen tensions (4). Based on the mechanisms of action of PI3K/Akt signaling, Myc, and

HIF-1, a key feature of the Warburg effect appears to be the upregulation of metabolic enzymes and GLUT1.

The metabolic difference between neoplastic and non-neoplastic cells is the basis for the development of a novel class of anti-neoplastic agents, the energy restriction- mimetic agents (ERMAs). By targeting glycolysis, ERMAs preferentially target and kill cells with high glycolytic rates (neoplastic cells) while largely sparing non-neoplastic cells that primarily generate adenosine triphosphate (ATP) via oxidative phosphorylation.

The efficacy of targeting energy metabolism for cancer treatment has been demonstrated by the fact that dietary caloric restriction or treatment with the ERMA 2-deoxyglucose

(2-DG) results in the inhibition of cancer glucose metabolism and suppression of tumorigenesis and the growth of xenograft tumors in a number of experimental models

(10-14).

We have previously shown that OSU-CG12, a peroxisome proliferator-activated receptor (PPAR) γ inactive derivative of the thiazolidinedione (TZD) ciglitazone, shares the abilities of glucose deprivation and 2-DG to elicit a starvation-associated cellular response in the human LNCaP prostate cancer cell line (15). The starvation-associated response is characterized in part by activation of the energy sensing protein adenosine monophosphate (AMP)-activated protein kinase (AMPK) and stimulation of endoplasmic reticulum (ER) stress leading to apoptosis and autophagy. Additionally, β-transducin repeat containing protein (β-TrCP), a Skp1/Cul1/F-box ubiquitin ligase, is upregulated, resulting in increased proteosomal degradation of cell cycle regulatory proteins, including

β-catenin, cyclin D1, and the transcription factor Sp1 (15-18). This ultimately results in 46 downregulation of the androgen receptor (AR) with decreased survival and proliferation of prostate cancer cells.

OSU-CG5 is a novel OSU-CG12 derivative. Although both compounds function as glucose uptake inhibitors, OSU-CG5 is more effective than OSU-CG12 at limiting cellular uptake of [3H]2-deoxyglucose (19). In this study, we characterized OSU-CG5’s cytotoxicity, its ability to induce the starvation-associated response, and its capacity to modulate protein biomarkers that promote the Warburg effect, such as phosphorylated-

Akt and Myc. The prostate cancer cell lines evaluated represent the spectrum of prostate cancer in human, namely: androgen-dependent prostate cancers, castration-resistant prostate cancers that express the AR, and castration-resistant prostate cancers that lack

AR expression. Briefly LNCaP cells are androgen-dependent human prostate cancer cells. This cell line was derived from a lymph node metastasis, has a mutated AR and wild-type p53, and expresses prostate-specific antigen (PSA) and cytokeratins 8 and 18

(20). The LNCaP-abl cell line was derived after chronic androgen deprivation of LNCaP cells in vitro (20-21). LNCaP-abl cells exhibit markedly higher levels of AR expression than LNCaP cells and are able to grow in vitro and in vivo without androgens (21-22).

The PC3 cell line was derived from a bone metastasis. This cell line does not express the

AR or PSA and is negative for p53 (20). Therefore, the LNCaP cell line was used to investigate the effect of OSU-CG5 on androgen-dependent prostate cancers, the LNCaP- abl cell line was used to model castration-resistant prostate cancers that express the AR, and the PC3 cell line was a model for castration-resistance without AR expression.

A previous study in our laboratory found that treatment of LNCaP cells with 48- hours of glucose deprivation suppressed the expression of a number of genes involved in promoting glycolysis and fatty acid biosynthesis (data not shown; genes listed in Table

1). Since upregulation of metabolic enzymes appears to be a feature of the Warburg effect, we sought to determine if OSU-CG5 treatment could suppress the expression of these metabolic enzymes, thus antagonizing the Warburg effect. Additionally, we also investigated the effect of OSU-CG5 on GLUT1 expression. We chose to evaluate

GLUT1 expression since upregulation of this glucose transporter is mediated by

PI3K/Akt signaling and Myc as part of the Warburg effect (3-4, 8). Additionally, it has been previously shown that the OSU-CG compounds inhibit GLUT1 (19).

In this study, we also characterized OSU-CG5’s in vivo activity, namely whether

OSU-CG5 could safely suppress the growth of a castration-resistant human prostate cancer cell line in xenograft mice. Specifically, we examined the effect of 100 mg/kg/day of OSU-CG5 on the growth of LNCaP-abl xenograft tumors. We also investigated whether treatment with OSU-CG5 could modulate a similar subset of biomarkers in vivo as in vitro.

MATERIALS AND METHODS

In vitro experiments:

Cell Cultures and Reagents: Human LNCaP and PC3 prostate cancer cells were obtained from the American Type Culture Collection (Manassas, VA) and LNCaP-abl cells were graciously provided by Qianben Wang (The Ohio State University, Columbus,

OH). PC3 and LNCaP cells were maintained with 10% fetal bovine serum and 1%

Penicillin/Streptomycin supplemented RPMI 1640 medium (Invitrogen, Carlsbad, CA), and the LNCaP-abl cells were grown in 10% charcoal–stripped fetal bovine serum and

1% Penicillin/Streptomycin supplemented phenol-red free RPMI 1640 medium

(Invitrogen). As previously described (23), cells were cultured at 37 °C in a humidified incubator containing 5% CO2. Our laboratory synthesized OSU-CG5 (data not shown).

OSU-CG5 was dissolved in DMSO and added to the culture medium with a final DMSO concentration of 0.1%.

Cell viability assays.

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

2H-tetrazolium bromide (MTT) assay. PC3, LNCaP, and LNCaP-abl cells were seeded in

96-well plates and incubated in their respective 10% fetal bovine serum-supplemented

RPMI 1640 media (Invitrogen) for 24 hours. This media was replaced with fresh media containing increasing concentrations of OSU-CG5. Cells were cultured in the presence of

OSU-CG5 for 72 hours. The drug-containing media was replaced with MTT (0.5 mg/ml in RPMI 1640) and incubated at 37°C for 2 hours. The MTT containing media was removed, and 200 µl of DMSO was added to each well. The absorbance at 570 nm was determined using a plate reader.

Cell counts to determine approximate cell viability after 48 hours of treatment with various concentrations of OSU-CG5 were also performed. PC3, LNCaP, and

LNCaP-abl cells were plated in 24-well plates and treated as described above for the 72

49 hours MTT assays. After 48 hours of exposure to OSU-CG5, cells were washed, trypsinized, and counted using a Z1 Coulter® Particle Counter (Beckman Coulter, Brea,

CA). All cell counts were performed in quadruplicate.

In vivo study:

Castrated male NCr athymic nude mice (5–7 weeks of age) were obtained from

Harlan Research Laboratories (Indianapolis, IN). The mice were group housed under conditions of constant photoperiod (12 h light/12 h dark). Mice received a standard rodent diet and water ad libitum. All procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of The Ohio

State University. Each mouse was inoculated s.c. in the right flank with 2x106 LNCaP-abl cells in a total volume of 0.1 mL serum-free medium containing 50% Matrigel (BD

Biosciences, San Jose, CA) under isoflurane anesthesia. As tumors became established

(mean tumor volume, 45.9± 5.3 mm3), mice were randomized to two groups (n=7 mice/group) that received the following treatments: a) vehicle or b) 100 mg/kg/day of

OSU-CG5.

As previously described (23), OSU-CG5 was prepared as a suspension in sterile water containing 0.5% methylcellulose (w/v) and 0.1% Tween 80 (v/v). Suspensions were prepared on a weekly basis. Mice received treatments via oral gavage under isoflurane anesthesia for 59 days. The duration of treatment was similar to that of other studies that have utilized this xenograft model (21). Tumors were measured weekly using calipers and their volumes calculated using the following standard formula: width2 x

50 length x 0.52. Body weights were measured weekly throughout the course of the study.

At the termination of the study (day 59), mice were euthanized via carbon dioxide asphyxiation approximately 2-4-hours after they received their last dose. Complete necropsies were performed. Blood was collected from 3 mice per group via cardiac puncture and submitted to the Comparative Pathology and Mouse Phenotyping Shared

Resource at the Ohio State University for complete blood counts (CBCs) and serum biochemistries for evaluation of hepatic parameters (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALKP], and gamma-glutamyl transpeptidase [GGT], and total bilirubin), renal parameters (blood urea nitrogen (BUN),

Creatinine [CREAT], calcium, and phosphorous), blood glucose, albumin, and cholesterol. At necropsy, the LNCaP-abl tumors were collected and divided in half. Half of each tumor was incubated in RNAlater (Qiagen, Valencia, CA) at 4°C for 24 hours.

The RNAlater was then removed and tumors were stored at -80°C prior to RNA extraction. The other half of each tumor was snap-frozen in liquid nitrogen and kept at

-80°C until needed for Western blot analysis.

The organs collected at necropsy were fixed in 10% neutral buffered formalin.

Five micrometer-thick, paraffin-embedded tissue sections of the liver, kidney, spleen, lung, heart, small intestine, brain, eye, and bone marrow were stained with hematoxylin and eosin (H&E) by standard procedures (n=5 mice/group). H&E-stained sections of the aforementioned tissues were examined with light microscopy by a board certified veterinary anatomic pathologist (LDBB) using an Olympus Model CHT (Olympus,

Center Valley, PA).

Protein isolation and western blot analysis.

For western blot analysis of in vitro biomarkers, LNCaP, LNCaP-abl, and PC3 cells were treated for 48 or 72-hours with various concentrations of OSU-CG5, and the cells were subsequently collected. For western blot analysis of intratumoral biomarkers, portions of the tumors were pulverized in liquid nitrogen using a ceramic mortar and pestle (n=5 mice/group). Cell pellets and pulverized tumor samples were lysed, protein concentrations were determined, and western blotting and densitometric analyses were performed as previously described (23).

The following primary antibodies were used for western blotting: mouse monoclonal antibodies: β-actin from MP Biomedicals (Irvine, CA), Cyclin D1 from

Santa Cruz (Santa Cruz, CA), and β-TRCP from Invitrogen; rabbit antibodies: insulin- like growth factor-1 receptor (IGF-1R), androgen receptor (AR), growth arrest and DNA damage-inducible gene (GADD)153, Sp1, cyclin E, and proliferating cell nuclear antigen

(PCNA) from Santa Cruz, and p-AMPK-Thr172, AMPK, glucose-regulated protein 78

(GRP78), Akt, p-Akt-Ser473, c-Myc, mammalian target of rapamycin (mTOR), p-mTOR from Cell Signaling (Beverly, MA).

RNA Isolation and Quantitative and Real-Time PCR (qRT-PCR) Analysis:

Total RNA was isolated from LNCaP, LNCaP-abl, and PC3 cells treated with either DMSO or 5µM of OSU-CG5 for 48 hours using TRIzol reagent (Invitrogen). A

Tissue Tearor, Model 985370-395, (BioSpec Products, Inc., Bartlesville, OK) and TRIzol reagent (Invitrogen) were used to homogenize and extract RNA from the portions of

52 xenograft tumors that were initially stored in RNAlater (n=3 mice/group). The tumor

RNA was subsequently purified using the RNAeasy Mini Kit (Qiagen). Purified total

RNA isolated from the tumors and from the cells was reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Primer sequences for qRT-PCR were obtained from

GeneCards® (http://www.genecards.org/) and Origene

(http://www.origene.com/qPCR/primers.aspx). qRT-PCR using the cDNA from the cell lines was carried out in the Bio-Rad CFX Connect™ Real-Time System (Bio-Rad) with

Bio-Rad SSO Advanced™ SYBR® green supermix (Bio-Rad). qRT-PCR using cDNA from the LNCaP-abl tumors was carried out in the Bio-Rad CFX96 Real-Time PCR

Detection System with iQ SYBR green supermix (Bio-Rad). Relative gene expression was normalized to 18s rRNA and calculated by using the 2(-Delta Delta C(T)) method.

Statistical Analysis:

Western blot and qRT-PCR analyses were performed in triplicate. Data were assessed for normality using the Shapiro Wilk test. All data were normally distributed except for the change in body weights and the hematologic and serum biochemical parameters. The normally distributed data were analyzed by the Student’s t test. Data that were not normally distributed were analyzed for statistical significance using the

Wilcoxon rank sum test. Differences were considered significant at P<0.05.

RESULTS

OSU-CG5 reduced LNCaP, LNCaP-abl, and PC3 cell viability and induced the starvation-associated response typical of TZD-derived ERMAs.

Treatment of prostate cancer cells with OSU-CG5 for 72-hours followed by the

MTT assay for cell viability revealed that the OSU-CG5’s cytotoxicity was modestly higher than that of OSU-CG12. The IC50 of OSU-CG5 in LNCaP cells was approximately 4 µM, compared to OSU-CG12’s previously reported IC50 of approximately 5.7 µM (15). The IC50 of OSU-CG5 in LNCaP-abl cells was approximately 4.3 µM, while the IC50 of OSU-CG12 was approximately 5.5 µM. The

IC50 of OSU-CG5 in PC3 cells was approximately 10 µM, while the IC50 of OSU-CG12 was greater than 10 µM (data not shown; Figure 1A). Based on cell counts after 48 hours of treatment with OSU-CG5, it appeared that approximately 50% of LNCaP and LNCaP- abl cells were viable after treatment with 5 μM of OSU-CG5, in contrast to approximately 65% of PC3 cells. Treatment with 10 μM of OSU-CG5 resulted in the viability of PC3 cells falling to 50% (Figure 1B).

To confirm that OSU-CG5’s mechanism of action was the same as that of glucose deprivation and OSU-CG12, LNCaP, LNCaP-abl, and PC3 cells were treated with OSU-

CG5 for 72 hours and western blots were performed. As previously described, the starvation-associated response involves upregulation of β-TRCP with degradation of downstream targets, activation of AMPK, and induction of an ER stress response (15).

As expected, OSU-CG5 treatment resulted in such a response (Figures 1C and 1D) with upregulation of β-TRCP and degradation of the downstream targets cyclin D1, β catenin,

54 and Sp1. The AR was downregulated, presumably due to decreased Sp1 mediated transcriptional activation. OSU-CG5 induced AMPK activation (Figures 1C and 1D), as determined by increased AMPK phosphorylation. AMPK activation resulted in the dephosphorylation and inhibition of mammalian target of rapamycin (mTOR), an effector that normally acts to promote protein synthesis, cell growth, and proliferation (11, 24).

OSU-CG5 treatment generated an ER stress response (Figures 1C and 1D), as determined by the upregulation of GRP78 and GADD153 (25).

OSU-CG5 modulated protein biomarkers that promote the Warburg effect, namely phosphorylated-Akt (Figures 1C and 1D) and Myc (Figure 1E). 72-hours of

OSU-CG5 treatment resulted in the decreased phosphorylation of Akt at the serine 473 position. This result was in accordance with previous research showing that energy restriction decreases signaling through IGF-1R, resulting in decreased pro-survival signaling through the PI3K/Akt pathway (11, 26). Decreased Akt phosphorylation was likely responsible in part for the decreased mTOR phosphorylation (11).When Myc protein expression was evaluated in the cell lines after 48 hours of treatment, a reduction in Myc protein was first noted when the cells were treated with 2.5 μM of OSU-CG5.

When cells were treated with 5 μM of OSU-CG5 for 48-hours, almost no Myc protein was detectable in all three cell lines (Figure 1E).

OSU-CG5 down-regulated the expression of metabolic enzymes and a glucose transporter that promote the Warburg effect in LNCaP, LNCaP-abl, and PC3 cells.

We examined the expression of a series of genes (Table 1) involved in glycolysis, lactate synthesis, and fatty acid synthesis in LNCaP, LNCaP-abl, and PC3 cells after treatment with 5 µM OSU-CG5 for 48 hours in order to understand the effect of OSU-

CG5 on cellular metabolism. As shown in figure 2A, treatment of LNCaP cells with 5

µM of OSU-CG5 for 48 hours down-regulated the expression of the listed metabolic enzymes by 36 to 92% (P<0.05). Treatment of LNCaP-abl cells with 5 µM of OSU-CG5 for 48 hours resulted in down-regulation of the majority of the metabolic enzymes by

15% to 92% (P<0.05), with the exception of ALDOA, AMACR, and LDH-A (P>0.05;

Figure 2B). Treatment of PC3 cells with OSU-CG5 at 5 µM for 48 hours resulted in statistically significant reductions in the gene expression levels of most of the enzymes in

Table 1 by 12 to 53%. Suppression of PC3 cell metabolic gene expression was statistically significant (P<0.05) for all genes except ACSL3, AMACR, HK2, LDHA,

PFK-1P, and PKM2 (Figure 2C). Evaluation of GLUT1 expression after 48 hours of treatment with 5 μM of OSU-CG5 revealed that OSU-CG5 down-regulated GLUT1 expression in the cell lines (P<0.05; Figure 2D). Specifically, OSU-CG5 reduced

GLUT1 expression by 58%, 51%, and 21% in LNCaP, LNCaP-abl, and PC3 cells, respectively.

100 mg/kg/day of OSU-CG5 via oral gavage suppressed the growth of LNCaP-abl xenograft tumors

To determine OSU-CG5’s in vivo efficacy, we investigated the effect of OSU-

CG5 treatment on the growth of LNCaP-abl xenograft tumors. Treatment of LNCaP-abl xenograft tumor baring mice with 100 mg/kg/day of OSU-CG5 for 59 days resulted in statistically significant suppression of tumor growth starting at day 7 of treatment (Figure

3A). At day 59, the average tumor volume for the vehicle-treated mice was 115.3±56.7 mm3, and the average tumor volume for the OSU-CG5 treated mice was 58.5±26 mm3

(P=0.033; Figure 3A). This translated to a 49% reduction in absolute tumor volume. The average change in tumor volume over the course of the study was +68.6±51.2 mm3 and

+13.3±26.2 mm3 (P=0.026) for the vehicle-treated and OSU-CG5 treated mice, respectively (Figure 3B), meaning that OSU-CG5 suppressed tumor growth by 81%.

Tumor growth suppression was due to decreased tumor cell proliferation. Western blots for PCNA demonstrated that OSU-CG5 treatment reduced intratumoral PCNA expression by 57% (P=0.030; Figure 3C & 3D).

Suppression of LNCaP-abl xenograft tumor growth by OSU-CG5 was not associated with any evidence of systemic toxicity. Daily oral administration of 100 mg/kg

OSU-CG5 did not result in weight loss, and there was no statistically significant difference between the amount of weight mice in the two groups gained over the course of the study (P>0.05). On average, the OSU-CG5-treated mice gained 1.44±0.75g (n=7), and the vehicle-treated mice gained 1.93±0.71g (n=7). Additionally, histological evaluation of H&E-stained sections of liver, kidney, spleen, lung, heart, small intestine,

57 brain, eye, and bone marrow from 5 mice per group did not reveal any lesions compatible with toxicity. Complete blood counts and serum chemistries did not reveal any significant hematologic abnormalities or any evidence of hepatic or renal dysfunction (P>0.05;

Table 2; n=3 mice/group). Additionally, there was no significant difference in blood glucose concentrations in the two groups of mice (Table 2), which was consistent with a previous experiment which demonstrated that OSU-CG5 does not affect glucose homeostasis (23).

