effects of pubertal exposure to dietary soy on estrogen ... · 12.04.2016 · mrna expression of...

Research Article

Effects of Pubertal Exposure to Dietary Soy onEstrogen Receptor Activity in the Breast ofCynomolgus MacaquesFitriya N. Dewi1,2, Charles E.Wood1, Cynthia J.Willson1, Thomas C. Register1,Cynthia J. Lees1, Timothy D. Howard3, Zhiqing Huang4, Susan K. Murphy4,Janet A. Tooze5, Jeff W. Chou5, Lance D. Miller6, and J. Mark Cline1

Abstract

Endogenous estrogens influence mammary gland develop-ment during puberty and breast cancer risk during adulthood.Early-life exposure to dietary or environmental estrogens mayalter estrogen-mediated processes. Soy foods contain phytoestro-genic isoflavones (IF), which have mixed estrogen agonist/antag-onist properties. Here, we evaluated mammary gland responsesover time in pubertal female cynomolgus macaques fed dietscontaining either casein/lactalbumin (n ¼ 12) or soy proteincontaining a human-equivalent dose of 120 mg IF/day (n ¼17) for approximately 4.5 years spanning menarche. We assessedestrogen receptor (ER) expression and activity, promoter meth-ylation of ERs and their downstream targets, and markers ofestrogen metabolism. Expression of ERa and classical ERaresponse genes (TFF1, PGR, andGREB1) decreased withmaturity,independent of diet. A significant inverse correlation was

observed between TFF1 mRNA and methylation of CpG siteswithin the TFF1 promoter. Soy effects included lower ERb expres-sion before menarche and lower mRNA for ERa and GREB1 aftermenarche. Expression of GATA-3, an epithelial differentiationmarker that regulates ERa-mediated transcription, was elevatedbefore menarche and decreased after menarche in soy-fed ani-mals. Soy did not significantly alter expression of other ER activitymarkers, estrogen-metabolizing enzymes, or promoter methyla-tion for ERs or ER-regulated genes. Our results demonstrategreater ER expression and activity during the pubertal transition,supporting the idea that this life stage is a critical window forphenotypic modulation by estrogenic compounds. Pubertal soyexposure decreasesmammary ERa expression aftermenarche andexerts subtle effects on receptor activity and mammary glanddifferentiation. Cancer Prev Res; 9(5); 1–11. �2016 AACR.

IntroductionEstrogen signaling plays a central role in the normal develop-

ment of the mammary gland, and the promotion of breast cancer(1, 2). Estrogen receptors (ER) are ligand-regulated transcriptionfactors consisting of subtypes a and b (3). Isoflavones (IF) are

bioactive components of soy foods that bind ERs, producingmixed estrogen agonist–antagonist effects (4). Epidemiologicevidence suggests that soy intake is inversely associated withbreast cancer risk, mortality, and recurrence, although thechemopreventive benefits may be limited to specific popula-tions (5, 6). The mechanism for this protective effect is notestablished. Many in vitro studies suggest that IFs may alterestrogen activity through ER-mediated effects and via modu-lation of estrogen synthesis and metabolism. IFs can also alterDNAmethylation (7, 8), affecting transcription of genes impor-tant to breast cancer (9, 10). Whether the epigenetic modula-tion by IF also involves genes associated with estrogen regula-tion has not been determined.

The early-life environment can establish trajectories of breastcancer risk extending into adulthood (11). Prepuberty and ado-lescence may be important windows for nutritional effects onlater-life susceptibility to cancer (12), as mammary gland mor-phogenesis occurs largely during the pubertal transition. Epide-miologic studies suggest that adolescent soy intake may have apreventive effect on breast cancer later in life (13). Rodent studiesindicate that IFs may interact with estrogen or ERs to alter breastdifferentiation, proliferation, and epithelial cell fate (14, 15). It isnot knownwhether these effects occur in the human breast. Thesegaps in knowledge are due in large part to the methodologic andethical limitations for evaluating soy effects on the breast ofhealthy pubertal girls. Here, we used a well-characterized primatemodel with highly comparable genetic, endocrine, and breast

1Department of Pathology, Section on Comparative Medicine, WakeForest School of Medicine, Winston-Salem, North Carolina. 2PrimateResearch Center, Bogor Agricultural University, Bogor, Indonesia.3Center for Genomics and Personalized Medicine Research, WakeForest School of Medicine,Winston-Salem, North Carolina. 4Depart-ment of Obstetrics and Gynecology, Division of Gynecologic Oncol-ogy, Duke University School of Medicine, Durham, North Carolina.5Department of Biostatistical Sciences,Wake Forest School of Med-icine,Winston-Salem, North Carolina. 6Department of Cancer Biology,Wake Forest School of Medicine,Winston-Salem, North Carolina.

