Dietary Tomato and Lycopene Impact Androgen Signaling- and ...€¦ · Introduction Prostate...

Research Article

Dietary Tomato and Lycopene Impact AndrogenSignaling- and Carcinogenesis-Related Gene Expressionduring Early TRAMP Prostate Carcinogenesis

Lei Wan1,2, Hsueh-Li Tan1,2, Jennifer M. Thomas-Ahner1, Dennis K. Pearl1,3, John W. Erdman Jr4,Nancy E. Moran1, and Steven K. Clinton1,5

AbstractConsumption of tomato products containing the carotenoid lycopene is associated with a reduced risk of

prostate cancer. To identify gene expression patterns associated with early testosterone-driven prostate

carcinogenesis, which are impacted by dietary tomato and lycopene, wild-type (WT) and transgenic

adenocarcinoma of the mouse prostate (TRAMP) mice were fed control or tomato- or lycopene-containing

diets from 4 to 10 weeks of age. Eight-week-old mice underwent sham surgery, castration, or castration

followed by testosterone repletion (2.5 mg/kg/d initiated 1 week after castration). Ten-week-old intact

TRAMPmice exhibit early multifocal prostatic intraepithelial neoplasia. Of the 200 prostate cancer–related

genesmeasured by quantitative NanoString, 189 are detectable, 164 significantly differ by genotype, 179 by

testosterone status, and 30 by diet type (P < 0.05). In TRAMP, expression of Birc5, Mki67, Aurkb, Ccnb2,

Foxm1, andCcne2 is greater compared withWT and is decreased by castration. In parallel, castration reduces

Ki67-positive staining (P < 0.0001) compared with intact and testosterone-repleted TRAMP mice. Expres-

sionof genes involved in androgenmetabolism/signaling pathways is reduced by lycopene feeding (Srd5a1)

and by tomato feeding (Srd5a2, Pxn, and Srebf1). In addition, tomato feeding significantly reduced

expression of genes associated with stem cell features, Aldh1a and Ly6a, whereas lycopene feeding

significantly reduced expressionof neuroendocrine differentiation–related genes,Ngfr and Syp. Collectively,

these studies demonstrate a profile of testosterone-regulated genes associated with early prostate carcino-

genesis that are potential mechanistic targets of dietary tomato components. Future studies on androgen

signaling/metabolism, stem cell features, and neuroendocrine differentiation pathways may elucidate the

mechanisms by which dietary tomato and lycopene impact prostate cancer risk. Cancer Prev Res; 7(12);

1228–39. �2014 AACR.

IntroductionProstate carcinogenesis is an extended process that is

likely impacted by a complex network of genetic, hor-monal, and environmental factors throughout the life

cycle, including the in utero development phase, theperiod of maximal growth during puberty, as well as theperiod of adulthood (1). Human prostate carcinogenesisclearly involves disruption of multiple molecular andbiologic processes, which contribute to its heterogeneityas a clinical entity (2). Normal prostate development,growth, and function, as well as the emergence of pros-tate cancer are androgen-dependent processes (3). Tes-tosterone, the predominant androgen in males, is con-verted by 5a-reductase (encoded by Srd5a1 and Srd5a2)into dihydrotestosterone (DHT), the high-affinity ligandfor the androgen receptor (AR) which, in conjunctionwith multiple cofactors, mediates transcriptional activity(3). While it is clear that androgens play a key role in theevolution of prostate cancer, the effects of dietary vari-ables hypothesized to impact prostate cancer risk onandrogen-driven processes are poorly understood. Tran-scriptomic analyses of human prostate cancer and non-cancerous tissue have revealed multiple dysregulatedpathways during the development of human primary,metastatic, and hormone-refractory prostate cancer (4).

1Comprehensive Cancer Center, The Ohio State University, Columbus,Ohio. 2Interdisciplinary Nutrition Program, The Ohio State University,Columbus, Ohio. 3Department of Statistics, The Ohio State University,Columbus, Ohio. 4Department of Food Science and Human Nutrition,University of Illinois, Urbana, Illinois. 5Division of Medical Oncology,Department of Internal Medicine, The Ohio State University, Columbus,Ohio.

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

L. Wan and H.-L. Tan contributed equally to this article.

Corresponding Author: Steven K. Clinton, Division of Medical Oncology,The Ohio State University, A456 Starling Loving Hall, 320 West 10thAvenue, Columbus, OH 43210. Phone: 614-293-2886; Fax: 614-293-7525; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-14-0182

�2014 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 7(12) December 20141228

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Published OnlineFirst October 14, 2014; DOI: 10.1158/1940-6207.CAPR-14-0182