OSU-CG5 mediated LNCaP-abl tumor growth suppression was associated with intratumoral modulation of biomarkers

Given that OSU-CG5 was able to invoke the starvation-associated response in

LNCaP-abl cells in vitro, western blotting for selected biomarkers was performed to determine if induction of a similar response occurred in vivo. Western blotting for the downstream targets of β-TRCP, namely Sp1, AR, cyclin D1, and cyclin E, was performed. The volumes of the 5 tumors per group utilized for western blotting are shown in Figure 4A. Western blotting revealed that OSU-CG5 treatment resulted in statistically significant decreases in Sp1, AR, cyclin D1, and cyclin E, with OSU-CG5 reducing the protein levels by 54% (P=0.017), 67% (P=0.010), 60% (P=0.014), and 54%

(P=0.015), respectively (Figures 4B and 4C). The reduction in the AR was confirmed to be due to downregulation of the gene, and not due to increased degradation of the pre- formed receptor. qRT-PCR performed using RNA isolated from the tumor homogenates

58 revealed that expression of the AR was reduced in OSU-CG5 treated tumors by 74.6%

(P=0.000) (Figure 4D).

Biomarkers with roles in promoting tumor cell survival, proliferation, and energy metabolism were also evaluated via western blotting and qRT-PCR (Figures 4B, 4C, and

4D). Since energy restriction modulates the IGF-1/IGF-1R signaling axis and downstream effector signaling via PI3K/Akt (11, 26), we investigated the expression of

IGF-1, IGF-1R, and phosphorylated-Akt. Western blotting revealed that OSU-CG5 treatment reduced the protein level of IGF-1R by 41% (P=0.028; Figures 4B and 4C).

Although phosphorylated Akt was decreased by 20%, this result was not statistically significant (P=0.05; Figures 4B and 4C). qRT-PCR confirmed the decrease in IGF-1R expression and revealed that OSU-CG5 also decreased intratumoral IGF-1 expression.

Namely, OSU-CG5 treatment reduced intratumoral IGF-1 and IGF-1R by 95.2%

(P=0.000) and 91.4% (P=0.009), respectively (Figure 4D). Given the important role of

Myc in enhancing the transcription of glycolytic enzymes, the protein level of Myc in the

LNCaP-abl tumors was also investigated. Western blotting showed that OSU-CG5 treatment reduced Myc expression by 54% (P=0.048) (Figures 4B and 4C). These data indicated that OSU-CG5 mediated LNCaP-abl tumor growth suppression involved activation of the starvation-associated response, as demonstrated by the downregulation of Sp1, AR, cyclin D1, and cyclin E, and modulation of cell survival and energy metabolism pathways, as established by the reductions in IGF-1, IGF-1R, and Myc.

OSU-CG5 mediated LNCaP-abl tumor growth suppression was associated with modulation of metabolic gene expression.

To determine if OSU-CG5 treatment could modulate the expression of metabolic enzymes in vivo in a similar manner to that observed in vitro, qRT-PCR was performed using RNA isolated from the LNCaP-abl tumor homogenates. OSU-CG5 treatment resulted in statistically significant suppression of the expression of a number of genes that promote glycolysis (Figure 5A). The intratumoral expression levels of PKM2, PDK3,

HK2, GPI, ALDOA, PFK-1M, PFK-1L, and LDH-A were reduced by 88.2% (P=0.000),

88.3% (P=0.000), 64.1% (P=0.011), 69.4% (P=0.003), 83.2% ( P=0.022), 51.9%

(P=0.02), 55.1% (P=0.047), and 92.7% (P=0.031), respectively, compared to the vehicle-treated controls. Although OSU-CG5 treatment reduced the expression of PFK-

1P and PFKFB4 by 49.5% and 76.2%, respectively, these results were not statistically significant (P>0.05). OSU-CG5 also suppressed the expression of enzymes involved in fatty acid metabolism (Figure 5B). These enzymes promote the Warburg effect by increasing the synthesis of macromolecules, namely phospholipids and fatty acids, needed for a cell to have a high rate of division. Specifically, OSU-CG5 treatment reduced the intratumoral expression of FASN, ACLY, ACACA, AMACR, and FADS2 by 77.8% (P=0.000), 64.4% (P=0.000), 70% (P=0.000), 65.1% (P=0.022), and 74.8%

(P=0.003), respectively, compared to the vehicle-treated control tumors.

DISCUSSION

Although the efficacy of energy restriction as an experimental chemotherapeutic strategy has been proven in multiple animal models of cancer (10-14, 27), actual dietary caloric restriction is not a realistic therapy for human cancer patients. Additionally, 2-

DG has a relatively low in vitro cytotoxicity (15) suggesting that a high doses would be necessary to achieve a significant effect in vivo. Finally, whenever a therapy targeting metabolically active cells is implemented, potential toxicity to other metabolically active organs, such as the liver, kidney, eye, and brain, must be considered. Such concerns are warranted given the fact that a clinical trial evaluating 2-DG (NCT00633087) was discontinued by the Food and Drug Administration due to the potential for liver toxicity

(28). Therefore, new ERMAs that are both highly effective and safe must be developed.

Here we demonstrate the in vitro and in vivo activity and efficacy of OSU-CG5, a novel TZD derived ERMA. In vitro, OSU-CG5 had a slightly improved cytotoxic profile compared to OSU-CG12. Like OSU-CG12, OSU-CG5 induced the starvation-associated response in prostate cancer cells. As part of this response in vitro, pro-survival signaling via the PI3K/Akt pathway was down-regulated. Additionally, 48 hours of OSU-CG5 treatment reduced Myc expression in all three cell lines.

As an anticipated part of the starvation-associated response, degradation of downstream targets of β-TRCP occurred in the LNCaP-abl xenograft tumors. OSU-CG5 also decreased pro-growth and survival signaling in these tumors as demonstrated by down-regulation of IGF-1, IGF-1R, AR, and Myc. OSU-CG5 treatment modulated the

PI3K/Akt pathway via a reduction in p-Akt in the xenograft tumors, although this result was not statistically significant (P=0.05).

As previously discussed, PI3K/Akt signaling and Myc activity are important for the Warburg effect (3-4). Therefore, modulation of these two pathways may have been responsible, at least in part, for the down-regulation of metabolic enzymes in vitro and in vivo and the reduction in GLUT1 expression in the 3 cell lines. The reduction in GLUT1 expression illustrated a likely mechanism through which OSU-CG5 reduces cancer cell glucose uptake. Although HIF-1α can also promote aerobic glycolysis (3, 9), OSU-

CG5’s effect on HIF-1α was not assessed in our xenograft tumors, since we would expect low oxygen tensions within the tumors themselves to result in stabilization of HIF-1α, thereby potentially abrogating any effect that OSU-CG5 could have had on its stability in vivo.

In LNCaP cells, metabolic enzymes modulated by OSU-CG5 included PFK-1, the enzyme responsible for the rate limiting step in glycolysis (1), HK2, the enzyme that catalyzes the first irreversible step in glycolysis (1), and LDH-A, the enzyme that converts pyruvate to lactate. Decreased expression of these three metabolic enzymes would be expected to decrease the glycolytic rate and reduce lactate synthesis.

Importantly, OSU-CG5 also reduced the expression of FASN and ACLY in all three cells lines. These enzymes are involved in fatty acid synthesis and are upregulated in a number of cancers (29). ACLY in particular is extremely important for promoting the Warburg effect, since it is responsible for converting citrate to acetyl-CoA for fatty acid synthesis, thereby preventing excessive citrate from accumulating within cells. High citrate levels 62 would result in feedback inhibition of glycolysis (29), and undermine the cell’s ability to have a high glycolytic rate. Because PI3K/Akt signaling has been shown to increase the expression of FASN and p-Akt activates ACLY(3), decreased signaling via PI3K/Akt, in part due to a reduction in IGF-1/IGF-1R, may partially explain OSU-CG5’s ability to reduce the expression of FASN and ACLY

PKM2 expression in LNCaP and LNCaP-abl cells and the xenograft tumors was also reduced by OSU-CG5 treatment. PKM2 is the enzyme responsible for converting phosphoenolpyruvate to pyruvate and has been described as a “molecular switch” that appears to be essential for promoting the Warburg effect (30), with experimental replacement of the embryonic PKM2 by the adult PKM1 isoform resulting in inhibition of the Warburg effect (30). PKM2 also function as a bridge between growth factor- receptor tyrosine kinase signaling and energy metabolism. Briefly, PKM2 exists as either a low activity dimer or highly active tetramer. The shift towards the low activity dimer form enables glycolytic intermediates to accumulate and be diverted into other synthetic pathways (3). The switch to the low activity state is mediated by PKM2 binding to proteins containing phosphorylated tyrosine residues (3, 31). This provides a mechanism through which growth factor signaling results in enhancement of the Warburg effect. By down-regulating PKM2 expression, OSU-CG5 interfered with the Warburg effect and dissociated one of the links between growth factor signaling and cell metabolism.

LNCaP and LNCaP-abl cells were more sensitive to OSU-CG5’s cytotoxic effects, modulation of biomarkers, and down-regulation of metabolic enzymes and

GLUT1 than PC3 cells. LNCaP cells and PC3 cells have different metabolic and gene 63 expression profiles (32-33) which could potentially influence their responses to an

ERMA. Alternatively, the differences observed between LNCaP and PC3 cells may relate to p53. PC3 cells are negative for p53, while LNCaP cells (and presumably LNCaP-abl) have wild-type p53 (20). Besides the fact p53 is essential for inducing cell cycle arrest and or apoptosis in response to cell stress (such as energy restriction), functional p53 is important for resisting the Warburg effect. p53 promotes oxidative phosphorylation over aerobic glycolysis by upregulating synthesis of cytochrome c oxidase 2 (SCO2) a protein necessary for oxidative phosphorylation (3, 6), and down-regulating the glycolytic enzyme phophoglycerate mutase (3). Additionally, p53 increases the expression of TP53- induced glycolysis and apoptosis regulator (TIGAR) which decreases the glycolytic rate.

TIGAR reduces the concentration of fructose-2,6-bisphosphate, a small molecule that activates PFK-1 (3, 6). Therefore, cells that lack p53 may have a more robust glycolytic rate than those with functional p53 and may, therefore, be more resistant to interventions that antagonize the Warburg effect. Based on this premise, the relative resistance of PC3 cells to OSU-CG5 may be due to the lack of functional p53. Studies comparing the effect of knocking-down p53 in LNCaP cells and expressing p53 in PC3 cells on cellular sensitivity to OSU-CG5 are needed to clarify the role of p53 in the differing susceptibilities of these lines.

Recently it has been proposed that tumors and cell lines that contain mutations that result in upregulation of PI3K/Akt signaling may be resistant to the effects of energy- restriction (34). Inactivating mutations in the PTEN gene are present in PC3 (22, 34-35),

LNCaP (22, 35), and LNCaP-abl cells (22). Since PTEN downregulates PI3K/Akt

64 signaling, such mutations would be expected to result in upregulation of the PI3K/Akt signaling axis and subsequent resistance to energy restriction (22). Interesting, all three of these cell-lines responded to OSU-CG5 treatment with the expected starvation-associated response and decreased cell viabilities. Additionally, OSU-CG5 treatment suppressed

LNCaP-abl xenograft tumor growth in vivo. OSU-CG5’s ability to overcome these cell lines’ purported resistance to energy restriction may be due to the fact that one of OSU-

CG5’s mechanisms of action is decreased phosphorylation and activation of Akt, thereby suppressing the PI3K/Akt signaling pathway.

OSU-CG5 treatment suppressed the growth of castration-resistant prostate cancer xenograft tumors in vivo. Based on the western blot results from the xenograft study, reduction in tumor growth was due to reduced cell proliferation, as evidenced by the 57% decrease in PCNA expression in the OSU-CG5 treated tumors. Decreased tumor cell proliferation was likely a result of the statistically significant down-regulation of intratumoral biomarkers that promote proliferation and survival including the AR, Myc,

IGF-1, and IGF-1R.

Tumor growth suppression not associated with toxicity. OSU-CG5- and vehicle- treated mice gained a similar amount of weight, which is a crude indication that OSU-

CG5 administration had no significant systemic side effects. More importantly, histologic examination of selected organs (liver, kidney, spleen, lung, heart, small intestine, brain, eye, and bone marrow) revealed no lesions consistent with toxicity, CBCs did not demonstrate any significant hematologic abnormalities, and serum chemistries did not reveal any evidence of significant hepatic or renal dysfunction. The lack of lesions in 65 the liver, kidney, brain, small intestine, eye, and bone marrow is reassuring since these organs contain populations of metabolically active cells that could, potentially, be negatively impacted by an ERMA.

In conclusion, OSU-CG5 induced the starvation-associated response, decreased

PI3K/Akt signaling and Myc activity, modulated the expression of metabolic enzymes that promote the Warburg effect, and reduced the expression of GLUT1 in vitro as well as suppressed the growth of LNCaP-abl xenograft tumors in vivo. Tumor growth suppression was due to decreased tumor cell proliferation evidenced by a reduction in

PCNA. Decreased tumor cell proliferation was likely due to OSU-CG5 reducing AR,

IGF-1, IGF-1R, and Myc concentrations and modulating metabolic enzyme expression.

Importantly, tumor growth suppression and biomarker modulation was not associated with any evidence of systemic or organ specific toxicity. Based on these results, OSU-

CG5 has great potential as a chemotherapeutic agent for castration-resistant prostate cancer, specifically, those cancers that express the AR.

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Figure 2.1

Evidence that OSU-CG5 reduced prostate cancer cell viability and modulated biomarker expression. Treatment of LNCaP, LNCaP-abl, or PC3 cells with OSU-CG5 for either 72

(A) or 48 hours (B) reduced cell viability. (C & D) Treatment of LNCaP (C), LNCaP-abl

(C), and PC3 cells (D) with OSU-CG5 for 72 hours resulted in modulation of biomarkers typical of the starvation-associated response. (E) Treatment of the three cell lines with

OSU-CG5 for 48 hours downregulated Myc expression. The bars in (B) represent the mean ± standard error of the mean (SEM). CG-5: OSU-CG5

Figure 2.1

Figure 2.2: Treatment of LNCaP (A), LNCaP-abl (B), and PC3 (C) cells with 5 μM of

OSU-CG5 downregulated the expression of many metabolic enzymes that promote the

Warburg effect. This downregulation was statistically significant (P<0.05) for all enzymes except ALDOA, AMACR, and LDH-A in the LNCaP-abl cells (B) and ACSL3,

AMACR, HK2, LDH-A, PFK-1P, and PKM2 in the PC3 cells (C). (D) Treatment of all three cell lines with 5 μM of OSU-CG5 for 48 hours resulted in statistically significant reductions in the expression of GLUT1 (P<0.05). Bars in all panels represent the mean ±

SD. CG5: OSU-CG5. PDK3, pyruvate dehydrogenase kinase isoenzyme 3, FADS2, fatty acid desaturase 2, ACSL3, Acyl-CoA synthase long-chain family member 3, LDHA, lactate dehydrogenase-A, ACAT2, Acetyl-CoA acetyltransferase 2, AMACR, Alpha- methylacyl-CoA racemase, PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6- biphosphatase 4, PFK-1P, 6-phosphofructo-1-kinase-platelet, PFK-1L, 6-phosphofructo-

1-kinase-liver, PFK-1M, 6-phosphofructo-1-kinase-muscle, GPI, glucose phosphate isomerase, HK2, hexokinase 2, ALDOA, fructose 1,6-bisphosphate aldolase A, PKM2, pyruvate kinase muscle splice isoform 2, ACLY, ATP citrate lyase, ACACA, Acetyl-

CoA carboxylase alpha, FASN, fatty acid synthase, GLUT1, glucose transporter 1.

7 3

Figure 2.2

Figure 2.3

(A) Treatment of athymic nude mice baring LNCaP-abl tumors with 100 mg/kg/day of

OSU-CG5 resulted in statistically significant tumor growth suppression (P<0.05) beginning at day 7 of treatment. (B) This dose of OSU-CG5 suppressed LNCaP-abl tumor by 81%. (C & D) The reduction in tumor growth was due to decreased cell proliferation within the xenograft tumors, as evidenced by the 57% decrease (P=0.030) in

PCNA compared to the vehicle-treated control mice. The bars in (A) represent the mean

± SEM, and the bars in (B) and (D) represent the mean ± SD.

7 5

Figure 2.3 72

Figure 2.4

Treatment of LNCaP-abl xenograft tumors with 100 mg/kg/day of OSU-CG5 resulted in reductions in intratumoral biomarker expression. (A) Volumes of the tumors utilized for western blotting. (B & C) The biomarkers modulated by OSU-CG5 were AR, Sp1, cyclin

D1, cyclin E, IGF-1R, p-Akt, and Myc compared to the vehicle-treated control (P<0.05 for all biomarkers except p-Akt). (D) qRT-PCR performed using RNA isolated from the

LNCaP-abl tumor homogenates revealed that the decreases in the protein levels of the

AR and IGF-1R were due to reduced gene expression. Additionally, expression of IGF-1 was also reduced by OSU-CG5 treatment. The bars in (C) and (D) represent the mean ±

SD. CG5: OSU-CG5

7 7

Figure 2.4 72

Figure 2.5

(A) OSU-CG5 treatment of LNCaP-abl xenograft tumors resulted in statistically significant downregulation of a number of genes involves in glycolysis, including PKM2,

PDK3, HK2, GPI, ALDOA, PFK-1M, PFK-1L, and LDHA (P<0.05). Although the expression of PFKFB4 and PFK-1P were reduced, these reductions were not statistically significant. (B) OSU-CG5 treatment of LNCaP-abl xenograft tumors resulted in statistically significant downregulation of a number of genes involves in fatty acid metabolism, including FASN, ACLY, ACACA, AMACR, and FADS2 (P<0.05). The bars in (A) and (B) represent the mean ± SD. CG5: OSU-CG5. 78

Gene Description Function PDK3 Pyruvate Promotes glycolysis by suppressing metabolism dehydrogenase kinase, through the TCA; a metabolic switch for cellular isozyme 3 adaptation to hypoxia (36)

FADS2 Fatty acid desaturase 2 Involved in polyunsaturated fatty acid metabolism; might represent a predictive factor of breast cancer risk (37) FASN Fatty acid synthase Catalyzes the synthesis of long-chain saturated fatty acids; Implicated in prostate carcinogenesis (38)

ACSL3 Acyl-CoA synthetase Converts long-chain fatty acids into fatty acyl- long-chain family CoA esters for lipid biosynthesis member 3 ACLY ATP citrate lyase Converts citrate to acetyl-CoA for fatty acid synthesis; involved in lung cancer pathogenesis associated with metabolic abnormality (39); ACLY inhibition reduced tumor growth (40)

ACACA Acetyl-Coenzyme A The rate limiting step in fatty acid synthesis; carboxylase alpha silencing of ACACA induces growth inhibition and apoptosis of prostate cancer cells (41)

LDHA Lactate dehydrogenase Upregulated by c-myc (9); Elevated LDH is A associated with poor survival in cancer patients (42), including those of prostate cancer (43)

ACAT2 Acetyl-CoA Synthesizes cholesterol esters acetyltransferase 2 AMACR -Methylacyl-CoA A racemase that interconverts pristanoyl-CoA racemase between its stereoisomers; a molecular marker for prostate cancer (44) PFKFB4 6-Phosphofructo-2- Its product, fructose-2,6-bisphosphate, mediates kinase/fructose-2,6- allosteric activation of PFK-1; a prognostic biphosphatase 4 marker in bladder cancer (45)

Continued

Table 2.1: A subset of metabolic genes involved in the regulation of glycolysis, lactate synthesis, and fatty acid metabolism

Table 2.1: Continued

PKM2 Pyruvate kinase, muscle Tumors exclusively express isoform 2 the embryonic M2 isoform of pyruvate kinase, of which the low activity allows upstream intermediates of glycolysis to accumulate to promote the Warburg effect (30). GPI Glucose phosphate isomerase Converts glucose-6-phosphate to fructose-6-phosphate

PFKP Phospho-fructokinase-1, A rate-limiting enzyme in platelet glycolysis PFKM Phospho-fructokinase-1, A rate-limiting enzyme in muscle glycolysis PFKL Phospho-fructokinase-1, liver A rate-limiting enzyme in glycolysis ALDOA Aldolase A, fructose- A glycolytic enzyme whose bisphosphate expression is upregulated in cancers HK2 Hexokinase 2 The first irreversible step in glycolysis

