Dietary Tomato and Lycopene Modulate Critical Androgen-Driven Mrna And

Dietary Tomato and Lycopene Modulate Critical Androgen-driven mRNA and

miRNA Expression in Early Prostate Carcinogenesis

DISSERTATION

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

Lei Wan, M.S.

Graduate Program in The Ohio State University Nutrition

The Ohio State University

2014

Dissertation Committee:

Professor Steven K. Clinton, Advisor

Professor Earl H. Harrison

Professor Dennis K. Pearl

Professor Susan Olivo-Marston

Lei Wan

2014

Abstract

Background: Epidemiological and laboratory studies suggest tomato and lycopene are dietary anti-cancer agents. Dietary tomato and lycopene have been shown to affect testosterone production and metabolism and vice versa. Testosterone plays critical roles in prostate carcinogenesis by activating the androgen receptor signaling pathway to modulate molecular processes. MicroRNAs (miRNA) are short non-coding RNAs and are deregulated in prostate carcinogenesis. Both diet and testosterone have been shown to target miRNA expression in prostate carcinogenesis. The TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model develops prostate cancer spontaneously during puberty. We hypothesized that 1) miRNA expression profiles are altered in early TRAMP prostate carcinogenesis, 2) that testosterone status modulates expression of genes in critical pathways and miRNA expression in early prostate carcinogenesis, 3) that consumption of tomato and lycopene leads to altered miRNA and mRNA expression, and 4) that dietary tomato and lycopene play a role in testosterone- driven mRNA and miRNA expression.

Methods: Two, 2x3 factorial studies were designed to investigate the effects of testosterone and tomato carotenoids on murine carcinogenesis gene expression (200 genes) and miRNA expression profile in early prostate carcinogenesis. TRAMP and wild type C57BL/6 mice (4-wk old) were randomly assigned to one of three diets: control, tomato powder, or lycopene beadlets diet. At 8-wk-old, mice were randomly assigned to ii

intact (sham surgery), castration, or castration + testosterone repletion groups. One week later, mice in the testosterone repletion group received 2.5 mg/kg/day testosterone propionate via osmotic pump. All mice were sacrificed 5 days after testosterone repletion. Body weight and tissue weight were recorded. Plasma was collected for lycopene analysis. Prostate lobes were collected for RNA isolation and histopathology analysis. MicroRNA profile (all known 602 miRNAs) and murine carcinogenesis gene codeset (200 genes) were assessed by NanoString® technique.

Results: In wild type mice, plasma lycopene was not detected in control fed mice, and did not differ significantly between tomato or lycopene fed mice ( 0.26 ± 0.02 vs. 0.27±

0.02 µmol/L, P=0.87). Diet and testosterone status did not alter changes of body weight

(6.5±0.2g, P=0.42) and UGT weight (252±14 mg, P=0.15). Castration significantly decreased UGT weight compared to sham-operation (124±7 vs. 345±8 mg, P<0.0001).

In TRAMP mice, plasma lycopene was not detected in control fed mice, and did not differ significantly between tomato or lycopene fed mice (0.32 ± 0.03 vs. 0.33 ± 0.03

µmol/L, P=0.81). Dietary tomato and lycopene did not cause changes of body weight and UGT weight. Castration decreased body weight gain (5.3±0.2 vs. 7.0±0.3 g), UGT weight (118±3 vs. 389±10 mg) and prostatic epithelial cell proliferation (0.32±0.1 vs.

3.8±0.4 %), compared to intact mice (P<0.0001).

Out of 200 mRNAs, 189 were detected in mouse prostatic tissue. Of these, 153 were regulated by a main effect of testosterone, 1 (Arntl) by diet alone, and 7 by testosterone and diet together (P<0.05). The effect of castration changed expression of genes related to cell growth, invasion and the aryl hydrocarbon receptor (AhR) signaling pathway.

iii

Tomato-feeding impacted molecules that are downstream of testosterone-regulated AhR signaling: Mapk1, Gstp1 and Jun in a testosterone-dependent manner (P<0.0001).

Moreover, dietary lycopene, but not tomato, decreased expression of neuroendocrine biomarkers nerve growth factor receptor (Ngfr) (-2.13 fold, P=0.01), synaptophysin (Syp)

(-2.29 fold, P=0.005), and a cell adhesion biomarker, cadherin-2 (Cdh2) (-1.82 fold,

P=0.03), compared to control diet. Furthermore, lycopene-feeding attenuated the reduced Birc5 expression by castration compared to control-feeding (P=0.01).

Out of 602 miRNAs, 78 were detected in mouse prostatic tissue. Compared to wild type,

41 miRNAs were up-regulated in TRAMP. TRAMP up-regulated miR-21 and miR-15a (8 and 9.23-fold, respectively, P< 0.0001). Castration down-regulated 38 miRNAs (>2 fold,

P < 0.05) and up-regulated 6 miRNAs (such as miR-150 and miR-1224, -2.94 and -2.11 fold, respectively, P<0.0001). The effect of testosterone-repletion restored miRNAs expression altered by castration to expression levels in intact mice (P<0.001).

Moreover, the impact of castration on miRNA expression is genotype specific. Castrated mice had down-regulated miR-200b and miR-15b in TRAMP but not in wild type, compared to intact mice (P<0.0001).

Tomato-fed mice had increased expression of miR-15a (2.1-fold, P =0.007) and miR-

200c (1.4 fold, P =0.047), compared to control-fed mice in both genotypes. Lycopene- fed TRAMP mice had greater expression of tumor suppressor miRNAs (let-7 family, miR-145/143, miR-15a/16 etc) than lycopene-fed wild type mice (2.1-79.7 folds, P <

0.05). Furthermore, castrated mice had decreased miR-429 (-7.0 and -7.2 folds,

P=0.029 and P=0.027) and miR-375 (-51.95 and -286.80 folds, P=0.007 and P<0.0001) than intact mice in tomato- and lycopene-, but not in control-fed mice.

Conclusion: Castration caused prostate growth regression and inhibition of proliferation in wild type and TRAMP mice. Testosterone status modulated gene expression in cell growth and AhR pathways in early prostate carcinogenesis. Further, dietary tomato and lycopene impacted testosterone-driven AhR signaling-targeted gene expression. The miRNA expression profile was deregulated in TRAMP mice compared to wild type mice in early prostate carcinogenesis. Testosterone was the dominant force regulating miRNA expression, whereas dietary tomato and lycopene play modest roles. Dietary tomato and lycopene independently regulated neuroendocrine biomarkers, a cell adhesion molecule and anti-cancer miRNA expression before causing morphological changes in early prostate carcinogenesis. The current study shows that dietary tomato and lycopene regulated testosterone-driven mRNA and miRNA expression in early prostate carcinogenesis. This supports the previous finding that dietary tomato and lycopene impact testosterone activity. The specific androgen-sensitive mRNA, miRNA, and signaling networks identified may be targets of tomato and lycopene bioactivity in prostate cancer inhibition.

Dedication

I dedicate this work to my family who motivates me to work harder and to keep pursuing my dream. Especially, I dedicate this to my parents and my sister, who always stand by

me and believe in me.

Acknowledgments

I especially thank my advisor, Prof. Steven Clinton, for his continuous guidance and support of my Ph.D. study and research. His enthusiasm, commitment, and knowledge inspire me in all aspects of my study and research. I express my great gratitude for him to accept me and train me to be a successful graduate student.

Besides my advisor, I would thank the rest of my committee members, Prof. Dennis

Pearl, Prof. Earl Harrison and Prof. Susan Olivo-Marston. I thank Prof. Pearl for his teaching and guidance on the statistical analysis of my research. I thank Prof. Harrison for his hard questions and insightful comments. My sincere thanks go to Prof. Olivo-

Marston for her encouragement and attention to my research.

I also want to express my thanks to Nancy Moran, for her time and attention on my research and my dissertation writing. My thanks also go to Jennifer Thomas-Ahner for her help and creative ideas for my research. I thank Kristen Roberts to always be there for me and help me get through hard time. I also thank all my current and previous

Clinton lab members: Ashley Schmitz, Beth Grainger, Colleen Spees, Besma Abbaoui and Shirley Tan.

Last but not least, I thank my family especially my parents and my sister for their love and support. I thank my boyfriend, Jing Wang, for taking care of me and keeping me company when I studied late. vii

Vita

2001 ...... B.S. Animal Science, Shenyang Agricultural University

2005 ...... M.A. Animal Nutrition, China Agricultural University

2008 to present ...... Graduate Research Associate, Department of Human Nutrition, The Ohio State University

Publications

Tan H., Thomas-Ahner J.M., Grainger E.M., Wan L, Francis D.M., Schwartz S.J., Erdman J.W Jr., Clinton S.K. (2010). Tomato-based food products for prostate cancer prevention: what have we learned? Cancer Metastasis Rev 29(3):553-68.

Fields of Study

Major Field: Human Nutrition

viii

Table of Contents

Abstract ...... ii

Dedication ...... vi

Acknowledgments ...... vii

Vita ...... viii

Table of Contents ...... ix

List of Tables ...... xii

List of Figures ...... xv

Chapter 1: Introduction ...... 1

Chapter 2: The Interface between Deregulated miRNA Expression and Testosterone during Early Prostate Carcinogenesis in the TRAMP model ...... 19

Abstract ...... 20

Introduction ...... 22

Materials and Methods ...... 24

Results ...... 27

Conclusions ...... 34

Conflict of Interest: ...... 35

Acknowledgments: ...... 35

Tables: ...... 36

Figures: ...... 43

References ...... 49

Chapter 3: Dietary Tomato and Lycopene Interrupt Testosterone-driven Gene

Expression in Early TRAMP Prostate Carcinogenesis ...... 52

Abstract: ...... 53

Introduction: ...... 55

Materials and Methods: ...... 57

Results: ...... 61

Discussion: ...... 65

Conclusions: ...... 70

Conflict of Interest: ...... 71

Acknowledgments: ...... 71

References: ...... 86

Chapter 4: Dietary Tomato and Lycopene Mediate miRNA Expression in Early Prostate

Carcinogenesis ...... 90

Abstract: ...... 91 x

Introduction ...... 93

Materials and Methods ...... 95

Results ...... 98

Discussion: ...... 102

Conclusion: ...... 106

Conflict of Interest: ...... 107

Acknowledgements: ...... 107

Tables: ...... 108

Figures ...... 115

References: ...... 123

Bibliography ...... 132

Appendix A. miRNAs detected in the Mouse Prostate ...... 146

Appendix B. Murine prostate carcinogenetic gene codeset...... 149

Appendix C. Correlation bewteen expression of miR-16 and Ccnd1.1 ...... 156

Appendix D. Correlation bewteen expression of miR-16 and Bcl2.1 ...... 157

Appendix E. Correlation bewteen expression of miR-16 and Fgf2.1 ...... 158

List of Tables

Table 2. 1 MicroRNA expression up-regulated in TRAMP compared to wild type mice. 36

Table 2. 2 MicroRNA expression down-regulated in TRAMP compared to wild type mice

...... 37

Table 2. 3 MicroRNA expression up-regulated in castrated compared to intact mice. ....38

Table 2. 4 MicroRNA expression down-regulated in castrated compared to intact mice.

...... 39

Table 2. 5 MicroRNAs up-regulated in testosterone repleted mice compared to castrated mice ...... 40

Table 2. 6 MicroRNAs down-regulated in testosterone repleted mice compared to castrated mice...... 41

Table 2. 7 MicroRNAs differently expressed between testosterone repletion and intact mice...... 42

Table 3. 1. Composition of diets employed in testosterone treatment in wild type and

TRAMP mice...... 72

xii

Table 3. 2 Summary of canonical pathways predicted to be regulated by castration compared to intact group...... 73

Table 3. 3 Twenty-six molecules impacted by castration which are consistent with the aryl hydrocarbon signaling pathway...... 74

Table 3. 4 Twenty molecules changed by castration in connected cell survival and cell movement networks...... 75

Table 3. 5 Expression of mRNAs changed by dietary tomato or lycopene...... 76

Table 4. 1 Composition of diets employed in testosterone treatment in wild type and

TRAMP mice...... 108

Table 4. 2 miRNA expression significantly increased by tomato feeding...... 109

Table 4. 3 Dietary lycopene significantly increased miRNA expression in TRAMP vs.

WT...... 110

Table 4. 4 Tomato and lycopene up-regulated miR-204 in TRAMP compared to WT. . 112

Table 4. 5 In tomato and lycopene feeding group, castration decreased miRNA expression compared to intact group...... 113

xiii

Table 4. 6 In tomato and lycopene feeding group, testosterone repletion restored miRNA expression...... 114

xiv

List of Figures

Figure 2. 1 H&E staining was assessed in prostate anterior lobes from WT and TRAMP mice fed with control diet for 10 weeks...... 43

Figure 2. 2 Heatmap of clustered microRNAs expression grouped by genotype and testosterone status...... 44

Figure 2. 3 MicroRNA expression significantly up-regulated in TRAMP mice compared to intact mice...... 45

Figure 2. 4 MicroRNAs up-regulated by castration, compared to sham-operation and testosterone-repletion...... 46

Figure 2. 5 MicroRNAs significantly down-regulated by castration compared to sham- operation and testosterone-repletion...... 47

Figure 2. 6 MicroRNA expression significantly changed by an interaction between genotype and testosterone...... 48

Figure 3. 1 Body weight gain and urogenital tract weight ...... 77

Figure 3. 2 Effect of diet and androgen status on final weights of four prostate lobes ....78

Figure 3. 3 Plasma lycopene was detected in mice fed tomato or lycopene diet...... 79

Figure 3. 4 The proliferation index of prostate tissue was measured by Ki67 staining in anterior lobe...... 80

Figure 3. 5 Number of miRNA expression changed by diet, genotype, or testosterone status...... 81

Figure 3. 6 Canonical pathways regulated by testosterone...... 82

Figure 3. 7 Cell growth and cellular movement networks was regulated by testosterone.

...... 83

Figure 3. 8 Gene mRNA expression changed by dietary tomato and lycopene...... 84

Figure 3. 9 Gene mRNA expression changed by diet x testosterone interaction...... 85

Figure 4. 1 Body weight gain of both wild type and TRAMP mice ...... 115

Figure 4. 2 Urogenital tract (UGT) weight of both wild type and TRAMP mice...... 116

Figure 4. 3 Plasma lycopene concentrations in wild type and TRAMP mice...... 117

Figure 4. 4 . Number of miRNAs changed by genotype, testosterone and diet...... 118

Figure 4. 5 MicroRNA expression changed by diet...... 119

xvi

Figure 4. 6 Dietary tomato and lycopene regulated miRNA in genotype specific manner.

...... 120

Figure 4. 7 Castration decreased miRNA expression in diet specific manner...... 121

Figure 4. 8 Correlation between expression of bcmo1 and miR-145 or miR-204 in

TRAMP mice ...... 122

xvii

Chapter 1: Introduction

Prostate Cancer Impact and risk factors

Prostate cancer is the most common non-cutaneous cancer and the second leading cause of death from cancers in the United States. Each year, 157 and 27 per 100,000 men are diagnosed and die, respectively of prostate cancer. [1] The treatments for prostate cancer normally include radiation, prostatectomy, androgen ablation therapy, and chemotherapy yet there are no cures for metastatic disease. The patients frequently face decreased quality of life and financial burden due to long term therapy.

Therefore there exists an urgency to develop strategies to lower risk and to prevent the development and recurrence of prostate cancer. Prostate cancer has a long latency period before clinical symptoms manifest, which provides an ideal opportunity for nutritional modulation in prostate cancer. [2]

The risk factors contributing to prostate cancer include age, ethnicity, family history, genetics, hormones and diet. [3] Prostate cancer occurs more often in men between ages 40 and 70 and is associated with aging. It is estimated that 60% of all prostate cancer cases occur in men 50 and older, 97% of which occur in men 65 and older. [4]

The prevalence of prostate cancer is higher in African Americans and lower in Asians.

The epidemiologic studies have suggested that men with a brother or father with prostate cancer are twice as likely to develop prostate cancer. [5] Moreover, the risk for the men with an affected brother is 2 times more than men with an affected father. It was suspected that environment shared by brothers is more similar than that shared by

fathers and sons. However, no high risk and highly penetrant genes have been discovered and the inherited risk is likely polygenetic. [6]

Testosterone is the primary androgen in males and is critical for normal prostate growth and differentiation. It also plays a critical role in prostate cancer progression.

Pharmacologic inhibition of 5a-reductase (finasteride and dutasteride) has been shown to decrease prostate cancer risk by 25% over 7 years by reducing the production of

DHT. [7] [8] Moreover, androgen ablation is the main treatment for hormone-sensitive prostate cancer, and results in reduced prostate size and limited cancer growth . [9]

Therefore, testosterone is essential in prostate cancer development.

Epidemiological studies further provide the evidence of an association between diet and cancer risk. Dietary components such as tomato carotenoids, pulses (legumes), milk/dairy, calcium, selenium and vitamin E have all been linked to the prevention of prostate cancer, yet conclusive evidence is still lacking. [10]

Tomatoes and Tomato Carotenoids in Prostate Carcinogenesis

Tomatoes are rich in carotenoids, a class of bioactive phytochemicals hypothesized to have anticancer activity. [11] [12] Lycopene is generally the most abundant carotenoid in red tomatoes, and is responsible for the red color of tomatoes. Epidemiological studies showed an inverse association between a diet high in tomato products and prostate cancer incidence. [13] [14] [15] Further, studies have shown that patients with prostate cancer have lower circulating and prostatic lycopene concentrations. [16] [17] [18]

Moreover, higher plasma concentrations of other carotenoids, lutein, zeaxanthin, and cryptoxanthin, were also modestly associated with lower prostate cancer incidence, which suggested anti-cancer activities of this class of compounds. [18]

The inhibitory effects of tomato carotenoids on prostate carcinogenesis were also suggested in human intervention studies.[19] [20] Consumption of lycopene-rich tomato extracts resulted in decreased tumor size and less involvement of surgical margins in patients with localized prostate cancer, compared to the control group. [21] Tomato products intake was found to protect DNA from damage from oxidative stress and lower

PSA level. [22] [23]

Both carcinogenesis (transgene or carcinogen induced) and tumorigenesis (xenograft) models have been employed to investigate the anti-cancer activities of tomatoes and lycopene. Lycopene beadlets were given to rodents by either incorporation into diet or by oral gavage. [24-31] Lycopene was detected in serum of study animals, which showed that the lycopene in the tomato powder and lycopene beadlets can be absorbed by rodent models to achieve blood concentrations similar to humans. Tomato and lycopene decreased prostate incidence, inhibited metastasis, and increased survival rate in animal models when interventions were initiated early in the carcinogenic process. [26, 29, 30]

In the tumorigenesis models, consumption of lycopene and tomato powder significantly decreased tumor weight and volume. [32, 33] However, we always need to be aware that publication bias may exist and studies showing no major effects may not be easily published.

In vitro cell culture studies have reinforced that carotenoids, especially lycopene, confer anti-cancer activity by inhibiting the growth of various cancer cells. [34, 35] Lycopene has been shown to suppress phosphorylation of retinoblastoma (Rb) to induce G0/G1 cell cycle arrest in both androgen-dependent and independent prostate cancer cells. [36]

Further, lycopene promotes apoptosis by inducing the release of mitochondrial cytochrome C and increasing mitochondrial potential in prostate cancer cells. [36] [37]

The inhibitory effect of lycopene on cancer migration was observed in a colon cancer cell line, such that the expression of E-cadherin was up-regulated by lycopene to inhibit the epithelial mesenchymal transition (EMT) in HT-29 cells. [38] Taken together, the mechanisms of the anti-cancer activities of lycopene include regulating oxidative stress, proliferation, apoptosis, invasion, and metastasis. [39] [37] [40]

Carotenoid metabolism and prostate carcinogenesis

There are two known oxygenases responsible for the oxidative cleavage of carotenoids in mammals. They are beta-carotene 15, 15’-monooxygenase (BCMO1, aka CMO-I or

BCO1) and beta-carotene 9’, 10’-dioxygenase enzyme (BCDO2, aka CMO-II or BCO2).

[41] [42] BCMO1 cleaves beta-carotene symmetrically at 15,15’ position to generate retinal. BCDO2 eccentrically cleaves beta-carotene and acyclic lycopene. [43] [44] The cleavage specificity of BCMO1 and BCDO2 on carotenoids has been studied in BCMO1 knockout (-/-) and BCDO2 knockout (-/-) mice. In BCMO1-/- mice, beta-carotene feeding with low vitamin A caused lower hepatic vitamin A concentration and lycopene-feeding with low vitamin A caused changes in lycopene biodistribution, compared to wild-type mice fed with beta-carotene and lycopene separately. [45] In BCDO2-/- mice, consumption of a lycopene-containing diet caused an increase in tissue and serum lycopene concentrations. [46] However, further studies are needed to assess the cleavage activity of BCMO1 and BCDO2 on other carotenoids.