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Yet it is more difficult to examine the molecular changesleading to the development of premalignancy in thehuman prostate, due to challenges of procuring suchpremalignant tissue samples. Rodent models of prostatecarcinogenesis provide an opportunity to define andro-gen-driven gene expression signatures contributing tothe emergence of premalignant histopathologic changes(5) and how dietary variables may modulate thesesignatures.Epidemiologic and experimental studies of prostate

cancer strongly suggest that disease risk is impacted bydietary patterns, specific nutrients, or phytochemicals (1).Notably, higher consumption of tomato products, esti-mated dietary lycopene intake, and circulating lycopeneconcentrations are inversely associated with prostate can-cer risk in several human cohort studies (6). Laboratorystudies provide evidence that tomato and lycopene maysuppress oxidative damage, modulate intracellular signal-ing resulting in reduced proliferation, and enhance sen-sitivity to apoptosis, among other mechanisms (6). Someevidence suggests that tomato or lycopene intake maymodulate testosterone production, serum concentrations,and metabolism, and may impact gene expression inhuman prostate cancer cells, normal rat prostate, andestablished prostate cancer xenografts (7–10). However,whether lycopene and/or other tomato phytochemicalsinhibit prostate cancer by targeting androgen signalingand metabolism in early stages of prostate carcinogenesishas not been thoroughly investigated.One of the key questions regarding the role of tomato

products in prostate carcinogenesis is the relative contri-bution of lycopene to the antiprostate cancer efficacy oftomato compared with other tomato phytochemicals.While tomato feeding was more effective than lycopenein improving survival in rats with carcinogen(NMU andandrogens)-induced prostate cancer (11), lycopeneappears to be nearly as effective as tomato componentsin reducing cancer incidence and in increasing survival intransgenic adenocarcinoma of the mouse prostate(TRAMP) mice (12). In addition, the timing of tomatoor lycopene intervention during the life cycle may also becritical. When tomato feeding is initiated at 4 weeks ofage, it delays prostatic intraepithelial neoplasia (PIN)formation in 12-week-old TRAMP mice (13). Similarly,tomato and lycopene feeding initiated at weaning and fedthroughout life reduces prostatic lesion incidence andseverity in TRAMP mice at 20 and 30 weeks of age(12–14). Intriguingly, recent cohort findings suggest astronger inverse association between tomato consump-tion reported at study enrollment and cumulative averagelycopene intake over many years than with lycopeneintakes recorded closer to the time of cancer diagnosis(15), possibly suggesting that longer term dietary tomatoproduct or lycopene intake could be important in humanprostate cancer prevention as well.Together, the experimental and human data warrant

further investigation of the impact of tomato and lyco-pene on the very early processes associated with prostate

carcinogenesis. Prostate cancer risk in the TRAMP modelis impacted by tomato, lycopene, and early castration(12–14, 16). In this model, the testosterone-drivenrat probasin promoter induces prostate-specific expres-sion of the SV40 antigen, initiating the carcinogenicprocess, that over a predictable time frame, progressesfrom a hyperplastic epithelium, to PIN (�6–10 weeks), toprostatic adenocarcinoma (�10–16 weeks), to poorlydifferentiated carcinoma with metastasis (�18–24 weeks;refs. 17, 18), recapitulating features of human androgen-dependent cancer progression. The primary objective ofthe current study is to define the impacts of dietarytomato or lycopene on prostatic gene expression duringearly, androgen-driven carcinogenesis. To accomplishthis objective, we first characterize the molecular path-ways deregulated in the early, premalignant stages ofTRAMP prostate carcinogenesis, and which pathways aresubject to androgen regulation as determined by removalof testosterone by castration, and then the impacts oftomato and lycopene feeding on expression of genesassociated with early androgen-driven prostate carcino-genesis are defined.

Materials and MethodsStudy design

All mouse experiments were performed in compliancewith the Ohio State University Institutional Animal Careand Use Committee. Two, 3 � 3 factorial studies wereutilized to investigate the effects of diet on androgen-driven prostate carcinogenesis in WT and TRAMP mice.Male TRAMP (þ/�) C57BL/6�FVB/N hybrid and maleC57BL/6 controls (Jackson Laboratory) were used. Toma-to and lycopene diets were formulated to provide bloodand tissue concentrations of lycopene similar to humans(19). TRAMP mice weaned at 4 weeks of age were ran-domized to one of three semipurified AIN-93G–baseddiets (ResearchDiets) with either 0.25% (w/w) placebobeadlets (DSM), 10% (w/w) tomato powder providing384 mg lycopene/kg diet [FutureCeuticals; with 0.25%(w/w) placebo beadlets], or 0.25% (w/w) lycopene bead-lets (RediVivo 10% lycopene, DSM) providing 462 mglycopene/kg diet, as previously described (19). Similardiets were fed to 4-week-old WT but contained 0.04% (w/w) lycopene beadlets in the lycopene diet providing 20mg lycopene/kg and 0.04% (w/w) control beadlets incontrol and tomato diets providing 40 mg lycopene/kgdiet, respectively. Eight-week-old mice within each dietgroup were randomized to undergo a sham (superficialincision only) surgery, castration, or castration followedby testosterone repletion (2.5 mg testosterone propio-nate/kg of body weight/day). Testosterone propionate(Sigma-Aldrich) dissolved in sterile cyclodextrin (Sig-ma-Aldrich) was administered 1 week after castration viamini osmotic pump implantation (Alzet), per the man-ufacturer’s instructions. All mice were sacrificed 5 daysafter pump implantation (12 days after surgery). Plasmawas collected, prostate glands were procured by