Vehicle OSU-CG5 ALT (U/L) 31 ± 11.9 27.9 ± 10 AST (U/L) 85.3 ± 10.8 105.1 ± 63.3 ALKP (U/L) 52.7 ± 10.5 65.1 ± 17.9 GGT (U/L) 1 ± 1 0 ± 0 Total bilirubin (mg/dL) 0.43 ± 0.1 0.7 ± 0.4 BUN (mg/dL) 23 ± 4.4 19.7 ± 1.5 CREAT (mg/dL) 0.33 ± 0.06 0.27 ± 0.06 Calcium (mg/dL) 9.4 ± 0.3 9.9 ± 0.5 Phosphorous (mg/dL) 6.7 ± 1.8 4.7 ± 0.4 Albumin (g/dL) 3.5 ± 0.1 3.6 ± 0.1 Cholesterol (mg/dL) 157.4 ± 21.1 161 ± 32.6 Glucose (mg/dL) 174.5 ± 40.3 198 ± 23 WBC # (K/µL) 6.5 ± 4.2 8 ± 0.3 Neutrophil # (K/µL) 1.3 ± 0.9 1.6 ± 0.4 Lymphocyte # (K/µL) 4.7 ± 3 5.8 ± 0.5 Monocyte # (K/µL) 0.5 ± 0.3 0.6 ± 0.1 Eosinophil # (K/µL) 0.03 ± 0.03 0.01 ± 0.01 Basophil # (K/µL) 0 ± 0 0 ± 0 nucleated RBCs # (K/µL) 0 ± 0 0 ± 0 Hematocrit (%) 41.3 ± 2.9 42.2 ± 1.1 RBC (M/µL) 9.1 ± 0.7 9.3 ± 0.3 Hemoglobin (g/dL) 13.9 ± 0.9 14.5 ± 0.6 MCV (fL)a 45.5 ± 2.1 45.4 ± 1.2 MCHC (g/dL)b 33.8 ± 0.8 34.3 ± 0.9 RDW (%)c 13.7 ± 0.2 12.3 ± 0.2 Reticulocyte # (K/µL) 14.7 ± 8.9 0 ± 0 Platelet # (K/µL) 875.7 ± 76 964.7 ± 148.3 *P>0.05 for all values aMCV: Mean corpuscular volume; bMCHC: Mean corpuscular hemoglobin concentration; cRDW: Red cell distribution width

Table 2.2: Treatment of LNCaP-abl xenograft tumor baring mice with 100 mg/kg/day

OSU-CG5 for 59 days did not result in any serum biochemical or hematological abnormalities (n=3 mice/group)*

CHAPTER 3: SUPPRESSION OF PROSTATE EPITHELIAL PROLIFERATION AND

INTRAPROSTATIC PRO-GROWTH SIGNALING IN TRANSGENIC MICE BY A

NEW ENERGY RESTRICTION-MIMETIC AGENT

ABSTRACT

Cells undergoing malignant transformation often exhibit a shift in cellular metabolism from oxidative phosphorylation to glycolysis. This glycolytic shift, called the

Warburg effect, provides a mechanistic basis for targeting glycolysis to suppress carcinogenesis through the use of dietary caloric restriction and energy restriction- mimetic agents (ERMAs). We recently reported the development of a novel class of

ERMAs that exhibits high potency in eliciting starvation-associated cellular responses and epigenetic changes in cancer cells though glucose uptake inhibition. The lead ERMA in this class, OSU-CG5, decreases the production of ATP and NADH in LNCaP prostate cancer cells. In this study, we examined the effect of OSU-CG5 on the severity of preneoplastic lesions in male transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Daily oral treatment with OSU-CG5 at 100 mg/kg from six to ten weeks of age resulted in a statistically significant decrease in the weight of urogenital tract and microdissected dorsal, lateral, and anterior prostatic lobes relative to vehicle controls. The suppressive effect of OSU-CG5 was evidenced by marked decreases in Ki-67

immunostaining and proliferating cell nuclear antigen (PCNA) expression in the prostate.

OSU-CG5 treatment was not associated with evidence of systemic toxicity. Microarray analysis indicated a central role for Akt, and Western blot analysis showed reduced phosphorylation and/or expression levels of Akt, Src, androgen receptor, and insulin-like growth factor-1 receptor in prostate lobes. These findings support further investigation of

OSU-CG5 as a potential chemopreventive agent.

INTRODUCTION

In 1924, Otto Warburg reported that cancer cells preferentially metabolize glucose via glycolysis to lactate, even in the presence of adequate oxygen. This phenomenon, termed

“aerobic glycolysis,” results in the net production of 2 adenosine triphosphate (ATP) molecules per molecule of glucose, in contrast to the approximately 36 molecules produced per molecule of glucose directed into the tricarboxylic acid cycle and used for oxidative phosphorylation (1-7). The metabolic shift toward aerobic glycolysis provides cancer cells with growth advantages (3-7). For example, limiting ATP production to the glycolytic pathway permits diversion of intermediates into anabolic pathways to synthesize the nucleic acids, proteins, and fatty acids needed for extensive cell proliferation (3-7).

Although this metabolic adaptation provides growth advantages to cancer cells, it also presents opportunities to exploit the peculiarities of tumor cell metabolism for therapeutic purposes. The proof-of-concept for targeting energy metabolism for cancer chemoprevention is provided by the fact that inhibition of glycolysis through dietary

caloric restriction or treatment with energy restriction-mimetic agents (ERMAs) such as

2-deoxyglucose (2-DG) suppresses the growth of tumor xenografts and carcinogenesis in various animal models (8-12). To date, most of the animal studies that have assessed the anticancer effects of ERMAs have focused on late stages of cancer development or tumor growth, while their effects on the development and progression of pre-neoplastic conditions, such as prostatic intraepithelial neoplasia (PIN), remain largely undefined.

The prostates of transgenic adenocarcinoma of mouse prostate (TRAMP) mice undergo a series of pathologic changes that mirror those which occur in men (13-15).

Lesions develop progressively following the testosterone-dependent activation of the rat probasin promoter and expression of the SV40 large and small T antigens (T Ag) in the prostatic epithelium. Transgene expression results in inhibition of p53 and Rb tumor suppressors and development of prostate tumors (14-17). Prostates from 6-week old intact TRAMP mice typically exhibit varying degrees of PIN with the development of well-differentiated adenocarcinomas by approximately 18-weeks of age (13-14, 16, 18) and the emergence of poorly differentiated cancers with neuroendocrine differentiation at later time points. Although controversy exists regarding the histiogenesis of the poorly differentiated carcinomas, that is, whether they originate from epithelial cells within PIN lesions or from a distinct neuroendocrine stem cell population (19-20), this has little relevance to studies that focus on the PIN lesions in lieu of carcinomas.

Prostate epithelial proliferation in the PIN lesions of TRAMP mice has been shown to be amenable to modulation by dietary energy-restriction. Specifically, calorically restricting 7-week-old, intact male TRAMP mice for 4 weeks reduces prostate

pathology and accessory sex gland weights (21). Therefore, we chose to utilize TRAMP mice to investigate the in vivo efficacy of the novel ERMA OSU-CG5 in modulating preexisting PIN lesions. Our hypothesis was that ERMA treatment would reduce the severity of lesions in the prostates of 10-week-old TRAMP mice.

OSU-CG5 is a derivative of OSU-CG12 (Fig. 1A), a previously described ERMA with a potency that is three orders-of magnitude higher than that of 2-DG in inducing cell death (22). Both OSU-CG compounds elicit energy restriction-associated cellular responses by inhibiting glucose transporters in tumor cells (23). OSU-CG5 exhibits an improved potency relative to OSU-CG12 in suppressing the [3H]2-deoxyglucose uptake

(IC50, 6 µM versus 9 µM) and viability of human LNCaP prostate cancer cells (IC50, 4.5 versus 6 µM) (23-24). Here, we examine OSU-CG5’s ability to suppress cancer cell energy production in vitro and OSU-CG5’s in vivo efficacy and safety in the TRAMP mouse model system of prostate neoplasia at the preneoplastic stage.

MATERIALS AND METHODS

Cell Cultures and Reagents

Human LNCaP prostate cancer cells were obtained from the American Type Culture

Collection (Manassas, VA) and maintained with 10% fetal bovine serum and 1%

Penicillin/Streptomycin supplemented RPMI 1640 medium (Invitrogen, Carlsbad, CA).

No authentication of this cell line was performed. All cells were cultured at 37°C in a humidified incubator containing 5% CO2. OSU-CG5 was synthesized by our laboratory

(data not shown). OSU-CG5 was dissolved in DMSO and added to the culture medium with a final DMSO concentration of 0.1%.

ATP and NADH bioassays

The EnzyLightTM ATP Assay Kit (EATP-100) for rapid bioluminescent quantification of ATP and the EnzyChromTM NAD+/NADH Assay Kit (E2ND-100) were obtained from BioAssay Systems (Hayward, CA). LNCaP cells were cultured in the presence of DMSO or OSU-CG5 at the indicated concentrations for 24 h. Bioassays were performed according to the manufacturer’s instructions in triplicate.

In vivo study

TRAMP mice (C57BL/6 TRAMP x FvB) were bred and housed, and the presence of the transgene in each mouse was confirmed by PCR, as previously reported (25). Mice received a standard rodent diet and water ad libitum. All procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of The Ohio State University.

At 6 weeks of age, intact male transgenic mice were randomized to two groups that received either vehicle (n=20) or 100 mg/kg/day of OSU-CG5 (n=19). Additionally, 6- week-old intact male wild-type littermates (n=6/group) were also randomized into the same two groups. The test article was prepared as a suspension in sterile water containing

0.5% methylcellulose (w/v) and 0.1% Tween 80 (v/v). Suspensions of OSU-CG5 were prepared on a weekly basis and stored at room temperature. Mice received treatments

once daily via oral gavage under isoflurane anesthesia for the duration of the study (4 weeks). Body weights were measured weekly and at necropsy. At the termination of the study (age 10 weeks) mice were euthanized via carbon dioxide approximately 2-4 hours after they had received their last dose, and complete necropsies were performed.

Urogenital tracts (UGTs) were removed from all mice and weighed, after which relative

UGT weights (UGT weight/terminal body weight x 100%) were determined. The livers, kidneys, hearts, spleens and testes of each wild-type mouse were weighed, and relative organ weights were determined as described above for the UGT.

The individual lobes of the prostate were microdissected at necropsy and immersed in either RNAlater (Qiagen, Valencia, CA; transgenic mice only, n=9 for vehicle; n=8 for

OSU-CG5) or PBS (n=11 for each transgenic mouse treatment group, n=6 for each wild- type mouse treatment group). The dorsal and lateral lobes of prostates microdissected in

RNAlater were stored at 4°C overnight in RNAlater and then transferred to empty vials for storage at -80°C for subsequent RNA extraction. The dorsal, lateral, ventral, and anterior lobes microdissected in PBS were weighed individually. One lobe of each pair was snap-frozen in liquid nitrogen and stored at -80°C until needed for Western blot analysis of relevant biomarkers, while the other was fixed in neutral buffered 10% formalin (NBF). All other tissues were fixed in NBF.

At necropsy, serum was collected from 5 transgenic mice per group and submitted to

Anilytics Incorporated (Gaithersburg, MD) for determination of free and total testosterone concentrations via radioimmunoassay. Serum was also collected from 5 transgenic mice in the OSU-CG5 group and 4 transgenic mice in the vehicle treated

group for determination of liver enzyme values, including alanine aminotransferase

(ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALKP), and gamma- glutamyl transpeptidase (GGT), and total bilirubin by the Comparative Pathology and

Mouse Phenotyping Shared Resource (CPMPSR) at the Ohio State University. Serum was collected from 3 wild-type mice per group for determination of serum glucose, IGF-

1, and insulin concentrations. Serum glucose was measured by the CPMPSR. IGF-1 and insulin concentrations were determined using the IGF1 Mouse ELISA kit (ab100695) from Abcam Inc (Cambridge, MA) and Insulin (Mouse) ELISA kit (80-INSMS-E01) from ALPCO Diagnostics (Salem, NH). Each ELISA was performed according to the manufacturers’ instructions in duplicate.

Histopathology and scoring of prostatic lesions in the TRAMP mice

Five-micrometer-thick, paraffin-embedded sections of the lobes of the prostate

(n=11 transgenic mice/group) as well as selected organs (n=5 transgenic mice/group: liver, kidney, spleen, lung, heart, small intestine, testes, thymus, brain, eye, and bone marrow) were stained with hematoxylin and eosin (H&E) by standard procedures. All tissues were examined via light microscopy by a board-certified veterinary anatomic pathologist (LDBB) using an Olympus Model CHT research microscope (Olympus,

Center Valley, PA). The H&E-stained sections of the four lobes of the prostate of each transgenic mouse were evaluated and scored separately using a grading scheme (19) in which the most severe and most common lesions in each lobe were determined and assigned numerical scores. The sum of the scores of the most severe lesion and the most

common lesion, termed the “sum of the adjusted lesion scores” (SALS), was then obtained for each lobe. The average SALS as well as the average most severe and average most common lesion scores of each lobe were compared between groups.

Immunohistochemistry

To evaluate prostate and small intestinal epithelial proliferation, 5-micrometer-thick, paraffin-embedded tissue sections of the lobes of the prostate and cross-sections of small intestine were immunostained for Ki67 using a commercially available rabbit anti-human monoclonal antibody (clone SP6; catalog#RM-9106-S0; Thermo Fisher Scientific,

Fremont, CA). This primary antibody was applied at a dilution of 1:180. The immunohistochemistry staining protocol utilized the Avidin-Biotin Complex method with

DakoCytomation Target Retrieval Solution and a Decloaking Chamber (Biocare Medical,

Concord, CA) according to the manufacturer’s instructions, following the application of a protein block (DakoCytomation Serum-Free Protein Block, Dako, Carpinteria, CA). For each lobe of the prostate and section of small intestine, the number of Ki67-positive cells in three randomly selected 400X (i.e., high-power) fields was counted and divided by the total number of cells (Ki67-positive and -negative) in those fields (n=5/group) to yield the percentage of Ki67 immunopositive cells.

RNA isolation and microarray analysis

Using a tissue homogenizer (Tissue Tearor, Model 985370-395; BioSpec

Products, Inc., Bartlesville, OK) and TRIzol reagent® (Invitrogen), total RNA was

isolated from the combined dorsal and lateral prostate lobes of control and OSU-CG5- treated transgenic mice (n=3/group). After purification using the RNAeasy Mini Kit®

(Qiagen), the RNA was submitted to the Microarray Shared Resource at The Ohio State

University Comprehensive Cancer Center (OSU-CCC) for RNA quantification and microarray analysis of gene expression using Affymetrix Genechip Mouse Genome 430

2.0 Arrays (Affymetrix, Santa Clara, CA). Microarray data was deposited in NCBI’s

Gene Expression Omnibus (GEO), and can be accessed via accession number GSE32422.

Protein isolation and western blot analysis

For western blot analysis of intra-prostatic biomarkers, the dorsal and lateral prostate lobes of individual transgenic mice were combined and pulverized in liquid nitrogen

(n=5/group). SDS lysis buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA) containing 1 × protease inhibitor cocktail (Sigma, St. Louis, MO) and PhoSTOP phosphatase inhibitor cocktail (Roche, Basel, Switzerland) was added to the crushed tissue. The resulting lysates were sonicated until clear and centrifuged at 16,100 x g for

15 minutes. Protein concentrations in the supernatants were determined using the Micro

BCATM protein assay (Pierce Chemical, Rockford, IL). Proteins were separated by 1- dimensional electrophoresis in 8 ~ 12% SDS polyacrylamide gels and transferred onto nitrocellulose membranes. After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies (see below) at 1:1,000 dilution in TBS-Tween 20 overnight at 4°C. The protein bands were developed using horseradish peroxidase- conjugated secondary antibodies at 1:5,000 dilution in the same buffer for 1 hour at room

temperature. Bands were visualized on X-ray film using an enhanced chemiluminescence system. Densitometric analysis of protein bands was performed using ImageJ software to determine the relative intensities of protein expression in drug-treated samples versus those of vehicle-treated controls after normalization to the internal reference protein β- actin.

The target proteins and commercial sources of the antibodies for several biomarkers are given here. Mouse monoclonal antibodies directed against β-actin were from MP

Biomedicals (Irvine, CA), Src from Calbiochem (Darmstadt, Germany), or SV40 T Ag from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies came from two sources. Reagents directed against insulin-like growth factor-1 receptor (IGF-

1R), androgen receptor (AR), and proliferating cell nuclear antigen (PCNA) were acquired from Santa Cruz. Antibodies directed against Akt, p-Akt-Ser473, PTEN, c-

Myc, glycogen synthase kinase 3β (GSK3β), and p-GSK3β were obtained from Cell

Signaling (Beverly, MA).

Statistical Analysis

Data was assessed for normality using the Shapiro-Wilk normality test. All data was found to be normally distributed except the Ki67 values for the ventral and anterior lobes of the prostate and the small intestine; the SALS for the dorsal, lateral, and anterior lobes of the prostates; the serum concentrations of total and free testosterone, glucose, IGF-1, and insulin; the serum activities of liver enzymes; and the relative weights of the heart, kidney, and spleen. For the normally distributed data, differences between group means

were analyzed for statistical significance using the Student’s t-test. For the data that was not normally distributed, statistical significance was evaluated using the Wilcoxon rank sum test. Differences were considered to be significant at P <0.05. Microarray data was submitted to The OSU-CCC Biomedical Informatics Shared Resource for statistical analysis, using the Bioconductor microarray analysis package, utilizing the Student’s t- test. Fold changes and differences in gene expression profiles were considered significant at P <0.05. Microarray pathways were analyzed using Ingenuity pathway analysis (IPA) software (Ingenuity Systems). Only genes with greater than 1.5-fold up- or down- regulation and P <0.05 were selected for pathway analysis. All p values were two-sided, except for those of the Western blots.

RESULTS

Suppressive Effects of OSU-CG5 on LNCaP cell ATP and NADH production

Previously, we demonstrated that OSU-CG5, a structurally optimized derivative of

OSU-CG12, blocked glucose uptake in LNCaP cells by blocking glucose transporters

(23-24). The consequent effect on energy production was manifested by its ability to lower the levels of ATP and NADH in a dose-dependent manner after 24 h of treatment.

At 5 µM, OSU-CG5 reduced ATP and NADH production by 58 ± 1% (P<0.0001) and 28

± 1% (P<0.0001), respectively, relative to the DMSO control (Fig. 1B).

OSU-CG5 decreased prostate weight and prostate epithelial cell proliferation in transgenic TRAMP mice

To investigate the effect of OSU-CG5 on the progression of pre-neoplastic lesions,

6-week-old TRAMP mice were treated once daily with OSU-CG5 (100 mg/kg, p.o., n=19) or vehicle (n=20) for 4 weeks. As UGT weight has previously been shown to correlate significantly with prostate lesion severity and progression (13, 26), the average

UGT weights of each group were compared. OSU-CG5 treatment resulted in 12% and

11% reductions in the absolute UGT weight (332 ± 40 mg versus 377 ± 37 mg in controls; P=0.001) and relative UGT weight (1.24 ± 0.14% versus 1.39 ± 0.11% in controls; P=0.001) (Fig. 2A). Moreover, the weights of the individual prostate lobes of the OSU-CG5-treated mice were decreased relative to vehicle controls (n=11 mice/group): dorsal, 25.7% (P=0.013); lateral, 31.5% (P=0.003); ventral, 10% (P=0.413); anterior, 16.5% (P =0.026) (Fig. 2B). Importantly, these reductions in weight were not associated with decreased SV40 large T Ag expression in the prostate (n=5 mice/group), as Western blot analysis indicated that the amount of SV40 oncoprotein in the combined dorsal and lateral lobes was comparable in both treatment groups (Fig. 2C).