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

Current address for C.E. Wood: Office of Research and Development, USEnvironmental Protection Agency, Research Triangle Park, NC; and currentaddress for C.J. Willson, Integrated Laboratory Systems, Inc., Research TrianglePark, NC.

Corresponding Author: Fitriya N. Dewi, Primate Research Center, Bogor Agri-cultural University, Jl. Lodaya II No. 5, Bogor 16151, Indonesia. Phone: 6225-1832-0417; Fax: 6225-1836-0712; E-mail: [email protected] [email protected]

doi: 10.1158/1940-6207.CAPR-15-0165

�2016 American Association for Cancer Research.

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development profiles to humans (16, 17) to comprehensivelyassess dietary soy effects on ER activity and estrogen regulation inthe breast across puberty.

Materials and MethodsDiet and animals

All animal procedures were performed at the Wake ForestSchool of Medicine (Winston-Salem, NC), which is fully accre-dited by the Association for the Assessment and Accreditation ofLaboratory Animal Care, in compliance with state and federallaws and standards of the US Department of Health and HumanServices and approved by theWake Forest University Animal CareandUse Committee. This study utilizedmammary gland samplesfrom 29 female cynomolgus macaques (Macaca fascicularis) dur-ing pubertal development. The experimental design has beendescribed previously (18). Briefly, animals were obtained fromthe Institut Pertanian Bogor at the approximate age of 1.5 yearsand randomized by body weight to receive one of two diets forapproximately 4.5 years: (i) control diet with casein and lactal-bumin as the protein source (CL, n¼ 12) or (ii) diet with isolatedsoy protein containing IFs (SOY, n ¼ 17) with the humanequivalent of 120 mg/day of IF (expressed as aglycone equiva-lents; soy protein isolate donated by Solae, LLC). Throughoutthe study, all animals were swabbed daily for vaginal bleeding;menarche was defined as the initiation of regularmonthly vaginalbleeding (18).

Breast biopsySerial breast biopsy samples were collected every 6 months

spanning the period of pubertal development (18, 19). Eachbiopsy samplewas divided; half was frozen for biomolecularwork,and half was fixed, embedded in paraffin, and sectioned for IHC.

To control for the high interindividual variation of pubertyonset, all outcomes were compared between monkeys of similardevelopmental stage across the pubertal transition. Thus, aftercompletion of the experiment, we were able to categorize thebiopsy samples into 8 time points relative to the onset of men-arche; from 18 to 23months premenarche up to 19 to 24monthspostmenarche. Serum concentrations of total isoflavonoids(genistein, daidzein, and the metabolite equol) were measuredat each time point by liquid chromatography electrospray ioni-zation mass spectrometry at the laboratory of Dr. Adrian Franke(University of Hawai'i Cancer Center; Honolulu, Hawaii) usingmethods described elsewhere (20); results are presented in Sup-plementary Fig. S1.

Quantitative gene expressionTotal RNA was extracted from frozen mammary tissues using

Tri Reagent (Molecular Research Center, Cincinnati, OH) andpurified using the RNeasy Mini Kit (Qiagen). Quantitative real-time reverse transcription PCR (qRT-PCR) was used to measuremRNA expression of ERs (ERa, ESR1; ERb, ESR2), classical estro-gen-induced genes (trefoil factor 1, TFF1; growth regulationby estrogen in breast cancer 1, GREB1; progesterone receptors Aand B, PGR-A, PGR-B), steroidogenic enzymes [steroid sulfatase,STS; aromatase,CYP19; estrogen sulfotransferase (EST) family 1E,SULT1E1; hydroxysteroid (17b) dehydrogenase 1 and 2,HSD17B1 and HSD17B2] and enzymes for estrogen catabolism(CYP1A1, CYP1B1, and CYP3A4) using methods described pre-

viously (21). qRT-PCR reactions were performed on the ABIPRISM 7500 Fast Sequence Detection System (Applied Biosys-tems), and relative expression was determined using the DCt

method calculated by ABI Relative Quantification 7500 Softwarev2.0.1 (Applied Biosystems). Human or macaque-specific Taq-Man primer-probe assays were used as targets (SupplementaryTable S1) and samples were normalized to mean values forhousekeeping genes (GAPDH and ACTB) using cynomolgusmacaque–specific primer-probe sets.