Tomato and Lycopene Impact Early Prostate Carcinogenesis

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microdissection and were separated into constituentlobes [anterior (AP), ventral (VP), dorsal (DP), and lateral(LP)], and 5 prostates per experimental group designatedfor histologic outcomes were fixed in 10% neutral buff-ered formalin overnight followed by processing and par-affin embedding. Three to 5 DP and LP designated forRNA isolation were preserved in RNAlater solution (LifeTechnologies).

Plasma lycopene measurementPlasma carotenoids were extracted and analyzed as

described previously (20).

HistopathologySections (5 mm) of prostate tissues were stained with

hematoxylin and eosin (H&E) and bright-field imageswere captured at 200� original magnification with aNikon Eclipse E800 microscope (Nikon Instruments Inc)equipped with a Nikon DS-Ri1 digital camera.

Immunohistochemistry and image analysisExpression of Ki67, the protein product of the Mki67

gene was validated using immunohistochemistry withantigen retrieval (1� Citra Plus Buffer, Biogenex; 30minutes in a steamer), monoclonal rat anti-mouse Ki67(TEC3; 1:50; 60 minutes; Dako) followed by biotiny-lated rabbit anti-mouse IgG (1:200; 30 minutes; Dako).Staining was visualized with streptavidin/HRP (1:200;30 minutes) and DAB (10 minutes; Dako) and counter-stained with Mayer’s hematoxylin (2 minutes; Sigma-Aldrich). Staining was quantified from scanned images(Aperio Scanscope XT scanner, Leica) using positivepixel counting [ImageScope 8.1 (Aperio) digital imageanalysis package]. The Ki67 protein expression is ex-pressed as a percentage of positive pixels relative to thetotal (positive þ negative) pixels for a whole AP cross-section.

RNA isolation and mRNA expressionRNA was extracted from fresh-frozen pooled DP and

LP preserved in RNALater at the time of dissection, usingthe RNeasy Mini Kit (Qiagen). A custom-designed murineprostate carcinogenesis gene codeset of 200 genes, select-ed on the basis of relevance to both human and murineprostate carcinogenesis as indicated by scientific lite-rature (Supplementary Table S1), was used to quantitategene expression using nCounter technology (NanoString)from 100 ng total RNA, without enzymatic amplifica-tion. NanoString nCounter technology was selected forquantitative gene expression analysis due to its documen-ted precision, sensitivity comparable with qRT-PCR, lackof reliance on enzymatic reactions and amplification, andmultiplex capabilities with limited starting material(21). Expression was analyzed by subtraction of the meanof the negative controls from the sample data, followedby natural log transformation and normalization toCcng1 (NM_009831.2), which was identified as a relevantnormalization factor based on low between-group vari-

ability (P < 0.05 by ANOVA), and high stability withingroups (mean SE ¼ 0.0105).

Statistical analysisTo define expression differences in response to the

main and interaction effects of genotype, testosterone,and diet, normalized mRNA data on the log scale wasanalyzed by a three-way ANOVA model assuming nointeractions with diet. The latter assumption was exam-ined and no evidence of important diet interactions wasfound (e.g., P values closely followed the uniform distri-bution expected under the null hypothesis and none ofthe 189 genes were affected by diet � genotype, diet �testosterone, diet � genotype � testosterone interactionsat the Bonferroni-adjusted significance level of 0.00026(0.05/189)], The model without diet interactions wasthen used to assess the prostatic gene expression changesby diet, testosterone, and genotype. Genes with a countnumber >100, and an expression fold-difference betweengenotypes (TRAMP vs. WT) or between testosterone sta-tuses (intact vs. castrated) in the top 5% of 189 detectedgenes were defined as biologically significantly regulated.Plasma lycopene concentration, body weight, tissueweight, and Ki67 protein expression were evaluated bythe same three-way ANOVA model and then by two-wayANOVA within genotypes. Pairwise comparisons ofgroups were evaluated using the post hoc Sidak test. Allstatistics were calculated using the Stata12 statistical anal-ysis package (StataCorp).

Pathway analysisThe Ingenuity Pathway Analysis application (http://

www.ingenuity.com)was used todetect prostaticmolecularpathways differences between TRAMP versus WT and intactversus castrated mice. The gene expression linear ratios andpairwise P values for TRAMP versus WT and castrationversus intact mice were used as data inputs. Significancecutoff criteria were set to P < 0.05. Genes significantlyimpacted by genotype and testosterone status were thenmapped onto the Global Ingenuity Knowledgebase Net-work and significance was calculated as previouslydescribed (19). The top 10 canonical pathways associatedwith early prostate carcinogenesis were identified on thebasis of P value for each pathway.