In mice, BCMO1 and BCDO2 have been hypothesized to contribute to the interaction between dietary tomato carotenoids and testosterone production in prostate carcinogenesis. Ford and colleagues observed significantly decreased serum and testicular testosterone levels in BCMO1-/- mice compared to wild-type mice, when fed a lycopene diet. [47] This suggested that lycopene metabolites generated by BCDO2

cleavage played a role in tomato carotenoid-regulated testosterone production. [47] Our previous studies showed that castrated mice had higher prostatic BCDO2 expression when compared to intact mice. Further, lycopene-fed BCDO2-/- TRAMP mice had higher prostate cancer lesion scores and incidences than lycopene-fed wild type mice.[48] This suggests that lycopene interventions may impact prostate physiology via BCDO2- generated lycopene metabolites. Lycopene metabolites have been shown to contribute to the chemopreventive effect of lycopene. [47, 49, 50] For example, apo-10’-lycopenoic acid has been shown to inhibit the proliferation of non-small lung cancer cells and tumorigenesis in vivo, as well as inhibit hepatocellular carcinogenesis and hepatic tumorigenesis in mice. [51] [52] However, the mechanisms underlying this phenotype have not been elucidated, and the direct effect of testosterone, dietary tomato and lycopene on the expression of BCMO1 and BCDO2 in prostate has not been investigated.

The mechanisms by which tomato carotenoids may inhibit prostate carcinogenesis are clearly multifaceted. [53] However, there is building evidence for the hypothesis that tomato phytochemicals including the carotenoids, and specifically lycopene, may influence prostate carcinogenesis by altering the production and metabolism of testosterone, or by disrupting androgen-driven carcinogenesis in the prostate.

Influence of Testosterone on Pathways Associated with Prostate Carcinogenesis

Biologically, testosterone is converted to dihydrotestosterone (DHT) by 5a-reductase in prostate tissue, which binds to androgen receptors to regulate gene expression affecting cell differentiation, function, cell growth and survival.[54] Activation of the androgen signaling pathway alters downstream genes involved in molecular pathways of prostate cancer progression. The influenced pathways include cell growth and proliferation,

apoptosis, DNA damage and reactive oxygen species (ROS) production, immunity and inflammation, angiogenesis, invasion and metastasis and citrate metabolism. [54] [55]

Androgen receptor signaling alters cyclins (cyclin A, cyclinB1) and cyclin dependent kinases (CDK1, CDK2) to regulate prostate cancer cell proliferation in the mouse and

LNCaP cells. [56] [57] A number of genes associated with the regulation of apoptosis have been shown to be modulated by testosterone. Castration reduced expression of

FLICE-like inhibitory protein (FLIP) to induce apoptosis of prostate epithelial cells. [58]

Androgen repletion after castration induced Bcl-2 expression in LNCaP prostate tumor model. [59] [60] [61]

It is speculated that oxidative stress contributes to deregulation of androgen signaling and progression of androgen dependent cancer to castration resistant prostate cancer.

[62] Androgen deprivation induced the expression of ROS generating enzyme, NADP (H) oxidase. [63] Moreover, castration and testosterone-repletion caused the alteration of enzymes involved in cellular redox regulation in rats. [64] Furthermore, androgen exposure in hormone sensitive cells increased DNA adduct levels and ROS production.

[65] However, little is known about the mechanisms by which oxidative stress impacts

AR signaling.

Although there is no direct causal mechanism between inflammation and prostate cancer, inflammation is hypothesized to be involved in etiology of prostate cancer. [66]

[67] [68] Androgen deprivation treatment (ADT) decreased levels of inflammatory factor

IL-6 and increased the expression of IL-1β, IL-8, TNF-α, and SDF-12 in patients with prostate cancer compared with the patients without ADT. [69] In terms of invasion and metastasis, androgen deprivation induced the over expression of invasion biomarkers of

N-cadherin, and Cadherin-11. [70] Therefore, interrupting androgen–driven pro-

carcinogenic cellular response may be one effective strategy in prostate cancer treatment.

MicroRNA as a potential target of tomato phytochemicals during prostate carcinogenesis

MicroRNAs (miRNA) are a class of small non coding RNAs. The deregulation of miRNA expression has been implicated in the carcinogenesis of many cancers. In prostate cancer, the association of miRNA alteration and progression of prostate cancer suggest that miRNAs may be investigated as therapeutic targets, as well as diagnostic and prognostic biomarkers of prostate cancer. However, this line of investigation and its translation to clinical care is only in its early phases. [71, 72] Mature miRNAs are 18-22 nucleotides long and cleaved from pri-miRNA by RNA polymerase II in the nucleus.

RNase III enzyme Drosha cleaves pri-miRNA into a 70 base pair imperfect, stem loop hairpin miRNA precursor called pre-miRNA. The pre-miRNAs are exported into the cytoplasm via Exportin-5, where they are further cleaved by RNase III Dicer into mature miRNA. The mature miRNA is incorporated into the RNA induced silencing complex and targets mRNA by binding to the seed sequence (2-8 nucleotides at the 3’ end of mRNA) in either a perfect complementary or an imperfect complementary manner. This induces the degradation of target mRNA or the suppression of protein translation

Deregulation of miRNA has been recently implicated in carcinogenesis by Croce and colleagues, who first identified the role of miRNA in cancer. [73] They discovered that miR-15 and miR-16 located in chromosome 13q14 were deleted or down-regulated in

68% of B cell chronic lymphocytic leukemia (B-CLL). Now regulation of miRNA expression has been considered as one of the strategies in carcinogenesis treatment.

[74] In prostate cancer tissue, deregulation of miRNA expression has been implicated in tumor progression and has been suggested as a potential therapeutic target.[75] In

prostate cancer, miR-106b-25 and its precursor MCM7 were up-regulated compared to non-tumor tissue. [76] Circulating miR-375 and miR-141 have been shown to correlate with prostate progression and were up-regulated in human prostate cancer tissues when compared to benign tissues.[77] [78] Moreover, the miRNA processing enzyme, Dicer, and several miRNA host genes were found to be up-regulated in primary prostate tumors when compared to non-tumor prostate tissue. [76] Genetic aberrations in miRNA locus copy numbers also cause abnormal miRNA expression, leading to prostate carcinogenesis. [79]

MicroRNAs are subject to androgen regulation in the normal prostate and prostate cancer. Androgen responsive elements have been detected in promoters of miR-21, miR-125b, miR-27a and miR-148a etc. [80] [81] Testosterone activates miRNA expression to induce proliferation in human prostate cancer cell lines. [81] [81-84]

Conversely, expression of miRNA was shown to mediate AR activity by suppressing expression of an AR co-repressor. [85] One mechanism of androgen regulation on miRNA expression has been proposed to regulate miRNA expression at both the transcriptional and the post-transcriptional level. [86] Therefore, modulation of miRNAs by androgen may be one of the characteristics of normal prostate function and may also have a role in prostate cancer progression.

Several dietary interventions inhibiting carcinogenesis have been recently found to act via miRNA regulation. [74, 87] Dietary zinc has been shown to down-regulate oncomir- miR-31 and miR-21 to inhibit esophageal cancer progression.[88] Curcumin up- regulated tumor-suppressor miR-15a/16 to induce apoptosis in breast cancer cell lines.[89] Lycopene has been shown to inhibit miRNA-21 expression in hepatic steatosis, a risk factor for liver cancer. [90] In prostate cancer cells, vitamin D also inhibited

proliferation by regulating miRNA expression. [87] However, the modulation of miRNA expression in prostate carcinogenesis by lycopene has not been reported yet.

Therefore, understanding miRNA expression patterns for development of chemopreventive targets, as well as diagnostic and prognostic biomarkers for prostate cancer should be an active focus of investigation.

Currently, miRNA expression and mechanisms are just beginning to be explored in experimental prostate carcinogenesis but the studies focused on miRNA in prostate cancer tissue and in vitro prostate cancer cells. To our knowledge, no studies have examined alterations of miRNA expression during the early stages of carcinogenesis.

We proposed the use of TRAMP (transgenic adenocarcinoma of the mouse prostate) mice to evaluate the interaction of dietary intervention and testosterone status on miRNA expression in early prostate carcinogenesis. The present study was the first study to examine testosterone, diet, and testosterone x diet interaction on miRNA expression in early prostate carcinogenesis.

TRAMP mice in prostate carcinogenesis

There remains continuing debate regarding the merit of various murine models of prostate carcinogenesis and no single model recapitulates all aspects of the complex and heterogeneous human disease. The TRAMP mouse is considered an appropriate candidate model to investigate prostate carcinogenesis. The TRAMP model was created by inducing expression of oncoprotein-SV-40Tag driven by a rat probasin (-426--

+28bp) promoter, especially in dorsal and lateral prostate epithelial cells in C57BL/6 inbred mice strain. The expression of the Tag transgene suppresses the tumor suppressor genes RB and p53. [91] The progression of cancer in TRAMP correlates with sexual maturity of the mice. Between 8 to 12 weeks, the TRAMP mice start to develop

epithelial hyperplasia. As the mice grow older, the hyperplasia become more severe and finally transforms to neoplasia around 24 weeks. By 30 weeks old, all mice develop metastasized tumors in lymph nodes, lungs, adrenal glands and bones. [92] TRAMP mice develop prostate cancer spontaneously, and recapitulate the progression of prostate cancer in human. [93, 94] Further, the dorsal and lateral lobes are most homologous to peripheral zone in prostate in humans, where 75% of prostate cancer originates. Circulating miRNAs in TRAMP mice have been shown to be over-expressed in human prostate tumor tissue compared to non-tumor tissue. [95] To this end, the

TRAMP model is a reasonable system to examine the effect of tomato powder and lycopene on androgen-driven miRNA and miRNA expression.

Androgen status impacts lycopene metabolism and vice versa, yet much more research is necessary to understand the interactions more clearly. Decreased testosterone levels in the rat resulted in increased serum concentrations of lycopene. [96, 97] In castrated lycopene-fed rats, serum lycopene concentration was 2 times higher than intact lycopene fed rat.[96] Campbell, et al. observed significantly decreased serum testosterone in F344 rats fed with lycopene than the group without lycopene. [97] Our previous study showed castration in C57BL/6 mice caused lower concentration of lycopene than sham operated mice.[48] Although the results are not consistent, all of these results indicate the interaction between the lycopene and androgen. The enzymes involved in steroid metabolism were shown to be regulated by lycopene consumption in the rat model.[98] Expression of 5a-reductase 2 was reduced in the lycopene-fed rats compared to the control group. [98] Prostate 17β-hydroxysteroid dehydrogenase 4 mRNA was up-regulated in tomato and lycopene fed adult rats compared to control-fed rats. However, in early prostate carcinogenesis, the effect of dietary tomato and lycopene on androgen signaling pathway is not known. Especially, the mechanism of 10

interaction between dietary tomato carotenoids and androgen signaling pathway in prostate carcinogenesis may provide evidence for chemopreventive strategies in prostate carcinogenesis.

Testosterone modulates miRNA expression and plays a role in prostate growth and prostate carcinogenesis. [99] Furthermore, depletion of androgens by castration in rats decreased a large set of miRNAs which was reversed by addition of testosterone. [100]

Tissue specific knock-outs of miRNA processing enzyme Dicer caused androgen insensitivity syndrome. [100] Regulation of miRNA is one of the proposed mechanisms in which dietary curcumin, vitamin D, and zinc play a role in cancer carcinogenesis. [87,

88] However, the effect of dietary tomato and lycopene on miRNA expression in prostate carcinogenesis is not reported, especially in the early stages of prostate carcinogenesis.

We hypothesized that early phases of prostate carcinogenesis involves androgen-driven deregulation of miRNA and mRNA expression. Therefore, we propose to use TRAMP mice to investigate miRNA expression profile in early prostate carcinogenesis and the effect of testosterone on deregulated miRNAs in early prostate carcinogenesis. In particular, the impact of dietary tomato carotenoids on androgen-driven miRNA expression is examined in early prostate carcinogenesis. Testosterone and dietary tomato and lycopene modulate critical mRNA and miRNA expression to inhibit the prostate carcinogenesis. Moreover, we hypothesized that dietary tomato and lycopene affected testosterone-driven mRNA and miRNA expression in early prostatic carcinogenesis.

Specific aim 1: 1) MicroRNA expression profile in early prostate carcinogenesis is different from control mice with a normal prostatic epithelium. 2) Testosterone

stimulation of the early prostate carcinogenesis is associated with deregulation miRNA expression.

Specific aim 2: 1) Testosterone modulates gene expression involved in early prostate carcinogenesis. 2) Dietary tomato and lycopene regulated critical androgen-driven pathways in early prostate carcinogenesis. 3) Dietary tomato is more potent than lycopene in regulating gene expression in early prostate carcinogenesis.

Specific aim 3: 1) Tomato carotenoids regulated miRNA expression in early prostate carcinogenesis, and 2) tomato and lycopene contributed to testosterone-modulated miRNA expression in early prostate carcinogenesis.

References:

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Chapter 2: The Interface between Deregulated miRNA Expression and Testosterone during Early Prostate Carcinogenesis in the TRAMP model

Authors:

Lei Wan1, Hsueh-Li Tan1, Jennifer M. Thomas-Ahner2, Nancy E. Moran2, Dennis K.

Pearl3, Steven K. Clinton 2,4*

Author affiliations:

1 The Interdisciplinary Ph.D. program of Nutrition, The Ohio State University

2 Comprehensive Cancer Center, The Ohio State University

3 Department of Statistics, The Ohio State University

4 Division of Medical Oncology, Department of Internal Medicine, The Ohio State University

Abstract

Background: Changes in non-coding regions of the genome including alterations of miRNA expression are hypothesized to be involved in prostate carcinogenesis. TRAMP mice demonstrate histopathologic progression similar to humans. We have employed wild-type and TRAMP mice to examine the interactions between testosterone and miRNA expression during the very early phases of prostate carcinogenesis.

Methods: TRAMP and wild type (C57BL/6) mice were assigned to intact (sham surgery), castration, or castration + testosterone repletion groups at 8 weeks of age

(n=3/genotype x testosterone group). One week after castration, the testosterone- repleted group received testosterone (2.5 mg/kg BW /day) via osmotic pump. Mice were sacrificed 5 days later and the prostate lobes were harvested for molecular and histological evaluation. All 602 known mouse miRNAs were analyzed by NanoString ® technique.

Results: TRAMP mice showed early signs of PIN lesions at necropsy. Out of all known

602 miRNAs, 78 were detected in mouse prostatic tissue. The TRAMP genotype significantly up-regulated expression of 41 miRNAs, compared to wild type. For example, miR-21 and miR-15a were up-regulated (8 and 9.23-fold, respectively,

P<0.0001). Castration decreased expression of 38 miRNAs (>2 fold, P<0.05) and significantly increased expression of miR-150 and miR-1224 (-2.94 and -2.11 fold, respectively, P<0.0001). Testosterone-repletion reversed castration-driven alterations of miRNAs expression. Moreover, the impact of castration on miRNA expression differed between wild type and TRAMP mice. In castrated mice, miR-200a and miR-15b were down-regulated in TRAMP (P<0.0001) but not in wild type, compared to intact mice.

Selected miRNAs impacted by genotype or testosterone in mice were found in human prostate cancer cells.

Conclusion: A subset of miRNAs is expressed in the mouse prostate and changes in miRNA are detected early in TRAMP carcinogenesis, prior to major histopathologic abnormalities. Many of these miRNAs are also androgen-driven suggesting that testosterone interacts with genetic deregulation of miRNA expression at the very earliest phases of prostate carcinogenesis.

Introduction

MicroRNAs (miRNAs) are a class of small, non-coding RNAs, and deregulated miRNA expression has been implicated in development of both solid and non-solid cancers, including prostate cancer. [1, 2] [3] Mature miRNAs are 20-22 nucleotides long and are cleaved from RNA precursor host genes. [4] MiRNAs are incorporated into RNA-induced silencing complexes, which bind to the seed sequence (2-8 nucleotides at the 3’-end of mRNA) in mRNA in either a perfect or imperfect, complementary manner to induce mRNA degradation and translation suppression. [5, 6] In prostate cancer tissue, miRNA expression is lower than in the benign peripheral zone. [7] [8] Moreover, the miRNA processing enzyme, Dicer, and several miRNA host genes were up-regulated in primary prostate tumors compared to non-tumor prostate tissue. [9] Genetic aberrations in miRNA locus copy numbers also caused abnormal miRNA expression leading to prostate carcinogenesis. [10] Therefore, understanding miRNA expression patterns for development of chemopreventive targets, as well as diagnostic and prognostic biomarkers for prostate cancer is an active area of investigation.

Androgen and androgen receptor (AR) signaling are the primary targets of prostate cancer therapy. Testosterone is the critical hormone of prostate growth and prostate carcinogenesis, and is the main androgen in males. Since Charles Huggins first found that prostate cancer is responsive to castration in the 1940s, anti-androgens have become the main therapy for androgen sensitive prostate cancer.[11] Inhibition of 5- alpha-reductase-driven conversion of testosterone to dihydrotestosterone (DHT) reduced prostate cancer incidence in several large prospective clinical trials [12] [13].

DHT binds to the AR to induce gene expression, impacting cell growth and proliferation,

apoptosis, DNA damage and oxidative stress, inflammation and immunity, and invasion and metastasis. [14] [15] [16] [17] [18]

MicroRNAs both respond to and mediate testosterone signaling in prostate carcinogenesis. In human prostate cancer cell lines, testosterone-induced miRNA expression promoted proliferation.[19] [19-22] Conversely, miRNA transfection in androgen-dependent prostate cancer cells also influenced the levels of AR. [21]

Moreover, dysfunction of miRNAs promoted the development of castration-resistant prostate cancer (CRPC) in a xenograft mouse model. [23] One mechanism of regulation of the AR by miRNA was through miRNA binding of the 3’ un-translated region of the AR gene. [22] Therefore, regulation of miRNAs by testosterone is a likely driver of prostate cancer progression.

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model is a useful tool to study prostate cancer development and prostate cancer. [24-26] In TRAMP, androgens activate the rat probasin promoter to induce prostatic expression of the SV40 oncogene, leading to spontaneous development of prostate cancer, which progresses from hyperplasia to neoplasia to metastatic cancer, recapitulating human disease progression [27]. Furthermore, in TRAMP mice with advanced cancer some of the same miRNAs found in patients with metastatic prostate cancer are found in the circulation, including miR-141 and miR-375.[28] Thus, the TRAMP model provides an opportunity to investigate testosterone and miRNA interactions in prostate carcinogenesis.

Previously, studies have focused on miRNAs profiles in human prostate cancer cells and in cancer versus non-malignant samples, and testosterone-responsive miRNAs have been identified in prostate cancer cells and samples. [29] However, the role of miRNAs and their response to testosterone in early prostate carcinogenesis remains unknown. 23

Therefore, we investigated miRNA signatures in early prostate carcinogenesis and responsiveness of miRNAs to testosterone regulation using the TRAMP model. We hypothesized that 1) prostate carcinogenesis in TRAMP mice involves alteration of miRNA expression and 2) testosterone modulates miRNA expression in early prostate carcinogenesis.

Materials and Methods

Animals

All animal experiments were performed in compliance with the Ohio State University

Institutional Animal Care and Use Committee (IACUC). TRAMP mice (Jackson

Laboratories, Bar Harbor, ME) and wild type C57BL/6 mice were bred to generate heterozygous, male TRAMP (+/-) x C57BL/6 mice, which were crossed with FVB/N (-/-) females to generate the F1 generation TRAMP (+/-) C57BL/6×FVB/N mice. The male F1 generation of TRAMP (+/-) C57BL/6×FVB/N and C57BL/6 mice was used in the study.

The probasin-SV40 T-antigen (PB-Tag) transgene was identified by using REDExtract-

N-Amp Tissue PCR kit (Sigma-Aldrich) following the manufacturer’s instructions. The pair of synthetic primers used was: 5’-CAGAGCAGAATTGTGGAGTGG-3’ and 5’-

GGACAAACCACAACTAGAATGCAGTG-3’ (Sigma-Aldrich). The internal positive control was β-catenin.

Study Design

A 2 x 3 x 3 factorial study was designed to investigate the effects of testosterone and diet on prostate carcinogenesis in wild type and TRAMP mice, but for the purpose of this report, the main effects of genotype and testosterone will be the focus and dietary effects will be addressed in subsequent reports. The mice were weaned at 4 weeks of age and randomized to one of three experimental diets: a purified AIN-93G diet 24

(ResearchDiets, New Brunswick, NJ) containing 0.25% (w/w) placebo beadlets (DSM,

Basel, Switzerland), 10% tomato powder (w/w) (TP; FutureCeuticals, Momence, IL ), or

0.25% (w/w) lycopene beadlets (10% lycopene beadlet, DSM) diet. The concentration of total LYC in the final prepared diet was 384 mg/kg for 10% TP and 486 mg/kg for 0.25%

LYC beadlets diet. Eight-week-old mice were randomized to undergo either a sham surgery, a castration surgery, or a castration surgery followed by testosterone repletion

(2.5 mg/kg of body weight) to study the effect of testosterone on miRNA expression.

Mice received testosterone at 2.5mg/kg/day based on preliminary studies in our lab showing 2.5 mg/kg/day restored prostatic proliferation rate and growth of prostate in

TRAMP mice.[30] One week after castration, testosterone propionate (Sigma-Aldrich, St.