IHCWe assessed protein expression and localization of ERs and

GATA-3, a transcription factor that regulates ERa-mediated tran-scription in the breast (22) and serves as a marker for luminal celldifferentiation (23), in mammary gland epithelium in tissuesections from a subset of samples (time points 0–11 monthspremenarche and 7–12 and 19–24 months postmenarche) usinga biotin–streptavidin staining method described previously (19).mAbs used were 1:15 anti-ERa (NCL-ER-LH1, Novocastra Labs),1:40 anti-ERb (Clone 14C8, Thermo Fisher Scientific), and 1:50anti-GATA-3 (CloneHG3-31: sc-268, Santa Cruz Biotechnology).Cell staining was quantified by a computer-assisted techniquewith a gridfilter; cells were scored on the basis of staining intensity(0,þ1,þ2,þ3) to obtain a semiquantitative H-score (19). On thebasis of the structures present, H-score data for immature/tran-sitional ducts andmature lobules were limited to premenarche orpostmenarche time points, respectively, whereas H-score data formature ducts and immature lobules were obtained for all timepoints. Morphologic criteria for immature, transitional, andmature mammary gland structures are described elsewhere(18). Representative IHC images for ERs are presented in Sup-plementary Fig. S2.

To evaluate changes in steroidogenic enzyme protein expres-sion, biopsies from two time points at 12–17 months preme-narche and postmenarche were used for IHC. Antibodiesand dilutions used were as follows: EST rabbit polyclonal(1:100, Biorbyt); HSD17B1 rabbitmonoclonal (1:50, Epitomics);HSD17B2 rabbit polyclonal (1:100, Proteintech); and STS rabbitpolyclonal (1:100, Sigma-Aldrich). Staining was scored qualita-tively based on intensity (0,þ1,þ2,þ3) in epithelium and stromaby two board-certified veterinary pathologists (C.J. Willson, J.MCline), and descriptive results are presented.

PyrosequencingWe used breast biopsy samples from three time points: 0–5

months premenarche and 7–12 and 19–24 months postme-narche, to assess promoter methylation. Genomic DNA wasextracted from the frozen specimens using DNeasy Kit (Qiagen)and treated with sodium bisulfite using the Zymo EZ DNAMethylation Kit (Zymo Research). Cynomolgus macaque–spe-cific pyrosequencing assays (Supplementary Table S2) weredesigned using PyroMark Assay Design software (Qiagen) forCpG sites around/near the estrogen responsive element (ERE) ofTFF1, GREB1, and PGR (half-site ERE), and CpG islands in thepromoter regions of ESR1 (promoter B) and ESR2 [up to 300 bpupstream of transcriptional start site (TSS), covering the regionthat corresponds to promoter 0N in human ERb (24)]. Bisulfite-converted DNA (40 ng) was amplified by PCR in a 25 mL reactionusing the PyroMark PCR Kit (Qiagen). Pyrosequencing was per-formed on Qiagen PyroMark Q96 MD Pyrosequencer with PyroQ-CpG software at the Duke Epigenetics Research Laboratory,

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Duke University Medical Center. The values shown represent themean methylation for the CpG sites contained within the ana-lyzed sequence.

Expression microarrayBreast gene expression profiles were obtained from four ani-

mals/group at two time points (7–12 and 19–24 months aftermenarche) utilizing the Affymetrix GeneAtlas System (Affyme-trix). Extracted RNA was assessed for quality and integrity using aNanodrop ND-2000 UV–VIS spectrophotometer (NanoDrop)and Agilent Bioanalyzer-2100 (Agilent Technologies). TheAmbion WT Expression Kit (Life Technologies) was used togenerate sense-strand cDNA, and fragmentation and labeling ofthe cDNA was done using the GeneChip WT Terminal LabelingandControls Kit (Affymetrix). Sampleswere hybridized toRhesusGene 1.1 ST WT Array Strips. Data analysis and quality controlwere performed using Partek Genomics Suite software (Partek)and the Limma package for R (25). RMA-normalized data wereanalyzed for difference in expression over time using a pairedt test; expression was also compared between diet groups usingempirical Bayesian analysis at the two different time points. Themicroarray data are available in the Gene Expression Omnibusrepository at the NCBI (accession no. GEO72940).