ResultsMouse body and tissue weights

Mouse urogenital tract (UGT) weights were not chan-ged by tomato or lycopene feeding, compared withcontrol feeding, regardless of genotype (SupplementaryFig. S1). Castration reduced UGT weight by 64% and 57%compared with those of intact and testosterone-repletedmice (P < 0.001 for both), respectively, in WT, and by69% and 60% compared with those of intact and testos-terone-repleted TRAMP (P < 0.001) mice, respectively.The body weights of WT mice were lower than thoseof TRAMP mice at the initiation of the study (14 � 0.2

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g vs. 20 � 0.2 g; P < 0.0001). Body weight gain frominitiation of experimental diets to the day of surgery(4 weeks) was not impacted by diet type. During the12-day testosterone manipulation phase, castrationcaused weight loss while sham surgery did not in bothWT (�0.05 � 0.2 g and 0.88 � 0.3 g, respectively, P ¼0.0002) and TRAMP (�0.23 � 0.1 g and 0.87 � 0.1 g,respectively, P < 0.0001) mice. This effect was reversed bytestosterone administration.

Plasma lycopene concentrationsLycopene was not detected in the plasma of either

control-fed WT or TRAMP mice (Supplementary Fig.S1). The achieved plasma lycopene concentrations weresimilar in tomato- and lycopene-fed WT and TRAMPmice. Compared with intact, castrated mice had lowerplasma lycopene concentrations in tomato- and lyco-pene-fed WT and TRAMP by 35% (P < 0.05) and 21%(N.S), respectively.

Histopathology of TRAMP prostate carcinogenesisTestosterone and genotype impacted prostatic mor-

phology in 10-week-old mice (Fig. 1), with intactTRAMP having multifocal early PIN lesions in DP.Castration caused glandular atrophy in both WT andTRAMP. Testosterone repletion stimulated glandularcell growth in both WT and TRAMP mice and wasassociated with early PIN lesions in the TRAMP. Thecurrent study focusing upon gene expression was notpowered to define the more subtle dietary impact onhistopathology.

Gene expression impacted by genotype, testosterone,and dietAmong the 200 genes in the codeset, expression of

189 genes was above the limits of detection, and expres-sion of genes strongly clustered into genotype- and

testosterone-responsive groups, with 164 being signifi-cantly changed by a main effect of genotype, 179 bytestosterone, and 30 by diet (P < 0.05; Fig. 2). There is aninteraction of genotype and hormone status for 155genes. The TRAMP mice had significantly upregulatedexpression of 139 genes and downregulated expressionof 25 genes, compared with WT.

Expression of genes associated with early prostatecarcinogenesis

The 5%of genesmost significantly impacted by genotypefell primarily in the functional groups of cell growth andcell-cycle regulation (Fig. 3 and Supplementary Table S2).The genes strongly upregulated in TRAMP compared withWT included baculoviral IAP repeat-containing 5 (Birc5,encoding survivin), antigen identified by monoclonal anti-body Ki67 (Mki67), aurora kinase B (Aurkb), cyclin B2(Ccnb2), secreted phosphoprotein-1 (Spp1), forkhead boxM1 (Foxm1), cyclin E2 (Ccne2), and E2F transcription factor1 (E2f1). Genes for which expression was biologicallydownregulated in TRAMP relative to WT were Egf andprobasin (Pbn).

Gene expression driven by testosteroneCastration significantly impacted expression of genes

involved in cancer growth and cell migration in both WTand TRAMP. Although insulin-like growth factor–bind-ing protein 3 (Igfbp3), CD44 antigen (Cd44), gap junc-tion protein a1 (Gja1), and colony-stimulating factor 1(Csf1) were not differentially expressed between WT andTRAMP, they were significantly increased in castratedcompared with intact mice (Fig. 4 and SupplementaryTable S3). In contrast, castration significantly loweredexpression of NK-3 transcription factor, locus 1 (Nkx3.1)and b2-microglobulin (B2m) expression, relative tointact mice.

WT

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Figure 1. Representative histologicimages (magnification ¼ 200�) ofH&E-stained 10-week-old mouseAP lobes from WT or TRAMP, aftereither sham surgery (intact),castration surgery (castration), orcastration surgery followed bytestosterone repletion (repletion).Arrow indicates an early PIN lesion(A–F).






Castration reversed expression of cell growth–relatedgenes which were induced in TRAMP compared with WTmice (Fig. 3).Castration reduced expressionofBirc5,Mki67,Aurkb, Ccnb2, Foxm1, and Ccne2 and enhanced expressionof Spp1, Egf, and Pbn, compared with that of the intactgroup.