The suppressive effect of OSU-CG5 on the UGT weight was linked to its ability to inhibit the proliferation of prostate epithelial cells. OSU-CG5 treatment led to a marked decrease in the proliferation index, as indicated by Ki-67 immunoreactivity, in all four lobes of the prostate (P<0.05 for all lobes; n=5 mice/group) (Fig. 3A & B). Western blot analysis indicated that OSU-CG5 decreased the expression level of PCNA in the

combined dorsal and lateral lobes by 32% (P=0.002) relative to the vehicle (n=5 mice/group) (Fig. 3C).

Histopathologic examination and scoring of each lobe of the prostate was performed

(n=11 mice/group), and the sum of the adjusted lesion score (SALS) for each lobe was determined. The average SALS of the dorsal, lateral, ventral, and anterior lobes of the prostates from the vehicle-treated mice were 9.5 ± 1.4, 9.0 ± 4.4, 6.0 ± 2.1, and 4.5 ± 3.0, respectively, while the average SALS for these lobes in OSU-CG5-treated mice were 9.1

± 1.8, 7.5 ± 1.0, 5.6 ± 2.5, and 5.5 ± 3.0. No statistical differences were found in the average SALS of each lobe or the average scores for the most severe or most common lesions in each lobe (Supplementary Table 1).

Testosterone radioimmunoassays indicated that the serum concentrations of free and total testosterone were 0.21 ± 0.11 pg/ml and 0.33 ± 0.09 ng/ml, respectively, for

OSU-CG5-treated mice (n=5 mice/group) versus 3.4 ± 4.6 pg/ml and 1.6 ± 2.0 ng/ml, respectively, for vehicle-treated mice (n=5 mice/group). Despite 93.7% and 79.7% decreases in the free and total testosterone, respectively, these reductions were not statistically significant (P>0.05), in part due to large variations in the testosterone concentrations of vehicle controls. This wide variation in the serum testosterone level, however, is in line with the reported range of 1–90 pg/ml in nude mice (27).

OSU-CG5 treatment was not associated with any evidence of systemic toxicity

No overt toxicity was noted with OSU-CG5, as the drug-treated transgenic group gained a similar amount of weight to that of the control group after 4 weeks of treatment

(Table 1). Similarly, there were no significant changes in the absolute or relative weights of the UGT, liver, kidneys, hearts, or testes (Table 1), in the wild-type mice (n=6 mice/group; P>0.05). Histological evaluation of H&E-stained sections of metabolically active organs including liver, kidney, spleen, lung, heart, small intestine, testes, thymus, brain, eye, and bone marrow (n=5 mice/group) did not reveal any lesions consistent with systemic or organ-specific toxicity. Although OSU-CG5 treatment was associated with mild splenomegaly with slight increases in the average absolute and relative weights of the spleen (P<0.05; Table 1), there were no associated histologic lesions. There was no biochemical evidence of hepatotoxicity as the concentrations of various hepatic biomarkers, including ALT, AST, ALKP, and GGT, and total bilirubin were not affected by 4 weeks of OSU-CG5 treatment (vehicle: n=4 mice; OSU-CG5: n=5 mice) (Table 1).

Moreover, analysis of the proliferation index of small intestinal epithelial cells found no significant differences in the percentage of Ki-67 immunopositive cells between these two groups of mice (n=5 mice/group) (Table 1). Together, these findings indicate that

OSU-CG5 treatment was not associated with any detectable systemic toxicity.

OSU-CG5-mediated suppression of prostate epithelial proliferation in transgenic

TRAMP mice was associated with modulation of gene expression in the combined dorsal and lateral lobes of the prostate

Earlier studies indicate that caloric restriction and 2-DG suppress carcinogenesis by perturbing cellular signaling and/or gene expression profiles (9-10, 12, 28-30).

Microarray analysis was performed using RNA from the combined dorsal and lateral

prostate lobes of OSU-CG5- and vehicle-treated animals (n=3 mice/group). Using IPA software, the top five gene networks affected by OSU-CG5 treatment were identified

(Supplementary Table 2). Of these, we focused on the “Cellular Assembly and

Organization, Cellular Function and Maintenance, Gene Expression” because of the likely role of genes in this network in maintaining cellular homeostasis and suppression of tumorigenesis (Supplementary Table 2). The pathway network is shown in Figure 4.

Akt appeared to have a central role in this constellation, with 5 genes in the network interacting directly with Akt and 5 genes interacting indirectly with Akt. In light of this information and due to the fact that an earlier study demonstrated upregulation of Akt signaling, as manifested by dramatically increased phosphorylation of Akt and GSK3, in proliferative prostate epithelium in TRAMP mice with PIN (25), we investigated the phosphorylation status of Akt and GSK3 in the combined dorsal and lateral lobes of prostates of OSU-CG5- versus vehicle-treated mice. As shown in Figure 5, OSU-CG5 treatment significantly reduced phospho-Ser473-Akt and phospho-GSK3β levels by 50%

(P=0.012) and 45% (P=0.043), respectively, relative to the vehicle-treated controls.

The microarray data also showed changes in the expression of genes encoding proteins of potential therapeutic relevance for prostate cancer. OSU-CG5 exhibited suppressive effects on the expression of the Rous sarcoma oncogene (v-src) (-1.44-fold,

P=0.007), and its cellular counterpart, the proto-oncogene c-src (-1.36-fold, P=0.012).

Down-regulation of Src represents a therapeutically relevant target in prostate cancer

(31). Additionally, OSU-CG5 decreased the expression of the Myc-like oncogene, s-myc

(-1.32-fold, P=0.007) and upregulated the expression of PTEN (1.25-fold, P=0.033).

Further review of the data revealed a number of other up-regulated genes with central roles in suppressing tumorigenesis and down-regulated genes with roles tumor development and progression. These genes, categorized according to mechanism of action, are found in supplementary Tables 3 and 4.

Considering the importance of Src, myc, and PTEN in prostate cancer pathogenesis, as well as the importance of growth factor signaling through IGF-1R and AR for prostate epithelial proliferation, we assessed the expression levels of these proteins by Western blotting (n=5/group). OSU-CG5 significantly reduced the expression of Src, AR, and

IGF-1R by 28% (P=0.001), 26% (P=0.036), and 47% (P=0.001), respectively, while no significant changes were noted in the protein levels of PTEN or c-Myc (Figure 5).

Despite reduced intraprostatic IGF-1R expression, OSU-CG5 did not affect glucose homeostasis as no significant changes (P>0.05) in the serum levels of glucose, IGF-1, or insulin were noted in drug-treated mice. Concentrations of these serum parameters in vehicle- and OSU-CG5-treated mice were as follows, respectively: glucose, 279 ± 11 and

288 ± 26 mg/dL; IGF-1, 2.7 ± 0 and 2.7 ± 0.1 ng/ml; insulin, 0.92 ± 0.23 and 0.94 ± 0.29 ng/ml. These results suggest that OSU-CG5’s ERMA activity was restricted to the proliferating cells within the PIN lesions and did not affect whole body energy metabolism.

DISCUSSION

The adaptation of cancer cells to preferentially utilize aerobic glycolysis is an early step in carcinogenesis and presents an opportunity for therapeutic exploitation. The

efficacy of energy restriction as a chemotherapeutic or chemopreventive strategy has been demonstrated by the finding that inhibiting glycolysis through dietary caloric restriction or by administration of 2-DG suppresses xenograft tumor growth and carcinogenesis in various animal models (8-12). However, dietary caloric restriction is not an easy intervention for human cancer patients, and previous work by our laboratory has demonstrated that 2-DG has relatively low in vitro cytotoxic activity (22).

Additionally, ERMAs currently in clinical trials have not been without serious side effects. For example, a clinical trial utilizing 2-DG to treat advanced prostate cancer

(NCT00633087) was stopped by the U.S. Food and Drug Administration because of concerns regarding hepatotoxicity (32). Therefore, new ERMAs with higher potency and improved in vivo safety are needed.

Although chronic dietary restriction (21, 33) or intermittent caloric restriction (34) has been shown to retard prostate lesion development in the TRAMP mouse model, we are aware of only one study that included an evaluation of caloric restriction on the progression of pre-neoplastic lesions (21). Studies in TRAMP mice have found that 20% caloric restriction initiated at approximately 7 weeks of age had a greater effect on the suppression of lesion development than 20% caloric restriction started at a later time point (20 weeks) (21, 33). This result implies that the age at which the dietary intervention is initiated influences its effectiveness. Additionally, intermittent caloric restriction delayed prostate tumor development longer than chronic caloric restriction when both were begun at the same age (34). Given the results from these studies, it seems

logical to examine the effect that an ERMA administered beginning at puberty could have on the severity of PIN lesions.

In this study, we showcase the ability of OSU-CG5, a novel glucose transporter inhibitor (23-24), to modify the early lesions of TRAMP mice. OSU-CG5 reduced the weights of the UGTs as well as the dorsal, lateral, and anterior lobes of prostates by suppressing prostate epithelial proliferation. As the UGT weight correlates with lesion severity and progression in TRAMP mice (13, 26), the effect of OSU-CG5 on UGT weights underscores this ERMA’s ability to reduce the prostate pathology within these mice. The lack of statistically significant changes in the histopathologic lesion scores of the individual prostate lobes in this study likely reflects the fact that the traditional scoring criteria for mouse prostatic cancer models are weighted to the assessment of later-stage neoplastic lesions rather than to the early PIN changes.

Importantly, our data indicate that OSU-CG5 is well tolerated. Chronic oral administration of OSU-CG5 resulted in no untoward (i.e., “toxic”) effects in metabolically active organs with naturally high glycolytic rates, including the liver, kidney, small intestine, brain, and eye, did not impact the proliferation of a non- neoplastic tissue (the small intestine), and did not induce elevations in serum activities of liver enzymes. Taken together, these animal data underscore OSU-CG5’s translational potential.

In this study, we also investigated multiple mechanisms by which OSU-CG5 acts to decrease prostate epithelial proliferation. For example, treatment with OSU-CG5 resulted in decreased AR expression, which accords well with our previous finding that

the ERMA OSU-CG12 suppressed AR expression in LNCaP cells by down-regulating expression of the transcription factor Sp1 (22). In light of the pivotal role of AR signaling in prostate carcinogenesis and tumor progression (35), down-regulation of AR expression likely contributed, at least in part, to the decreased prostate epithelial proliferation observed in this study.

Despite decreased AR expression levels in the prostate, there was no evidence of testicular atrophy in OSU-CG5-treated wild-type mice as suggested by lack of histologic lesions within the testes or changes in testicular weights. Together with the finding that

OSU-CG5 did not reduce the wild-type UGT weights, these data imply that OSU-CG5 does not act as an anti-androgen. Although the decrease in serum testosterone concentrations in OSU-CG5-treated mice was not statistically significant due to a high degree of inter-individual variability, caloric restriction has been reported to decrease circulating testosterone (36). Therefore, the decreased testosterone level in this study might represent a cellular response to OSU-CG5 as it mimics caloric restriction in vivo.

Any decrease in serum testosterone may have potentiated the effect of the decreased AR.

Further studies are needed to determine any effect that OSU-CG5 may have had on the hypothalamic-pituitary hormonal axis that control gonadal function, especially after prolonged therapy. Interestingly, lesion progression in TRAMP mice has previously been shown to be relatively resistant to reductions in serum testosterone, with 80% of TRAMP mice castrated by 12 weeks of age developing prostate tumors by 24 weeks of age (37).

Moreover, OSU-CG5 inhibited the IGF-1R/PI3K/Akt signaling axis in the combined dorsal and lateral lobes of prostates. The microarray data suggested a central

100

role for Akt in the top network modulated by OSU-CG5, and we subsequently showed the suppressive effect of OSU-CG5 on phosphorylation of Akt and its target GSK3.

This finding is in accordance with a recent report that calorically restricting Hi-Myc transgenic mice reduces the incidence of prostatic adenocarcinomas and the phosphorylation level of Akt (38).

From a mechanistic perspective, the ability of OSU-CG5 to inhibit Akt signaling might, in part, be attributable to the observed downregulation of intraprostatic IGF-1R expression, which would have led to decreased IGF-1/IGF-1R signaling. Down- regulation of IGF-1/IGF-1R signaling has been shown to be a major mechanism underlying the effect of caloric restriction on tumor suppression (39). Despite reduced intraprostatic IGF-1R expression, there was no significant change in the serum level of

IGF-1 in the OSU-CG5-treated wild-type cohort. Together with the lack of changes in serum glucose and insulin, this finding lends credence to our hypothesis that OSU-CG5 targets neoplastic cell energy metabolism and does not affect global glucose homeostasis.

There is also conflicting information regarding the effect of caloric restriction on IGF-1 levels in TRAMP mice. Although intermittent caloric restriction has been shown to reduce serum IGF-1 levels (34), no significant changes in serum IGF-1 was reported in

TRAMP mice receiving chronic caloric restriction (33).

Although we believe that we have demonstrated the efficacy of OSU-CG5 in suppressing PIN epithelial proliferation in vivo, our study was not without limitations.

Since the focus of this study was PIN and the mice were euthanized before tumors developed, we were unable to evaluate how OSU-CG5 treatment might influence prostate

101

carcinoma development and the pattern and rate of metastasis. Such an investigation of later-stage neoplastic lesions is currently underway. In addition, the TRAMP model may not be the best transgenic mouse model in which to evaluate the development of prostate carcinomas since controversy exists regarding the exact histogenesis of the poorly differentiated carcinomas that represent the majority of end-stage tumors. Furthermore, although statistically significant, the effect of OSU-CG5 on the weights of UGT and prostate lobes were relatively modest compared to the effect on prostate epithelial cell proliferation (as determined by Ki-67 and PCNA) and biomarker modulation. A possible explanation for this disconnect is that OSU-CG5-mediated changes in proliferation may precede more substantial differences in lobe weights. Further work is needed to define the regimen and manner in which ERMAs like OSU-CG5 may be introduced into the clinical armamentarium for treating prostatic and other cancers.

In summary, our study demonstrated that oral administration of OSU-CG5 suppressed the proliferation of prostate epithelial cells in TRAMP mice without evidence of toxicity or modulation of whole body glucose homeostasis. Microarray and Western blot data revealed that OSU-CG5 targeted proliferating pre-neoplastic cells via multiple mechanisms that include the modulation of cell survival and proliferation pathways and interference with cellular energy metabolism. This range of antitumor activities in the absence of toxicity suggested the translational potential of OSU-CG5 as a chemopreventive agent, warranting further investigation in this regard.

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expression of a heterologous gene specifically to the prostate in transgenic mice. Molecular Endocrinology. 1994;8:230-9. 18. Greenberg NM. Transgenic Models for Prostate Cancer Research. Urol Oncol. 1996;2:119-22. 19. Berman-Booty LD, Sargeant AM, Rosol TJ, Rengel RC, Clinton SK, Chen CS, et al. A Review of the Existing Grading Schemes and a Proposal for a Modified Grading Scheme for Prostatic Lesions in TRAMP Mice. Toxicol Pathol. 2012; 40 (1): 5-17. 20. Chiaverotti T, Couto, S.S., Donjacour, A., Mao, J.H., Nagase, H., Cardiff, R.D., Cunha, G.R., Balmain, A. Dissociation of Epithelial and Neuroendocrine Carcinoma Lineages in the Transgenic Adenocarcinoma of Mouse Prostate Model of Prostate Cancer. The American Journal of Pathology. 2008;172:236-46. 21. Suttie A, Nyska, A., Haseman, J.K., Moser, G.J., Hackett, T.R., Goldsworthy, T.L. A Grading Scheme for the Assessment of Proliferative Lesions of the Mouse Prostate in the TRAMP Model. Toxicologic Pathology. 2003;31:31-8. 22. Wei S, Kulp SK, Chen CS. Energy restriction as an antitumor target of thiazolidinediones. J Biol Chem. 2010;285:9780-91. 23. Wang D, Chu PC, Yang CN, Yan R, Chuang YC, Kulp SK, et al. Development of a novel class of glucose transporter inhibitors. J Med Chem. 2012;55:3827-36. 24. Lin HY, Kuo YC, Weng YI, Lai IL, Huang TH, Lin SP, et al. Activation of silenced tumor suppressor genes in prostate cancer cells by a novel energy restriction- mimetic agent. Prostate. 2012; 72(16):1767-78 25. Sargeant AM, Klein, R.D., Rengel, R.C., Clinton, S.K., Kulp, S.K., Kashida, Y., Yamaguchi, M., Wang, X., Chen, C.S. Chemopreventive and bioenergetic signaling effects of PDK1/Akt pathway inhibition in a transgenic mouse model of prostate cancer. Toxicologic Pathology. 2007;35:549-61. 26. Kee K, Foster BA, Merali S, Kramer DL, Hensen ML, Diegelman P, et al. Activated polyamine catabolism depletes acetyl-CoA pools and suppresses prostate tumor growth in TRAMP mice. J Biol Chem. 2004;279:40076-83. 27. van Steenbrugge GJ, van Dongen JJ, Reuvers PJ, de Jong FH, Schroeder FH. Transplantable human prostatic carcinoma (PC-82) in athymic nude mice: I. Hormone dependence and the concentration of androgens in plasma and tumor tissue. Prostate. 1987;11:195-210. 28. Kritchevsky D. Caloric restriction and experimental carcinogenesis. Hybrid Hybridomics. 2002;21:147-51. 29. Zhu Z, Jiang W, McGinley JN, Thompson HJ. 2-Deoxyglucose as an energy restriction mimetic agent: effects on mammary carcinogenesis and on mammary tumor cell growth in vitro. Cancer Res. 2005;65:7023-30. 30. Liao Z, Wang S, Wiegers BS, Clinton SK. Energy balance alters dunning R3327- H prostate tumor architecture, androgen receptor expression, and nuclear morphometry in rats. Prostate. 2006;66:945-53. 31. Fizazi K. The role of Src in prostate cancer. Ann Oncol. 2007;18:1765-73. 32. Omar HA, Berman-Booty L, Kulp SK, Chen CS. Energy restriction as an antitumor target. Future Oncol. 2010;6:1675-9.

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33. Suttie AW, Dinse GE, Nyska A, Moser GJ, Goldsworthy TL, Maronpot RR. An investigation of the effects of late-onset dietary restriction on prostate cancer development in the TRAMP mouse. Toxicol Pathol. 2005;33:386-97. 34. Bonorden MJ, Rogozina OP, Kluczny CM, Grossmann ME, Grambsch PL, Grande JP, et al. Intermittent calorie restriction delays prostate tumor detection and increases survival time in TRAMP mice. Nutr Cancer. 2009;61:265-75. 35. Dutt SS, Gao AC. Molecular mechanisms of castration-resistant prostate cancer progression. Future Oncol. 2009;5:1403-13. 36. Levay EA, Tammer AH, Penman J, Kent S, Paolini AG. Calorie restriction at increasing levels leads to augmented concentrations of corticosterone and decreasing concentrations of testosterone in rats. Nutr Res. 2010;30:366-73. 37. Gingrich JR, Barrios, R.J., Kattan, M.W., Nahm, H.S., Finegold, M.J., Greenberg, N.M. Androgen-independent Prostate Cancer Progression in the TRAMP Model. Cancer Research. 1997;57:4687-91. 38. Blando J, Moore T, Hursting S, Jiang G, Saha A, Beltran L, et al. Dietary energy balance modulates prostate cancer progression in Hi-Myc mice. Cancer Prev Res (Phila). 2011;4:2002-14. 39. Powolny AA, Wang S, Carlton PS, Hoot DR, Clinton SK. Interrelationships between dietary restriction, the IGF-I axis, and expression of vascular endothelial growth factor by prostate adenocarcinoma in rats. Mol Carcinog. 2008;47:458-65.