For each diet group, gene set enrichment analysis (GSEA) wasperformed using prerankedmethod in the GSEA software version2.0.13 with default parameters (26) on the gene lists generatedfrom pairwise comparisons by time. Each list of 16,915 genes wasranked on the basis of fold change, and sets were compared withcurated KEGG gene sets available from molecular signature data-base (MsigDB) v4.0 (27). Enriched sets with FDR <5% wereconsidered significant.

Statistical analysisLogarithmic or square-root conversions were used where

appropriate to improve normality of the residuals. Data werebacktransformed to original scale for presentation as least squaresmeans (LSM)� SEMor LSM (LSM� SEM, LSMþ SEM)when SEswere asymmetric. All analyses were done across the pubertaltransition, and separately for pre- and postmenarche. For post-menarche, the menstrual cycle stage of the animals (follicular orluteal) during each biopsy was determined retrospectively basedon themenstrual bleeding calendar for each animal and used as acovariate. We used JMP (version 10.0.0, SAS Institute) to fit amixedmodel ANOVAwith a randomanimal effect tomodelmainand interactive effects of diet and time adjusted for body weightand menstrual cycle stage (postmenarche) to estimate and com-pare differences in relative mRNA expression, protein expression,andmethylation ratios in SOY and CL groups over time. Multiplepairwise comparisons were done with Tukey Honestly SignificantDifference (HSD) test. Relationships betweenmRNA and proteinor DNA methylation levels were examined by Spearman rankcorrelation.

ResultsExpression of ERs

Mammary ESR1 mRNA levels decreased across the pubertaltransition (P < 0.05; Fig. 1A), and were inversely associated withbody weight (P < 0.001; data not shown). ESR1 expression waslower in the SOY group only after menarche (P < 0.01 for dieteffect), and higher in the follicular versus luteal phase indepen-

dent of diet (P < 0.05; data not shown). Before menarche, ERaprotein in immature ducts showed a diet x time interaction (P <0.05) in which expression was higher in SOY versus CL at 0–5months premenarche (Fig. 1B). Followingmenarche, ERa expres-sion in mature ducts (P < 0.05) and immature lobules (P¼ 0.07)decreased with time independent of diet, showing positive cor-relation with mRNA expression (Spearman r ¼ 0.50, P < 0.0001for mature ducts; Spearman r ¼ 0.25, P < 0.05 for immaturelobules). Nodiet or time effectwas observed for ERa expression intransitional ducts or mature lobules. We assessed methylation ofCpG sites around the ESR1promoter region B (Fig. 1C) and foundno diet effect. Two CpG sites showed modest but significantincreases in methylation over time (P < 0.05). Regardless oftreatment, methylation levels were low at all assessed CpG sitesacross pubertal development (<5%).

For ERb, we observed amain effect of diet but not time on ESR2expression across the pubertal transition (P<0.05; Fig. 2A). Beforemenarche, therewasmarginally lower ESR2 expression in the SOYgroup (P ¼ 0.08). After menarche, no diet or time effects wereobserved. ERb protein expression was lower following SOY treat-ment in transitional ducts beforemenarche andmature ducts aftermenarche (P < 0.05 for both; Fig. 2B). Similar to ESR1, promotermethylation of ESR2 was low (<3%) and did not differ by diet ortime (Fig. 2C). Only 1 of 16 CpG sites showed increasing meth-ylation with time (P < 0.01).

ER activity markersRelative expression of ER-regulated gene markers TFF1, PGR,

and GREB1 decreased across the pubertal transition (P < 0.01for time effect in all genes; Fig. 3A–D). GREB1 was marginallydecreased after menarche (P ¼ 0.08) and showed a significantdiet effect in which expression was lower in the SOY grouppostmenarche (P < 0.05 vs. CL). Menstrual cycle stage had asignificant effect on TFF1 (P < 0.01), GREB1 (P < 0.05), andPGR-B (P < 0.0001); these markers were higher in the follicularphase versus the luteal phase (data not shown). Body weight, asurrogate for age, had an inverse association with postmenar-chal TFF1 (P < 0.05), PGR-A (P ¼ 0.05), PGR-B (P < 0.01), andGREB1 (P < 0.05).