Louis, MO) was administered via Alzet® mini-osmotic pump implantation (Durect,

Cupertino, CA), following the manufacturer’s instructions. The mice were sacrificed by cervical dislocation 5 days after pump implantation or 12 days after castration surgery.

Prostate glands were procured by micro-dissection, and prostatic tissues were allocated to either RNA isolation (n=3/group) or for histological analysis (n=4 mice/ group). For the mRNA analysis group, the dorsal and lateral prostate lobes were immersed in RNAlater

® solution (Life technologies, Grand Island, NY) overnight at 4°C. The next day,

RNAlater® solution was removed and then the sample was stored at -80°C until RNA isolation. Micro-dissected prostates from mice designated for histological outcomes were fixed in 10% neutral buffered formalin overnight followed by processing and paraffin embedding.

Histology and Image analysis

Prostate samples underwent hematoxylin and eosin (H&E) staining. The 5um thick tissue slices were baked for 60 min before processing slides in Leica Autostainer XL

(Leica Microsystemsleica XL, Buffalo Grove, IL). Images were captured at 200X by 25

bright-field microscopy with a Nikon Eclipse E800 microscope (Nikon Instruments Inc,

Melville, NY) and Nikon Ds Ri1 digital camera (Nikon Instruments Inc).

RNA isolation

Total RNA was extracted from the fresh frozen prostate lobes preserved with RNAlater® using the RNeasy Mini kit (Qiagen, Valencia, CA) following the manufacturer’s instructions.

NanoString®

A mouse microRNA codeset (602 at the time of analysis, Version 1.0) was analyzed using NanoString nCounter technique (NanoString, Seattle, WA). Total RNA (100 ng) was incubated with specific reporter and capture probes at 64˚C for 18 h. After incubation, excess probes were removed, and the remaining hybridized miRNAs were immobilized on a streptavidin-coated cartridge using nCounter Prep Station

(NanoString), and miRNAs were counted using an nCounter Digital Analyzer

(NanoString®).

NanoString Data Analysis

Detectable miRNAs were identified by subtraction of the mean of the negative controls from the sample value. Data were normalized by natural log transformation followed by subtraction of the natural log-transformed endogenous reference, mmu-miR-720

(MIMAT0003484), which was selected based on low between-group variability (P >0.05 in ANOVA), and high stability within groups [mean standard error (MSE) =0.0356]. To define expression homology between the murine and human prostatic miRNA expression, detectable mouse miRNAs were compared to published human prostate cancer tissue and cell miRNA expression. [8]

In order to identify miRNAs that were differentially expressed in response to genotype and testosterone, normalized miRNA data was analyzed by three-way (genotype, hormone and diet) analysis of variance (ANOVA) using Stata12 statistical analysis package (StataCorp, College Station, TX). The significance level was set as P<0.05. The dietary main effects and diet interactions with other variables will be presented elsewhere.

To verify the pattern of changes in miRNA expression by genotype and hormone identified by NanoString®, we performed qRT-PCR on select miRNAs, based on the following criteria: 1) the count number was larger than 50, 2) the overall P<0.05 for the main effect of genotype or testosterone status on miRNA expression (ANOVA), and 3) fold-change between the treatment (genotype or testosterone) and respective control was greater than 2. qRT- PCR

TaqMan MicroRNA Assays Kit (Life Technologies) was used to quantify miR-200a and miR148a, which were normalized to miR-720. Samples were analyzed in 3 replicates.

Mmu-miR-720 was used as the reference and fold changed was calculated using 2-ΔΔCT method. [31]

Results

TRAMP prostate carcinogenesis

By 10 weeks old, TRAMP mice had visible prostatic intraepithelial neoplasia (PIN) lesions in prostate lobes (Figure 2.1d). Castration caused glandular atrophy in both wild type and TRAMP mice (Figure 2.1b&e). Testosterone repletion (2.5mg/kg) stimulated glandular cell growth in both wild type and TRAMP mice and resulted in a greater

number of PIN lesions in TRAMP mice (Figure 2.1c&f). H&E staining shows that castration caused significant prostate gland atrophy while testosterone repletion restores the glandular morphology in wild type mice.

MicroRNAs involved in prostate carcinogenesis in TRAMP mice are found in human tissue

The current study investigated the effect of testosterone and genotype on miRNA expression in early prostate carcinogenesis using the TRAMP model. Of the 602 known mouse miRNAs measured, 78 were detectable in mouse prostate tissue (Figure 2.2,

Appendix A), and 61 were differentially expressed between TRAMP and wild type mice

(P<0.05). The TRAMP genotype significantly up-regulated expression of 41 miRNAs compared to wild type (fold change>2, P<0.05). Castration significantly decreased expression of 38 miRNAs compared to sham-operation (fold change>-2, P<0.05) and significantly increased expression of 2 miRNAs (fold change>2, P<0.05). Out of the 78 miRNAs detected in mouse prostate, 64 have been previously found in human prostate cancer tissue.[8]

MicroRNAs are differently expressed between wild type and TRAMP mice

In TRAMP mice, 41 miRNAs were differently expressed compared to wild type mice

(Table 2.1). The highly up-regulated miRNAs by the effect of genotype (fold change>2, count number>50) included mmu-miR-15a (fold change=9.23, P<0.0001), mmu-miR-93

(fold change=65.67, P<0.0001), mmu-miR-21 (fold change=8, P<0.0001), mmu-miR-204

(fold change=2.26, P<0.0001) and mmu-mir-23b (fold change =2.09, P=0.002) (Figure

2.3). Eight miRNAs were down-regulated in TRAMP compared to wild type mice (Table

2.2), but none by greater than a 1.5-fold change.

MicroRNAs are differently expressed depending on testosterone status 28

Castration up-regulated expression of 6 miRNAs (Table 2.3) and down-regulated expression of 38 miRNAs compared to sham-surgery. There were 10 miRNAs down- regulated by greater than 2-fold by castration (Table 2.4). Testosterone-repletion up- regulated expression of 10 miRNAs (Table 2.5), and down-regulated expression of 2 miRNAs (Table 2.6), compared to castration. Mmu-miR-150 and mmu-miR-1224 were strongly up-regulated in castrated mice (2.11 and 2.74 fold, P<0.0001, respectively) compared to intact mice and down-regulated in testosterone-repleted mice (-2.94 and -

2.11 fold, P<0.0001, respectively), compared to castrated mice (Figure 2.4). Mmu-miR-

375 and mmu-miR-148a-3p were the most significantly down-regulated miRNAs by castration (-58.27 and -14.43 fold, P<0.0001 respectively) and up-regulated with testosterone-repletion (50.77 and 3.05 fold P<0.0001, respectively) (Figure 2.5). As expected, most miRNAs were expressed similarly between intact and testosterone- repleted mice, except mmu-miR-146a and mmu-miR-148a (Table 2.7). Therefore, compared to the sham-operation, castration down-regulated expression of a large set of miRNAs and testosterone-repletion restored miRNA expression.

Genotype-specific changes to miRNA expression in response to testosterone status

Expression of 16 miRNAs was significantly regulated by testosterone in a genotype- specific manner. Several of the most significant genotype x testosterone interaction- regulated miRNAs included mmu-miR-15b, mmu-miR-25, mmu-mir-200b, and mmu- miR-16 (Figure 2.6). Castration significantly decreased expression of miR-15a, miR-25, miR-200b and miR-16 in TRAMP mice but not in wild type mice (P<0.0001).

MicroRNAs that are not regulated by genotype or testosterone

Genotype, testosterone and genotype x testosterone interaction effects did not alter expression of mmu-miR-29b and mmu-miR-133a significantly.

Discussion

This study investigated alterations in miRNA expression in early prostate carcinogenesis by comparing TRAMP mice and healthy wild type mice, and identified miRNAs which are present in the mouse prostate and subject to testosterone regulation. Alteration of miRNA signatures by TRAMP genotype and testosterone status suggested that 1) expression of a large (41) miRNA set was up-regulated in TRAMP compared to wild type mice, 2) prostatic miRNA expression profile is subject to testosterone regulation, and 3) the effect of testosterone status on miRNA expression is in part dependent on genotype.

Overall, the 10 week old TRAMP mouse is a useful model for investigation of miRNA expression changes in early prostate carcinogenesis. The miRNAs detected here in the mouse prostate have also been previously reported to be present in human prostate cancer tissue. Our results suggest that the differing androgen-responsiveness and profiles of miRNA expression in TRAMP versus wild type mice contribute to the early prostate cancer initiation seen in TRAMP mice. Therefore, development of strategies targeting miRNAs utilizing the TRAMP model deserves further investigation in the context of chemoprevention.

MicroRNAs over-expressed in early prostate carcinogenesis compared to healthy prostate tissue.

We found a number of differences in miRNA expression between TRAMP and wild type mice, suggesting that deregulation of miRNA expression occurs in the very early phases of prostate carcinogenesis. Specifically, miRNA-21 and miR-93, which have been previously found to play roles in prostate carcinogenesis, were up-regulated in TRAMP compared to wild type mice (Figure 2.3/Table 2.1). Previous studies have shown that miR-21 is over-expressed in human prostate cancer cells, compared to normal prostate

cancer cells. [23] Moreover, a study in androgen-sensitive prostate cancer cells demonstrated that miR-21 over-expression may contribute to angiogenesis and invasion.

[32], [33] Our results supports the hypothesis that miR-21 functions as a oncogenic miRNA, and does so in early prostate carcinogenesis. While in the current study miR-93 was not highly expressed in mouse prostate tissue (count number<50), it was 65-fold up- regulated in TRAMP compared to wild type mice. Previously, miR-93 and its host gene were found to be up-regulated in prostate cancer tissue compared to healthy tissue, and miR-93 is believed to act by targeting Caspase-7, Bim and p21 to inhibit apoptosis and increase prostate cancer progression. [34] [9] In addition to oncogenic microRNAs, miR-

15a, which is believed to act as a tumor suppressor, was up-regulated in TRAMP compared to wild type mice. Previously miRNA-15a was found to function as a tumor suppressor in chronic lymphocytic leukemia, gastric cancer, breast cancer, and prostate cancer. [35] [36] [37] [38] In humans, it is found to be deleted in 80% prostate tumor samples, and its deletion is associated with decreased apoptosis, increased proliferation and cancer invasion. [38] [39] However, the role of miR-15a in early carcinogenesis remains unknown. We speculate that the up-regulation of miR-15a may be indicative of an adaptive response to inhibit carcinogenic processes driven by oncogenic miRNAs, but future studies on the expression and function of miR-15a in early carcinogenesis should be pursued.

Comparison of overall up-regulation of miRNA expression in TRAMP mice to that reported in advanced human prostate cancer.

A full characterization of miRNA profiles in human prostate carcinogenesis does not exist at this time, thus we can only speculate on comparisons between our study and human data. We found miRNA expression to be generally up-regulated in TRAMP mice

(prior to the development of carcinoma), compared to wild type mice (Table 2.2). This is 31

consistent with a previous report of differing circulating miRNAs in TRAMP vs. WT mice, in which TRAMP mice had generally greater miRNA expression than TRAMP negative control. [28] Lu, et al. (2005) suggested that miRNA expression is generally down- regulated in human prostate cancer tissue.[40] However, George and Croce (2006) showed that both up- and down-regulation of miRNA expression is observed in cancer tissues compared to non-cancer tissues. Several critical miRNAs identified in human prostate cancer cells, such as miR-29b and miR-133a [41] [42] [43], were unchanged in

TRAMP mouse early carcinogenesis. How miRNA expression patterns during different stages of TRAMP prostate carcinogenesis change from early to late malignancy should be investigated to better understand the temporal roles of miRNA expression in carcinogenic processes.

To determine which miRNA expression patterns are driven by androgen status, and how androgens drive miRNA changes in early carcinogenesis, both TRAMP and wild type mice were exposed to three different androgen conditions. Reduction of circulating androgens by castration reduced TRAMP-induced miRNA expression (Figure 2.3, Table

2.4). Compared to the intact and the testosterone-repleted mice, castrated mice had lower expression of miR-21 and miR-15a, regardless of genotype. It has been previously shown that miR-21 has an AR-binding domain and is induced by testosterone in LNCaP cells. [23] In our study, another strongly up-regulated miRNA in TRAMP vs. wt mice was miR-93. As expected, castration strongly down-regulated miR-93, compared to sham- operation and testosterone repletion groups. Androgen deprivation therapy, such as anatomical or pharmacologic castration, is a strategy to treat prostate cancer, and androgen signaling is a target in prostate cancer treatment. Critical genes in prostate carcinogenesis have androgen binding sites or are regulated by androgen receptor signaling. [44] In early prostate carcinogenesis, castration was found to decrease PIN 32

area in TRAMP mice (Figure 2.1). Thus, one of the inhibitory mechanisms of castration on prostate cancer may be the reduction of pro-oncogenic miRNA expression.

The role of androgens in miRNA expression

The current study is the first to report the testosterone sensitivity of murine miRNAs in early prostate carcinogenesis. The most strongly down-regulated miRNA by castration was miR-375 (fold change=-58, P<0.001). A previous study showed that miR-375 was up-regulated in both TRAMP with advanced prostate cancer compared to WT and in patients with metastatic, castration-resistant prostate cancer compared to healthy subjects or patients with localized androgen-sensitive prostate cancer. [28, 45]

Moreover, miR-375 was associated with shorter relapse-free duration in prostate cancer.

It has been shown that mRNA-375 is a tumor suppressor microRNA in oral cancer.

However, the function of miR-375 in prostate carcinogenesis has not yet been studied.

The current study showed that miR-375 is testosterone-sensitive and is down-regulated with testosterone-depletion.

Furthermore, this study is the first to show that castration up-regulates expression of miR-150 during early prostate carcinogenesis in TRAMP mice. A previous study showed miR-150 was deregulated in cancer compared to non-cancer tissue in gastric and colorectal cancer [46], but the expression of miR-150 in human prostate cancer has not yet been described. In addition, the effect of testosterone on miRNA expression differed between TRAMP mice and wild type mice. In wild type mice, testosterone status did not change expression of either miR-25 or miR-15b/16b and miR200b. However, in

TRAMP mice, castration significantly reduced expression of miR-25 and miR-16b, and testosterone repletion up-regulated their expression compared to the intact group. In nearly every case, testosterone repletion restored expression of miRNAs changed by

castration (Figure 2.4, Table 2.5). The regulated miRNA expression was restored to levels in intact mice when receiving 2.5 mg/kg/day testosterone, with concurrent stimulation of cell growth (Figure 2.1).

One question regarding the TRAMP model may be whether prostate carcinogenesis driven by SV-40 oncogene models miRNA profile changes seen in human prostate cancer. We found expression of miR-200a, miR-148a, and miR15a/15b in TRAMP mice to be consistent with expression patterns found in human LNCaP cells. Testosterone- repletion caused down-regulation of these miRNAs compared to sham-operation in mice and testosterone treatment caused down-regulation in human LNCaP cells compared to hormone-free media. To date, there is no report in humans of miRNA alterations in early prostate carcinogenesis, and therefore a direct comparison between our model of early carcinogenesis and human early carcinogenesis cannot be made.

The current study identified a number of miRNAs that occur with early prostate carcinogenesis, that are sensitive to testosterone, and identified which are androgen- dependent or independent. Future studies should be conducted to elucidate the biological functions of these miRNAs in early prostate carcinogenesis. Previously, it was reported that testosterone regulation of miRNA expression can be either AR-dependent or independent-manner. [47] Therefore determination of AR binding sites in miRNA regulatory regions will provide additional information on the mechanisms of androgen regulation of miRNA expression.

Conclusions

The current study used TRAMP mice as a model to study miRNA expression changes contributing to early stages of prostate carcinogenesis for further pharmaceutical and

dietary intervention study. Furthermore, testosterone-sensitivity of miRNAs in early prostate carcinogenesis provides targets for prostate cancer treatment. Understanding the changes in miRNA expression throughout prostate cancer progression provides opportunities to develop targeted prevention therapies and develop biomarkers of early carcinogenesis. Several dietary compounds, such as curcumin and resveratrol, have shown promise and these along with others should be investigated. [48] [49] Early cancer prevention by pharmacologic or dietary interventions targeting miRNA expression warrant further research.

Conflict of Interest:

The authors declare no conflicts of interest

Acknowledgments:

We thank the Nucleic acid core facility in the Ohio State University Comprehensive

Cancer Center (NIH/NCI P30 016058) to measure miRNA profiles using NanoString® nCounter technique. This work was supported by National Institutes of Health (NCI-

R01125384) and Dr. Erdman was the principal investigator.

Tables:

fold- P- Accession Gene ID 1 change value MIMAT0000540 mmu-miR-93 65.67 <0.0001 MIMAT0000141 mmu-miR-130a 13.48 <0.0001 MIMAT0004536 mmu-miR-151-5p 11.98 <0.0001 MIMAT0000128 mmu-miR-30a 11.76 <0.0001 MIMAT0003782 mmu-miR-676 10.86 <0.0001 MIMAT0000526 mmu-miR-15a 9.23 <0.0001 MIMAT0000530 mmu-miR-21 8 <0.0001 MIMAT0000158 mmu-miR-146a 7.12 <0.0001 MIMAT0003454 mmu-miR-423-3p 5.92 <0.0001 MIMAT0000655 mmu-miR-100 5.87 <0.0001 MIMAT0000534 mmu-miR-26b 5.37 0.003 MIMAT0000652 mmu-miR-25 5.34 <0.0001 MIMAT0000546 mmu-miR-103 4.72 0.004 MIMAT0000536 mmu-miR-29c 4.39 0.007 MIMAT0000590 mmu-miR-342-3p 3.9 0.003 MIMAT0000739 mmu-miR-375 3.82 0.001 MIMAT0000519 mmu-miR-200a 3.22 <0.0001 MIMAT0005460 mmu-miR-1224 3.14 <0.0001 MIMAT0000219 mmu-miR-24 2.92 0.018 MIMAT0000162 mmu-miR-152 2.8 0.045 MIMAT0000595 mmu-miR-345-5p 2.76 <0.0001 MIMAT0000233 mmu-miR-200b 2.62 <0.0001 MIMAT0000138 mmu-miR-126-3p 2.61 0.035 MIMAT0000237 mmu-miR-204 2.26 <0.0001 MIMAT0000122 mmu-let-7i 2.14 0.026 MIMAT0000125 mmu-miR-23b 2.09 0.002 MIMAT0000161 mmu-miR-151-3p 1.97 0.003 MIMAT0000135 mmu-miR-125a-5p 1.75 <0.0001 MIMAT0000247 mmu-miR-143 1.68 <0.0001 MIMAT0000537 mmu-miR-27a 1.64 <0.0001 MIMAT0000210 mmu-miR-181a 1.59 <0.0001 Table 2. 1 MicroRNA expression up-regulated in TRAMP compared to wild type mice. 1 3-way ANOVA analysis for genotype, hormone, and dietary main effects and interactions (alpha=0.05). (n=3/group).

Accession Gene Fold change1 P-value MIMAT0004894 mmu-miR-574-3p -1.95 0.51 MIMAT0011287 mmu-miR-2183 -1.44 0.55 MIMAT0000238 mmu-miR-205 -1.42 0.05 MIMAT0000656 mmu-miR-139-5p -1.38 0.31 MIMAT0004883 mmu-miR-466g -1.38 0.2 MIMAT0011217 mmu-miR-2141 -1.35 0.59 MIMAT0000565 mmu-miR-328 -1.08 0.44 MIMAT0000711 mmu-miR-365 -1 0.99 Table 2.2 MicroRNA expression down-regulated in TRAMP compared to wild type mice 1miRNA expression changed by genotype ≥|1.0| fold. P -values are according to 3-way ANOVA analysis for genotype, hormone, and dietary main effects and interactions. (alpha=0.05). (n=3/group).

Castration/Intact

Accession Gene Fold Change P-value MIMAT0003782 mmu-miR-676 4.87 0.005 MIMAT0003130 mmu-miR-486 4.48 0.001 MIMAT0000656 mmu-miR-139-5p 3.15 0.001 MIMAT0005460 mmu-miR-1224 2.71 <0.0001

MIMAT0000160 mmu-miR-150 2.11 <0.0001 MIMAT0000524 mmu-let-7e 1.96 <0.0001 Table 2. 3 MicroRNA expression up-regulated in castrated compared to intact mice. miRNA expression significantly different between castrated and intact mice, according to Tukey’s post hoc test following 3-way ANOVA analysis for genotype, testosterone, and dietary main effects and interactions (alpha=0.05). (n=3/group). (fold change>1.5 and count number>50)

Castration/Intact Accession Gene Fold Change P-value MIMAT0000739 mmu-miR-375 -58.27 <0.0001 MIMAT0000536 mmu-miR-29c -20.92 <0.0001 MIMAT0000516 mmu-miR-148a -14.43 <0.0001

MIMAT0000212 mmu-miR-183 -13.84 <0.0001 MIMAT0000540 mmu-miR-93 -12.99 <0.0001

MIMAT0000534 mmu-miR-26b -8.96 0.005 MIMAT0001537 mmu-miR-429 -4.83 <0.0001

MIMAT0000595 mmu-miR-345-5p -4.26 <0.0001 MIMAT0000711 mmu-miR-365 -3.85 <0.0001

MIMAT0000130 mmu-miR-30b -3.33 <0.0001

Table 2. 4 MicroRNA expression down-regulated in castrated compared to intact mice. miRNA expression significantly different between castrated and intact groups according to Tukey’s post hoc tests following 3-way ANOVA analysis for genotype, testosterone, and dietary main effects and interactions (alpha=0.05) (n=3/group) (fold change<-2 and count number >50).