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Figure 3.1

Evidence that OSU-CG5 reduces energy production (A) Chemical structures of OSU-

CG12 and OSU-CG5. (B) Dose-dependent suppressive effects of OSU-CG5 on ATP and

NADH levels in LNCaP cells after 24 h of treatment. The analyses were performed in triplicate according to procedures described under Materials and Methods.

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Figure 3.2

OSU-CG5 reduced prostate weight in TRAMP mice. Six-week-old, intact male TRAMP mice were treated orally with OSU-CG5 (100 mg/kg) once daily for 4 weeks. The weights of UGTs from each mouse were determined, as well as the weights of individual prostate lobes after microdissection. Treatment with OSU-CG5 caused statistically significant reductions in (A) absolute and relative UGT weights and (B) the weights of the dorsal, lateral, and anterior lobes of the prostate. (C) Drug treatment had no effect on the expression level of the SV40 large T antigen in the combined dorsal and lateral prostates (5 mice/group) as determined by western blotting. In (A) and (B), bars represent the mean ± SD. DP, dorsal lobe of the prostate; LP, lateral lobe of the prostate; AP, anterior lobe of the prostate; VP, ventral lobe of the prostate; CG5, OSU-CG5.

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Figure 3.3: OSU-CG5 reduced prostate epithelial proliferation in TRAMP mice.

Proliferation within the prostates of TRAMP mice treated with OSU-CG5

(100/mg/kg/day) or vehicle for 4 weeks was assessed by immunohistochemistry for Ki67

(A, B) and western blotting for PCNA (C). (A) Photomicrographs showing Ki67 immunoreactivity in the dorsal and lateral lobes of representative vehicle- and OSU-

CG5-treated TRAMP mice (400X). (B) The percentages of cells that were immunopositive for Ki67 in each prostate lobe were reduced in OSU-CG5-treated

TRAMP mice relative to vehicle-treated controls (n = 5 mice/group). (C) Western blot analysis shows that OSU-CG5 treatment reduced the expression levels of PCNA in the combined dorsal and lateral lobes prostates of TRAMP mice relative to vehicle-treated controls (5 mice/group). In (B) and (C), bars represent the mean ± SD. DP, dorsal lobe of the prostate; LP, lateral lobe of the prostate; AP, anterior lobe of the prostate; VP, ventral lobe of the prostate; Ctl, vehicle control; CG5, OSU-CG5.

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Figure 3.3

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Figure 3.4: Gene network in the combined dorsal and lateral lobes of prostate of

TRAMP mice treated for 4-weeks with 100mg/kg/day of OSU-CG5 was created using

Ingenuity pathway analysis software. We focused on the network that was described as being involved in “Cellular Assembly and Organization, Cellular Function and

Maintenance, Gene Expression.” Akt appears to have a central role in this pathway.

Genes are represented as nodes, with up-regulated genes colored red and down-regulated genes colored green. The intensity of the color indicates the degree of up-or down- regulation. Genes that are not colored were not in the analyzed data set. The biological relationships between genes are indicated by lines; solid lines represent direct interactions whereas dashed lines represent indirect interactions. Arrowheads indicate the directionality of interaction, and lines without arrowheads indicate binding. Lines that begin and end at the same gene indicate auto-regulation. =Transporter, =Enzyme,

=Kinase, =Phosphatase, =Transcription regulator, =Other,

=Complex/group, =Peptidase.

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Figure 3.4

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Figure 3.5

(A) Western blot analysis of the effects of OSU-CG5 on the phosphorylation of Akt-Ser-

473 and GSK3β and the expression of several pro-survival factors (Src, AR, IGF-1R, c-

Myc) and a tumor suppressor (PTEN) in the combined dorsal and lateral lobes of prostates in TRAMP mice treated with OSU-CG5 (100/mg/kg/day) or vehicle for 4 weeks. (B) Densitometric analysis of protein bands was performed to calculate relative phosphorylation/expression levels of individual proteins. Bars represent the mean ± SD.

Ctl, vehicle control; CG5, OSU-CG5; AR, Androgen Receptor; IGF-1R, Insulin-like growth factor-1 receptor. 112

Vehicle-treated OSU-CG5-treated TRAMP mice: Body weight gain (g): 2.0 ± 1.0 (n = 20) 2.2 ± 1.1 (n = 19)

Wild-type littermates of TRAMP mice: Organ weight (mg) (n = 6 for both groups) UGT 351 ± 60 355 ± 53 (% body weight) (1.31 ± 0.15) (1.29 ± 0.15) Liver 1303 ± 124 1372 ± 124 (% body weight) (4.89 ± 0.31) (5.00 ± 0.33) Kidney 408 ± 28 450 ± 36 (% body weight) (1.54 ± 0.14) (1.64 ± 0.04) Heart 164 ± 19 173 ± 41 (% body weight) (0.62 ± 0.07) (0.63 ± 0.13) Spleen 72 ±6 82 ± 8 (% body weight) (0.27 ± 0.02) (0.3 ± 0.02) Testes 204 ± 21 221 ± 16 (% body weight) (0.76 ± 0.07) (0.81 ± 0.09)

TRAMP Mice: Liver function (n = 4 for vehicle; n = 5 for OSU-CG5) ALT (U/L) 22 ± 5 19 ± 2 AST (U/L) 89 ± 75 50 ± 16 ALKP (U/L) 54 ± 6 63 ± 12 GGT (U/L) 4 ± 2 7 ± 2 Total bilirubin (mg/dL) 0.3 ± 0.1 0.3 ± 0.1

TRAMP Mice: Cell proliferationa in non-neoplastic tissues (n = 5 for both groups) Small intestinal epithelium 48.2 ± 4.1 52.9 ± 6.8 * Statistically equivalent for OSU-CG5-treated and vehicle-treated mice, p> 0.05, for all values except absolute and relative weights of the spleen a Proliferation evaluated as % of Ki-67-immunopositive cells

Table 3.1: Evidence that OSU-CG5 caused no systemic toxicity in young adult mice after 4 weeks of treatment

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CHAPTER 4: THE NOVEL ENERGY-RESTRICTION MIMETIC AGENT OSU-CG5

REDUCES PROSTATE CANCER SEVERITY IN A TRANSGENIC MOUSE MODEL

OF PROSTATE CANCER

ABSTRACT

Dietary caloric restriction (CR) is one of the most effective experimental chemotherapeutic and chemopreventive strategies to date. However, because CR is not a practical treatment strategy for human cancer patients, drugs that can mimic CR at the cellular level are needed. These compounds are termed energy restriction-mimetic agents

(ERMAs). We have previously shown that 100 mg/kg/day of the ERMA OSU-CG5 decreases prostate epithelial proliferation within prostatic intraepithelial neoplasia lesions of transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, resulting in decreased urogenital tract and prostate lobe weights. In our current study, we evaluated

OSU-CG5’s ability to act as a chemopreventive agent. While administration of 1286 ppm

(approximately 100 mg/kg/day) of OSU-CG5 via an AIN-76A diet to intact male

TRAMP mice for 18 weeks did not alter the incidence of poorly differentiated prostate carcinomas or the age at which these tumors developed, OSU-CG5 reduced prostate disease severity, as determined by tumor volume and mass. Namely, the tumors that

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developed in OSU-CG5-treated mice were 54.6% smaller by volume and 54.1% smaller by mass than the control mouse tumors. The reduction in tumor size was associated with decreased tumor cell proliferation, as determined by Ki67, and reductions in androgen receptor and phosphorylated-Akt levels within the OSU-CG5 treated tumors. OSU-CG5 administered in the diet was well tolerated and did not cause systemic toxicity. Given

OSU-CG5’s ability to reduce prostate tumor size, OSU-CG5 may have potential as a chemotherapeutic agent for men with prostate cancer.

INTRODUCTION

Dietary caloric restriction (CR) is one of the most effective experimental chemotherapeutic and chemopreventive strategies. In multiple studies, CR decreases the growth of xenograft tumors, suppresses the development of spontaneous and chemically induced tumors, and modulates carcinogenesis in genetically engineered mice (1-9).

Although most experimental work with CR has been performed utilizing rodent models of cancer, recently similar results have been observed in non-rodent species. Namely, chronic 30% CR reduces the incidence of spontaneous gastrointestinal adenocarcinomas in rhesus macaques (5). The mechanisms through which caloric restriction suppresses carcinogenesis have not been fully elucidated, but they likely include decreased cell proliferation and increased apoptosis. Decreased proliferation and increased apoptosis have been suggested to be due in part to down-regulation of the insulin like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) signaling axis with resultant decreased

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phosphatidylinositol-3 kinase (PI3K)/Akt signaling and concurrent activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) (1-3). These mechanisms have been more fully evaluated in vitro. For example, in prostate and breast cancer cells, glucose deprivation results in downregulation of cell surface receptors involved in cell survival and growth, such as the androgen receptor (AR) and epidermal growth factor receptor (EGFR), decreased activation of pro-growth kinases including

Akt, induction of an endoplasmic reticulum (ER) stress response, activation of AMPK, and increased Sirtuin 1 (Sirt1) expression (10).

Typically, animals in most CR studies are fed between 60-80% of the calories that the ad libitum fed controls receive. In other words, they are calorically restricted by approximately 20-40% (1). Although this extent of CR may be feasible in a healthy individual, this degree of CR is not practical in human cancer patients. Therefore, compounds that can induce a response that mimics that of actual CR at the cellular level and results in decreased tumor growth at the whole animal level without concurrent weight loss need to be developed.

Compounds that can induce a cellular response that mimics energy deprivation are termed energy restriction-mimetic agents (ERMAs). ERMAs that have been used in animal models of cancer include 2-deoxyglucose (2-DG) (11), resveratrol (12), and

OSU-CG5 (13). 2-DG inhibits the glycolytic enzyme phosphohexose isomerase, thereby downregulating glycolysis and mimicking energy restriction (1,14). Resveratrol is a phytoalexin that reduces cancer cell glucose utilization in part by activating AMPK (14), inhibiting the mammalian target of rapamycin (mTOR), and decreasing the expression of 116

the glycolytic enzyme pyruvate kinase M2 (15). OSU-CG5, like other members of the

OSU-CG family of compounds, is a glucose uptake inhibitor (16). In vitro, the OSU-CG compounds are more potent than 2-DG and resveratrol in reducing prostate cancer cell viability. Additionally, the ability of OSU-CG compounds to induce a response similar to that of glucose deprivation is well documented (10).

In addition to its in vitro activity, OSU-CG5 has demonstrated efficacy in both xenograft (data not shown) and transgenic mouse models of prostate cancer (13), namely the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Briefly, TRAMP mouse prostates progressively develop a series of lesions similar to those of adult and elderly men, but at a much more rapid rate (17-19). Specifically, the initial lesions are various degrees of prostatic intraepithelial neoplasia (PIN), which first appear at approximately 6 weeks of age. As the mice age, there is neoplastic transformation and local invasion with the development of well differentiated adenocarcinomas (by approximately age 18 weeks of age) and poorly differentiated carcinomas with both local and systemic metastases (by approximately 24 weeks of age) (17-18,20-22). Prostate lesions arise in these mice due to the prostate epithelial specific expression of the transgene, the SV40 large T antigen (T Ag), which is controlled by the testosterone- dependent rat probasin promoter. The T Ag inhibits the tumor suppressors p53 and Rb in the prostate and thus results in prostate tumor development (13,17-19,21,23).

Since multiple studies have shown this model’s responsiveness to dietary CR (8-

9,24), we utilized TRAMP mice for one of our initial studies with OSU-CG5. In that study we treated 6-week-old, intact, male TRAMP mice with 100 mg/kg/day of OSU- 117

CG5 for 4 weeks and then euthanized them. We found that 4 weeks of OSU-CG5 treatment decreased prostate epithelial cell proliferation within PIN lesions, resulting in statistically significant reductions in urogenital tract and prostate lobe weights without evidence of systemic toxicity (13). However, that study only focused on OSU-CG5’s effect on the preneoplastic PIN lesions, since mice were sacrificed before prostate tumors developed. Therefore, in our current study, we aimed to determine OSU-CG5’s effect on

TRAMP mouse prostate tumorigenesis. We investigated if OSU-CG5 could reduce the viability of TRAMP-C2 cells, a cell line derived from a TRAMP mouse prostate tumor

(25), and, most importantly, if OSU-CG5 could modulate TRAMP mouse prostate tumor development when treatment was begun at puberty (age 6 weeks) and continued until most mice should have prostate tumors (age 24 weeks).

MATERIALS AND METHODS

Cell Culture and Reagents

TRAMP-C2 cells were purchased from the American Type Culture Collection

(Manassas, VA), and were grown in glucose free Dulbecco's Modified Eagle Medium

([DMEM], Gibco Life Technologies, Grand Island, NY) that was supplemented with 2 grams/liter of glucose. The working DMEM solution also contained 10% fetal bovine serum and 1% Penicillin/Streptomycin. Cells were cultured in a 37°C humidified incubator with 5% CO2. OSU-CG5 was produced in our laboratory (data not shown).

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Cell viability assays.

Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-

2H-tetrazolium bromide (MTT) assay, as previously described (13). Briefly, TRAMP-C2 cells were plated in 96-well plates and incubated in the aforementioned DMEM. After 24 hours of growth, OSU-CG5 was solubilized in DMSO and added to the culture medium.

Cells were cultured in the OSU-CG5 containing DMEM for either 48 or 72 hours. After the allotted time elapsed, OSU-CG5-containing media was replaced with MTT containing media (0.5 mg/ml) and incubated at 37°C. After 2 hours, the MTT containing media was discarded, and 200 µl of DMSO was added to each well. A plate reader was used to determine the absorbance at 570 nm.

Formulation and Feeding of the diet containing 1286 ppm OSU-CG5

Control and experimental AIN-76A diets were obtained from Harlan

Laboratories, Inc (Madison, WI). So that the average daily intake of each mouse would be approximately 100 mg/kg/day of OSU-CG5, the dose that was used in our previous study with OSU-CG5 (13), 1286 ppm was selected for the concentration of OSU-CG5 in the experimental AIN-76A diet. This concentration of OSU-CG5 was based on the estimated daily dietary intake of each mouse and average mouse body weight from a previous dietary chemoprevention study that utilized a different compound (data not shown). Diets were stored in the dark at 4°C. Both the control and OSU-CG5 containing diets were weighed and replenished twice a week. The daily dietary intake of each mouse was determined by calculating the amount of diet consumed per cage each week and then 119

dividing that value by the number of mice per cage and the number of days per week. The daily dietary intake of OSU-CG5 was estimated by multiplying the calculated average daily dietary intake of the mice receiving OSU-CG5 in the diet by 1286 ppm and dividing this value by the average body weight of the OSU-CG5 mice. The daily intake of OSU-

CG5 was calculated on a weekly basis.

Animal Study Design

TRAMP mice (C57BL/6 TRAMP x FvB) were bred at The Ohio State University

(OSU), housed, and genotyped as heterozygous transgenic by PCR, as previously reported (26). Mice were provided access to water at all times. All procedures and protocols were approved by the Institutional Animal Care and Use Committee of OSU.

At 6 weeks of age, intact male transgenic mice were randomized to two groups that were fed either the control AIN-76A diet (n=31) or the AIN-76A diet formulated to contain 1286 ppm of OSU-CG5 (n=31). Mice were provided their respective diets ad libitum. Mice were weighed weekly and at necropsy. Mice were gently transabdominally palpated once a week by LDBB to assess for prostate tumor development. Mice continued on the study for either 18 weeks (until age 24 weeks) or approximately 2 weeks after the detection of a palpable prostate tumor (Control: 2.1 ± 1.4 weeks; OSU-

CG5: 2.1 ± 1 weeks), whichever came first. Once mice reached either of the aforementioned study endpoints, they were euthanized via carbon dioxide asphyxiation and complete necropsies were performed. Urogenital tracts (UGTs) were removed en bloc from all mice and weighed, and the relative UGT weights (UGT weight/terminal 120

body weight x 100%) were subsequently calculated. When grossly identifiable amongst the abdominal adipose tissue, the iliac lymph nodes were collected at necropsy. Some of these lymph nodes were lost in processing, resulting in 13 iliac lymph nodes from each group being available for histologic examination.

At necropsy, the UGTs were microdissected in PBS to separate out the four individual paired lobes of the prostates and any grossly apparent prostate tumors. The lobe of origin of each tumor was noted at necropsy. All grossly apparent tumors

(n=16/group) and the four individual paired lobes of the prostate were weighed, and tumor volumes were determined using calipers (tumor length x width x height). Sections of each tumor and one lobe of each pair were snap-frozen in liquid nitrogen and stored at

-80°C. Sections of each tumor and the other lobe of each pair were placed in tissue cassettes (Thermo Fisher Scientific, Fremont, CA) and fixed in neutral buffered 10% formalin (NBF). All other tissues were fixed in NBF. Formalin fixed epididymises, hearts, kidneys, livers, spleens, and testes were weighed (n=6 mice/group), and relative organ weights were determined as described above for the UGT.

At necropsy, blood was collected from 3 mice per group. The serum was submitted to the Comparative Pathology and Mouse Phenotyping Shared Resource (CPMPSR) at

OSU for determination of liver enzyme values (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALKP], and gamma-glutamyl transpeptidase [GGT], and total bilirubin), renal parameters (blood urea nitrogen (BUN),

Creatinine [CREAT], calcium, and phosphorous), cholesterol, albumin, and blood glucose. Whole blood was submitted to the CPMPSR for complete blood counts (CBCs).

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Histopathology and scoring of prostatic lesions in the TRAMP mice

Five-micrometer-thick, paraffin-embedded sections of the lobes of the prostate, prostate tumors, as well as a subset of organs were stained with hematoxylin and eosin

(H&E) by standard procedures (n=31/group). Due to losses during tissue processing, all four lobes of the prostate were available for histological examination in 29 of the control mice and 30 of the mice that had received the OSU-CG5 containing diet. H&E stained sections of heart, kidney, liver, lung, seminal vesicle, spleen, and urinary bladder were examined in each animal (n=31 mice/group). Testes were examined histologically in 30 of the control mice and 31 of the OSU-CG5 treated mice. Thirteen iliac lymph nodes per group were available for histologic examination. Based on the recommendations of the

Society of Toxicologic Pathology (27), the following tissues were examined in 5 mice per group: bone with bone marrow, brain, colon, epididymis, esophagus, eye, gall bladder, harderian gland, pancreas, peripheral lymph node, salivary gland, skeletal muscle, skin, small intestine, stomach, thymus, and trachea. Additionally, the following endocrine organs were also evaluated: adrenal gland (n=4/group), thyroid gland (n=7/control group; n=6/OSU-CG5 group), and pituitary (n=5/control group; n=7/OSU-CG5 group) All tissues were examined using an Olympus Model CHT research microscope (Olympus,

Center Valley, PA) by a blinded board-certified veterinary anatomic pathologist (LDBB).

The H&E-stained sections of the four lobes of the prostate were evaluated and scored separately by the blinded board-certified veterinary pathologist (LDBB) using a published grading scheme (22). Prostate tumors were graded as part of their respective lobes of origin. To briefly review, in this grading scheme, each lobe’s most severe and

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most common lesions were identified and given numerical scores. The sum of these two scores (the “sum of the adjusted lesion scores” [SALS]), was then calculated for each lobe. The average SALS, most severe, and most common lesion scores of each lobe were compared between groups.

Immunohistochemistry

To evaluate tumor cell proliferation and apoptosis, 5-micrometer-thick, paraffin- embedded tissue sections of the prostate tumors (n=7 tumors/group) were immunostained for Ki67 using a commercially available rabbit monoclonal antibody (catalog#RM-9106-

S0; Thermo Fisher Scientific) and for cleaved caspases 3 using a commercially available rabbit polyclonal antibody (catalog#9661L; Cell Signaling Technology, Danvers, MA).