A significant time effect on TFF1 promoter methylation wasobserved (P < 0.0001; Fig. 4A). Seven CpG sites flanking thepromoter ERE showed increased methylation levels with develop-ment (by�5%–7%), and therewas an inverse correlation betweenTFF1 methylation and mRNA expression (Spearman r ¼ �0.31,P < 0.01). We assessed CpGmethylation around an ERE located at1.6 kb upstream of the GREB1 TSS but did not find a diet or timeeffect (Fig. 4B) or association between methylation of this regionandGREB1 expression. The CpG sites within the promoter regionsfor PGR-A and PGR-B (Fig. 4C) showed low levels of methylationacross the pubertal transition (<10%and<5% for PGR-A and PGR-B, respectively) independent of dietary treatment. PGR-A showed amodest increase in methylation level with maturity (P < 0.05 fortime effect) and body weight (P < 0.05; data not shown). Amongthe 17 CpG sites assessed within the PGR-A promoter, 6 showed asignificant changewith time and1 showed a diet x time interaction(P < 0.05).Methylation of PGR-A and PGR-Bdid not correlatewiththeir respective mRNA expression.

ER regulation and luminal cell differentiationGATA-3 expression was localized to the luminal epithelium of

mammary ductal and lobular structures. In immature ducts,

Soy Effects on Estrogen Pathways in the Pubertal Breast

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GATA-3 did not differ by diet, whereas in transitional ducts, therewas a diet x time interaction (P < 0.05) with increased expressionprior tomenarche only in the SOY group (P<0.05). PremenarchalGATA-3 expression (Fig. 5A) in mature ducts and immaturelobules showed a marginal increase with time (P ¼ 0.06 in bothstructures) independent of diet. Aftermenarche (Fig. 5B), GATA-3expression in immature lobules was lower in SOY (P < 0.05);

however, this effect disappeared with adjustment for menstrualcycle stage. Formature lobules, therewas amain effect of time (P<0.05) and a diet x time interaction (P < 0.05) in which increasedGATA-3 expression was only observed in the CL group. Except inthe transitional duct, GATA-3 expression was significantly corre-lated with expression of ERa protein (Spearman r > 0.3, P < 0.05)and mRNA (Spearman r > 0.2, P < 0.05).

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Figure 1.ERa expression and promoter methylation in the mammary gland across the pubertal transition. ESR1 mRNA level decreased with maturity, with a significantsoy effect after menarche (A). ERa protein expression did not differ by diet but was lower postmenarche (B). Methylation of CpG sites within promoter Bof ESR1 did not differ by diet (C). Values are LSM for n ¼ 11–17 monkeys/group (bars ¼ SEM). Significant main effects are indicated in each panel.

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Global gene expressionWe did not find a significant difference in the transcriptional

profiles between the two diet groups and across 7–12months and19–24 months postmenarche using data globally adjusted formultiple comparisons. We used the unadjusted P values to iden-tify genes that changed over time in each diet group, and genesthat differed by diet at each time point (Supplementary Fig. S3).Results from this relaxed analysis showed that breast develop-ment from 7–12 months to 19–24 months postmenarche across

the two dietary groups involved different sets of genes associatedwith distinct KEGG pathways.

Estrogen-metabolizing enzymesWe previously reported no difference in serum estradiol (E2)

concentration between SOY and CL groups (18). Here, we alsofound no diet effect on mammary mRNA expression of genesinvolved in estrogen conjugation, synthesis, bioactivation, andcatabolism (Fig. 6A–E). A significant time effect was observed for

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Figure 2.ERb expression and promoter methylation in the mammary gland across the pubertal transition. ESR2 mRNA did not change over time; the overall expressionwas lower with soy (A). ERb protein expression was lower with soy in transitional ducts (before menarche) and mature ducts (after menarche; B). There wasno effect of diet or time on methylation of CpG sites in the promoter upstream of ESR2 (C). Values are LSM for n ¼ 11–17 monkeys/group (bars ¼ SEM). Significantmain effects are indicated in each panel.






mRNA expression of several steroidogenic enzymes. For example,STS (Fig. 6A) and SULT1E1 (Fig. 6B) expression decreased overtime (P < 0.01), whereas HSD17B1 (Fig. 6C) increased across thepubertal transition, particularly aftermenarche (P < 0.05). Expres-sion of aromatase (CYP19) was generally low with an increaseafter menarche (Fig. 6E; P < 0.01) independent of diet. Nosignificant time or diet effects were observed for expression ofHSD17B2 (Fig. 6D), or for CYP1A1, CYP1B1, and CYP3A4 (datanot shown).