A significant interaction between genotype and testos-terone impacted expression of Ccne2 and E2f1 (Fig. 4).Ccne2 [fold change (FC) ¼ 6.13 � 1.21, P < 0.0001] andE2f1 (FC ¼ 5.56 � 1.21, P < 0.0001) were significantlyupregulated in TRAMP compared with WT. They weredownregulated in castrated TRAMP (P < 0.0001) but notin castrated WT, compared with intact TRAMP and WT,respectively.

Molecular pathways impacted by genotype andtestosterone

Out of the 10 most significantly genotype-regulatedcanonical pathways, 9 were also impacted by castra-tion compared with intact mice (Fig. 5 and Supplemen-tary Table S4). While GADD45 signaling was withinthe top 10 pathways impacted by genotype, it was not inthe top 10 pathways changed by castration, though itwas still significantly impacted by castration [P ¼ 4.71E-18 or �Log(P) ¼ 17.4]. The NF-kB signaling pathway

consequently fell into the top 10 canonical pathwaysaltered by testosterone status.

Gene expression impacted by tomato and/or lycopenefeeding

Tomato and lycopene feeding modulated expressionof genes related to several biologic targets. Remarkably,out of 189 detectable prostatic genes, tomato and lyco-pene similarly impacted expression of 26 genes and dif-ferentially impacted only 4 genes, those encoding nervegrowth factor receptor (Ngfr), synaptophysin (Syp), B2m,and vitamin D receptor (Vdr; Fig. 6). Among the 30 genesimpacted by diet, 4 genes were increased by and 26were decreased by tomato and lycopene. The genes canbe functionally grouped into (i) androgen metabolismand signaling: 5a-reductase 1 and 2 (Srd5a1/2), paxillin(Pxn), and sterol response element–binding factor1 (Srebf1); (ii) MAPK signaling: TGF1 and TGF2(Tgfbr1/2), fibroblast growth factor 2 (Fgf2), Egfr, Kras,and Nfkb; (iii) p53 signaling: Igf, caspases 2, 8, and 9(Casp2/8/9); (iv) cell adhesion molecules: P selection(Selp), cadherin 1 and 2 (Cdh1/2), and vimentin (Vim);(v) TR/RXR and VDR/RXR activation: solute carrierfamily 2 (facilitated glucose transporter), member 1(Slc2a1), retinoid X receptor a (Rxra), and vitamin D

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Figure 2. Hierarchical clustering of189 detectable prostatic genes inresponse to genotype andtestosterone status. I, intact; C,castration; R, castration followedby testosterone repletion (A). Venndiagram depicting the numbers ofprostatic genes for whichexpression was significantly (P �0.05) impacted by either the maineffects of genotype, testosterone,or diet (nonoverlapping portions) ora combination of main effects(overlapping portions; B).

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receptor (Vdr); (vi) phase II detoxifying enzymes: gluta-thione peroxidase 3 (Gpx3) and superoxide dismutase 2(Sod2); (vii) complement and coagulation cascades: plas-minogen activator, urokinase receptor (Plaur); viii)endocytosis: vascular epithelial growth factor receptor1(Vegfr1); (ix) neuroendocrine differentiation: nervegrowth factor receptor (Ngfr) and synaptophysin (Syp);and (x) stem cell features: aldehyde dehydrogenase 1A1(Aldh1a1) and lymphocyte antigen 6a (Ly6a).Tomato and lycopene feeding downregulated androgen

metabolism/signaling–related gene expression, relative tocontrol feeding (Fig. 6). Tomato- and lycopene-fed mice

had lower expression of genes encoding the isoforms of 5a-reductase, Srd5a1 (lycopene vs. control FC¼�1.18� 1.08,P ¼ 0.03) and Srd5a2 (tomato vs. control FC ¼ �1.25 �1.09, P ¼ 0.04). Two androgen receptor coregulators weredownregulated in tomato-fed, compared with control-fedmice: Pxn (tomato vs. control FC¼�1.19� 1.09, P¼ 0.04)and Srebf1 (tomato vs. control FC ¼ �1.21 � 1.11, P ¼0.05).

Neuroendocrine differentiation- and stem cell-relatedgenes were downregulated by tomato and lycopene feed-ing. Lycopene-fed mice had lower expression of prostaticNgfr (FC¼�1.61� 1.16, P¼ 0.02) and Syp (FC¼�1.72�

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Figure 3. Gene expression wasstrongly upregulated (A–D) ordownregulated (E and F) in TRAMPcompared with WT mice with asignificant main effect of genotype(P < 0.05), in intact, castration,or castration followed bytestosterone repletion (repletion)groups, and was significantlydecreased in castration comparedwith intact and testosteronerepletion group (P < 0.0001; notdenotedon figures). Fold changeofexpression is relative to the intact,WT group. Data are expressed asmean � SE (n ¼ 3–5/group).��, significant between-genotype,within surgical treatment difference(P < 0.0001). Castrationsignificantly decreased Ki67protein expression of AP in WT (G)and TRAMP (H). ��, significantdifferences in castration comparedwith intact and repletion in bothWTand TRAMP, regardless of diettype.