The immunohistochemical staining protocol was as previously described (13). Three random images from each slide were obtained at 400X (i.e., high-power) magnification.

The manual tag feature of the Image-Pro Plus 7.0 (Media Cybernetics, Inc., Bethesda,

MD) image analysis software was used to quantify immunopositive and immunonegative cells. For each tumor section and antibody, the number of immunopositive cells in the three randomly selected 400X fields was counted and divided by the total number of cells

(immunopositive and -negative) in those fields to yield the percentage of either Ki67 or cleaved caspases 3 immunopositive cells.

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Protein isolation and western blot analysis

Because many of the tumors contained large central areas of necrosis, accurate western blotting for biomarker expression could only be performed in a limited number of tumors (n=4/control group; n=5/OSU-CG5 group). Sections of tumors that had been stored at -80°C were pulverized in liquid nitrogen. Protein extraction, quantification, and subsequent western blotting were done as previously described (13). The relative intensities of protein expression were determined by densitometric analysis of the protein bands using ImageJ software. β-actin was used as the internal reference protein for normalization. Antibodies utilized for western blotting included both mouse monoclonal antibodies and rabbit polyclonal antibodies. Mouse monoclonal antibodies included those directed against β-actin from MP Biomedicals (Irvine, CA), and SV40 T Ag from Santa

Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies included those directed against insulin-like growth factor-1 receptor (IGF-1R), and androgen receptor

(AR) from Santa Cruz, and Akt, p-Akt-Ser473, glycogen synthase kinase 3β (GSK3β), and p-GSK3β from Cell Signaling (Beverly, MA).

Statistical Analysis

Data was assessed for normality using the Shapiro-Wilk normality test. All data was normally distributed except for tumor volumes, absolute and relative UGT weights, weights of the lateral lobes of the prostate, the absolute and relative kidney weights, change in body weight over the course of the study, length of time on the study, the

SALS, most severe and most common lesions scores for each lobe, percentage of cells

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that were immunopositive for cleaved caspases 3, the serum chemistry and CBC values, and the average daily dietary intake. The statistical significance of the normally distributed data was determined using the Student’s t-test. Differences in lesion incidence, metastases, and percentage of mice that survived until 24 weeks of age were evaluated using the Chi-squared test. Survival until age 24 weeks was evaluated via the

Kaplan–Meier survival curve. The statistical significance of the non-parametric data was evaluated using the Wilcoxon rank sum test. Differences were considered to be significant at P <0.05, and all t-tests were 2-tailed.

RESULTS

OSU-CG5 reduced the viability of TRAMP-C2 cells

To assess OSU-CG5’s ability to reduce the viability of TRAMP-C2 cells in vitro and confirm that OSU-CG5 could modulate the growth of cells derived from TRAMP prostate tumors, TRAMP-C2 cells were treated with varying concentrations of OSU-CG5 for either 48 or 72 hours, and the MTT assay was performed. After 48 hours of treatment with approximately 4.25 μM of OSU-CG5, cell viability was reduced by approximately

50% (Figure 1A). A similar reduction in viability was observed after 72 hours of treatment with 3.5 μM of OSU-CG5 (Figure 1A).

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1286 ppm of OSU-CG5 did not significantly reduce UGT weights, alter the prostate tumor incidence, or delay prostate tumor development

To investigate OSU-CG5’s effect on prostate tumor development in TRAMP mice, intact, male TRAMP mice (n=31/group) were fed either a control AIN-76A diet

(control mice) or an equivalent AIN-76A diet modified by the addition of 1286 ppm of

OSU-CG5 (OSU-CG5 mice). Mice were randomized to their respective groups at age 6 weeks and continued on the study for either 18 weeks (until age 24 weeks) or two weeks after palpation of a prostate tumor, whichever came first. At necropsy, the UGT tracts were weighed and the absolute and relative UGT tract weights were compared between groups (Table 1). Although the average absolute and relative UGT weights of OSU-CG5 mice were reduced by 29.2% and 32%, respectively, these reductions were not statistically significant (Figures 2A & 2B; P>0.05). Additionally, OSU-CG5 did not significantly reduce the weights of the individual lobes of the prostate (Table 1).

1286 ppm of OSU-CG5 in the diet did not alter the incidence of prostate tumors or significantly delay their development. Seventeen poorly differentiated carcinomas were identified in the control mice and eighteen in the OSU-CG5 mice. Although the

OSU-CG5 mice were, on average, older than the control mice when the tumors were first discovered (19.8 ± 2.8 weeks versus 17.8 ± 3.2 weeks), this difference was not statistically significant (P >0.05). Consequently, although the OSU-CG5 mice survived on the study longer than the control mice (until age 22.6 ± 2.1 weeks versus age 21.7 ± 3 weeks), this difference was also not statistically significant (P >0.05; Figure 2C). There was also no statistically significant difference in the percentage of mice in each group 126

that stayed on the study for the full 18 weeks, with 54.8% of the control mice and 58.1% of the OSU-CG5 mice surviving until 24 weeks of age (P>0.05).

1286 ppm of OSU-CG5 administered in an AIN-76A diet reduced prostate disease severity, as determined by the average tumor volume and weight

Although OSU-CG5 did not reduce the prostate tumor incidence, the tumors that developed in the OSU-CG5 mice were significantly smaller than those in the control mice. Sixteen tumors in each group were grossly identifiable at necropsy and able to be weighed and measured. The average volume of the prostate tumors in the control mice was 2.8 ± 1.43 cm3 compared to an average volume of 1.27 ± 0.63 cm3 in the OSU-CG5 mice (P <0.001; Figure 3A). This represented a 54.6% reduction in tumor volume. The average weight of the prostate tumors in the control mice was 1.63 ± 0.67 g compared to an average weight of 0.75 ± 0.4 g in the OSU-CG5 mice (P<0.001; Figure 3B). This translated to a 54.1% reduction in tumor weight. The reduction in tumor size was not due to decreased transgene expression. Western blotting using protein extracted from the prostate tumors revealed that the two groups of mice expressed similar levels of the T ag

(Figure 3C). Additionally, the length of time between tumor discovery and euthanasia did not influence tumor size at necropsy since there was no significant difference between the lengths of time mice in each group remained on the study after tumors were discovered. Namely, the control and OSU-CG5 mice stayed on the study 2.1 ± 1.4 weeks and 2.1 ± 1 weeks after tumor discovery, respectively (P>0.05).

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Histopathologic examination and scoring of each lobe of the prostate was performed (n=29 mice/control group; n=30 mice/OSU-CG5 group). Tumors were examined and scored as part of their respective lobe of origin. The most severe and most common lesions in each lobe were identified and the sum of the adjusted lesion score

(SALS) for each lobe was calculated (Table 2). No statistically significant differences were found in the average most severe lesion score, most common lesion score, or the

SALS of each lobe. When the type, number, and incidences of the most severe lesions in each lobe were compared between the two groups, no statistically significant differences were identified (P>0.05 for all; Table 3).

The reduction in prostate tumor size was due to suppression of tumor cell proliferation

To evaluate whether decreased tumor cell proliferation or increased tumor cell apoptosis was responsible for the reduction in tumor size, the percentages of cells within each tumor that were proliferating or undergoing apoptosis were determined using immunohistochemistry (n=7 tumors/group). The reductions in tumor weight and volume were most likely due to decreased tumor cell proliferation as determined by the percentage of tumor cells that were immunopositive for Ki67 (the proliferation index).

Administration of 1286 ppm of OSU-CG5 in the diet reduced the percentage of tumors cells that were immunoreactive for Ki67 by 16.6% (P=0.014; n=7 tumors/group; Figures

4A & 4B). Immunohistochemistry for cleaved caspases 3 was performed to determine the percentage of cells undergoing apoptosis (apoptotic index). While administration of

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OSU-CG5 in the diet increased the apoptotic index, this increase was not statistically significant, with 0.22 ± 0.17 % and 0.41 ± 0.31% of tumor cells immunopositive for cleaved caspases 3 in the control and OSU-CG5 mice, respectively (P>0.05; n=7 tumors/group; Figure 4C & 4D).

Reduced tumor cell proliferation was associated with modulation of biomarkers that promote cell growth and survival

Since OSU-CG5 has previously been shown to down-regulate phospho-Ser473-

Akt (p-Akt), IGF-1R, the AR, and p-GSK3β within TRAMP mouse PIN lesions (13), we evaluated the levels of these biomarkers within the prostate tumors via western blotting

(n=4/control group; n=5/OSU-CG5 group). We found that 1286 ppm of OSU-CG5 administered in the diet reduced p-Akt and AR levels by 35% (P=0.014) and 53%

(P=0.031), respectively (Figures 5A & 5B). Treatment with OSU-CG5 did not significantly alters IGF-1R or p-GSK3β levels within the prostate tumors (P>0.05).

1286 ppm of OSU-CG5 administered via an AIN-76A diet did not reduce the incidence of metastases

OSU-CG5 treatment did not alter the incidence of metastases to the iliac lymph nodes, lungs, or liver. Thirteen iliac lymph nodes were available for histologic evaluation in each group. All of these lymph nodes contained tumor metastases. Sections of liver

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and lung were examined from each mouse (n=31/group). Three of the prostate tumor baring mice in each group had pulmonary micrometastases, which translated to incidences of 17.6% and 16.7% for the control and OSU-CG5 mice, respectively.

Additionally, when sections of liver were evaluated, no control mice had hepatic metastases while one prostate tumor baring OSU-CG5 mouse had a hepatic metastasis.

The addition of 1286 ppm OSU-CG5 to the diet did not alter feed consumption

Given the fact that caloric restriction has proven to be one of the most effective means to suppress tumor growth (1-2), we needed to ensure that the reduction in prostate tumor size was not due to the OSU-CG5 mice voluntarily calorically restricting themselves. Therefore, we determined the average dietary intakes of the TRAMP mice in both groups over the course of the study. We found that the addition of 1286 ppm of

OSU-CG5 to the standard AIN-76A diet did not significantly alter daily food intake or result in voluntary caloric restriction. The average daily intake for the control mice was

2.53 ± 0.13 g, and the average daily intake for the OSU-CG5 mice was 2.59 ± 0.11 g (P

>0.05). Based on the daily intake of the OSU-CG5 containing diet, the daily intake of

OSU-CG5 was determined on a weekly basis and found to be 112.4 ± 11.5 mg/kg/day

(Figure 6A). This intake was close to our target of 100 mg/kg/day, the dose that proved effective in our previous study utilizing TRAMP mice (13).

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Administration of 1286 ppm of OSU-CG5 in a diet for 18 weeks was not associated with any evidence of systemic toxicity

No obvious evidence of systemic toxicity was identified in the mice that received

1286 ppm of OSU-CG5 in the AIN-76A diet. OSU-CG5 mice and control mice gained a similar amount of weight over the course of the study, with OSU-CG5 mice gaining 7.08

± 3.01 g and control mice gaining 7.27 ± 2.83 g (P>0.05; n=31 mice/group; Figure 6B).

Similarly, when the absolute and relative weights of the epididymises, heart, kidney, liver, spleen, and testes were evaluated (Table 4), no significant differences were apparent between the two groups of mice (n=6 mice/group).

To evaluate further for any evidence of toxicity, H&E stained sections of heart, kidney, liver, lung, seminal vesicle, spleen, and urinary bladder were evaluated histologically (n=31 mice/group) as were sections of testes (n=30/control group; n=31/OSU-CG5 group). An extended list of organs were also evaluated from 5 mice/group (See Materials and Methods for the detailed list of tissues). There were no histological changes consistent with toxicity in any of the organs examined.

Additionally, to further assess these mice for any evidence of hepatic or renal toxicity, serum chemistries were performed (n=3 mice/group), and no significant abnormalities were identified (Table 5). Blood glucose values for the two groups were also compared, and no significant differences were identified (Table 5). This was in accordance with our previous study that found that OSU-CG5 did not modulate whole body glucose metabolism (13). Finally, to assess the mice for any hematologic abnormalities, complete

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CBCs were performed (n=3 mice/group), and no significant changes were notes (Table

5).

DISCUSSION

In this study we evaluated OSU-CG5’s ability to act as a chemopreventive agent in an aggressive mouse model of prostate cancer. The TRAMP model is one of the few mouse models of prostate cancer that develop PIN lesions that progress to locally invasive carcinomas that can metastasize both locally to the iliac lymph nodes and systemically to the lung and liver within a relatively short time period (28). In other words, TRAMP mice can be used to model every stage of prostate carcinogenesis in men.

Another mouse model that develops prostate tumors with metastases is the PB-

Cre4xPTENloxP/loxP model in which the PTEN tumor suppressor is conditionally knocked- out in the prostate (28). This is in contrast to many of the other mouse models of prostate cancer, such as the Hi-Myc model, that only develop PIN and invasive carcinomas, but lack metastases (28).

A review of the literature revealed that TRAMP mice have been utilized in four dietary CR studies. In three of these studies, the dietary intervention was initiated at around the time of puberty (9,24,29) while one was delayed until 20 weeks of age when advanced prostate lesions were present (8). Studies ranged from chronic 20% (8,24) to

25% CR (9) or 50% intermittent CR with mice fed 50% of the calories as the controls for two weeks followed by two weeks of being fed the same amount as the control mice 132

(9,29). In the two studies that utilized chronic CR only, lesion progression was reduced by CR, and the age at which the dietary intervention was initiated affected the degree to which CR inhibited lesion development (8,24). Two of the studies compared the effect of chronic CR to intermittent CR, and these studies found that intermittent CR had a greater ability to delay tumorigenesis than chronic CR (9,29). None of these studies provided comparisons of tumor volume or weights between groups. These studies also did not assess the effect of CR on prostate tumor cell proliferation. Therefore, the effect of actual

CR on TRAMP prostate tumor weights, volumes, and proliferative index is unknown.

Either UGTs and or accessory sex glands were weighed in the studies (8-9,24,29). The average UGT weight was reduced in the 20% calorically restricted mice in one of the chronic CR studies (8), and statistically significant reductions in absolute and relative weights of the accessory sex glands were described in the other 20% CR study (24).

These results were in contrast to the studies that evaluated the difference between chronic

25% CR and intermittent 50% CR (9,29). Those two studies found no statistically significant differences between the average relative UGT weights of the control, chronically calorically restricted, and intermittently calorically restricted groups (9,29).

Our study had a similar finding, with 1286 ppm of OSU-CG5 not inducing a statistically significant reduction in the absolute or relative UGT weight.

In our study we found that 1286 ppm of OSU-CG5 administered via an AIN-76A diet did not reduce tumorigenesis in TRAMP mice as determined by the incidence of poorly differentiated prostate carcinomas or the age at which the tumors developed.

However, OSU-CG5 significantly reduced the average weight and volume of the tumors

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that developed, without affecting transgene expression. This reduction in tumor size was most likely due to the inhibitory effect of OSU-CG5 on tumor cell proliferation. In contrast, OSU-CG5 did not appear to significantly induce tumor cell apoptosis, as determined by immunostaining for cleaved caspases 3. Although the observed decrease in

Ki67 immunostaining was relatively modest compared to the reductions in average tumor volume and weight, reduced cell proliferation was likely the primary driving force behind the more substantial reduction in tumor size. This is because the relationship between a reduction in cell proliferation and its contribution to tumor size is most likely non-linear.

Additionally, our previous study with OSU-CG5 in TRAMP mice showed that OSU-

CG5’s anti-proliferative activity begins during the PIN lesion stage and the effect is evident after as little as 4 weeks of treatment (13). Therefore, although the reduction in

Ki67 was relatively modest, we would expect that suppression of cell proliferation in the prostates of the TRAMP mice in the current study began soon after treatment was initiated. Consequently, a modest reduction in prostate cell proliferation over approximately 18 weeks could translate to large scale reductions in tumor volume and weight.

Reductions in tumor cell proliferation may have been due to the reduced p-Akt and AR levels within the prostate tumors from OSU-CG5 treated mice. This was in accordance with our previous study in which we demonstrated that OSU-CG5 suppresses phosphorylation of Akt and AR expression within TRAMP mouse PIN lesions (13). The

PI3K/Akt signaling axis promotes cell proliferation, growth, and survival (30). Therefore, decreased phosphorylation of Akt should result in suppression of tumor cell proliferation.

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Modulation of Akt phosphorylation has therapeutic relevance in human prostate cancer since the tumor suppressor PTEN, which down-regulates signaling via the PI3K/Akt pathway, is frequently mutated in human prostate cancers, resulting in inappropriate upregulation of this pathway (31). Therefore, suppression of Akt phosphorylation by

OSU-CG5 could potentially antagonize a constitutively active PI3K/Akt signaling axis and limit cell proliferation.

Because AR signaling plays an extremely important role in prostate cancer development and tumor cell proliferation (32), suppression of the AR may be another mechanism through which OSU-CG5 reduced tumor cell proliferation. Importantly, despite the fact that OSU-CG5 downregulated the AR in our study, it did not appear to function as an anti-androgen or induce a castration-like state. Namely, there was no reduction in testicular weight and the testes did not contain histologic changes consistent with atrophy. Although we have previously shown that OSU-CG5 treatment does not to reduce serum testosterone concentrations (13), we could not confirm that in this study because not enough serum was available for testosterone analysis.

A recent study highlighted the importance of the AR in TRAMP mouse disease progression (33). That study tested ASC-J9, a molecule that enhances the degradation of the AR, in TRAMP mice. Those researchers found that intraperitoneal administration of

75 mg/kg/day of ASC-J9 reduces the prostate tumor incidence, AR level, and tumor cell proliferation, concurrent with an increase in tumor cell apoptosis (33). Those results were similar to ours in that OSU-CG5 also reduced tumor cell proliferation and the AR level.

However, unlike ASC-J9, OSU-CG5 did not affect tumor incidence or tumor cell 135

apoptosis. This difference may be due to the fact ASC-J9 was administered via intraperitoneal injection (33), while OSU-CG5 was administered via a diet. ASC-J9 may have been able to reach a higher serum concentration than OSU-CG5, thereby rendering it more effective at suppressing prostate cancer tumorigenesis and inducing tumor cell apoptosis. It would be interesting to evaluate the efficacy of OSU-CG5 when administered via intraperitoneal injection and determine if the mode of administration affects OSU-CG5’s efficacy. The difference between the effects of ASC-J9 and OSU-

CG5 may also relate to the fact that ASC-J9 specifically targets the AR at the post- transcriptional level, resulting in AR degradation (33) while OSU-CG5 down-regulates the AR by suppressing AR transcription (data not shown).

Despite the fact that OSU-CG5 reduces IGF-1R levels within PIN lesions of

TRAMP mice (13), no such reduction was seen in the prostate tumors in the current study. Interestingly, although down-regulation of the IGF-1/IGF-1R axis has been associated with CR (1-2), the role of IGF-1/IGF-1R signaling in TRAMP mouse prostate tumor development is controversial. First of all, CR has not been consistently associated with reductions in IGF-1 in TRAMP mice (8-9). Additionally, a recent study found that reducing systemic IGF-1 levels by knocking out the IGF-1 gene in the liver does not inhibit TRAMP mouse prostate tumor development (34). Finally, another study demonstrated that conditionally knocking-out IGF-1R in the prostatic epithelium of

TRAMP mice accelerates tumorigenesis (35). Given this surprising information about

IGF-1/IGF-1R in the TRAMP model, it is difficult to interpret the lack of IGF-1R suppression in our current study. Additionally, the serum IGF-1 concentration in this

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study could not be determined not enough serum was available. We presume that serum

IGF-1 concentrations were unaffected by treatment, since OSU-CG5 has previously been shown not to affect serum IGF-1 concentrations (13).