Repletion/Castration

Fold Accession Gene P- value Change MIMAT0000739 mmu-miR-375 50.77 <0.0001 MIMAT0000536 mmu-miR-29c 15.89 <0.0001 MIMAT0000540 mmu-miR-93 15.14 <0.0001 MIMAT0000212 mmu-miR-183 12.93 <0.0001 MIMAT0000162 mmu-miR-152 7.98 <0.0001 MIMAT0000534 mmu-miR-26b 5.80 0.030 MIMAT0000141 mmu-miR-130a 5.60 0.014 MIMAT0001537 mmu-miR-429 4.78 <0.0001 MIMAT0000648 mmu-miR-10a 4.52 0.043 MIMAT0000655 mmu-miR-100 4.08 0.042 Table 2. 5 MicroRNAs up-regulated in testosterone repleted mice compared to castrated mice miRNA expression significantly different testosterone repletion group and castration group according to Tukey’s post hoc tests following 3-way ANOVA analysis for genotype, hormone, and dietary main effects and interactions (alpha=0.05). (n=3/group). (fold change >2)

Repletion/Castration Fold Accession Gene P- value Change MIMAT0000160 mmu-miR-150 -2.94 <0.0001 MIMAT0005460 mmu-miR-1224 -2.11 <0.0001 Table 2. 6 MicroRNAs down-regulated in testosterone-repleted mice compared to castrated mice. miRNA expression significantly different testosterone repletion group and castration group according to Tukey’s post hoc tests following 3-way ANOVA analysis for genotype, hormone, and dietary main effects and interactions (alpha=0.05) (n=3/group).

Repletion/Intact Fold Accession Gene P- value Change MIMAT0000158 mmu-miR-146a 4.60 0.015 MIMAT0000516 mmu-miR-148a -1.59 0.021 Table 2. 7 MicroRNAs differently expressed between testosterone repletion and intact mice. miRNA expression significantly different testosterone repletion group and intact group according to Tukey’s post hoc tests following 3-way ANOVA analysis for genotype, hormone, and dietary main effects and interactions (alpha=0.05). (n=3/group).

Figures:

Intact Castration Repletion

a b c

d e f

TRAMP

Figure 2. 1 H&E staining was assessed in prostate anterior lobes from WT and TRAMP mice fed with control diet for 10 weeks. Tomato and lycopene fed mice had similar histology with control fed mice and images were not shown. (Image 200X). H&E, haematoxylin-eosin. In 10 wk TRAMP, PIN area was already developed (arrow).

Figure 2. 2 Heatmap of clustered microRNA expression grouped by genotype and testosterone status. Cas, Castration; Int, intact; Rep, testosterone repletion. Eighty detected miRNAs in prostate were clustered using cluster 3.0 and Java Treeview. Each column is an individual mouse the rows are miRNAs. The range is -3 to 3.

*** *** *** ***

***

Figure 2. 3 MicroRNA expression significantly up-regulated in TRAMP mice compared to intact mice. Values are expressed as mean fold change ± se. Expression of miRNA in intact, wild-type was designated as “0” and expression of other treatments is expressed as fold changed above or below that of wild-type intact group (n=9/group). * P<0.05, ***P<0.0001.

* ** ***

***

Figure 2. 4 MicroRNAs up-regulated by castration, compared to sham-operation and testosterone-repletion. Values are expressed as mean fold change ± se. Expression of miRNA in intact, wild-type was designated as “0” and expression of other treatments is expressed as fold changed above or below that of wild-type intact mice (n=9/group). * P<0.05, ** P<0.01, ***P<0.0001(compared to intact group in wild type and TRAMP separately).

***

*** *** ***

Figure 2. 5 MicroRNAs significantly down-regulated by castration compared to sham- operation and testosterone-repletion. Values are expressed as mean fold change ± se. Expression of miRNA in intact, wild-type was designated as “0” and expression of other treatments is expressed as fold changed above or below that of wild-type intact mice (n=9/group). ** P<0.01, ***P<0.0001(compared to intact group in wild type and TRAMP separately).

***

Figure 2. 6 MicroRNA expression significantly changed by an interaction between genotype and testosterone. Values are expressed as mean fold change ± se. Expression of miRNA in intact, wild-type was designated as “0” and expression of other treatments is expressed as fold changed above or below that of wild-type intact mice (n=9/group). ***P<0.0001(compared to intact TRAMP mice).

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Chapter 3: Dietary Tomato and Lycopene Interrupt Testosterone-driven Gene Expression in Early TRAMP Prostate Carcinogenesis

Authors:

Lei Wan1, Hsueh-Li Tan1, Jennifer M. Thomas-Ahner2, Nancy E. Moran2, Dennis K.

Pearl3, Steven K. Clinton 2,4*

Author affiliations:

1 The Interdisciplinary Ph.D. program of Nutrition, The Ohio State University

2 Comprehensive Cancer Center, The Ohio State University

3 Department of Statistics, The Ohio State University

4 Division of Medical Oncology, Department of Internal Medicine, The Ohio State University

Abstract:

Background: Epidemiological and laboratory studies suggest tomato and lycopene possess anti-prostate cancer activities. Testosterone is the critical hormone in prostate growth and prostate carcinogenesis. Thus, we hypothesized that tomato and lycopene feeding would inhibit prostate carcinogenesis by interrupting androgen-driven gene expression in prostate carcinogenic pathways and, therefore, morphological changes in early prostate carcinogenesis.

Methods: TRAMP (transgenic adenocarcinoma of the mouse prostate model) mice (4- wk-old) were fed one of the three diets (AIN-93G+0.25% control beadlets (w/w),

AIN93G+10% tomato powder (w/w) and AIN93G+0.25% lycopene beadlets (w/w)). Four weeks later, mice were assigned to either intact (sham surgery), castration, or castration with testosterone repletion groups. One week after castration, the castration with testosterone-repletion group received testosterone (2.5 mg/kg BW /day) via osmotic pump. Mice were sacrificed 5 d after pump implantation and the prostate was reserved for molecular and histological evaluation. Body weight and urogenital tract weight (UGT) were recorded and plasma lycopene was analyzed by HPLC-PDA. Prostatic mRNA expression was assessed using a customized NanoString ® murine prostate carcinogenesis codeset (200 genes).

Results: Early sign of PIN lesions were observed at 10-wk-old TRAMP mice. Plasma lycopene was not detected in control-fed mice, and were increased but did not differ significantly between tomato- or lycopene-fed mice (P=0.81). Castration decreased body weight gain (5.3±0.2 g vs. 7.0±0.3 g), UGT weight (118±3 mg vs. 389±10 mg), and proliferation rate (0.32±0.1% vs. 3.8±0.4%) in the prostate anterior lobe, compared to intact mice (all P<0.0001). Out of 200 measured mRNAs, 189 were detected in mouse 53

prostatic tissue, and of these, 153 were changed by testosterone status, 1 by diet alone, and 7 by both testosterone and diet (P<0.001) main effects. Castration induced genetic changes consistent with activation of the cell growth and invasion, aryl hydrocarbon receptor (AhR) signaling, and down-stream Tgfb1/2 (fold change=3.4, P<0.0001), as determined by IPA network analysis. Dietary tomato impacted several testosterone- sensitive down-stream AhR signaling molecules including Mapk1, Gstp1 and Jun

(P<0.0001) in a testosterone-dependent manner. Moreover, dietary lycopene but not tomato decreased expression of neuroendocrine biomarkers: never growth factor receptor (Ngfr) (-2.13 fold, P=0.02), synaptophysin (Syp) (-2.29 fold, P=0.005), and a cell adhesion biomarker, cadherin-2 (Cdh2) (-1.82 fold, P=0.03), compared to control diet.

Lycopene-feeding also strengthened the down-regulation of Birc5 which was dramatically reduced by castration to a greater extent than the control-feeding (P=0.01).

Conclusion: Castration caused prostate growth regression and proliferation inhibition in

TRAMP in parallel with changes in gene expression patterns. Prostatic expression of genes involved in the AhR signaling pathway are androgen-dependent in early TRAMP prostate carcinogenesis, and this expression is further impacted by dietary tomato and lycopene. Dietary lycopene more strongly regulated neuroendocrine and cell adhesion biomarkers than dietary tomato in early prostate carcinogenesis. Therefore, dietary tomato and lycopene modulated androgen-driven mRNA expression before impacting morphological changes in early prostate carcinogenesis TRAMP mice.

Introduction:

Consumption of foods rich in tomato carotenoids, especially lycopene, is associated with lower prostate cancer risk. [1] The mechanisms by which lycopene inhibits prostate cancer development include reducing oxidative stress, decreasing proliferation, increasing apoptosis, improving gap junction communication, inhibiting angiogenesis, and regulating androgen signaling. [2] [3] [4] [5] [6] [7] Testosterone is the primary androgen in males, and is essential for prostate growth and prostate carcinogenesis. [8]

Testosterone is mainly generated from cholesterol under the control of the hypothalamus-pituitary-testis axis, and is locally converted by 5α-reductase into dihydrotestosterone (DHT), which binds to the androgen receptor to regulate genes involved in critical prostate carcinogenesis pathways. [9]

Lycopene impacts testosterone metabolism and vice versa. Consumption of lycopene resulted in decreased circulating testosterone, and castrated rats accumulated twice the hepatic lycopene as intact control rats. [10, 11] Furthermore, in androgen-sensitive prostate cells, lycopene was 4.5 times more concentrated than in androgen-independent prostate cells. [12] Finally, lycopene consumption impacted the activity of enzymes involved in testosterone metabolism and production in mice, including 5α-reductase and

Cyp7B1.[13] Taken together, these studies indicate that androgen sensitivity is correlated with increased lycopene uptake, and lycopene interrupts androgen production and signaling, all of which may impact prostate carcinogenic processes.

Consumption of tomato and lycopene is a potential prostate cancer prevention strategy.

The inhibition of DHT conversion to testosterone was associated with lower prostate cancer incidence. It was previously shown that tomato feeding disrupts androgen-driven processes in early TRAMP (transgenic adenocarcinoma of the mouse prostate) 55

carcinogenesis to prevent progression from proliferative intraepithelial neoplasia to carcinoma, the mechanisms remain unclear. [14] Furthermore, whether lycopene, the red carotenoid in tomatoes, is the primary bioactive compound in tomatoes or if the mixture of phytochemicals present in the whole tomato contributes to anti-cancer activity is under investigation. Dietary tomato had a stronger effect on inhibiting prostate carcinogenesis in rats than lycopene-alone. [15] In contrast, Konjeti, et al. showed that lycopene but not tomato inhibited prostate carcinogenesis in TRAMP. [16] Tomatoes are rich in vitamins and phytochemicals including carotenoids and polyphenols. [17] [18]

Recent studies have suggested non-lycopene carotenoids and polyphenols in tomatoes also possess anti-cancer activity. [14, 19-22] For instance phytoene and phytofluene, colorless precursors of lycopene, have been shown to decrease inflammatory prostglandin E2 levels in vitro, and phytofluene-fed rats had lower serum testosterone concentrations compared to control-fed rats. [23] [10] Furthermore, tomato polyphenols were shown to counteract IGF-1 in prostate cancer cells. [24] While the efficacy of tomato vs. lycopene in reduction of prostate cancer incidence has shown mixed results in preclinical models [15] [16], several studies in TRAMP mice indicated that tomato- feeding inhibits prostate carcinogenesis. In TRAMP mice, tomato-feeding decreased prostatic neoplasia area, prostate cancer incidence, and increased survival rate compared to control diet.[14] Zuniga, et al. showed tomato-feeding initiated at 4-wk-old led to increased apoptotic index and decreased proliferation index compared to control diet in 18-wk-old TRAMP mice. [25] Tan, et al. also showed that tomato-feeding initiated upon weaning reduced the histopathologic lesion score in prostate lobes in 19-wk-old

TRAMP mice.[26] These studies indicate that early dietary intervention may be critical.

However, the comparative efficacy of tomato vs. lycopene in inhibition of early carcinogenic gene expression and morphological changes is unknown.

Which androgen-driven processes in early carcinogenesis, such as growth and proliferation, apoptosis, and invasion, are disrupted by tomato- or lycopene-feeding remain unknown. In the current study, we defined androgen-driven morphological and gene expression changes, examined the impacts of tomato and lycopene, and therefore identified the testosterone-dependent dietary effects on expression of 200 prostate cancer-related genes in early TRAMP prostate carcinogenesis. We hypothesized that 1) testosterone drives changes in morphology and gene expression in critical molecular pathways involved in early prostate carcinogenesis, 2) dietary tomato and lycopene impact critical androgen-driven gene expression changes in early prostate carcinogenesis, and 3) dietary tomato is more potent than lycopene in regulating androgen-driven gene expression in early prostate carcinogenesis.

Materials and Methods:

Animal and diets

All animal experiments were performed in compliance with the Ohio State University

Institutional Animal Care and Use Committee (IACUC). Male TRAMP mice (Jackson

Laboratories, Bar Harbor, ME) were maintained on a C57BL/6 background then crossed with FVB/N females to generate the male TRAMP (+/-) C57BL/6×FVB/N mice. The SV transgene was confirmed by genotyping using the REDExtract-N-Amp Tissue PCR kit

(Sigma-Aldrich, St. Louis, MO) following the manufacturer’s instructions.

Study Design

A 3 x 3 factorial study was utilized to investigate the effects of diet and testosterone status on prostate carcinogenesis in TRAMP mice. Mice were weaned at 4 weeks of age and randomized to one of the three diets: purified AIN-93G diet (ResearchDiets, New

Brunswick, NJ) containing 0.25% (w/w) placebo beadlets (DSM, Basel, Switzerland), 57

10% tomato powder (LOT 18190, FutureCeuticals, Momence, IL), or 0.25% (w/w) lycopene beadlets (RediVivo 10% lycopene, DSM, Heerlen, Netherlands) (Table 3.1).

The concentration of total lycopene in the final prepared diet was 384 mg/kg for 10% tomato powder and 482 mg/kg for 0.25% lycopene beadlet diet. Eight-week-old mice within each diet group were randomized to undergo either a sham (superficial incision only) surgery, a castration surgery, or a castration surgery followed by testosterone repletion (2.5 mg/kg of body weight). One week after castration, testosterone propionate

(Sigma-Aldrich, St. Louis, MO) was administered via Alzet® mini osmotic pump implantation (Durect, Cupertino, CA), following the manufacturer’s instructions. The mice were sacrificed by cervical dislocation 5 d after pump implantation or 12 d after castration surgery. Plasma was collected for lycopene concentration analysis. Prostate glands were procured by micro-dissection, and were separated in constituent lobes: anterior (AP), ventral (VP), dorsal (DP), and lateral (LP). Five micro-dissected prostates/experimental group were designated for histological outcomes, and were fixed in 10% neutral buffered formalin overnight followed by processing and paraffin embedding. The dorsal and lateral prostate lobes were immersed in RNAlater ® solution

(Life technologies, Grand Island, NY) overnight at 4 °C. The next day, RNAlater® was removed and the sample was stored at -80 °C until RNA isolation.

Plasma lycopene analysis

Plasma carotenoids (n=6/group) were extracted and analyzed by high pressure liquid chromatography by photodiode array detection, as previously described. [27]

Immunohistochemistry

Proliferation index was estimated by Ki67 staining. Paraffin-embedded tissue sections (5

µm) were baked for 60 min at 60 °C followed by deparaffinization and stepwise 58

rehydration in 2 washes of xylene, exposure to 100% ethanol (twice), 95% ethanol, and

70% ethanol (3 min each). After breaking protein cross-linkages by steamer incubation for 30 min for antigen retrieval, the slides were transferred to a humid chamber to inactivate endogenous peroxidase with peroxide (15 min). The primary antibody was monoclonal rat anti-mouse Ki67 antigen clone TEC3 (1:50) (Dako, Produktionsvej,

Denmark) (60 min), and the secondary antibody was biotinylated rabbit anti-mouse IgG

(1:200) (Dako) (30 min). After antibody binding, tissues were stained in streptavidin/HRP

(1:200) (30 min), and DAB (Dako) for 10 min. Lastly, the tissue sections were stained with Mayer’s hematoxylin for 2 min and dehydrated in 70% ethanol, 95% ethanol, 100% ethanol (twice), and xylene (twice) (3 min each). Glass coverslips were placed on the slides on the mounting medium and were air-dried in the fume hood.

Image analysis

Stained slides were scanned at the Virtual Microscopy core facility at the Ohio State

University for Aperio® imageScope (Aperio®, Vista CA) analysis. Proliferation index was indicated by positive Ki67staining. In 10-wk-old TRAMP mice, both intact and repletion groups had prostatic intraepithelial neoplasia (PIN) areas. Traditionally, proliferation rate is presented as percentage of positive staining of total staining in three selected area

(400 x image).[28] In the current study, the proliferation rate had dramatic variation depending on if selected counting areas had PIN lesion, which cause bias in statistic and mask the differences between treatments. To solve this problem, we assessed the percentage of positive pixel counting percentage to the total staining in the whole anterior lobe. We used the positive pixel counting 8.1 (Aperio ® ImageScope) in the whole tissue to quantify the Ki67 staining.

RNA isolation

The RNeasy Mini kit (Qiagen, Valencia, CA) was used to extract the total RNA from the fresh frozen prostate lobes preserved with RNAlater® following the manufacturer’s instructions. mRNA expression

We custom-designed a murine prostate carcinogenesis gene codeset of 200 genes implicated in prostate carcinogenesis and androgen sensitive, The gene codeset also included carotenoid metabolizing enzymes beta-carotene 15,15’- monooxygenase

(Bcmo1), beta-carotene 9’,10’-dioxygenase (Bcdo2), and three housekeeping genes

(Actb, B2m and Gapdh) (Appendix B). Gene expression was examined by NanoString® technique (NanoString®, Seattle, WA). For analysis, extracted total RNA (100 ng) was incubated with specific reporter and capture probes at 64 ˚C for 18 h. After incubation, the excess probes were removed, and the remaining hybridized miRNAs were immobilized on a streptavidin-coated cartridge using the nCounter Prep Station

(NanoString®). Last, miRNAs were counted by an nCounter Digital Analyzer

(NanoString®). TRAMP prostate tissue mRNA expression was analyzed by subtraction of negative controls from the sample NanoString data, followed by natural log transformation and normalization to Ccng1 (NM_009831.2) which has low between- group variability (P<0.05 by ANOVA), and high stability within groups [mean standard error (MSE) = 0.0105].

Statistics

In order to identify mRNAs that were differentially expressed in response to diet and testosterone, normalized miRNA data was analyzed by 2-way (hormone status and diet type were the independent variables) ANOVA. The significance level was set as α=0.05.

Body weight, tissue weight and plasma lycopene concentrations were also analyzed by

2-way ANOVA. The Sidak post hoc analysis was used for pair-wise comparisons after

ANOVA. All statistics were calculated using Stata12 statistical analysis package

(StataCorp, College Station, TX)

IPA pathway analysis

IPA software (http://www.ingenuity.com) was used to detect androgen-dependent changes consistent with alterations in signaling networks and pathways. The gene expression ratios and pair wise P-values for castration vs. intact groups were used as data inputs for IPA pathway analysis. Cutoff standard were set to P<0.05. Molecules which were significantly impacted by castration status were overlaid onto global molecular networks and significance was calculated as previously described. [29] [30]

Results:

Animal weight and tissue weight

Tomato powder- and lycopene-fed TRAMP mice had similar changes in body weight as control-fed TRAMP mice over 6 weeks (5.9 ±0.3 g vs. 6.1 ±0.3 g vs. 6.3 ±0.3 g, respectively) (Figure 3.1). Castration significantly decreased body weight gain (5.3 ±0.2 g vs. 7.0 ±0.3 g, P<0.0001) and UGT weight (118.6 ±3.7 mg vs. 389.2 ± 10 mg,

P<0.0001) compared to intact mice (Figure 3.1). Compared to castration, testosterone- repletion returned the body weight gain and UGT weight to be similar to that of the intact group (P<0.0001) (Figure 3.1). Castration dramatically decreased prostate lobe (AP

8.2±0.3 mg; VP, 2.3±0.1; DP, 5.4±0.2 mg and LP 2.8 ± 0.5 mg) weights compared to intact group (AP 33.2 ±1.3 mg; VP, 27.9±0.4; DP, 13.7±0.6 mg and LP 8.2 ± 0.9 mg)

(P<0.0001) (Figure 3.2). Tomato-feeding numerically reduced AP (16.4 ±1.4 mg) and statistically reduced DP (7.7 ±0.5) weight compared to control diet (AP, 21.5 ±1.8 mg;

DP, 9.7 ±0.6 mg) (P=0.07 and P=0.02, respectively) (Figure 3.2). Lycopene led to 61

increased LP (7.5 ±1.1 mg) weight compared to control diet (4.4 ±0.7 mg) (P=0.06)

(Figure 3.2).