1.18, P¼ 0.02) than control-fedmice. Tomato-fedmice hadlower prostatic expressionofAldh1a1 (FC¼�1.14�1.15,P¼ 0.04) and Ly6a (FC ¼ �1.34 � 1.14, P < 0.001) thancontrol-fed mice.

Protein expression of Ki67Ki67 protein expression was lower in castrated compared

with intact mice in bothWT and TRAMP (1.1% vs. 0.7% forWT and 3.6% vs. 0.2%, P < 0.0001 for TRAMP), as predictedby mRNA patterns (Fig. 3). Dietary intervention did not

result in a significant impact on Ki67 protein expression atthis early time point.

Discussion

It is clear from studies in multiple laboratories thattomato components or lycopene reduce the progression ofprostate carcinogenesis in TRAMP and other rodent models(9, 11–14). As in human prostate cancer, the progression inTRAMP is initially driven by testosterone and evolves into

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Figure 4. Prostatic gene expressionsignificantly impacted by androgenstatus (A–F) and a significantgenotype � testosterone statusinteraction (G and H). ��,significant differences betweenandrogen status groups, withingenotype (P < 0.0001 comparedwith intact and repletion groups).Fold change of expression isrelative to intact, WT. Data areexpressed as mean � SE (n¼ 3–5/group).

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poorly differentiated castration-resistant disease (22). Inthis study, we utilized a system of castration and testoster-one-repletion in WT and TRAMP mice to examine howdietary tomato or lycopene impact androgen-driven geneexpression early in prostate carcinogenesis, before thedevel-opment of significant histopathologic changes. As antici-pated, prostate weight, histologic features, and gene expres-sion profiles were dramatically changed in response totestosterone status, with 95% of the detectable genes in ourgene codeset being impacted by testosterone and 87% bygenotype. With this foundation, these molecular signaturesin testosterone-driven early prostate carcinogenesis can beused to address which processes are impacted by the dietaryvariables of tomato and lycopene. We observed that 16%(30 genes) of themeasured genes were significantly impact-ed by the dietary interventions. Interestingly, 29 of thesediet-sensitive genes are also subject to androgen regulation(Fig. 1), and this marked overlap indicates that tomato andlycopene feeding modulate expression of androgen-regu-lated genes in early prostate carcinogenesis. In the context ofsimilar plasma lycopene concentrations in the tomato- andlycopene-fed mice, regardless of genotype, the effects ofdietary tomato or lycopene are remarkably similar, both inthe genes impacted and the magnitude of changes. Theseresults suggest that lycopene accounts for much of theimpact of tomato feeding; although a few very intriguingdifferences emerged. Interestingly, the dietary interventionsdownregulated gene expression associated with androgenmetabolism and signaling, neuroendocrine and stem cellfeatures.

Molecular signature of early prostate carcinogenesisin the TRAMP model

Although minimal histopathologic changes are notedat 10 weeks of age, expression of the SV40 construct in theTRAMP prostate yields a unique molecular signaturecompared with WT (Figs. 1 and 2). As predicted, basedupon known inhibitory functions of T-antigen on expres-sion of tumor suppressors p53 and Rb, expression ofdownstream genes controlling cell-cycle regulation/pro-liferation and p53 signaling pathways are activated inearly prostate carcinogenesis (Figs. 3 and 5). Many ofthese early changes observed in our study are known topersist in established cancer in 30-week-old TRAMP mice,and indeed hyperproliferation is characteristic of theTRAMP system throughout its progression (23, 24). Theupregulation of Birc5 (encoding survivin), Mki67, Ccnb2,Foxm1, Ccne2, and E2f1 and downregulation of Egf andPbn (encoding probasin) are consistent with thesechanges. Ki67 expression is frequently used as a histo-pathologic biomarker of proliferative status and highexpression is associated with decreased survival inpatients with prostate cancer (25). Survivin is an apopto-sis inhibitor, and prostatic expression is correlated withcancer progression in humans (26). FOXM1 is a regulatorof the cell cycle, which is upregulated in metastaticprostate cancer tissue (4). These genes are included inthe p53, cell cycle, and checkpoint regulation pathways(Supplementary Table S4), and these early changes areconsistent with changes reported in more advancedTRAMP cancers (23).

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Figure 5. The top 10 canonicalpathways for which associatedgene expression most significantlydiffered [increased or decreased;as indicated by �Log(P value)] bygenotype (A) and castration status(B), as calculated from geneexpression data by IPA.