In our study, we evaluated the efficacy and mechanism of action of OSU-CG5, a relatively new ERMA. Resveratrol is the only other ERMA that has been evaluated in

TRAMP mice (12). That study utilized 625 mg/kg of resveratrol in an AIN-76A diet, was initiated when the mice were 5 weeks old, and continued until the mice were 28 weeks old. Those researchers found that resveratrol significantly reduced the frequency of poorly differentiated carcinomas and increased the incidence of well differentiated adenocarcinomas. However, they found no significant differences in the tumor weight or the age at which mice developed tumors (12). They also showed that dietary resveratrol reduced cell proliferation within PIN lesions of 12 week old TRAMP mice, but they did not assess the effect on cell proliferation within the poorly differentiated carcinomas (12).

When comparing the results of this study with our study, resveratrol appeared to be able to suppress tumor development in the TRAMP model resulting in fewer poorly differentiated carcinomas, while OSU-CG5 reduced tumor size, resulting in smaller poorly differentiated tumors. Based on these results, resveratrol may be a better chemopreventive agent than OSU-CG5, while OSU-CG5 may be a better chemotherapeutic agent. Since most men will not initiate treatment until a prostate tumor is detectable, OSU-CG5 is likely the more relevant compound for translation into clinical prostate cancer therapy.

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In our study we found that 1286 ppm of OSU-CG5 in the diet did not reduce the incidence of tumor metastases to the iliac lymph nodes, lungs, or liver. Interestingly, neither intermittent CR, nor chronic CR, nor resveratrol have been found to affect the incidence of metastasis in TRAMP mice (9,12). This suggests that neither caloric restriction nor ERMAs can suppress the molecular events involved in metastasis.

The TRAMP mice in this study were supplied with a diet containing 1286 ppm of

OSU-CG5. Based on weekly evaluation of the mice’s intake of the diet, mice consumed on average slightly more than 100 mg/kg/day, the target dose. 100 mg/kg/day was chosen as the target dose given its proven efficacy at reducing proliferation within PIN lesions of

10 week TRAMP mice (13). Our current study confirmed that this dose of OSU-CG5 could reduce cell proliferation beyond the PIN stage. Since OSU-CG5 did not affect PIN severity in the previous short-term study (13), and it did not change the incidence of poorly differentiated carcinomas in the current study, we concluded that OSU-CG5 reduced cell proliferation without modulating tumor development.

Importantly, 1286 ppm of OSU-CG5 was well tolerated by the mice. The mice on the experimental OSU-CG5 containing diet consumed a similar amount of food and gained a similar amount of weight as the control mice. Since ERMAs preferentially target metabolically active and proliferating cells, in depth evaluation of multiple organ systems was performed to ensure that OSU-CG5 treatment did not result in off-target effects or toxicities. No histologic lesions suggestive of toxicity were found in any of the organs examined, including organs with metabolically active populations of cells, such as the bone marrow, brain, eye, gastrointestinal tract, kidney, liver, and skin. The lack of bone 138

marrow toxicity was supported by the fact that no abnormalities were apparent on the

CBCs. Serum chemistries confirmed that OSU-CG5 had no untoward effects on hepatic and renal function. In accordance with our previous study (13), blood glucose levels were unaffected by OSU-CG5 treatment. Serum cholesterol was also unaffected. Taken together, the blood glucose and cholesterol data support the fact that OSU-CG5 did not modify the treated mice’s energy metabolism.

In conclusion, while 1286 ppm of OSU-CG5 administered in an AIN-76A diet did not inhibit tumor development in TRAMP mice, it reduced prostate disease severity, as determined by prostate tumor weight and volume. The reductions in tumor weight and volume were attributable to OSU-CG5’s antiproliferative effect. OSU-CG5 was well tolerated, as TRAMP mice that received the experimental diet for 18 weeks had no histologic, biochemical, or hematologic evidence of toxicity. OSU-CG5’s ability to significantly reduce prostate tumor growth without adversely affecting metabolically active organs may make it a good candidate for translation into clinical trials as a treatment for disseminated prostate cancer or a therapy for men who are not good surgical candidates.

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Figure 4.1

(A) OSU-CG5 reduced the viability of TRAMP-C2 cells after either 48 or 72 hours of treatment. Bars represent SD.

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Figure 4.2

1286 ppm of OSU-CG5 in a control AIN-76A diet reduced the absolute (A) and relative

(B) UGT weights of TRAMP by mice by 29.2% and 32%, respectively. However, these reductions were not statistically significant (P>0.05). (C) OSU-CG5 did not significantly alter TRAMP mouse survival. In (A) and (B) bars represent the mean ± SD. UGT, urogenital tract; CG5, 1286 ppm OSU-CG5

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Figure 4.3

1286 ppm of OSU-CG5 in an AIN-76A diet resulted in statistically significant reductions in prostate tumor volume (A) and prostate tumor weight (B). N= 16 tumors/group. (C)

The reduction in tumor size was not due to decreased transgene expression, as transgene protein levels were equivalent in the two groups of mice. In (A) and (B) bars represent the mean ± SD. CG5, 1286 ppm OSU-CG5.

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Figure 4.4

1286 ppm of OSU-CG5 in an AIN-76A diet reduced tumor cell proliferation in prostate tumors from TRAMP mice but did not significantly increase the apoptotic index (n = 7 mice/group). (A) Photomicrographs showing Ki67 immunoreactivity in representative tumors from control and OSU-CG5 mice (400X). (B) The percentage of tumor cells that were immunopositive for Ki67 was reduced in the OSU-CG5 fed mice relative to the controls. (C) Photomicrographs showing cleaved caspases 3 immunoreactivity in representative tumors from control and OSU-CG5 mice (400X). (D) The percentage of tumors cells that were immunopositive for cleaved caspases 3 was not significantly increased in the OSU-CG5 mice. In (B) and (D) bars represent the mean ± SD. CG5,

1286 ppm OSU-CG5

146

14 7

Figure 4.4

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Figure 4.5

(A) Western blot analysis of the effects of dietary administration of OSU-CG5 on Akt-

Ser-473 and GSK3β phosphorylation and AR and IGF-1R expression in the prostate tumors of TRAMP mice. (B) Densitometric analysis of p-Ser473-Akt, p-GSKβ, AR, and

IGF-1R protein bands to determine relative phosphorylation/expression levels. The bars in (B) represent the mean ± SD. AR, Androgen Receptor; IGF-1R, Insulin-like growth factor-1 receptor.

148

Figure 4.6

(A) Dietary intake of OSU-CG5 over the course of the study, as determined on a weekly basis. (B) Mouse body weights over the course of the study.

149

Control Diet 1286 ppm OSU-CG5 Absolute UGT (mg)* 1443 ± 803.2 1022.3 ± 397.7 Relative UGT* 4.66 ± 2.83% 3.17 ± 1.29% Dorsal Lobe (mg)†a 52.1 ± 17.6 49.8 ± 17.5 Lateral Lobe (mg)†b 28.9 ± 6.5 27.3 ± 9.2 Ventral Lobe (mg)†c 23.4 ± 3.7 27.5 ± 8 Anterior Lobe (mg)†d 82 ± 20.5 83 ± 26 *N=31 mice/group †Given the presence of tumors, individual lobe weights could not be accurately assessed in all of the mice. The number of mice for which the lobes could be weighed is given below: a Dorsal Lobe: N=27 control mice and 28 OSU-CG5 mice bLateral Lobe: N=14 control mice and 16 OSU-CG5 mice cVentral Lobe: N=14 control mice and 15 OSU-CG5 mice dAnterior Lobe: N=25 control mice and 30 OSU-CG5 mice

Table 4.1: 1286 ppm of OSU-CG5 administered via an AIN-76A diet did not significantly reduce the average weights of the UGT or individual lobes of the prostate

(P>0.05)

150

Prostate Lobe Dorsal Lateral Ventral Anterior Control 22.4 ± 9.4 34.1 ± 10 25.7 ± 18.7 11.1 ± 10.7

SALS* OSU-CG5 19.3 ± 6.8 32.5 ± 10.6 26.1 ± 17.9 8.7 ± 6.8 Control 11.5 ± 4.8 18.1 ± 3.5 13.5 ± 8.8 9 ± 4.2 Most severe lesion score OSU-CG5 10 ± 3.6 17.4 ± 4 14 ± 8.5 8 ± 3.6 Most Control 10.9 ± 4.8 16 ± 6.6 12.2 ± 10 2.2 ± 6.5 common lesion score OSU-CG5 9.3 ± 3.6 15.1 ± 6.9 12.1 ± 9.7 0.7 ± 3.8 *SALS: Sum of the adjusted lesion score N=29 for the control mice; N=30 for the OSU-CG5 mice

Table 4.2: The average SALS and scores for the most severe and most common lesions in individual lobes of prostates of TRAMP mice that received either a control AIN-76A diet or an experimental AIN-76A diet with 1286 ppm of OSU-CG5 for 18 weeks

151

Group* PD Caa MD Acab WD Acac PL tumord High PINe Moderate Low PINe PINe Control DP 6 (20.7%) 0 (0%) 0 (0%) 4 (13.8%) 19 (65.5%) 0 (0%) 0 (0%) LP 17 (58.6%) 0 (0%) 12 (41.4%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) VP 16 (55.2%) 0 (0%) 2 (6.9%) 0 (0%) 0 (0%) 3 (10.3%) 8 (27.6%) AP 3 (10.3%) 0 (0%) 0 (0%) 0 (0%) 26 (89.7%) 0 (0%) 0 (0%)

OSU-CG5 DP 3 (10%) 0 (0%) 0 (0%) 1 (3.3%) 26 (86.7%) 0 (0%) 0 (0%) LP 15 (50%) 0 (0%) 14 (46.7%) 0 (0%) 0 (0%) 1 (3.3%) 0 (0%) VP 17 (56.7%) 0 (0%) 2 (6.7%) 0 (0%) 0 (0%) 4 (13.3%) 7 (23.3%) AP 1 (3.3%) 1 (3.3%) 0 (0%) 1 (3.3%) 24 (80%) 0 (0%) 3 (10%) *N= 29 mice/control group; N= 30 mice/OSU-CG5 group 1

5 The number in parentheses represents the percentage of mice for which this was the most severe lesion in that lobe 2

aPD Ca: Poorly differentiated carcinoma; bMD Aca: Moderately differentiated adenocarcinomal cWD Aca: Well differentiated adenocarcinoma; dPL tumor: Phyllodes-like tumor; ePIN: Prostatic intraepithelial neoplasia

DP: dorsal lobe; LP: lateral lobe; VP: ventral lobe; AP: anterior lobe

Table 4.3: The number and frequency of the most severe lesion in each lobe of the prostate of TRAMP mice that received either a

control AIN-76A diet or an experimental diet containing 1286 ppm of OSU-CG5 for 18 weeks

152

Organs*(mg) Control Diet 1286 ppm OSU-CG5 Liver (% body weight) 1385 ± 309.1 (4.23 ± 0.28) 1395 ± 225.2 (4.12 ± 0.4) Kidneys (% body weight) 436.7 ± 78.1 (1.34 ± 0.08) 451.7 ± 57.8 (1.34 ± 0.17) Spleen (% body weight) 83.3 ± 16.3 (0.26 ± 0.04) 91.7 ± 19.4 (0.27 ± 0.06) Heart (% body weight) 156.7 ± 23.4 (0.48 ± 0.05) 145 ± 13.8 (0.44 ± 0.08) Testes (% body weight) 241.7 ± 22.3 (0.75 ± 0.1) 250 ± 25.3 (0.75 ± 0.1) Epididymus (% body weight) 176.7 ± 23.4 (0.55 ± 0.1) 165 ± 69.2 (0.51 ± 0.2) *P> 0.05 for all values

The number in parentheses represents the relative weight of these organs as a percentage of body weight.

Table 4.4: Absolute and relative organ weights of the TRAMP mice that received either the control AIN-76A diet or the AIN-76A with 1286 ppm of OSU-CG5 for 18 weeks

(n=6 mice/group)

153

Control OSU-CG5 ALT (U/L) 25.5 ± 2.7 33.5 ± 19.4 AST (U/L) 50.9 ± 1.3 70.7 ± 35 ALKP (U/L) 87.6 ± 29.3 78.1 ± 8.3 GGT (U/L) 4.3 ± 1.5 5.7 ± 1.2 Total bilirubin (mg/dL) 0.37 ± 0.06 0.37 ± 0.06 BUN (mg/dL) 21.3 ± 3.2 17.7 ± 3.8 CREAT (mg/dL) 0.2 ± 0.1 0.23 ± 0.06 Calcium (mg/dL) 9.2 ± 0.4 9.1 ± 0.7 Phosphorous (mg/dL) 5.8 ± 0.7 7.3 ± 0.8 Albumin (g/dL) 3.5 ± 0.1 3.5 ± 0.2 Total protein (g/dL) 6 ± 0.3 5.5 ± 0.5 Cholesterol (mg/dL) 189.2 ± 45.4 164.7 ± 63.2 Glucose (mg/dL) 204.7 ± 6.8 202 ± 29.1 WBC # (K/µL) 7.9 ± 2 5.6 ± 1.7 Neutrophil # (K/µL) 1.5 ± 0.3 1.2 ± 0.3 Lymphocyte # (K/µL) 5.8 ± 1.7 4 ± 1.4 Monocyte # (K/µL) 0.5 ± 0.1 0.5 ± 0.2 Eosinophil # (K/µL) 7 x 10-3± 6 x 10-3 7 x 10-3± 6 x 10-3 Basophil # (K/µL) 0 ± 0 3 x 10-3± 6 x 10-3 nucleated RBCs # (K/µL) 0 ± 0 0 ± 0 Hematocrit (%) 42.8 ± 2.2 42.5 ± 0.8 RBC (M/µL) 10.1 ± 0.5 10.2 ± 0.1 Hemoglobin (g/dL) 14.8 ± 0.6 14.6 ± 0.4 MCV (fL)a 42.2 ± 0.8 41.8 ± 0.3 MCHC (g/dL)b 34.7 ± 0.4 34.4 ± 0.4 RDW (%)c 12.3 ± 0 12.7 ± 0.5 Continued *P>0.05 for all values aMCV: Mean corpuscular volume; bMCHC: Mean corpuscular hemoglobin concentration; cRDW: Red cell distribution width; dMPV: Mean platelet volume; ePDW: Platelet distribution width

Table 4.5: Administration of OSU-CG5 in an experimental diet for 18 weeks did not result in any detectable abnormalities in serum chemistries or complete blood counts (n=3 mice/group).* 154

Table 4.5: Continued

Reticulocyte # (K/µL) 0 ± 0 0 ± 0 Reticulocye % 0 ± 0 0 ± 0 Platelet # (K/µL) 948.3 ± 80.1 1054.7 ± 224.2 MPV (fL)d 4.9 ± 0.06 5.1 ± 0.1 PDW (%)e 50.1 ± 0.8 48.4 ± 1.6

155

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APPENDIX A: SUPPLEMENTAL DATA TABLES FROM CHAPTER 3

Prostate Lobe Dorsal Lateral Ventral Anterior Vehicle-treated 9.5 ± 1.4 9.0 ± 4.4 6.0 ± 2.1 4.5 ± 3.0 SALS* OSU-CG5-treated 9.1 ± 1.8 7.5 ± 1.0 5.6 ± 2.5 5.5 ± 3.0 Most severe Vehicle-treated 6.5 ± 1.4 5.8 ± 4.4 3.9 ± 1.0 4.5 ± 3.0 lesion score OSU-CG5-treated 6.1 ± 1.8 4.4 ± 0.7 3.8 ± 1.1 5.7 ± 2.6 Most common Vehicle-treated 3 ± 0 3.2 ± 0.6 2.1 ± 1.4 0 lesion score OSU-CG5-treated 3 ± 0 3.2 ± 0.6 1.9 ± 1.5 0 * Sum of the Adjusted Lesion Scores

N=11 mice/group

Table A.1: SALS* and averages scores for the most severe and most common lesions in individual lobes of prostates in vehicle-treated versus OSU-CG5-treated TRAMP mice

176

Associated Network Functions Score Network Genesa Cellular Assembly and 33 Akt, Alpha tubulin, ↑ATM, ↑Calm1 (includes Organization, Cellular Function others),↑Calmodulin, ↑CENPJ, ↑CLIP1, ↑Creb, and Maintenance, Gene ↓CSF1R, ↓CTXN1, ↑CYTH3, ↑DYNC1H1, Expression Dynein, ↑EPB4.1, ↑FAM48A, ↑GABPB2, ↑GNB1, Histone h3, ↑Hsp70, ↑HTT, ↑INTS7, ↑Mediator, ↑MICU1, ↑MTM1, ↑NDEL1, ↑NRF1, ↑NSD1, ↑PDE4B, PDGF BB, ↓PDGFRB, ↓STRAP, ↑THRAP3, ↑TP53BP1,↓TTN (includes EG:22138),↑WHSC1L1 Drug Metabolism, Endocrine 33 Alpha catenin, ARL6IP5, ↑BOP1/LOC727967, System Development and ↑CCNL2, ↓COL6A2, ↑CREBZF, ↑CTNND1, Function, Lipid Metabolism ↑DTL, ↓DUSP3, ↑EEA1, ↑Enah, ↑ERCC6, FSH, ↑GIT2, ↑Gm5117/Tgs1, hCG, ↓ITGB5, ↑KIF1B, ↑LETMD1, Lh, ↑MAPK6, ↓MB, ↑MED15, ↑MED1 (includes EG:19014), MED25 (includes EG:292889), ↑PNN, ↑PTPN21, ↑PTS, ↑RAB1A, ↑RASAL2, RNA polymerase II, SH3BP4, ↑SLC9A8, ↑SORBS1, Sos Developmental Disorder, 27 14-3-3, ↑ABCD3, ↑AEBP2, AMPK, ↑CCAR1, Neurological Disease, Collagen type I, ↓Creatine Kinase, ↑ELF1, ↑ERK, Cardiovascular System ERK1/2, ↓FLT4, ↓HFE, ↑HLTF, ↓HSPB6, Development and Function ↑MAP3K8, ↑MAT2A, ↑MDM4, Mi2, ↑NISCH, ↑OGT, p85 (pik3r), ↑PARD3, ↓PDE10A, Proinsulin, Ras, ↑RASGRF1, ↑RBBP4, ↓SMAD1, ↑SMG1, ↓SULF2, TCR, ↓UCP2, ↑USP7, Vegf, ↑WWOX Cell Death, Cancer, Inflammatory 24 ↑ATF2, ↑BCL2, BCR, ↑BRAF, ↑BTRC, Caspase, Response CD3, ↑CRK, Cytochrome c, ↑EIF2AK2, Focal adhesion kinase, Growth hormone, Gsk3, ↓ICAM1, IKK (complex), ↓IL1RL1, ↑ITCH, Mlc, NFkB (complex), ↑NFKBIZ, ↑PI3K (complex), ↑Pkc(s), ↓PP2A, ↑PRKCD, ↑RAB1A, Rap1, ↑RBM5, ↓RHOB, ↓RIOK3, ↑ROCK1, ↓SHC1 (includes EG:20416), ↑TBK1, ↑TLN1, ↑TOP2A, ↑YES1 Cellular Growth and 22 ↑26s Proteasome, Actin, ↑CCDC76, ↑CDC7 Proliferation, Cell Cycle, Cell- (includes EG:12545), Cyclin A, Cyclin E, mediated Immune Response ↑DUSP16, E2f, ↑EIF4G1, ↑ELAVL1, ↑EP400, ↑EWSR1, ↑FUBP1, ↓GAS2L1, Gm-csf, ↓GPI, ↓HLA-DQA1, IFN Beta, Ige, IgG, IL12 (complex), IL12 (family), Immunoglobulin, Interferon alpha, ↑NASP, P38 MAPK, ↓PPP2R1A, ↑RBL1, ↓S100A11, ↑SKP2 (includes EG:27401), ↑STAT3, STAT5a/b, ↑TNPO1, ↑TRPM3, ↓VIM ↑, ↓: up-or down-regulated genes in OSU-CG5 treated TRAMP mice a Genes in the analyzed data set are shown in bold Table A.2: Top 5 affected gene networks generated by IPA in OSU-CG5 treated TRAMP mice 177

Pathway Gene Fold Description P value Function Grouping (Public ID) change Regulation of the Fusion, derived from A RNA-binding protein and putative tumor suppressor; Fus facilitates cell cycle arrest in prostate cancer cells by cell cycle and t(12;16) malignant 3.37 0.046 proliferation BE985138 modulating the expression of cyclin D1, CDK6, and liposarcoma p27 (1). Fubp1 Far upstream element Functions as an ATP-dependent DNA helicase; 2.78 0.044 participates in the negative regulation of MYC BB488001 (FUSE) binding protein 1 expression (2) Ccar1 Cell division cycle and A cell cycle- and apoptosis-regulatory protein via 2.58 0.029 transcriptional down-regulation of c-myc and cyclin B1 AI503765 apoptosis regulator 1 (3). Involved in spermatogenesis; enhances the degradation Gmcl1 Germ cell-less homolog 1 2.53 0.028 of MDM2 and increases the amount of p53 by BM239632 modulating the nucleoplasmic transport (4). 1

78 Ubiquitin specific peptidase Usp7 2.25 0.020 Stabilizes p53 via deubiquitylation and promotes p53 BM247366 7 DNA binding(5) Epidermal growth factor Involved in the internalization of activated EGFR and Eps15 receptor pathway substrate 2.25 0.025 Met receptor tyrosine kinase, thereby dampening AI649374 15 growth factor signaling (6) Dusp16 Dual specificity phosphatase 2.21 0.018 Inactivates MAPK signaling (7). BB119893 16 RAN GTPase activating Rangap1 2.12 0.003 Activates RaN’s (a Ras related protein) GTPase AI481700 protein 1 activity, resulting in RaN inactivation(8). Growth arrest and DNA- Suppresses the progression of the cell cycle Gadd45gip1/CRIF1 damage-inducible, gamma progression by inhibiting cyclin and cyclin dependent 1.55 0.041 BG072462 interacting protein 1/CR6- kinase complexes activity; also acts to repress AR interacting factor 1 transactivation (9). Continued

Table A.3: Select upregulated genes in the dorsal and lateral prostates of TRAMP mice treated with OSU-CG5 (N = 3).