Circulating lycopene concentration under effect of testosterone and diet

Lycopene was not detected in mice fed the control diet. Mice fed tomato or lycopene had similar plasma lycopene concentrations (0.27± 0.02 µmol/L and 0.27 ±0.02 µmol/L,

P=0.81). Testosterone status did not change plasma lycopene concentrations in tomato- or lycopene-fed mice. Moreover, testosterone and diet did not significantly interact to impact plasma lycopene concentration (Figure 3.3).

Testosterone status altered proliferation rate of prostate tissue

Immunohistochemistry of Ki67 was used to measure the proliferation index in the AP lobe. Castration significantly decreased proliferation rate (0.3 ±0.07 %, P <0.0001) and repletion restored the proliferation rate (2.6±0.3 %, P <0.0001), compared to intact group

(3.7±0.4 %). Dietary tomato and lycopene did not significantly alter the proliferation rate, compared to control-feeding. Moreover, a testosterone x diet interaction did not influence proliferation (Figure 3.4).

Testosterone and diet affect prostatic gene expression

Out of 200 genes, 189 were detected in TRAMP prostate tissue. Testosterone status uniquely impacted expression of 153 genes, and diet type uniquely impacted expression of 1 gene (P<0.05). Testosterone and diet impacted expression of 7 of the same genes, and a testosterone x diet interaction impacted expression of 10 genes. Expression of 18 genes was not altered under the effect of testosterone and dietary intervention (Figure

3.5). The strongly up-regulated genes in castration group included tumor necrosis factor

(Tnf) (fold change=33.71, P<0.0001), insulin-like growth factor binding protein 3 (Igfbp3)

(fold change=24.48, P <0.0001), prostaglandin-endoperoxide synthase 2 (Ptgs2) (fold 62

change=10.37, P <0.0001) and sonic hedgehog (Shh) (fold change=10.04, P<0.0001).

Castration strongly down-regulated E2F transcription factor 1 (E2f1) (fold change=-

10.07, P<0.0001), NK-3 transcription factor, locus 1 (Drosophila) (Nkx3-1) (fold change=-10.98, P<0.0001), cell division cycle 25C (Cdc25c) (fold change=-11.73,

P<0.0001), antigen identified by monoclonal antibody Ki-67 (Mki67) (fold change=-13.02,

P<0.0001), and probasin (Pbsn) (fold change=-28.70, P<0.0001).

Androgen-regulated genes were involved in aryl hydrocarbon reporter signaling

Genes differentially expressed between intact and castration group were mapped onto canonical pathways in the Ingenuity Pathways Knowledge Base in IPA. Androgen- dependent gene expression changes were most strongly associated with altered aryl hydrocarbon receptor signaling (AhR) (Figure 3.6). Out of the 189 genes detected in mouse prostate, androgen status regulated expression of 26 genes involved in AhR signaling (Table 3.2). Based on Fisher’s exact test, the probability between the genes in the data set and the canonical pathway is 4.56E-7, which suggested the strong association between genes in the dataset and canonical pathway (Table 3.2).

Cell survival and death, cellular movement is the most influenced networks

Twenty significantly regulated genes by testosterone status were consistent with changes in cell survival, death, and cellular movement regulatory pathways based on

IPA analysis (Figure 3.7). Castration down-regulated mRNA expression of the proliferation biomarker, Ki67 (fold change=-13.02, P<0.0001) and angiogenic vascular endothelial growth factor A (Vegfa) (fold change=1.58, P<0.0001), whereas castration up-regulated caspase 1 (Casp1) (fold change=7.92, P<0.0001), vimentin (Vim) (fold change=3.38, P<0.0001) and transforming growth factor, beta 1 (Tgfb1) (fold change=3.06, P<0.0001), compared to sham operation.

Dietary tomato and lycopene altered expression of genes involved in proliferation and invasion

Dietary tomato significantly up-regulated aryl hydrocarbon receptor nuclear translocator- like (Arntl) compared to control group (fold change=1.27, P=0.01) in early prostate carcinogenesis in TRAMP mice. Dietary lycopene decreased expression of nerve growth factor receptor (TNFR super family, member 16) (Ngfr) (fold change=-2.13, P =0.01), synaptophysin (Syp) (fold change=-2.02, P =0.005), and cadherin 2 (Cdh2) (fold change=-1.60, P =0.026), compared to control diet. Moreover, compared to dietary tomato, dietary lycopene down-regulated Ngfr (fold change=-2.13, P =0.019), Syp (fold change=-2.29, P =0.005) and Cdh2 (fold change=-1.82, P =0.030) (Figure 3.8, Table

3.5).

The effect of androgen-deprivation on mRNA expression was dependent on tomato- or lycopene-feeding

Dietary tomato and lycopene impacted the effect of androgen-depletion on expression of androgen-regulated mitogen-activated protein kinase 1 (Mapk1) (P=0.0256), baculoviral

IAP repeat-containing 5 (Birc5) (P=0.0168), glutathione S-transferase, pi 1 (Gstp 1)

(P=0.0213) and Jun oncogene (Jun) (P=0.0336) (Figure 3.9). Castrated mice had lower

Birc5 expression compare to intact and testosterone-repleted mice and this difference was accentuated in lycopene- followed by tomato- and control diet-fed mice

(lycopene>tomato>control diet, fold changes are -14.00, -9.66, -5.55 respectively

P<0.05). However, while the effect of castration on Jun and Mapk1 expression differed by dietary treatment in the overall model, expression did not differ by diets within castrated mice based on Sidak post hoc multi-comparison. Castration significantly up- regulated Gstp 1 (P<0.0001) compared to intact mice, when fed control or lycopene diets, but not tomato diet. 64

Discussion:

Anti-cancer activities of androgen deprivation and tomato bioactive compounds have been reported in prostate cancer. However, the molecular mechanisms of the preventative effect of dietary intervention, and testosterone x diet interaction in early prostate carcinogenesis are not yet known. The current study is the first to investigate the impact of dietary tomato carotenoids on androgen-driven gene expression in prostate carcinogenesis. In TRAMP early prostate carcinogenesis, testosterone and dietary tomato and lycopene regulated genes involved in cell growth and invasion pathways of prostate carcinogenesis. Dietary tomato and lycopene strengthened the effect of castration on mRNA expression in early prostate carcinogenesis, which suggested that tomato carotenoids impact on androgen-driven gene expression.

Castration led to morphological changes of prostate tissue

It is well-known that testosterone is essential to maintain growth of prostate gland.

Depletion of testosterone by castration significantly decreased body weight gain and

UGT weight compared to sham operation in 10-wk-old TRAMP mice. Testosterone repletion (2.5mg/kg/body weight) returned body weight gain and UGT weight to levels of sham-operated mice. Dietary tomato- and lycopene-feeding did not independently change body weight or UGT weight. Moreover, dietary tomato and lycopene did not impact the castration-modulated body weight gain and UGT weight. It has been shown that tomato-fed TRAMP mice displayed less PIN area even at 12-wk-old, compared to control fed mice. [14] However, in the current study, tomato and lycopene did not reduce

PIN area compared to control-feeding. The disparity may because TRAMP mice developed overall PIN area at 12-wk old whereas in 10-wk old TRAMP mice developed

PIN area sparsely. Castration decreased prostate lobe weights compared to sham

operation and testosterone repletion. Dietary tomato decreased AP and DP weights whereas, dietary lycopene increased LP weight compared to control diet. The differences may be as a result of cell-type specific response to dietary tomato and lycopene. The inhibitory effect of castration on prostate growth was confirmed by reduced proliferation index in anterior lobes compared to intact and testosterone- repleted mice. However, dietary tomato and lycopene did not cause differences in proliferation rate compared to control diet. Taken together, testosterone status decreased prostate tissue growth in 10-wk-old TRAMP mice, but dietary tomato and lycopene did not influence castration modulated body weight and proliferation rate. Our current study utilized a 10-wk old early prostate carcinogenesis model fed with tomato and lycopene for 6 weeks. The function of tomato carotenoids may need longer period to be present in physiological change as shown in 18-wk old TRAMP mice intervened at 4- wk old. [14, 25]

Castration did not impact on plasma lycopene concentration

The concentration of plasma lycopene was similar between tomato-fed and lycopene-fed mice. Castration did not alter plasma lycopene concentration. Reports on the impacts of castration on lycopene concentration have been mixed. In one report, castration led to increased hepatic lycopene compared to sham operation. [11] However, we previously found that castration in wild type mice decreases serum lycopene concentrations. [26]

The genotype and species differences may be one of the reasons of this disparity.

Castration inhibited prostate carcinogenesis

Castration significantly decreased Mki67 expression and Ki67 protein expression to inhibit proliferation in prostate tissue in TRAMP mice. Another strongly decreased gene by castration was mouse Pbsn mRNA expression. Rat probasin is a promoter which

drives expression of the SV-40 oncogene in TRAMP mice to induce prostate carcinogenesis. [31] Mouse probasin has strong sequence conservation with rat

Probasin, and is lost in poorly differentiated prostate tumor tissue in TRAMP mice. [32]

Mouse probasin has been shown to be reduced by castration and re-expressed by androgen treatment. [32] The two strongly up-regulated genes are Tnf and Igfbp3. TNF activation was previously shown to induce apoptosis in normal and prostate cancer cells.

[33] [34] In the mouse prostate, TNF is up-regulated and is essential for castration induced prostate regression.[35] Lower expression of IGFBP-3 is associated with greater risk of prostate cancer.[36] It has been shown that IGFBP-3 knockout mice have greater metastatic prostate cancer and serum IGFBP-3 was not changed 72 hr post-castration in wild type mice. [37] However, in TRAMP mice, castration decreased serum IGFBP-3 compared to intact mice. The increased IGFBP-3 expression in castrated mice may contribute to prostate atrophy in prostate tissue. Taken together, androgen deprivation

(castration) modulates expression of genes implicated in cell growth in early prostate carcinogenesis.

Castration activated canonical pathways in early prostate carcinogenesis

Castration significantly changed expression of genes involved in the aryl hydrocarbon receptor (AhR) signaling pathway, epithelial mesenchymal transition (EMT) regulation, cyclins and cell cycle regulation, cell growth regulation, and cell survival signaling.

Previous studies have shown that castration decreases prostate tumor growth and cyclin dependent kinase 1 and 2, cyclin A and cyclin B mRNA levels in a xenograft mouse model. [38] [39] Androgen depletion has been proposed to promote angiogenesis. [40]

In human prostate tumor tissue, expression of androgen receptor was associated with

Vegf and Hif1 expression. [41]

The current study showed that castration activated the AhR signaling pathway in prostate carcinogenesis. AhR is the transcription factor that is activated by xenobiotic exposure or endogenous ligands. [42] Ligand-bound AhRs translocate into nuclei and form a heterodimer with aryl hydrocarbon receptor nuclear translocator (ARNT). AhR and ARNT were previously shown to be expressed in prostate tissue, and ablation of

AhR in mice reduced prostate weight compared to wild type mice. [43] Prenatal activation of AhR by xenobiotic ligands is associated with increased prostate cancer risk, whereas the prostatic activation of AhR in human adults and mice was associated with decreased prostate cancer risk. [43] [44]

Dietary tomato and lycopene regulated neuroendocrine and cell adhesion biomarkers expression

We found that dietary lycopene significantly decreased Ngfr, Cadh2 and Syp compared to the control diet (Figure 3.8). Nerve growth factor functions as an autocrine and paracrine regulator in prostate cancer progression by binding to receptor NGFR and

TrkA.[45] [46] Imbalance of NGFR /TrkA was involved in prostate carcinogenesis. [46]

Our current study only assessed Ngfr expression, and future studies are required to measure the effect of tomato and lycopene on the ratio of Ngfr/TrkA. Cadehrin-2 is a cell adhesion molecule and increased expression of CADH2 was detected in castration- resistant and metastatic prostate cancer.[47] [48] Therefore, decreased Cadh2 expression may lead to reduced invasion potential in early prostate carcinogenesis in

TRAMP. SYP is a neuroendocrine marker and is expressed in metastatic prostate cancer tissue in human and TRAMP mice. [49] [50] Our current study suggests that dietary lycopene decreased neuroendocrine biomarkers Ngfr and Syp in TRAMP mice.

Thus, our current study suggests that dietary lycopene but not dietary tomato inhibited

neuroendocrine and invasion biomarkers during early prostate carcinogenesis in

TRAMP.

Dietary tomato and lycopene contributed to castration activated AhR signaling pathway.

The effect of testosterone on downstream genes in AhR signaling is diet-specific. The diet x testosterone interaction regulated Mapk1, Jun, and Gstp 1 and they were included in AhR signaling activated by castration (Table 3.3). Our current study showed that castration down-regulated Mapk1 expression in mice regardless of diet. It has been shown that activation of MAPK1 is associated with prostate cancer progression and targeting of MAPK1 inhibits hormone-refractory prostate cancer in mice. [51] [52]

Castration increased Gstp 1 expression with control- and lycopene-feeding but not tomato-feeding, compared to sham-operation (Figure 3.9). Hypermethylation of GSTP1 has been proposed as a prognostic biomarker of prostate cancer. [53] Methylation of the

CpG islands of GSTP1 was found to cause loss of GSTP1 protein expression and was detected in prostate cancer cases but not in normal prostate tissue or benign prostate hyperplasia. [54] [55] In our current study, 10-wk-old TRAMP mice developed areas of

PIN in prostate tissue and Gstp1 was detected. However, the effects of diet and testosterone on GSTP 1 methylation and protein levels should be further assessed in the future. Therefore, we suspected that one mechanism that dietary tomato and lycopene impacted on androgen-driven mRNA expression is through epigenetic histone methylation.

Dietary tomato and lycopene differently regulate prostatic gene expression

The debate on the efficacy of a whole food vs. a single compound has drawn a lot of attention. In the current study, we showed castration inhibits Birc5 mRNA expression, with the greatest inhibition exhibited in lycopene-fed, then tomato-fed, then control-fed

mice (Figure 3.9). BIRC5 is the smallest member of the inhibitor of apoptosis family and prostatic mRNA expression of BIRC5 has been correlated with progression of prostate cancer. [56] [57] Previous studies showed that DHT stimulation increased BIRC5 expression, and activation of BIRC5 confers resistance to anti-androgen treatment in

LNCaP cells. [58] Our current study is the first to show that castration down-regulates

Birc5 expression during early prostate carcinogenesis in 10-wk-old TRAMP. In addition, our current study is the first to show that tomato and lycopene enhanced the inhibitory effect of castration on Birc5 expression, and that lycopene had a greater effect than tomato on enhancing castration impact on Birc5 expression. Therefore, early dietary intervention by consuming tomato lycopene and tomato may inhibit potential of prostate initiation and development.

The current study has some limitations. To reduce the probability of experiment-wise errors, we selected 200 genes to measure based on a priori relationships with prostate carcinogenesis, instead of using a whole genome array to assess the impact of testosterone and dietary effect on gene expression and pathways in early prostate carcinogenesis. While this approach guarded us from unintended false positives, it does not provide a comprehensive data set to fully characterize all pathways changed in early carcinogenesis. For the pathways regulated by testosterone in early prostate carcinogenesis, genomic profile analysis would have provided more candidate pathways. However, the approach of the current study did identify dietary tomato- and lycopene-regulated androgen-driven gene expression in early prostate carcinogenesis.

Conclusions:

Our current study showed that dietary tomato and lycopene play roles in castration mediated proliferation and cell growth in early prostate carcinogenesis in 10-wk-old 70

TRAMP mice. We found that dietary lycopene is more effective in regulating expression of neuroendocrine and cell adhesion biomarkers than tomato in early prostate carcinogenesis. Moreover, testosterone is more potent in impacting prostate physiology and gene expression than dietary factors in early prostate carcinogenesis. The results of our current study provide evidence that dietary tomato and lycopene regulate androgen- driven gene expression in early prostate carcinogenesis, by impacting aryl hydrocarbon receptor signaling and cell cycle control signaling, thus explaining the impact of chronic tomato- and lycopene-feeding on reduced TRAMP carcinogenesis.

Conflict of Interest:

The authors declare no conflicts of interest

Acknowledgments:

We thank the Nucleic acid core facility in the Ohio State University Comprehensive

Cancer Center (NIH/NCI P30 016058) to measure miRNA profiles using NanoString® nCounter technique. This work was supported by National Institutes of Health (NCI-

R01125384) and Dr. Erdman was the principal investigator.

Tables:

Ingredient Control Tomato Lycopene Powder

g/kg diet Casein 200 185 200 L-Cystine 3 3 3 Corn starch 397 326 397 Maltodextrin 132 132 132 Sucrose 100 100 100 Soybean oil 70 65 70 Mineral mix1 35 35 35 Vitamin mix2 10 10 10 Cellulose 50 50 50 Choline bitartrate 2.5 2.5 2.5 Tomato powder 0 100 0 Lycopene beadlets 0 0 2.5 Control beadlets 2.5 2.5 0 Table 3. 1. Composition of diets employed in testosterone treatment in wild type and TRAMP mice. 1 AIN93G-MX formulation [59] 2 AIN93G-VX formulation[59]

Pathways1 Ratio2 -log(P-value)

Aryl hydrocarbon receptor signaling 26/163=0.16 26.341

Epithelial-mesenchymal transition 24/192=0.145 20.667

P 53 signaling 19/96=0.198 20.113

IL-K signaling 23/194=0.119 19.051

Cyclins and cell cycle regulation 16/67=0.239 18.763

PPAR signaling 18/105=0.171 18.528

Pten signaling 19/133=0.143 18.112

Glucocorticoid receptor signaling 25/294=0.085 18.023

IL-8 signaling 22/208=0.106 17.803

Table 3. 2 Summary of canonical pathways predicted to be regulated by castration compared to intact group. 1Canonical pathways generated from castration vs. intact dataset in IPA. Ratio is the number of genes in dataset to total number in Ingenuity Pathways Knowledge Base. 2The ratio of impacted genes which were measured by NanoString® compared to the total number of genes known to be part of that signaling pathway.

Accession Symbol Fold Change P-value NM_007891.4 E2f1 -10.17 <0.0001 NM_001037134.1 Ccne2 -8.77 <0.0001 NM_001040654.1 Cdkn2a -5.12 0.0003 NM_013467.3 Aldh1a1 -3.28 <0.0001 NM_016756.4 Cdk2 -2.31 <0.0001 NM_007499.1 Atm -1.7 <0.0001 NM_011949.3 Mapk1 -1.56 <0.0001 NM_009870.3 Cdk4 -1.45 <0.0001 NM_009029.2 Rb1 -1.35 <0.0001 NM_011952.2 Mapk3 -1.24 0.0003 NM_008302.3 Hsp90ab1 -1.14 0.0001

NM_013693.1 Tnf 33.72 <0.0001 NM_011243.1 Rarb 4.09 <0.0001 NM_009829.3 Ccnd2 3.95 <0.0001 NM_009367.1 Tgfb2 3.75 <0.0001 NM_009873.2 Cdk6 3.4 <0.0001 NM_011577.1 Tgfb1 3.06 <0.0001 NM_007631.1 Ccnd1 3.03 <0.0001 NM_010591.2 Jun 2.74 <0.0001 NM_010554.4 Il1a 2.49 0.0002 NM_008678.2 Ncoa2 1.44 <0.0001 NM_013541.1 Gstp1 1.37 <0.0001 NM_010358.5 Gstm5 1.27 0.012 NM_011305.3 Rxra 1.22 0.001 NM_008689.2 Nfkb1 1.18 0.0002 NM_008679.3 Ncoa3 1.12 0.012 Table 3. 3 Twenty-six molecules impacted by castration which are consistent with the aryl hydrocarbon signaling pathway. Genes were significantly changed by castration compared to intact group.

Fold Accession Gene P-value Change NM_001081117.2 Mki67 -13.02 <0.0001 NM_001040654.1 Cdkn2a -5.12 0.001 NM_001025250.3 Vegfa -1.58 <0.0001 NM_007610.1 Casp2 -1.53 <0.0001 NM_175283.3 Srd5a1 -1.51 <0.0001

NM_009807.2 Casp1 7.92 <0.0001 NM_009851.2 Cd44 7.12 <0.0001 NM_010427.4 Hgf 6.67 <0.0001 NM_008006.2 Fgf2 4 <0.0001 NM_011701.4 Vim 3.38 <0.0001 NM_007556.2 Bmp6 3.07 <0.0001 NM_011577.1 Tgfb1 3.06 <0.0001 NM_001079908.1 Fgfr1 2.85 <0.0001 NM_010554.4 Il1a 2.49 0.002 NM_001111274.1 Igf1 1.95 <0.0001 NM_009864.2 Cdh1 1.45 <0.0001 NM_008402.2 Itgav 1.39 <0.0001 NM_007393.1 Actb 1.34 <0.0001 NM_016769.3 Smad3 1.33 <0.0001 NM_009805.4 Cflar 1.09 0.029 Table 3. 4 Twenty molecules changed by castration in connected cell survival and cell movement networks. Focus molecules generated in castration vs. intact gene set in IPA library.