Testosterone-driven gene expression in early prostatecarcinogenesis

Modulation of testosterone activity remains a key thera-peutic intervention extending the lives of patients withprostate cancer (3). The use ofweak antiandrogens targeting5a-reductase, which inhibits the conversion of testosteroneto DHT, has shown significant activity in human chemo-prevention studies with acceptable toxicity (27, 28). Inparallel, early castration (4-week-old) in TRAMP decreasesprostate cancer formation and growth (16). One of theobjectives of the current study is to define patterns of genesassociated with early prostate carcinogenesis impacted bytestosterone status, as these genes may provide insight intomechanisms and serve as targets for diet and chemopreven-tive agents. Castration suppressed cell-cycle progression-related genes (Mki67, Birc5, Aurkb, Ccnb2, and Foxm1) and

the protein product of Mki67, Ki67 (Fig. 3). These findingssupport the concept that disruption of testosterone-drivensignaling impacting cell cycle and proliferation shouldcontinue to be pursued for prostate cancer prevention.

In parallel, castration also activates expression of genesassociated with suppression of cellular growth, such as,Igfbp3 and Gja1 (Fig. 4). IGFBP3 inhibits the growth-stimulating functions of IGF by sequestering IGF1, andmay impact autocrine, paracrine, and endocrine actionsof IGF (29). The inverse association between IGFBP3 andprostate cancer risk remains somewhat speculative, yetthis finding illustrates the importance of the intimateorchestration that exists between steroid and proteinhormone/growth factor action in prostate carcinogenesis(29). In the current study, castration increased expressionof Gja1 (encoding a gap junction communication

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Wan et al.





protein, connexin 43), which is consistent with the effectof castration on the WT rat prostate (30). The putativefunction of connexin 43 in prostate cancer developmentis stage-specific: in primary prostate cancer cells, loss ofconnexin promotes metastatic potential, and in a humanprostate cancer cell line, connexin 43 inhibits prolifera-tion, independent of gap junction function (31). Overall,castration reduced/reversed expression of genes associat-ed with the canonical pathways induced in early prostatecarcinogenesis (Fig. 5), with a marked reduction of pro-liferation and cell-cycle regulation pathways.

Tomato and lycopene similarly impact geneexpressionThe plasma lycopene concentrations were compar-

able in both WT and TRAMP mice (0.34 and 0.36mmol/L in WT and TRAMP, respectively), were within theachievable range of plasma concentrations found inhumans (32), and tomato and lycopene similarly alteredexpression of 26 genes, with no effect on 159 genes. Only4 genes showed divergent expression, yet these are alsovery interesting: Ngfr, Syp, Vdr, and B2m (Fig. 6). Thus, itseems that lycopene largely contributed to the impactof tomato feeding on gene expression in early prostaticcarcinogenesis. Genes encoding proteins related toandrogen metabolism/signaling, MAPK signaling, p53signaling, and phase II detoxification enzymes largelyconstituted the genes similarly impacted by lycopene andtomato feeding.Interestingly, the neuroendocrine-related genes, Syp

and Ngfr, are expressed in the 10-week-old mouse pros-tate, and expression is inhibited by dietary lycopene.Neuroendocrine cells (identified by synaptophysin) havealso been detected in PIN lesions of 12-week-old TRAMPand in advanced, poorly differentiated TRAMP tumors(33, 34), and it is important to recognize that neuroen-docrine cells are present in normal human prostates andbenign prostatic hyperplasia (35). High-grade and late-stage prostate tumors, especially castration-refractory can-cers, often exhibit a neuroendocrine phenotype, as iden-tified by SYP staining (36). Higher intake of lycopene isassociated with lower risk of aggressive and lethal prostatecancer (15). To our knowledge, this is the first report toshow lycopene significantly downregulates Syp and Ngfrin prostate carcinogenesis, suggesting lycopene mayattenuate prostate carcinogenesis by reducing neuroen-docrine features associated with poorly differentiated andmore aggressive cancer.

Impacts of tomato and lycopene feeding on androgenmetabolism- and stem cell feature-related geneexpressionRegulation of androgen metabolism and signaling are

among the proposed mechanisms by which tomato andlycopene exert inhibitory effects in prostate cancer. Weobserved that tomato and lycopene feeding decreasedSrd5a1 and Srd5a2 (encoding 5a-reductase 1 and 2) expres-sion (Fig. 6). Other studies have also suggested similar

effects in various, yet quite different, types of scenarios(8–10, 37). Pharmacologic inhibition of SRD5A enzymeactivity by finasteride and dutasteride reduced overall pros-tate cancer incidence by 23% to 25% compared with pla-cebo in brief chemoprevention trials (27, 28, 38), and thusit may be possible that lycopene or tomato exhibit antican-cer activity by reducing overall enzyme content and sub-sequent activity. Thus, the effect of dietary tomato andlycopene on functional changes and enzymatic activity of5a-reductase warrant future mechanistic investigation.