178

Table A.3: Continued

Arhgap12 Rho GTPase activating Inactivates Rac1, and negatively regulates HGF- 2.68 0.030 Inhibition of cell BB159907 protein 12 mediated invasive growth response (10). invasion and or A putative tumor suppressor by inhibiting Rac1 Nisch metastasis Nischarin 2.13 0.015 oncogenic activity (11); epigenetically silenced in lung AK014314 cancer patients (12). DNA damage A chromatin remodeling enzyme involved in the repair response of DNA double-stranded breaks by altering chromatin Ep400 E1A binding protein p400, 2.5 0.019 structure; alterations in chromatin structure induced by AW541237 mRNA Ep400 are needed to recruit the DNA repair enzyme BRCA1 and TRP53BP1(13) Trp53bp1 Transformation related Involved in tumor suppression by acting in the DNA 2.21 0.035 BB119893 protein 53 binding protein 1 damage response pathway (14). Non-POU-domain- Nono containing, octamer binding 2.14 0.048 Involved in repair of double-stranded DNA breaks (15). AK013444 1 protein 7

9 Involved in the DNA damage sensing/repair pathway. Nek1 NIMA (never in mitosis gene 2.02 0.048 NEK-deficient cells transform and acquire the ability to BB418199 a)-related expressed kinase 1 grow in anchorage-independent conditions (16). Xpa Xeroderma pigmentosum, 1.90 0.020 Participates in nucleotide-excision repair(17) BM117916 complementation group A Involved in the response to DNA double-stranded ATM Ataxia telangiectasia 1.59 0.027 breaks, by causing cell cycle arrest and inducing DNA NM_007499 mutated homolog (human) repair(18). Continued

179

Table A.3: Continued

Acts as a molecular scaffold for the IFN-α mediated Flnb activation of the JNK signaling pathway for the Filamin, beta 2.26 0.027 BM206272 induction of apoptosis; apoptosis is prevented if filamin B is knocked-down (19) Interacts with apoptosis signal-regulating kinase 1 Modulation or Gspt1 (ASK1) and enhances its ability to induce apoptosis; G1 to S phase transition 1 2.16 0.036 induction of BB162021 GSPT1 also promotes apoptosis by binding to and apoptosis inhibiting the activity of inhibitors of apoptosis (20). ELAV (embryonic lethal, Elavl1 (huR) A RNA binding protein; under conditions of cellular abnormal vision, Drosophila) 2.06 0.027 BB284404 stress, it is cleaved and promotes apoptosis (21). like 1 (Hu antigen R) Rbm6 A tumor suppressor that modulates apoptosis; it is RNA binding motif protein 6 2.02 0.034 BB031290 down-regulated in metastatic solid tumors (22) Nucleoside Adk 180 Adenosine kinase 2.41 0.040 Activation of chemotherapeutic agents (23). metabolism BB053697 Hyaluronidase that removes β-N-acetylglucosamine Mgea5 Meningioma expressed residues from proteins; decreased expression has been 2.61 0.035 BB555250 antigen 5 (hyaluronidase) found in poorly differentiated and metastatic breast tumors(24). Mediates ER-Golgi transit of glycosylated proteins; Lman1 LMAN1 mutational inactivation is a frequent and early Lectin, mannose-binding, 1 2.33 0.030 BE981934 event contributing to microsatellite unstable colorectal Cell homeostasis cancer (25). InaD-like (Drosophila)/Pals- Inadl/PATJ Important for epithelial cell polarization and tight associated tight junction 2.31 0.028 BB273436 junction formation(26). protein Edem3 ER degradation enhancer, 2.31 0.042 Degrades misfolded glycoproteins (27). BB549831 mannosidase alpha-like 3 Adar Involved in adenosine-to-inosine RNA editing; reduced RNA adenosine deaminase 1 2.04 0.008 BB308291 expression in human cancers (28). 1.87 Upregulation of β-TRCP is associated with the Modulation of Btrc (β-TRCP) Beta-transducin repeat 0.029 starvation-associated response in LNCaP prostate energy restriction BB136156 containing protein cancer cells(29) 180

Pathway Grouping Gene Public ID Description Fold change P value Function Development of an A mesenchymal marker; overexpression invasive phenotype Vim contributes to the invasive phenotype of Vimentin -1.84 0.020 AV147875 androgen-independent prostate cancer (30). Flt4 Promotes invasion and metastasis of FMS-like tyrosine kinase 4 -1.66 0.004 AI323512 cancer cells (31) Upregulation associated with increased Rhob Ras homolog gene family, -1.59 0.048 prostate cancer motility and invasion BC018275 member B (32). Aberrant activation of the Wnt signaling Wnt5b Wingless-related MMTV pathway in human cancer leads to more -1.54 0.008 AV303043 integration site 5B malignant phenotypes, such as invasion, and metastasis (33) Efnb2 Stimulate invasiveness in prostate

1 Ephrin B2 -1.51 0.041 cancer cells by activating Eph receptors 81 U30244 (34) Continued

Table A.4: Select downregulated genes in the dorsal and lateral prostates of TRAMP mice treated with OSU-CG5 (N = 3).

181

Table A.4: Continued

Tumor cell survival, Integrin v5 acts as a survival factor Itgb5 progression, Integrin 5 -1.98 0.034 for PC-3 cells cultured in serum-free BB543979 protection, and conditions (35). proliferation; Hspb6 Heat shock protein, alpha- A small heat shock protein; tumor -1.68 0.024 inhibition of BB755506 crystallin-related, B6 protective (36) apoptosis Csf1r Colony stimulating factor 1 Overexpression promotes prostate -1.68 0.029 AI323359 receptor cancer progression (37) Ube2K Ubiquitin-conjugating Encodes a protein (Hip2) that blocks -1.64 0.048 AI551201 enzyme E2K apoptosis mediated by Smac/Diablo(38) Ckmt1 Ubiquitous mitochondrial Facilitates prostate cancer progression -1.55 0.005 NM_009897 creatine kinase 1 (39). Dual specificity Expression is increased in prostate Dusp3/Vhr phosphatase 3/vaccinia cancer, and can inhibit c-Jun NH(2) -1.54 0.038 BC016269 virus phosphatase VH1- terminal kinase (JNK)-mediated

1 related apoptosis in LNCaP cells (40). 82 Activation results in increased Notch1

signaling and enhanced cell survival; Ddr1 Discoidin domain receptor -1.54 0.030 increased expression described in BB234940 family, member 1 multiple human cancers, including esophageal, breast, and lung (41). Shc1 SH2 domain-containing Plays a critical role during ErbB2- -1.54 0.047 BB753533 transforming protein C1 induced tumor progression (42) Increased expression in a number of human cancers, including pancreatic S100a11 S100 calcium binding -1.52 0.023 (43) and lung cancer (44); may be BC021916 protein A11 (calgizzarin) associated with increased proliferation (44). Increases survival and decreases Sulf2 apoptosis in hepatocellular carcinoma Sulfatase 2 -1.51 0.038 AU020235 cells through the PI3K/Akt signaling pathway(45) Continued

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Table A.4: Continued

Development of Transforming growth factor An androgen receptor co-activator; androgen- Tgfb1i1/Ara55 beta 1 induced transcript increased expression may be associated -1.68 0.034 independence NM_009365 1/Androgen receptor with the development of castration- associated protein 55 resistant prostate cancer (46-47) A downstream component of TGF; Smad1 Mothers Against DPP might be involved in the development of -1.55 0.037 BB257769 Homolog 1 androgen-independence in prostate cancers (48). Involved in cellular uptake of steroids; Solute carrier organic anion Slco2b1 increased expression identified in the transporter family, member -1.51 0.037 metastases of castration-resistant BB553107 2b1 prostate cancer (49).

1 Glucose metabolism Gpi1 Glucose phosphate

83 -1.75 0.004 A key enzyme in glycolysis BE992059 isomerase 1

Increased expression in colon cancer(50); increased expression may impart chemoresistance to cancer Uncoupling protein 2 Ucp2 cells(51); acts as a pyruvate transporter (mitochondrial, proton -1.56 0.018 AW108044 and thereby limits the amount of carrier) pyruvate used for oxidative phosphorylation, thereby promoting the Warburg effect (51).

Tumor associated

genes/genes Pdgfrb Platelet derived growth A potential therapeutic target for -1.75 0.029 upregulated in AA499047 factor receptor beta metastatic prostate cancer (52) various cancers Psca A cell surface marker in prostate cancer Prostate stem cell antigen -1.55 0.027 AF319173 (53)

183

Gene Public ID Description Fold change P value Akap9 A kinase (PRKA) 1.79 0.024 C79026 anchor protein (yotiao) 9 Angel2 Angel homolog 2 1.64 0.039 AA200940 (Drosophila) Arih2 Ariadne homolog 2 2.03 0.0033 BG066446 (Drosophila) Atrx Alpha 2.00 0.024 BB825830 thalassemia/mental retardation syndrome X-linked homolog (human) Cdadc1 Cytidine and dCMP 1.63 0.046 BC006901 deaminase domain containing 1 Enah Enabled homolog 2.16 0.030 AK020248 (Drosophila) Luc7l2 LUC7-like 2 (S. 1.68 0.018 BI076494 cerevisiae) Msi2 Musashi homolog 2 1.66 0.027 AV319863 (Drosophila) Mycbp2 MYC binding protein 1.54 0.046 AW107953 2 Ncor1 Nuclear receptor co- 1.57 0.022 AI481996 repressor 1 Pabpn1 Poly(A) binding 2.56 0.020 AV028400 protein, nuclear 1 Pdk1 Pyruvate 1.63 0.0061 BB391928 dehydrogenase kinase, isoenzyme 1 Phip Pleckstrin homology 1.56 0.023 BB425239 domain interacting protein Continued Table A.5: Genes that have previously been shown to be modulated by caloric restriction (54) that are up- or down-regulated in the dorsal and lateral prostates of TRAMP mice treated with OSU-CG5 (N = 3)

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Table A.5: Continued

Ptgr2 Prostaglandin 1.59 0.034 BB209194 reductase 2

Ptpn21 Protein tyrosine 1.83 0.013 AK013777 phosphatase, non- receptor type 21 Rbm6 RNA binding motif 2.02 0.034 BB031290 protein 6 Rock1 Rho-associated 1.72 0.028 BI662863 coiled-coil containing protein kinase 1 Sec61a1 Sec61 alpha 1 1.99 0.011 BF148622 subunit (S. cerevisiae) Sfrs11 Splicing factor, 1.57 0.033 AW261583 arginine/serine-rich 11 Sfrs2 Splicing factor, 1.95 0.042 AF250135 arginine/serine-rich 2 (SC-35) Sltm SAFB-like, 1.87 0.015 BB206801 transcription modulator Son Son DNA binding 1.71 0.0021 BG067046 protein

Sorbs1 Sorbin and SH3 2.14 0.014 BB259710 domain containing 1 Spnb2 Spectrin beta 2 1.64 0.045 AW550681 Stat3 Signal transducer 2.10 0.0099 BG069527 and activator of transcription 3, mRNA (cDNA clone IMAGE:3665873) Tlk2 Tousled-like kinase 1.59 0.048 BM198864 2 (Arabidopsis) Continued

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Table A.5: Continued

Tsc22d1 TSC22 domain 1.98 0.025 BM230984 family, member 1 Ttc3 Tetratricopeptide 1.92 0.049 BM116591 repeat domain 3 Tug1 Taurine upregulated 1.58 0.039 D50523 gene 1 Uba5 Ubiquitin-like 2.06 0.020 BI438002 modifier activating enzyme 5 Xpa Xeroderma 1.90 0.020 BM117916 pigmentosum, complementation group A Zfp800 Zinc finger protein 1.92 0.019 BG063017 800 C1qb Complement -2.06 0.019 BB111335 component 1, q subcomponent, beta polypeptide Col6a2 Collagen, type VI, -1.75 0.0092 BI455189 alpha 2 Ddr1 Discoidin domain -1.54 0.029 BB234940 receptor family, member 1 Itgb5 Integrin beta 5 -1.98 0.034 BB543979 Pde10a Phosphodiesterase -1.52 0.017 AK014090 10A Pltp Phospholipid transfer -1.57 0.023 AI591480 protein Slc25a39 Solute carrier family -1.59 0.0044 BB514802 25, member 39

186

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APPENDIX B: SUPPLEMENTAL DISCUSSION OF THE MICROARRAY DATA FROM CHAPTER 3

SUPPLEMENTAL DISCUSSION

The top 10 genes upregulated by OSU-CG5 treatment were: Fus (fusion, derived from t(12;16) malignant liposarcoma, 3.372-fold), Nsd1 (Nuclear receptor-binding SET- domain protein 1, 2.932-fold), Cep192 (Premature mRNA for mKIAA1569 protein,

2.902-fold), Herc4 (hect domain and RLD 4, 2.877-fold), Ubap2l (Ubiquitin associated protein 2-like, 2.866-fold), Fubp1 (far upstream element (FUSE) binding protein 1,

2.776-fold), Arhgap12 (Rho GTPase activating protein 12, 2.676-fold), Hltf (helicase- like transcription factor, 2.638-fold), Ccdc76 (coiled-coil domain containing 76, 2.63- fold), and Mgea5 (meningioma expressed antigen 5 [hyaluronidase], 2.612-fold).

Supplementary table 2 contains a more comprehensive list of selected upregulated genes.

The top 10 genes downregulated by OSU-CG5 treatment were Myh1 (myosin, heavy polypeptide 1, skeletal muscle, adult, -3.033-fold), C1qb (complement component 1, q subcomponent, beta polypeptide, -2.056-fold), Itgb5 (integrin beta 5, -1.979-fold),

Laptm5 (lysosomal-associated protein transmembrane 5, -1.849-fold), Vim (vimentin, -

1.839-fold), Ppp1r11 (protein phosphatase 1, regulatory (inhibitor) subunit 11, -1.805- fold), H2-aa/Hla-dqa1 (histocompatibility 2, class II antigen A, alpha, -1.766-fold), Gpi1

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(glucose phosphate isomerase 1, -1.752-fold), Pdgfrb (platelet derived growth factor receptor, beta polypeptide, -1.751-fold), and Col6a2 (collagen, type VI, alpha 2, -1.747- fold). Supplementary table 3 contains a more comprehensive list of selected downregulated genes.

When the top ten up-regulated genes were examined, at least three had important roles in the suppression of tumorigenesis. Namely, Fus facilitates cell cycle arrest in prostate cancer cells by modulating the expression of cyclin D1, CDK6, and p27 (1).

Fubp1 participates in the negative regulation of Myc expression (2) and thereby regulates cell proliferation. Arhgap12 is involved in suppressing invasion; it inactivates Rac1 and negatively regulates the HGF-mediated invasive growth response(3). Of the remaining seven genes, four have roles related to tumor suppression and homeostasis. The products of Hltf and Mgea5 have been reported to be down-regulated or hypermethylated in various cancers, suggesting that they may function as yet undefined tumor suppressors.

Two genes had roles in maintaining cellular homeostasis; Herc4 is an ubiquitin ligase and

Nsd1 is involved in histone methylation. Further review of the data revealed a number of other up-regulated genes with central roles in suppressing tumorigenesis (Supplementary table 2).

When the top ten down-regulated genes were examined, at least three had important roles in promoting carcinogenesis. Vim is involved in the development of an invasive phenotype (4). Itgb5 acts as a survival factor for PC-3 cells, a castration-resistant human prostate cancer cell line, cultured in serum-free conditions (5). Gpi1 may promote

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cancer cell growth by promoting the Warburg effect; this enzyme catalyzes the second enzymatic reaction in glycolysis, and its upregulation is, therefore, expected to increase the glycolytic rate. Moreover, another of the top down-regulated genes was Pdgfrb which has been identified as a potential therapeutic target for metastatic prostate cancer (6).

Further review of the microarray data revealed that a number of other genes involved in prostate cancer tumorigenesis were down regulated. These included genes involved in the development of castration-resistance such as Tgfb1i1/Ara55 (transforming growth factor beta 1 induced transcript 1/androgen receptor associated protein 55, -1.68- fold, P=0.034) (7), Smad1 (Mothers Against DPP Homolog 1, -1.55-fold, P=0.037) (8), and Slco2b1 (solute carrier organic anion transporter family, member 2b1, -1.51-fold,

P=0.037) (9) as well as genes associated with prostate cancer progression such as Csf1r

(colony stimulating factor 1 receptor, -1.68-fold, P=0.029) (10), and Ckmt1 (ubiquitous mitochondrial creatine kinase 1, -1.55-fold, P=0.005) (11). A more complete list of down-regulated genes with functions related to tumorigenesis is in supplementary table 3.

Interestingly, studies of caloric restriction in mice have identified genes that are consistently modulated in multiple mouse tissues(12). Although these studies did not include prostate tissue (12), a comparison with our microarray data showed that the expression levels of a number of the genes listed in those studies were modified in OSU-

CG5-treated TRAMP mouse prostates (supplementary Table 5). For example, 32 (7.4%) out of 402 upregulated genes (≥ 1.5-fold) and 7 (9.2%) out of 76 downregulated genes (≥

1.5-fold) by OSU-CG5 were also modulated by caloric restriction in multiple mouse 193

tissues. Although this gene expression data comparison is interesting, because the effect of caloric restriction on gene expression in the prostate was not assessed in that study

(12), we cannot fully evaluate how closely our gene expression profile resembled that of the calorically restricted mouse prostate.

194

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