Fold Change P-value

Tom vs. Lyc vs. Lyc vs. Tom vs. Lyc vs. Lyc vs.

Con Con Tom Con Con Tom NM_033217.3 Ngfr 1.17 -1.82 -2.13 0.769 0.010 0.019 NM_009305.2 Syp 1.14 -2.02 -2.29 0.987 0.005 0.005 NM_007489.3 Arntl 1.27 1.16 -1.10 0.010 0.084 0.362 NM_007664.4 Cadh2 1.14 -1.60 -1.82 0.993 0.026 0.030 Table 3. 5 Expression of mRNAs changed by dietary tomato or lycopene. mRNAs were statistically changed by dietary tomato or lycopene (Sidak post hoc comparison, n=11/group) Tom: tomato, Con: control, Lyc: lycopene.

Figures

***

Figure 3. 1 Body weight gain and urogenital tract weight They were measured in TRAMP mice fed with three diets (Control, tomato and lycopene) for 6 weeks. Values are expressed as mean ±se (n=23/group). Dietary tomato and lycopene did not impact either body weight gain over 6 weeks or final UGT weight compared to control diet. ***Castration significantly decreased body weight gain and UGT weight (P <0.0001, 2-way ANOVA). No interaction was observed.

*** ***

Figure 3. 2 Effect of diet and androgen status on final weights of four prostate lobes (AP, anterior lobe; VP, ventral lobe; DP, dorsal lobe; LP lateral lobe). Values are expressed as mean ±se (n=23/group). Dietary tomato decreased AP and DP weight compared to control diet (P<0.05). Dietary lycopene increased LP weight (P<0.05). *** indicates that Castration significantly decreased AP, VP, DP and LP weight compared to intact group (*** P<0.0001).

Figure 3. 3 Plasma lycopene was detected in mice fed tomato or lycopene diet. Values are expressed as mean ± se (n=6/group).

* * *

Figure 3. 4 The proliferation index of prostate tissue was measured by Ki67 staining in anterior lobe. Ki67 quantification is presented by percentage of strong positive staining to total staining in Aperio imageScope (Positive Pixel Count, version 8.1, Aperio®). Values are expressed as mean ± se (n=3/group) Two-way ANOVA (STATA 12) was used to evaluate the effect of testosterone (P<0.0001), diet (P=0.1269), and testosterone x diet interaction (P=0.3416).

Figure 3. 5 Number of miRNA expression changed by diet and testosterone status. Out of 200 measured genes, 189 were detected in TRAMP prostate tissue. Testosterone uniquely changed expression of 153 genes, diet uniquely changed expression of 1, and both testosterone and diet changed expression of 7. An interaction between testosterone and diet impacted expression of 10 genes (2-way ANOVA, n=3/group).

Figure 3. 6 Canonical pathways regulated by testosterone. Castration-driven changes in gene expression were significantly consistent with changes in canonical pathways, as identified by IPA. These 9 canonical pathways are the most significant to the data set, based on Fischer’s exact test (P <10-6). EMT, epithelial mesenchymal transition.

Figure 3. 7 Cell growth and cellular movement networks was regulated by testosterone. Measured gene expression changed by castration vs. sham-operated that was consistent with changes in cell survival and death and cellular movement networks (which have 20 connected genes). Red, down-regulated by castration vs. sham operation group; Green, up-regulated by castration.

* *

Figure 3. 8 Gene mRNA expression changed by dietary tomato and lycopene. Expression of Arntl, Ngfr, Syp, and Cadh2 are differently expressed in control-, tomato powder- and lycopene diet-fed mice (Sidak post hoc comparison, n=3 or 4 /group, * P<0.05 vs. control)

*** ***

* * * ***

* * *

Figure 3. 9 Gene mRNA expression changed by diet x testosterone interaction. MAPK1, Birc5, Gstp1 and Jun mRNA are differently expressed under the diet x testosterone interaction (2-way ANVOA, STATA 12, n=3/group)

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Chapter 4: Dietary Tomato and Lycopene Mediate miRNA Expression in Early Prostate Carcinogenesis

Authors:

Lei Wan1, Hsueh-Li Tan1, Jennifer M. Thomas-Ahner2, Nancy E. Moran2, Dennis K.

Pearl3, Steven K. Clinton 2,4*

Author affiliations:

1 The Interdisciplinary Ph.D. program of Nutrition, The Ohio State University

2 Comprehensive Cancer Center, The Ohio State University

3 Department of Statistics, The Ohio State University

4 Division of Medical Oncology, Department of Internal Medicine, The Ohio State

University

Abstract:

Background: Epidemiological and laboratory studies suggest dietary tomato and lycopene possess anti-cancer activities in prostate cancer. MicroRNAs bind to the 3’

UTR of mRNA to degrade mRNA or suppress translation. Deregulation of miRNAs is implicated in prostate cancer progression. Testosterone is the critical hormone of prostate carcinogenesis. We hypothesized that dietary tomato and lycopene impacted on testosterone-driven miRNA expression in early prostate carcinogenesis.

Methods: Two, 2x3 factorial studies were designed to investigate effect of testosterone, diet and genotype on miRNA expression profiles in early prostate carcinogenesis.

TRAMP and healthy wild type C57BL/6 mice (4-wk old) were randomly assigned to one of three diets: control, tomato powder (TP), or lycopene beadlets diet (LYC). At 8-wk-old, mice were randomly assigned to intact (sham surgery), castration, or castration + testosterone repletion groups. One week later, mice in the testosterone repletion group received 2.5 mg/kg/day testosterone propionate via osmotic pump. All mice were sacrificed 5 days after testosterone repletion. Body weight and urogenital weight (UGT) was recorded. Plasma was collected for lycopene analysis and prostate lobes were collected for RNA isolation. MicroRNA profile and expression of all known 602 miRNAs were assessed by NanoString® technique.

Results: In wild mice, diet and testosterone status did not change body weight gain. In

TRAMP mice, castration significantly decreased body weight gain (P <0.0001). UGT weight was significantly decreased by castration in both WT and TRAMP (P<0.0001).

Plasma lycopene was not detected in control-fed mice and concentration was similar between TP and LYC-diet fed mice (WT, 0.33 ± 0.003 µmol/L; TRAMP, 0.27±0.002

µmol/L) within genotypes. In wild type mice, castration significantly lowered plasma 91

lycopene concentration compared to intact operation, regardless of diet (P=0.02).

Dietary tomato significantly up-regulated anti-apoptotic miR-15a (2.05-fold, P=0.0007) and anti-metastatic miR-200c (1.4-fold, P=0.047), compared to control-fed mice in both genotypes. Dietary lycopene significantly up-regulated tumor suppressor miRNAs (let-7 family, miR-145/143, miR-15a/16 etc) in TRAMP mice, compared to lycopene-fed wild type mice (2.08-79.67 folds, P <0.05). Furthermore, testosterone significantly decreased miR-429 (-7.04 and -7.16 folds, P=0.029 and P=0.027) and miR-375 (-51.95 and -286.80 folds, P=0.007 and P<0.0001) in tomato- and lycopene- but not in control-fed mice, compared to intact mice.

Conclusions: Dietary tomato and lycopene enhanced tumor-suppressor miRNA expression in TRAMP mice. Effect of castration on miRNA expression was strengthened in dietary tomato and lycopene. Moreover, castration reduced lycopene concentration in a genotype-specific manner.

Introduction

Consumption of tomato and lycopene, the red carotenoid pigment in tomatoes, is inversely associated with prostate cancer incidence. In 1995, Giovannucci et al. first showed the inverse association between high intake of tomato products and incidence of prostate cancer. [1] Moreover, lower relative risk of prostate cancer (RR=0.79, P=0.04) was observed for the highest quintile intake of lycopene. [1] [2] Patients with prostate cancer have lower circulating carotenoids (lycopene, lutein, beta-cryptoxanthin, zeaxanthin) and prostatic lycopene concentrations. [3] [4] [5] The anti-cancer mechanisms of lycopene include decreasing proliferation [6, 7].[8], increasing apoptosis

[9], reducing oxidative stress [10] [11] [12] [13], and inhibiting invasion and metastasis

[14] in vitro.

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model is a useful model to study mechanisms of dietary tomato carotenoids in vivo. The TRAMP model is easily manipulated and develops prostate cancer in prostate epithelial cells spontaneously and specifically.[15] In TRAMP mice, the rat probasin promoter drives expression of oncogene SV40 expression under the regulation of androgen. TRAMP mice absorb dietary tomato and lycopene and accumulate carotenoids in prostate tissues. Consumption of tomato and lycopene decreased prostate cancer incidence in

TRAMP models. [16] [17] [18] Moreover, it is suggested that the inhibitory effect of tomato and lycopene on prostate cancer progression was greater when it was initiated on mice less than 8 weeks old mice, a time near the onset of cancer initiation. [19]

MicroRNAs are 18-22 nucleotide long, non-coding RNAs. Deregulation of miRNAs has been implicated in prostate carcinogenesis. It has been shown that miRNAs bind to

3’UTR of mRNA to induce degradation and translation suppression. [20, 21] The 93

modulation of miRNAs by 1, 25(OH)2D, curcumin, selenium, zinc, folic acid, soy isoflavones and resveratrol has been studied, and suggest that dietary components alter miRNA transcription which consequently regulate carcinogenesis by targeting genes critical to cancer development and progression.[22] [23] [24] [25] [26] For example, curcumin up-regulated miR-15a and miR-16 to decrease anti-apoptotic Bcl-2 expression and increase apoptosis in breast cancer cells. [24] It has also been shown that lycopene induces miR-21 to reduce fatty acid-binding protein 7 to inhibit hepatic steatosis, a condition that often precedes hepatocellular carcinoma. [27] However, the effect of lycopene on miRNA expression in prostate cancer remains unknown.

It has been shown that tomato lycopene impacts on testosterone production, the driving hormone of prostate carcinogenesis. Before prostate cancer becomes androgen insensitive, androgen signaling is the main target in treating prostate cancer. [28] The concentration of circulating testosterone differed between mice consuming and not consuming tomato or lycopene. [29] Genes encoding enzymes involved in testosterone synthesis, such as 17β-hydroxylsteroid dehydrogenase 1, may also be regulated by dietary consumption of tomato and lycopene. [30] MicroRNAs are subject to testosterone-regulation in prostate carcinogenesis. [31] [32] We previously found that castration modulated miRNAs expression in the early carcinogenesis (Chapter 2).

Carotenoid metabolism enzymes, BCMO1 and BCDO2, influenced interaction between dietary tomato carotenoids and testosterone activity. BCMO1-/- mice had decreased serum and testicular testosterone compared to intact wild-type mice. [33, 34] Our previous studies showed that castration induced higher BCDO2 expression, compared to intact operation. Moreover, greater prostate cancer lesion occurs in lycopene-fed

BCDO2-/-TRAMP mice compared to wild type mice.[35] Moreover, previous studies

suggested anti-cancer activities of lycopene metabolites. [34, 36, 37] Apo-10 lycopenoic acid has been shown to inhibit the proliferation of non-small lung cancer cells and tumorigenesis in vivo. [38] Taken together regulation of BCMO1 and BCDO2 may be one of the mechanisms of interaction between tomato carotenoids and testosterone in inhibiting prostate carcinogenesis.

The influence of tomato and lycopene on androgen-regulated miRNA expression, especially in early prostate carcinogenesis is not known yet. Therefore, we assessed the effect of dietary tomato and lycopene, testosterone, and the interaction of these two variables on miRNA expression in early prostate carcinogenesis. We hypothesize that 1) tomato carotenoids regulate miRNA expression in early prostate carcinogenesis to prevent prostate cancer development, and 2) tomato and lycopene play a role in testosterone-modulated miRNA expression in early prostate carcinogenesis.

Materials and Methods

Animals and Diets

All animal experiments were performed in compliance with the Ohio State University

Institutional Animal Care and Use Committee (IACUC). Heterozygous TRAMP (+/-) x

C57BL/6 mice were bred from TRAMP mice (Jackson Laboratories, Bar Harbor, ME) and wild type C57BL/6 mice, then crossed with FVB/N females to generate the male

TRAMP (+/-) C57BL/6×FVB/N mice. The male TRAMP (+/-) C57BL/6×FVB/N and healthy control C57BL/6 mice were used in the study. The presence of the SV transgene was confirmed using PCR by REDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, St.

Louis, MO) following the manufacturer’s instructions.

Study Design

Two 3x 3 factorial studies were designed to investigate the effects of diet and testosterone status on prostate carcinogenesis in wild type and TRAMP mice. The main effects of testosterone status and TRAMP genotype have been addressed in Chapter 2.

TRAMP mice were weaned at 4 weeks of age and randomized to one of the three diets: purified AIN-93G diet (ResearchDiets, New Brunswick NJ) containing 0.25% (w/w) placebo beadlets (DSM, Basel, Switzerland), 10% tomato powder (TP; FutureCeuticals,

Momence, IL ), or 0.25% (w/w) lycopene beadlets (RediVivo10% lycopene, DSM,

Netherlands) diet (Table 4.1) in TRAMP. In wild type, the diet composition is similar except the percentage of lycopene beadlets was 0.04% (w/w). The concentration of total

LYC in the final prepared diet was 20 mg/kg (WT) and 384 mg/kg (TRAMP) for 10% TP and 40 mg/kg (WT) and 462 mg/kg (TRAMP) for 0.25% LYC beadlets diet. Eight-week- old mice within each diet group were randomized to undergo either a sham (superficial incision only) surgery, a castration surgery, or a castration surgery followed by testosterone repletion (2.5 mg/kg of body weight) to study the effect of testosterone on miRNA expression. One week after castration, testosterone propionate (Sigma-Aldrich) was administered via Alzet® mini osmotic pump implantation (Durect, Cupertino, CA), following the manufacturer’s instructions. The mice were sacrificed by cervical dislocation 5 days after pump implantation or 12 days after castration surgery. Plasma was collected for lycopene concentration analysis. Prostate glands were procured by micro-dissection. The dorsal and lateral prostate lobes were immersed in RNAlater ® solution (Life technologies, Grand Island, NY) overnight at 4°C. The next day, RNAlater® solution was removed and then the sample was stored at -80°C until RNA isolation.

Plasma lycopene analysis

Plasma carotenoids were extracted and analyzed in the lab of J.W.E. as previous described. [39] 96

RNA isolation

RNeasy Mini kit (Qiagen, Valencia, CA) was used to extract the total RNA from the fresh frozen prostate lobes preserved with RNAlater® following the manufacturer’s instructions.

NanoString® on microRNA analysis

The mouse microRNA codeset (Version 1.0) (602 at the time of analysis) was analyzed using NanoString nCounter technique (NanoString®, Seattle, WA). For analysis, extracted total RNA (100 ng) was incubated with specific reporter and capture probes at

64˚C for 18 h. After incubation, the excess probes were removed, and the remaining hybridized miRNAs were immobilized on a streptavidin-coated cartridge using the nCounter Prep Station (NanoString®). Last, miRNAs were counted by an nCounter

Digital Analyzer (NanoString®).

NanoString Data Analysis

Detectable miRNAs were identified by subtracting the mean of the negative controls from the sample NanoString® data. The data were natural log-transformed and then normalized to the natural log-transformed endogenous reference, mmu-miR-720

(MIMAT0003484). The endogenous reference was selected based on low between- group variability (P <0.05 by 1-way ANOVA), and high stability within groups [mean standard error (MSE) =0.0356].

TRAMP prostate tissue mRNA expression was analyzed by subtraction of negative controls from the sample NanoString data, followed by natural log transformation and normalization to Ccng1 (NM_009831.2) which has low between-group variability (P<0.05 by ANOVA), and high stability within groups [mean standard error (MSE) =0.0105].

In order to identify miRNAs that were differentially expressed in response to diet, testosterone, and genotype, normalized miRNA data was analyzed by three-way

(genotype, hormone and diet) ANOVA using the Stata12 statistical analysis package

(StataCorp, College Station, TX). The significance level was set as P<0.05. The dietary main effects and diet interactions with testosterone and genotype are presented. The effect of testosterone and genotype on miRNA expression has been reported in previous study.

Statistics

Plasma lycopene concentration, diet effect, and dietary interaction with testosterone or genotype on miRNA expression were analyzed by ANOVA (1, 2, or 3 way ANOVA). The

Tukey’s post hoc test was used to make pair-wise comparisons between distinct diet x genotype and diet x testosterone combinations after ANOVA. All statistics were operated in Stata12 (StataCorp, College Station, TX).

Prediction of miRNAs targeting mRNAs

TargetScan (Mouse) and miRNA.org were used to predict mRNAs that are targeted by specified miRNAs. The association between miRNA expression and targeted mRNA expression was assessed by Spearman correlation (Stata12). If P of correlation is <

0.05, the mRNA was considered to be a target of a specific, diet-regulated miRNA.

Results

Animal body and tissue weights

Mice body weight was not changed by dietary tomato and lycopene, compared to control diet, regardless of genotype (P>0.05) (Figure 4.1). Body weight gain was decreased in castrated mice compared to intact and testosterone repleted mice (P<0.001) (Figure

4.2). Urogenital tract weight (UGT) was significantly lower in castrated mice compared to intact and testosterone-repleted mice, regardless of genotype (P<0.001) (Figure 4.2).

Dietary tomato and lycopene accumulation

Plasma lycopene was not detected in either wild type and TRAMP mice fed control diet

(Figure 4.3). Plasma lycopene was not significantly different between dietary tomato and dietary lycopene within each genotype (P>0.05, 2 way ANOVA). In wild type, plasma lycopene was 0.32±0.03 µmol/L and 0.33±0.03 µmol/L in tomato-and lycopene-fed mice respectively. In TRAMP mice plasma lycopene was 0.26±0.02 and 0.27±0.02 µmol/L in tomato and lycopene fed mice respectively. There was no genotype effect on plasma lycopene concentrations in either tomato- or lycopene-fed mice (P>0.05).

Effects of diet, and diet x testosterone, diet x genotype on miRNA expression

Of 80 detectable miRNAs, dietary tomato and lycopene independently changed expression of 2 miRNAs, miR-15a and miR-200c. Genotype and testosterone were previously found to independently change expression of 20 and 13 miRNAs, respectively

(Chapter 2). Twenty-five miRNAs were impacted by both diet and genotype, 33 miRNAs were impacted by both genotype and testosterone, and 2 miRNAs were impacted by genotype, testosterone and diet (Figure 4.4). Tomato and lycopene feeding interacted with genotype to significantly change expression of 32 miRNAs (P <0.05). Tomato- and lycopene-feeding interacted with testosterone status to impact 25 miRNA expression (P

<0.05). In addition, 46 miRNAs were significantly changed under the interaction of genotype, testosterone and diet.

Dietary tomato powder and lycopene significantly regulated miR-15a and miR-200c expression

Tomato and lycopene significantly impacted expression of 7 miRNAs (P<0.05), and 2 of them were fragments of tRNA (mmu-miR-2183 and mmu-miR-2141). When miRNA expression was compared between dietary interventions, only miR-200c and miR-15a were significantly changed by either dietary tomato. Tomato-feeding significantly increased miR-200c (fold change=1.47, P =0.047) and miR-15a (fold change=3.50,

P=0.007) expression compared to control-feeding (Table 4.2). However, dietary lycopene alone did not significantly change miRNA expression.

Diet and genotype interacted to modulate miRNA expression during early prostate carcinogenesis

Dietary tomato and lycopene significantly up-regulated miRNA expression in TRAMP compared to wild type mice (Table 4.3). Dietary lycopene significantly increased expression of miR-423-5p (fold change=2.37, P =0.002), miR-145 (fold change=2.08, P

=0.036), miR-143 (fold change=2.82, P =0.010), miR-98 (fold change=3.42, P =0.0001), miR-15a (fold change=10.02, P =0.036 ), miR-16 (fold change=3.42, P <0.0001), miR-

204 (fold change=3.81, P <0.0001), and let-7 family (fold change=2.15-2.73, P <0.0001) in TRAMP mice compared to wild type mice and the expression of miR-145/143, miR-16, and miR-204 under the diet x genotype interaction (Figure 4.6). Compared to lycopene, dietary tomato impacted expression of fewer miRNAs between genotype types. Dietary tomato significantly increased expression of miR-204 (fold change=2.63, P =0.021) in

TRAMP compared to WT mice (Table 4.4).

Diet and Testosterone interacted to modulate miRNA expression during early prostate carcinogenesis

An interaction between testosterone status and diet type impacted miRNA expression

(Table 4.5, Figure 4.7). Testosterone status altered expression of miR-429 and miR-

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375 in tomato- and lycopene-fed mice, but not in control-fed mice. Compared to intact mice, castration decreased miR-429 in tomato-fed (fold change=-7.04, P =0.029) and lycopene-fed (fold change=-7.16, P- =0.027) but not control-fed mice. Moreover, compared to intact group, castration down-regulated miR-375 in tomato (fold change=-

13.27, P =0.007) and lycopene group (fold change=-286.8, P <0.001) but not in control fed mice. Furthermore, testosterone repletion restored expression of miR-429 and miR-

375 in tomato- and lycopene-fed mice to expression in tomato and lycopene-fed intact mice (P<0.001). (Table 4.5, Table 4.6).