Expression of AR coregulators were also impacted bytomato and lycopene feeding in early prostate carcinogen-esis, suggesting another mechanism by which tomato andlycopenemay reduce androgen signaling. Previously, toma-to and lycopene were reported to inhibit prostatic steroid–binding protein mRNA expression, AR coregulator (ProteinDJ1), and an androgen receptor–stabilizing chaperone pro-tein (HSP90) in the prostate of healthy rats, theDunning ratmodel, and human primary prostate epithelial cells (9,10, 39). That Pxn is impacted by tomato and lycopene isa novel finding. SREBP (encoded by Srebf1) and paxillin(encoded by Pxn), are AR coactivators which bind to the ARpromoter region in human prostate cancer cells (40, 41).SREBP overexpression enhances proliferation and prostatecancer progression and is associated with progression ofhuman prostate cancer (42). Therefore, the inhibitory effectof tomato and lycopene on Srd5a1/2, Srebf1, and/or Pxnmight contribute to the inhibition of prostate carcinogen-esis by impacting key coregulators of androgen signaling.

The prostatic glandular epithelium includes a subpopu-lation of cells with self-renewal potential (43). In normaland cancerous human and murine prostate, stem cell anti-gen-1(Sca1, encoded by Ly6a gene), has been used as abiomarker of prostate stem/progenitor cells (44). Expres-sion of ALDH1A1 is also increased in progenitor cells inboth human prostate cancer and rodent models (45). Inhuman clinical samples, ALDH1A1 is positively correlatedwith Gleason score and pathologic stage and is inverselyassociated with patient survival (45, 46). The inhibitoryeffect of tomato and lycopene on Ly6a and Aldh1a expres-sion (Fig. 6) indicates that inhibition of stem cell featuresand therefore promotion of terminal differentiation, mightbe a novel mechanism by which tomato consumptionreduces prostate cancer risk, warranting investigationbeyond the expression of these two relevant genes.

ConclusionIn conclusion, gene expression profiles are clearly

deregulated as early as 10 weeks-of-age in the TRAMPmodel, a time representing the emergence of morpho-logic changes in the mouse prostate. In addition, tes-tosterone regulation of carcinogenic gene expressionpatterns likely mimics early stages of human carcino-genesis. While the impacts of tomato and lycopenefeeding on gene expression were of a lesser magnitudethan those observed between genotypes or androgenstatuses, this was to be expected of the physiologically






relevant dietary intervention, as opposed to the muchlarger impacts of the TRAMP genotype and castration.Nonetheless, the differences are illustrative of the broadimpacts of tomato and lycopene feeding on androgen-driven early carcinogenic gene expression. Our evidencesuggests that the cancer preventive effects of tomato andlycopene are partially mediated by their inhibition ofandrogen activity and coregulators of androgen receptoractivation. Novel mechanisms of tomato and/or lyco-pene on neuroendocrine markers and cancer stem cellbiology deserve further investigation. Remarkably, ourgene expression signatures in early carcinogenesis indi-cate that lycopene and tomato have nearly equivalentimpact on androgen-driven gene expression in theTRAMP model, suggesting that lycopene is the majorbioactive tomato component impacting early TRAMPcarcinogenesis.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Wan, H.-L. Tan, J.W. Erdman Jr, S.K. ClintonDevelopment of methodology: L. Wan, H.-L. Tan, J.M. Thomas-Ahner,D.K. Pearl, S.K. ClintonAcquisitionofdata (provided animals, acquired andmanagedpatients,providedfacilities,etc.):L.Wan,H.-L.Tan, J.M.Thomas-Ahner,S.K.Clinton

Analysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): L. Wan, H.-L. Tan, J.M. Thomas-Ahner,D.K. Pearl, N.E. Moran, S.K. ClintonWriting, review, and/or revision of the manuscript: L. Wan, H.-L. Tan,J.M. Thomas-Ahner, D.K. Pearl, J.W. Erdman Jr, N.E. Moran, S.K. ClintonAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. Wan, H.-L. Tan, S.K. ClintonStudy supervision: S.K. Clinton

AcknowledgmentsThe authors thank Amy Elsen for her technical contribution to the serum

carotenoid analysis.

Grant SupportThis work was supported by The National Institutes of Health [NCI-

R01125384; Principal Investigator (PI): J.W. Erdman Jr, co-investigator: S.K.Clinton] and the American Institute for Cancer Research (AICR; PI: S.K.Clinton) with additional resources provided by the Ohio State UniversityMolecular Carcinogenesis and Chemoprevention Program, Center forAdvanced Functional Food Research, Food Innovation Center, and TheJamesDevelopment Prostate Cancer Prevention Fund (302024).N.E.Moranwas fundedby a Pelotonia Postdoctoral Fellowship (OSU-CCC). TheNucleicAcid and the Comparative Pathology Shared Resources supported by TheOhio State University Comprehensive Cancer Center (NIH/NCI P30016058) were utilized.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 6, 2014; revised September 5, 2014; accepted October 3,2014; published OnlineFirst October 14, 2014.

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2014;7:1228-1239. Published OnlineFirst October 14, 2014.Cancer Prev Res Lei Wan, Hsueh-Li Tan, Jennifer M. Thomas-Ahner, et al. Prostate CarcinogenesisCarcinogenesis-Related Gene Expression during Early TRAMP Dietary Tomato and Lycopene Impact Androgen Signaling- and

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