Dietary tomato and lycopene altered expression of miRNAs predicted to target anti- apoptotic genes

The correlations between expression of miR-16 and target mRNAs (Bcl-2, Ccnd1 and

Fgf2) were estimated by Spearman correlation. Expression of miR-16 was negatively correlated with targeted Bcl-2 (tomato r=- 0.78, P=0.0125; lycopene r= - 0.73,

P=0.0246), Ccnd1 (tomato r= - 0.83, P=0.0053; lycopene r= - 0.63, P=0.0671), and Fgf2

(tomato r=0.82, P=-0.0072; lycopene r= - 0.67, P=0.0499) mRNA expression in tomato- fed and lycopene fed (Appendix C-E).

Dietary tomato and lycopene altered expression of miRNAs predicted to target Bcmo1

Two carotenoid metabolism enzyme genes, BCMO1 and BCO2 were included in 200- gene codeset. MicroRNAs targeting Bcmo1 and Bco2 were predicted using TargetScan and microRNA.org. Three miRNAs were both predicted to target Bcmo1 and were altered by dietary tomato and lycopene (mmu-miR-204, mmu-miR-145, and mmu-miR-

143). In wild type, tomato-fed mice, expression of Bcmo1 was negatively associated with miR-204 expression (r= - 0.68, P<0.05). In TRAMP, lycopene-fed mice, expression of

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Bcmo1 was positively associated with miR-145 expression (r=0.67, P<0.05) (Figure

4.8).

Discussion:

Dietary tomato and lycopene have been suggested to inhibit prostate cancer progression by regulating critical genes and pathways in prostate carcinogenesis. [41] [42] The interaction between dietary tomato/lycopene and testosterone was observed in prostate carcinogenesis. [43] [34] The important role of miRNAs in cancer progression was not recognized until recently. [44] In the current report we have found that 1) dietary tomato and lycopene feeding impacted miRNA expression in early prostate carcinogenesis in mice, 2) dietary tomato and lycopene impacted testosterone-driven miRNA expression, and 3) dietary tomato and lycopene influenced miRNAs targeting genes involved in carotenoid metabolism and cancer progression. The TRAMP model provides a means to investigate miRNA expression changes in response to dietary tomato and lycopene. In particular, we found that dietary tomato and lycopene enhanced tumor suppressor miRNA expression involved in anti-proliferation in early prostate carcinogenesis in

TRAMP mice and altered miRNAs predicted to regulate carotenoid metabolic enzyme transcriptions.

Dietary tomato enhanced tumor suppressor miRNAs

The current findings suggest that dietary tomato and lycopene up-regulate tumor suppressor miRNA expressions in early prostate carcinogenesis in TRAMP mice.

Although concentration of lycopene in 10%TP and LYC beadlet diet were different, plasma lycopene concentrations were similar in mice fed with tomato and lycopene.

Dietary tomato significantly increased miR-15a and miR-200c expression. MicroRNA-

15a is pro-apoptotic microRNA and is lost in 80% prostate cancer samples. [45] [46] 102

MicroRNA-200c is a member of the miR-200 family, [47] and expression of the miR-200 family has been shown to inhibit the epithelial to mesenchymal transition (EMT). [48] [49]

Therefore, up-regulation of miR-15a and miR-200c expression by tomato-feeding might contribute to decreased prostatic carcinogenesis as previously reported in tomato-fed,

TRAMP mice. [18] [50] Compared to control diet, lycopene did not significantly change miRNA expression. However, dietary lycopene feeding did impact miRNA expression in a genotype-specific manner.

Dietary lycopene modulates anti-cancer miRNA expression

Dietary lycopene activation of tumor suppressor networks observed in the current study likely underlie the previously reported inhibition of prostate carcinogenesis in TRAMP by lycopene-feeding.[18] Based on previous literature in human prostate cancer, the miRNAs up-regulated by dietary tomato and lycopene in TRAMP mice function as tumor suppressors which increase apoptosis and decrease proliferation in prostate cancer

(Table 4.3). In lycopene-fed mice, tumor suppressor miRNA expression was significantly up-regulated in TRAMP mice, compared to wild type mice. Lycopene-feeding strongly up-regulated miR-15a and miR-16 expression in TRAMP mice compared to wild type mice. In human prostate cancer, miR-15a and miR-16 are in the same cluster, and are associated with inhibited proliferation and cancer invasion. [46] [51] Dietary lycopene feeding significantly up-regulated miR-145 and miR-143 in TRAMP mice compared to wild type. MicroRNA-145 and microRNA-143 belong to same microRNA cluster and were down-regulated in human prostate cancer sample compared to normal prostate tissue. [52] Over-expression of miR-145 and miR-143 decreased cell growth and induced apoptosis in human prostate cancer. [53] [54] In mouse prostate carcinogenesis, let-7a, let-7b, let-7c, let-7d, let-7e, and let-7f were significantly up- regulated in TRAMP mice, compared to wild type mice fed with lycopene (Table 4.3). 103

The Let-7 family functions as tumor suppressor miRNAs in multiple cancers. [55] [56]

[57] Let-7a, 7c, and 7d were shown to be down-regulated in human prostate cancer samples and prostate cancer cells. The expression of let-7a and let-7c inhibited cell proliferation and increased cell-cycle arrest. [58] [59] [60] [61] In the current study, dietary lycopene-activated expression of the let-7 family may contribute to the inhibition of prostate carcinogenesis initiation TRAMP mice. Tumor suppressor p53 was previously found to activate miR-15a/16, miR-145/143, and the let-7 family in response to DNA damage and genomic instability. [62] [63] Thus it is plausible that dietary lycopene feeding attenuates prostate carcinogenesis in TRAMP mice.

Dietary tomato and lycopene regulate cell growth and cellular movement

Dietary tomato and lycopene regulated miRNA expression involved in suppressing proliferation and enhancing apoptosis in early prostate carcinogenesis in TRAMP mice.

Previous studies showed that miR-15a/16 degrades transcripts of anti-apoptotic Bcl-2 and Ccnd1 in human prostate cancer cell lines.[46, 51] In addition, it was shown that miR-145/143 decreased expression of oncogene c-Myc to inhibit growth of prostate cancer cells. [52-54] [62] In lycopene fed mice, TRAMP mice had significantly increased miR-16 expression than wild type (Figure 4.3). The negative correlation between miR-16 and target mRNA (Bcl-2, Ccnd1 and Fgf2) in lycopene fed TRAMP suggested dietary tomato and lycopene increased pro-proliferation Bcl-2, Ccnd1 and Fgf2 mRNA expression (Appendix C-E). Although dietary tomato up-regulated miR-204 expression in TRAMP compared to wild type, negative correlation was not observed between miR-

204 and target Bcl-2 in TRAMP mice. Taken together, dietary lycopene may inhibit the early prostate carcinogenesis by suppressing proliferative genes in 10 wk old TRAMP mice.

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Dietary tomato and lycopene enhance the effect of castration on prostate carcinogenesis.

Castration decreased miRNA expression in a diet specific manner. Castration significantly suppressed expression of miR-429 and miR-375 in tomato and lycopene-fed mice, but not in control-fed mice (Figure 4.7). We previously found that testosterone significantly decreased expression of miR-375 and miR-429, compared to the intact and testosterone repletion group in early prostate carcinogenesis in mice. As a member of the miR-200 family, increased miR-429 has been shown to be associated with the mesenchymal to epithelial transition in prostate cancer cells. [64] To elucidate the function of miR-429 in early prostate carcinogenesis, the future studies are required to assess the down-stream genes and pathways. We have previously reported the testosterone sensitivity of miR-375, however, the function of miR-375 in prostate cancer is now known yet. The next step is to evaluate function of miR-375 in prostate carcinogenesis under testosterone and dietary tomato or lycopene. In a whole, we reported that dietary tomato and lycopene strengthen inhibitory effect of castration on miR-375 and miR-429.

Dietary tomato and lycopene enhanced miRNAs predicted to regulate carotenoid metabolism

Beta-carotene 15, 15’-monooxygenase (BCMO1) and beta-carotene 9’, 10’-bioxygenase

2 (BCDO2) are the two main enzymes responsible for carotenoid cleavage. BCMO1 mainly cleaves beta-carotene at the central double bond to generate retinal, and BCDO2 mainly cleaves acyclic pro-vitamin A such as lycopene eccentrically to generate apo- lycopenal. [65] Bcmo1 had a significant negative correlation with miR-145 in tomato-fed

TRAMP mice and a significant positive correlation with miR-204 in lycopene-fed TRAMP mice (Figure 4.8). We previously showed that Bcmo1 was below detection level in wild 105

type mice, but it was detected in TRAMP prostatic tissue and significantly correlated with altered miRNAs by dietary tomato and lycopene in current study. [35] Current study is the first to show that dietary tomato and lycopene impacted on Bcmo1 expression through regulating miRNA expression. Previous studies showed that miRNAs regulated mRNA in two ways: 1) degradation of transcripts level, 2) accumulation of target mRNA levels when miRNAs inhibited translation. [66] In current study, both the significant correlation suggest the regulation between miRNA and carotenoid metabolism enzyme, however, the mechanism of the regulation need further investigation.

It is noted that dietary effect is not as strong as genotype and testosterone effect. Large portion of miRNA expression was altered by either genotype or testosterone effect, which indicates genotype and testosterone have stronger effect on miRNA expression than dietary intervention (Figure 4.4). However, dietary interaction with genotype and testosterone were observed to inhibit prostate carcinogenesis in current study. The goal is to use miRNAs as biomarker in response to dietary tomato and lycopene in human prostate cancer prevention. The current study provides evidence of effect of dietary tomato or lycopene on miRNA expression in prostate carcinogenesis for human dietary intervention study.

Conclusion:

In conclusion, the current study is the first to report dietary tomato and lycopene regulated miRNA expression in early prostate carcinogenesis in TRAMP mice. These candidate miRNAs may contribute to the effects of dietary tomato and lycopene observed in prostate cancer attenuation found in clinical and pre-clinical trials. Future studies to investigate miRNA expression as a target of prostate cancer chemoprevention by diet will provide a better understanding of dietary prevention mechanisms. 106

Conflict of Interest:

The authors declare no conflict of interest.

Acknowledgements:

We thank the Nucleic acid core facility in the Ohio State University Comprehensive

Cancer Center (NIH/NCI P30 016058) to measure miRNA and mRNA expression by

NanoString® nCounter. This work was supported by National Institutes of Health (NCI-

R01125384; PI: John W. Erdman, Jr.).

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Tables:

Ingredient Control Tomato Lycopene Powder g/kg diet Casein 200 185 200 L-Cystine 3 3 3 Corn starch 397 326 397 Maltodextrin 132 132 132 Sucrose 100 100 100 Soybean oil 70 65 70 Mineral mix1 35 35 35 Vitamin mix2 10 10 10 Cellulose 50 50 50 Choline bitartrate 2.5 2.5 2.5 Tomato powder 0 100 0 Lycopene beadlets3 0 0 2.5/0.4 Control beadlets 2.5 /0.4 0 0 Table 4. 1 Composition of diets employed in testosterone treatment in wild type and TRAMP mice. 1 AIN93G-MX formulation [67] 2 AIN93G-VX formulation[67]. The concentration lycopene/control beadlet is 0.4 g/kg in wild type and 2.5g/kg in TRAMP mice.

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Tomato vs. Control

Fold Accession Gene P-value Change MIMAT0000657 mmu-miR-200c 1.47 0.047 MIMAT0000526 mmu-miR-15a 3.50 0.007 Table 4. 2 miRNA expression significantly increased by tomato feeding. Expression of miR-200c and miR-15a were significantly up-regulated by tomato feeding, compared to control feeding group. (P < 0.05, Tukey’s multi-comparison, n=18/group)

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TRAMP TRAMP LYC LYC WT LYC Function Target Cancer References WT LYC Accession miRNAs (p mRNAs type (FC) value) MIMAT0004825 mmu-miR-423-5p 2.37 0.002 no MIMAT0000157 mmu-miR-145* 2.08 0.036 Cell growth c-Myc Epithelial Spizzo et al 2010 suppressor , p-53 cancers [54] apoptosis Mucin 1 Zhang et al 2013 promoter MDM2 [52] Sachdeva et al 2010 [53] 109 Suzuki et al 2009

[62]

MIMAT0000247 mmu-miR-143* 2.82 0.010 In the same cluster with miR-145

MIMAT0000523 mmu-let-7c 2.73 0.003 Cancer Prostate Nadiminty et al growth cancer 2012[68] suppressor

MIMAT0000521 mmu-let-7a 2.43 0.044 Proliferation E2F2 Prostate Dong et al inhibitor CCND2 cancer 2010[58] Colon Akao et al 2006 cancer [69]

Table 4. 3 Dietary lycopene significantly increased miRNA expression in TRAMP vs. WT.

MicroRNAs differently expressed between TRAMP mice and wild type (WT) mice in lycopene feeding group (P< 0.05,

Tukey’s comparison n=9/group) CON: control; TOM: tomato; LYC: lycopene. FC: fold change.

110 Continued

Cont

Table 4. 4 Continued

MIMAT0000383 mmu-let-7d 2.28 0.023 Steroidogene PBX3 Prostate Ramberg et al sis cancer 2011[61] Down- regulated in tumor tissue

MIMAT0000545 mmu-miR-98 3.42 0.001 Cell cycle CCNJ Prostate Ting et al 2012[70] arrest cancer

MIMAT0000522 mmu-let-7b 2.15 0.001 No

110 MIMAT0000524 mmu-let-7e 2.51 0.045 No

MIMAT0000527 mmu-miR-16* 5.97 0.000 Cell cycle Bcl-2 Prostate Bonci et al arrest CCND1 cancer 2008[46] Apoptosis WNT3A Musumeci et al suppressor FGF2 2011 [51]

MIMAT0000237 mmu-miR-204 3.81 0.000 Apoptosis Bcl-2 Gastric Sacconi et al suppressor cancer 2012[71]

MIMAT0000526 mmu-miR-15a 10.02 0.036 15a/16 are in a cluster

MIMAT0004865 mmu-miR-297c 4.70 0.048 No report

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TRAMP Tom vs. WT Tom TRAMP Lyc vs. WT Lyc Accession Gene Fold Fold P-value P-value Change Change MIMAT0000237 mmu-miR-204* 2.63 0.021 3.81 <0.0001 Table 4. 5 Tomato and lycopene up-regulated miR-204 in TRAMP compared to WT. MicroRNA-204 is differently expressed between TRAMP mice and wild type (WT) mice in tomato and lycopene feeding group (P < 0.05, Tukey’s comparison n=9/group) Tom, tomato; Lyc, Lycopene

112

Castration/control Castration/tomato Castration/lycopene vs. intact/control vs. intact/tomato vs. intact/lycopene Accession Gene Fold P- Fold P- Fold P- value change value change value change MIMAT0001537 mmu-miR-429 -2.24 0.879 -7.04 0.029 -7.16 0.027 MIMAT0000221 mmu-miR-191 -1.08 1.000 -3.40 0.021 -1.69 0.831 MIMAT0000739 mmu-miR-375 -13.27 0.217 -51.96 0.007 -286.80 <0.001 Table 4. 6 In tomato and lycopene feeding group, castration decreased miRNA expression compared to intact group. In tomato and lycopene feeding group, castration significantly down-regulated miR-429, miR-191 and miR-375, compared to intact group. (P<0.05, Tukey’s multi-comparison, n=6/group)

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repletion/control repletion /tomato repletion /lycopene vs. vs. vs. castration/control castration /tomato castration/lycopene Accession Gene Fold P- Fold P- Fold P- Change value Change value Change value MIMAT0001537 mmu-miR-429 1.59 0.995 6.61 0.039 10.37 0.004 MIMAT0000221 mmu-miR-191 -1.08 1.000 2.94 0.064 2.24 0.331 MIMAT0000739 mmu-miR-375 7.04 0.579 47.75 0.009 389.43 <0.001 Table 4. 7 In tomato and lycopene feeding group, testosterone repletion restored miRNA expression. In tomato and lycopene feeding group, testosterone repletion significantly up-regulated miR-429 and miR-375, compared to castration group. (P<0.05, Tukey’s multi-comparison, n=6/group)

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Figures

***

Figure 4. 1 Body weight gain of both wild type and TRAMP mice Bars represent the mean±se (n=5/group in wild type, and in TRAMP, n=12 in intact and repletion group and n=37/group in castration) (***P<0.0001, compared to intact and testosterone repletion).

115

*** ***

Figure 4. 2 Urogenital tract (UGT) weight of both wild type and TRAMP mice. Bars represent the mean ± se (n=5/group in wild type. in TRAMP, n=12 in intact and repletion group and n=37/group in castration). (***P<0.0001 compared to intact and castration repletion).

116

Figure 4. 3 Plasma lycopene concentrations in wild type and TRAMP mice. It was measured in both wild type and TRAMP mice fed with diet for 6 weeks. Value was expressed as mean ± se (n=4/group, wild type and n=6/group TRAMP) (*P<0.05).

117

Figure 4. 4 . Number of miRNAs changed by genotype, testosterone and diet. Venn diagram of miRNAs significantly (P<0.05 in ANOVA) regulated by main effects of genotype, testosterone, and diet.

118

Figure 4. 5 MicroRNA expression changed by diet. Dietary tomato and lycopene significantly increased miR-15a and miR-200c compared to AIN93G feeding diet (P <0.05, 3-way ANOVA n=18/group).

119

* *

* * *

Figure 4. 6 Dietary tomato and lycopene regulated miRNA in genotype specific manner. Dietary lycopene feeding significantly increased expression of miR-145, miR-143, miR-16 and miR-204 in TRAMP vs. WT (P < 0.05, Tukey’s multi-comparison after 2-way ANOVA, n=9/group). Fold change is compared to wild t type AIN93G group and designated as 0 in graph.

120

* *

Figure 4. 7 Castration decreased miRNA expression in diet specific manner. In dietary tomato and lycopene group, castration significantly decreased expression of miR-375 and miR-429 compared to intact group but not in AIN93G feeding group (P < 0.05, Tukey’s multi- comparison after 2-way ANOVA, n=9/group) (* P<0.05). Fold change is compared to intact AIN93G group and designated as 0 in graph.

121

Figure 4. 8 Correlation between expression of bcmo1 and miR-145 or miR-204 in TRAMP mice. Expression of bcmo1 is negatively associated with miR-204 in wild type tomato fed TRAMP (Spearman correlation, Stata12, n=9/group). Expression of Bcmo1 is positively associated with miR-145 in lycopene fed TRAMP group (Spearman correlation, Stata12, n=9/group). Normalized Bcmo1 and microRNA expression data was used to plot the correlation.

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Chapter 5: Conclusions

The inhibitory effect of dietary tomato and lycopene on prostate carcinogenesis has been suggested by epidemiologic studies and a growing array of rodent studies of carcinogenesis or tumorigenesis. However, the mechanisms remain poorly understood.

One hypothesis is that tomato phytochemicals may act through altering the androgen signaling pathways, given the importance of testosterone in prostate biology and cancer therapy. Furthermore, the importance of miRNA deregulation in prostate carcinogenesis is being elucidated by comparing the miRNA expression profile between normal human prostate tissue and prostate cancer tissue. Testosterone status modulated miRNA expression in prostate cancer cells and in vivo animal cancer model. A few studies are showing that diet and nutrients display effects on miRNA expression that may be involved in prostate carcinogenesis. To promote the anti-cancer activities of dietary tomato carotenoids in human prostate cancer, we elucidate mechanisms that may mediate this process. In particular, we proposed that lycopene containing foods may have an impact very early on the prostate carcinogenic process. In the current study, we investigated the impact of dietary tomato carotenoids and testosterone on prostate growth, histopathologic change, and a murine prostate carcinogenesis gene codeset

(200 genes) and miRNA expression in both wild type and TRAMP mice.

The results show that testosterone status has a profound and dominant effect on prostate growth by decreasing prostate epithelial cells proliferation in early prostate 128

carcinogenesis. Moreover, testosterone status strongly influenced murine carcinogenesis gene expressions. In particular, apoptotic Tnf and Igfbp3 were strongly up-regulated whereas proliferation biomarker (Mki67), and Prbn were strongly down regulated in castrated mice compared to intact mice. In addition, cell growth and AhR signaling pathway were significantly modulated. The expression of AhR down-stream genes such as Gstp1, Jun, Mapk1 were regulated by castration in a diet specific manner. Although dietary tomato and lycopene did not cause a measureable impact on early age prostate growth, they modulated gene expression involved in prostate carcinogenesis. In particular, dietary lycopene decreased expression of neuroendocrine biomarkers (Ngfr and Syp) and a cell adhesion biomarker (Cadh2). Further, the extent of castration-reduced Birc5 expression was greater in lycopene fed mice than tomato fed mice. Therefore, tomato and lycopene differently modulated androgen-driven mRNA expression in early prostate carcinogenesis.

Dietary tomato carotenoids and testosterone status altered miRNA expression in early prostate carcinogenesis. Early prostate carcinogenesis in TRAMP mice resulted in widespread up-regulated miRNA expression compared to wild type mice. It is noted that both oncomir (miR-21) and tumor suppressor miRNAs (miR-15/16) were up-regulated in

TRAMP mice compared to wild type mice. Testosterone depletion (castration) resulted in more down-regulated miRNAs (miR-15a, miR-93, miR-148a et al.) than up-regulated miRNAs (miR-150 and miR-1224). Moreover, testosterone-modulated miRNA expression was in genotype specific manner. Expression of miR-15b/16, miR-200b and miR-25 were down-regulated in TRAMP mice but not in wild type. Taken together, testosterone status plays role in miRNA expression in genotype specific manner.

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Dietary tomato carotenoids up-regulated miR-200c and apoptotic miR-15a expression.

Moreover, dietary lycopene-fed TRAMP mice had increased anti-cancer let-7 family, miR-145/143, miR-15a/16 expression compared to lycopene-fed wild type mice. Dietary tomato carotenoids enhanced inhibitory effect of castration on miR-429 and miR-375.

Carotenoid metabolism enzyme Bcmo1 was inversely correlated with increased miR-145 expression in lycopene-fed TRAMP mice and positively correlated with miR-204 in tomato-fed TRAMP mice. Therefore, dietary tomato and lycopene modulated miRNA expression in genotype specific manner and interrupted androgen-regulated miRNA expression in prostate carcinogenesis.

In conclusion, the current study identified that castration modulated cell growth and AhR molecular pathways which are impacted by dietary tomato and lycopene in early prostate carcinogenesis. Early prostate carcinogenesis exhibited miRNA deregulation and imbalanced miRNA expression was predominantly modulated by testosterone status in a genotype specific manner. Dietary tomato and lycopene up-regulated anti- carcinogenic miRNAs in TRAMP, compared to WT mice. Dietary tomato and lycopene strengthened the effect of castration on microRNA expression. Further, dietary lycopene had greater effect in regulating genetic changes in early prostate carcinogenesis.

To elucidate the molecular mechanism of impact of testosterone and tomato carotenoids, we will define the function of specific genes and miRNAs in early prostate carcinogenesis in future studies. The current study assessed the mRNA expression of genes regulated by testosterone and tomato carotenoids and protein expression measurement of these genes are needed to define the modulation level (transcriptional

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vs. translational). The ultimate goal is to define the mechanism of dietary tomato and lycopene in inhibiting prostate carcinogenesis and apply these findings to human clinical trial. We will assess expression of critical miRNAs between human prostate cancer tissue and normal prostate tissue. The function of critical miRNAs in early prostate carcinogenesis will be answered in miRNAs over-expression or knock out models.

MicroRNA binds to mRNA to induced transcript degradation or translation suppression.

We identified miRNAs involved in initiation of early prostate carcinogenesis and tomato carotenoids modulated androgen-driven miRNA expression. The next step is to explore the function of miRNA on mRNA by analyzing the correlation between miRNA expression and mRNA expression or predicting mRNA from computational algorithm tools such as TargetScan and miRNA.org. Ultimately, we will be able to identify a genetic network regulated by tomato carotenoids impacted androgen-driven modulation in early prostate carcinogenesis.

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Appendix A. miRNAs detected in the Mouse Prostate

Previously Reported in Accession Gene Human Prostate[8] MIMAT0000523 mmu-let-7c MIMAT0000522 mmu-let-7b MIMAT0000157 mmu-miR-145 MIMAT0000136 mmu-miR-125b-5p MIMAT0003484 mmu-miR-720 MIMAT0000521 mmu-let-7a MIMAT0000124 mmu-miR-15b yes MIMAT0000383 mmu-let-7d MIMAT0000657 mmu-miR-200c yes mmu-miR-1937a MIMAT0009401 +mmu-miR-1937b MIMAT0000233 mmu-miR-200b yes MIMAT0000135 mmu-miR-125a-5p MIMAT0009409 mmu-miR-1944 MIMAT0000535 mmu-miR-29a yes MIMAT0000238 mmu-miR-205 yes MIMAT0000121 mmu-let-7g yes MIMAT0000210 mmu-miR-181a yes MIMAT0000516 mmu-miR-148a yes MIMAT0000527 mmu-miR-16 yes MIMAT0009429 mmu-miR-1937c MIMAT0000247 mmu-miR-143 yes MIMAT0000652 mmu-miR-25 yes MIMAT0000532 mmu-miR-23a yes

146

MIMAT0000524 mmu-let-7e yes MIMAT0000519 mmu-miR-200a yes MIMAT0000531 mmu-miR-22 yes MIMAT0001537 mmu-miR-429 yes MIMAT0000237 mmu-miR-204 yes MIMAT0000160 mmu-miR-150 yes MIMAT0000131 mmu-miR-99a yes MIMAT0005460 mmu-miR-1224 MIMAT0000537 mmu-miR-27a yes MIMAT0000221 mmu-miR-191 yes MIMAT0000711 mmu-miR-365 yes MIMAT0000545 mmu-miR-98 yes MIMAT0000125 mmu-miR-23b yes MIMAT0000230 mmu-miR-199a-3p yes MIMAT0004825 mmu-miR-423-5p yes MIMAT0000739 mmu-miR-375 yes MIMAT0000565 mmu-miR-328 yes MIMAT0000515 mmu-miR-30d yes MIMAT0001081 mmu-miR-196b yes mmu-miR- MIMAT0000385 yes 106a+mmu-miR-17 MIMAT0000132 mmu-miR-99b yes MIMAT0000526 mmu-miR-15a yes MIMAT0000530 mmu-miR-21 yes MIMAT0000122 mmu-let-7i yes MIMAT0000138 mmu-miR-126-3p yes MIMAT0004894 mmu-miR-574-3p yes MIMAT0000130 mmu-miR-30b yes MIMAT0000590 mmu-miR-342-3p yes

147

MIMAT0000656 mmu-miR-139-5p yes MIMAT0000595 mmu-miR-345-5p yes MIMAT0000161 mmu-miR-151-3p yes MIMAT0004865 mmu-miR-297c MIMAT0004883 mmu-miR-466g MIMAT0000212 mmu-miR-183 yes MIMAT0000546 mmu-miR-103 yes MIMAT0003130 mmu-miR-486 MIMAT0004536 mmu-miR-151-5p yes MIMAT0011217 mmu-miR-2141 MIMAT0000162 mmu-miR-152 yes MIMAT0000525 mmu-let-7f yes MIMAT0000648 mmu-miR-10a yes MIMAT0000540 mmu-miR-93 yes MIMAT0000655 mmu-miR-100 yes MIMAT0000128 mmu-miR-30a yes MIMAT0000536 mmu-miR-29c yes MIMAT0003151 mmu-miR-378 yes MIMAT0003454 mmu-miR-423-3p yes MIMAT0000145 mmu-miR-133a yes MIMAT0000158 mmu-miR-146a yes MIMAT0000127 mmu-miR-29b yes MIMAT0000141 mmu-miR-130a yes MIMAT0000219 mmu-miR-24 yes MIMAT0000534 mmu-miR-26b yes MIMAT0000165 mmu-miR-155 MIMAT0000386 mmu-miR-106b yes MIMAT0003782 mmu-miR-676 MIMAT0011287 mmu-miR-2183

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Appendix B. Murine prostate carcinogenetic gene codeset

Accession Gene NM_008935.1 prominin 1 (Prom1)

NM_017471.2 probasin (Pbsn)

NM_010113.3 epidermal growth factor (Egf)

NM_010921.3 NK-3 transcription factor, locus 1 (Drosophila) Nkx3-1

NM_009735.3 beta-2 microglobulin (B2m)

NM_013467.3 aldehyde dehydrogenase family 1, subfamily A1 (Aldh1a1)

NM_011488.2 signal transducer and activator of transcription 5A (Stat5a)

NM_008267.3 homeobox B13 (Hoxb13)

NM_013891.4 SAM pointed domain containing ets transcription factor (Spdef) NM_010570.4 insulin receptor substrate 1 (Irs1)

NM_008969.3 prostaglandin-endoperoxide synthase 1 (Ptgs1)

NM_011254.5 retinol binding protein 1, cellular (Rbp1)

NM_008160.5 Glutathione peroxidase-1 (Gpx1) NM_011169.5 prolactin receptor (Prlr)

NM_024264.3 cytochrome P450, family 27, subfamily a, polypeptide 1 (Cyp27a1)

NM_007988.3 fatty acid synthase (Fasn)

NM_133360.2 acetyl-Coenzyme A carboxylase alpha (Acaca)

NM_008255.1 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (Hmgcr)

NM_001025250.3 vascular endothelial growth factor A (Vegfa)

149

NM_011952.2 Mitogen-activated protein kinase 3 (Mapk3) NM_007956.4 estrogen receptor 1 (alpha) (Esr1)

NM_009805.4 CASP8 and FADD-like apoptosis regulator (Cflar)

NM_007393.1 actin, beta, cytoplasmic (Actb)

NM_007912.4 epidermal growth factor receptor (Egfr)

NM_008402.2 integrin alpha V (Itgav)

NM_008810.2 pyruvate dehydrogenase E1 alpha 1 (Pdha1)

NM_008084.1 Gadph NM_009391.3 RAN, member RAS oncogene family (Ran)

NM_010411.2 histone deacetylase 3 (Hdac3)

NM_011697.2 vascular endothelial growth factor B (Vegfb)

NM_010664.1 keratin 18 (Krt18)

NM_009875.4 cyclin-dependent kinase inhibitor 1B (Cdkn1b)

NM_031869.2 protein kinase, AMP-activated, beta 1 non-catalytic subunit (Prkab1)

NM_001003817.1 v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian) (Erbb2)

NM_008714.2 Notch gene homolog 1 (Drosophila) (Notch1)

NM_008540.2 SMAD family member 4 (Smad4)

NM_019827.3 glycogen synthase kinase 3 beta (Gsk3b)

NM_177821.6 E1A binding protein p300 (Ep300) NM_008562.3 myeloid cell leukemia sequence 1 (Mcl1) NM_009864.2 cadherin 1 (Cdh1) NM_013476.3 androgen receptor (Ar) NM_008678.2 nuclear receptor coactivator 2 (Ncoa2)

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NM_009743.4 BCL2-like 1 (Bcl2l1) NM_008679.3 nuclear receptor coactivator 3 (Ncoa3) NM_008689.2 nuclear factor of kappa light polypeptide gene enhancer in B cells 1, p105 (Nfkb1) NM_019812.1 sirtuin 1 (silent mating type information regulation 2, homolog) 1 (S. cerevisiae) (Sirt1) NM_007614.3 catenin (cadherin associated protein), beta 1 (Ctnnb1) NM_133915.2 paxillin (Pxn) NM_016769.3 MAD homolog 3 (Drosophila) (Smad3) NM_011305.3 retinoid X receptor alpha (Rxra) NM_010559.2 interleukin 6 receptor, alpha (Il6ra) NM_008591.1 met proto-oncogene (Met) NM_009367.1 transforming growth factor, beta 2 (Tgfb2) NM_011486.4 signal transducer and activator of transcription 3 (Stat3), transcript variant 3 NM_007544.3 BH3 interacting domain death agonist (Bid) NM_011113.3 plasminogen activator, urokinase receptor (Plaur) NM_028835.1 autophagy-related 7 (yeast) (Atg7) NM_001177352.1 myelocytomatosis oncogene (Myc) NM_019739.2 forkhead box O1 (Foxo1) NM_001122733.1 kit oncogene (Kit) NM_015733.4 caspase 9 (Casp9) NM_007913.5 early growth response 1 (Egr1) NM_007836.1 growth arrest and DNA-damage-inducible 45 alpha (Gadd45a) NM_010568.2 insulin receptor (Insr) NM_007527.3 BCL2-associated X protein (Bax) NM_021284.5 v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (Kras) NM_011400.3 solute carrier family 2 (facilitated glucose transporter), member 1 (Slc2a1) NM_010358.5 glutathione S-transferase, mu 1 (Gstm1) NM_008960.2 phosphatase and tensin homolog (Pten) NM_010513.2 insulin-like growth factor I receptor (Igf1r) NM_011480.1 sterol regulatory element binding factor 1 (Srebf1) NM_053069.5 autophagy related 5 (Atg5) NM_010431.2 hypoxia inducible factor 1, alpha subunit (Hif1a)

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NM_009652.2 thymoma viral proto-oncogene 1 (Akt1) NM_008228.2 histone deacetylase 1 (Hdac1) NM_008229.2 histone deacetylase 2 (Hdac2) NM_013671.3 superoxide dismutase 2, mitochondrial (Sod2) NM_008259.3 forkhead box A1 (Foxa1) NM_009812.2 caspase 8 (Casp8) NM_011308.2 nuclear receptor co-repressor 1 (Ncor1) NM_009029.2 retinoblastoma 1 (Rb1) NM_007610.1 caspase 2 (Casp2) NM_011640.1 transformation related protein 53 (Trp53) NM_001111075.2 cell division cycle 25 homolog B (S. pombe) (Cdc25b) NM_009870.3 cyclin-dependent kinase 4 (Cdk4) NM_007415.2 poly (ADP-ribose) polymerase family, member 1 (Parp1) NM_011045.2 proliferating cell nuclear antigen (Pcna) NM_010066.3 DNA methyltransferase (cytosine-5) 1 (Dnmt1) NM_007971.2 enhancer of zeste homolog 2 (Drosophila) (Ezh2) NM_016756.4 cyclin-dependent kinase 2 (Cdk2) NM_009810.2 caspase 3 (Casp3) NM_007499.1 ataxia telangiectasia mutated homolog (human) (Atm) NM_009765.2 breast cancer 2 (Brca2) NM_010881.2 nuclear receptor coactivator 1 (Ncoa1) NM_001110208.1 thymoma viral proto-oncogene 2 (Akt2) NM_207225.1 histone deacetylase 4 (Hdac4) NM_020009.2 mechanistic target of rapamycin (serine/threonine kinase) (Mtor) NM_010413.3 histone deacetylase 6 (Hdac6) NM_007669.4 cyclin-dependent kinase inhibitor 1A (P21) (Cdkn1a) NM_008360.1 interleukin 18 (Il18) NM_133217.3 beta-carotene oxygenase 2 (Bco2) NM_007522.2 BCL2-associated agonist of cell death (Bad) NM_010157.3 estrogen receptor 2 (beta) (Esr2) NM_201645.1 UDP glucuronosyltransferase 1 family, polypeptide A1 (Ugt1a1) NM_173371.3 hexose-6-phosphate dehydrogenase (glucose 1- dehydrogenase) (H6pd)

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NM_139294.5 Braf transforming gene (Braf) NM_011146.1 peroxisome proliferator activated receptor gamma (Pparg) NM_009504.3 vitamin D receptor (Vdr) NM_008302.3 heat shock protein 90 alpha (cytosolic), class B member 1 (Hsp90ab1) NM_175283.3 steroid 5 alpha-reductase 1 (Srd5a1) NM_011434.1 superoxide dismutase 1, soluble (Sod1) NM_001033988.1 nuclear receptor coactivator 4 (Ncoa4) NM_019584.3 beclin 1, autophagy related (Becn1) NM_011949.3 mitogen-activated protein kinase 1 (Mapk1) NM_001081117.2 antigen identified by monoclonal antibody Ki 67 (Mki67) NM_007630.2 cyclin B2 (Ccnb2) NM_009689.2 baculoviral IAP repeat-containing 5 (Birc5) NM_011496.1 aurora kinase B (Aurkb) NM_008021.4 forkhead box M1 (Foxm1) NM_011497.3 aurora kinase A (Aurka) NM_001037134.1 cyclin E2 (Ccne2) NM_009764.3 breast cancer 1 (Brca1) NM_007891.4 E2F transcription factor 1 (E2f1) NM_009860.2 cell division cycle 25C (Cdc25c) NM_007659.3 cyclin-dependent kinase 1 (Cdk1) NM_177382.3 cytochrome P450, family 2, subfamily r, polypeptide 1 (Cyp2r1) NM_001040654.1 cyclin-dependent kinase inhibitor 2A (Cdkn2a)

NM_009354.1 telomerase reverse transcriptase (Tert) NM_008342.2 insulin-like growth factor binding protein 2 (Igfbp2) NM_172301.3 cyclin B1 (Ccnb1) NM_001163028.1 beta-carotene 15,15'-monooxygenase (Bcmo1) NM_007489.3 aryl hydrocarbon receptor nuclear translocator-like (Arntl) NM_008970.3 parathyroid hormone-like peptide (Pthlh) NM_008161.2 glutathione peroxidase 3 (Gpx3) NM_013541.1 glutathione S-transferase, pi 1 (Gstp1) NM_001012335.1 midkine (Mdk)

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NM_010738.2 lymphocyte antigen 6 complex, locus A (Ly6a) NM_011577.1 transforming growth factor, beta 1 (Tgfb1) NM_009371.2 transforming growth factor, beta receptor II (Tgfbr2) NM_007616.3 caveolin 1, caveolae protein (Cav1) NM_011701.4 vimentin (Vim) NM_008816.2 platelet/endothelial cell adhesion molecule 1 (Pecam1) NM_001111059.1 CD34 antigen (Cd34) NM_010288.3 gap junction protein, alpha 1 (Gja1) NM_008610.2 matrix metallopeptidase 2 (Mmp2) NM_008006.2 fibroblast growth factor 2 (Fgf2) NM_001083316.1 platelet derived growth factor receptor, alpha polypeptide (Pdgfra) NM_009851.2 CD44 antigen (Cd44) NM_008343.2 insulin-like growth factor binding protein 3 (Igfbp3) NM_001113530.1 colony stimulating factor 1 (macrophage) (Csf1) NM_001079908.1 fibroblast growth factor receptor 1 (Fgfr1) NM_010591.2 Jun oncogene (Jun) NM_009829.3 cyclin D2 (Ccnd2) NM_010427.4 hepatocyte growth factor (Hgf) NM_009662.2 arachidonate 5-lipoxygenase (Alox5) NM_009741.3 B cell leukemia/lymphoma 2 (Bcl2) NM_007960.3 ets variant gene 1 (Etv1) NM_013599.2 matrix metallopeptidase 9 (Mmp9) NM_011333.3 chemokine (C-C motif) ligand 2 (Ccl2) NM_009365.2 transforming growth factor beta 1 induced transcript 1 (Tgfb1i1) NM_207668.2 acid phosphatase, prostate (Acpp) NM_008809.1 platelet derived growth factor receptor, beta polypeptide (Pdgfrb) NM_013693.1 tumor necrosis factor (Tnf) NM_009263.3 secreted phosphoprotein 1 (Spp1) NM_011347.1 selectin, platelet (Selp) NM_009807.2 caspase 1 (Casp1) NM_033217.3 nerve growth factor receptor (TNFR superfamily, member 16) (Ngfr) NM_009305.2 synaptophysin (Syp) 154

NM_007664.4 cadherin 2 (Cdh2) NM_010518.2 insulin-like growth factor binding protein 5 (Igfbp5) NM_015775.2 transmembrane protease, serine 2 (Tmprss2) NM_007631.1 cyclin D1 (Ccnd1) NM_011641.2 transformation related protein 63 (Trp63) NM_009170.3 sonic hedgehog (Shh) NM_027011.2 keratin 5 (Krt5) NM_009873.2 cyclin-dependent kinase 6 (Cdk6) NM_007556.2 bone morphogenetic protein 6 (Bmp6) NM_011243.1 retinoic acid receptor, beta (Rarb) NM_008815.2 ets variant gene 4 (E1A enhancer binding protein, E1AF) (Etv4) NM_011198.3 prostaglandin-endoperoxide synthase 2 (Ptgs2) NM_133659.2 avian erythroblastosis virus E-26 (v-ets) oncogene related (Erg) NM_013614.2 ornithine decarboxylase, structural 1 (Odc1) NM_001146100.1 hexokinase 1 (Hk1) NM_010554.4 interleukin 1 alpha (Il1a) NM_053188.2 steroid 5 alpha-reductase 2 (Srd5a2) NM_001111274.1 insulin-like growth factor 1 (Igf1) NM_007825.2 cytochrome P450, family 7, subfamily b, polypeptide 1 (Cyp7b1) NM_010228.3 FMS-like tyrosine kinase 1 (Flt1)

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Appendix C. Correlation bewteen expression of miR-16 and Ccnd1.1

1Dietary tomato and lycopene caused expression of miR-16 and target Ccnd1 differnetly correlated in wild type and TRAMP mice (spearman correlation, n=9/group).

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Appendix D. Correlation bewteen expression of miR-16 and Bcl2.1

1Dietary tomato and lycopene caused expression of miR-16 and target Bcl2 differnetly correlated in wild type and TRAMP mice (spearman correlation, n=9/group).

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Appendix E. Correlation bewteen expression of miR-16 and Fgf2.1

1 Dietary tomato and lycopene caused expression of miR-16 and target Fgf2 differently correlated in wild type and TRAMP mice (spearman correlation, n=9/group).

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