The results for steroidogenic enzyme immunoreactivityin the mammary gland are summarized in SupplementaryTable S3. Briefly, EST showed cytoplasmic expression in stro-mal cells which was moderate intensity before menarche and

weaker intensity after menarche. A subset of animals (8/15premenarche and 3/15 postmenarche) displayed weak tomoderate cytoplasmic staining in ductal and lobular epithelialcells for STS. HSD17B1 expression was cytoplasmic and mostintense in the myoepithelial cells and stroma immediatelysurrounding lobules and ducts. Generally <20% of epithelialcells in either lobules or ducts showed weak cytoplasmicimmunoreactivity for HSD17B1; there was no appreciabletrend in the expression between pre- and postmenarche. Sim-ilarly, no developmental pattern was observed in HSD17B2expression between the two time points; the staining wascytoplasmic and intense in stromal cells, and weak in <10%of epithelial cells.

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Figure 3.Expression of ER-regulatedmarkers inthe mammary gland during thepubertal transition. mRNA levels forTFF1 (A) and PGR (B and C) decreasedwithmaturity but did not differ bydiet.GREB1 (D) was lower in the soygroup after menarche. Values areLSM for n ¼ 11–17 monkeys/group(bars¼ SEM). Significant main effectsare indicated in each panel.

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DiscussionDiet during adolescence can alter developmental signaling

networks and influence later-life susceptibility to cancer. Herewe evaluated the effects of a high-soy dietwithphytoestrogenic IFson ER expression and ER-dependent activity in the breast duringpubertal development. Soy treatment resulted in amodest down-regulation of ERa transcription after menarche, which appearedto be independent of ER promoter methylation. This changeoccurred alongside a decrease in expression of GREB1, which isa classic estrogen-induced marker, suggesting a mild bufferingeffect of soy on ER activity for select targets after menarche.Expression of the GATA-3 differentiation marker, which regulatesERa-mediated transcription, tended tobehigher beforemenarcheand lower following menarche in soy-fed compared with CL-fedanimals. Our findings demonstrate that there are high levels of ERsignaling in the pubertal breast, and that pubertal soy exposuremayhave subtle effects on this activity, potentially influencing ER-dependent responses later in life.

Endogenous estrogens are key regulators of mammary glandmorphogenesis and important risk factors for breast cancerduring adulthood (1). In humans (28) and macaques (18), alarge increase in mammary lobular differentiation can be seenaround the time of menarche. In human breast, ERs are presentfrom the fetal stage onward, although little is known about thedynamics of this expression over time (29). An autopsy studyshowed that ERa mRNA level was higher in the breast ofpremenarchal compared with perimenarchal girls (30); we pre-viously reported a similar finding in monkeys (21). The currentreport is the first to longitudinally illustrate the developmentalprofile of ER expression and activity markers in the breast acrosspuberty in the same subjects. We found decreasing ERa but notERb across the menarchal transition. Classic ERa-regulatedmarkers TFF1, PGR, and GREB1 also decreased over time, whichsupports the idea that estrogen responsiveness in the breast ishighest during early puberty and decreases with adulthood.Our findings suggest that adolescence may be a critical periodof susceptibility to hormonal disruption by environmental

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Figure 4.Promoter methylation of ER-regulated markers. Methylation of 7CpG sites near the EREwithin the TFF1promoter increased with time (A).There was no diet or time effect on4 CpG sites near the ERE proximal toGREB1 (B). The level of CpGmethylation within PGR promoters Aand Bwas lowwith no effect of diet ortime (C). Values are LSM for n ¼ 11–17monkeys/group (bars ¼ SEM).Significant main effects are indicatedin each panel.






estrogens through both direct ER interactions and alterations ofendogenous estrogen metabolism.

Structural similarities between soy IFs and E2 allow IFs to bindto ERs but with weaker affinity. In previous studies in bothpremenopausal and postmenopausal monkeys, dietary doses ofIFs (�129 mg/day human equivalent dose) did not elicit clearestrogenic effects, while having modest selective ER-inhibitoryeffects when given with exogenous estrogen (31, 32). Here, weshowed that pubertal soy intake resulted in lower mRNA expres-sion of ERa (postmenarche) and ERb (premenarche). Further-more, GREB1 expression was also lower in the soy group aftermenarche, whereas other ER-regulated markers (i.e., TFF1 andPGR) were not altered. These findings suggest that adolescent soyintake may produce a subtle decrease in estrogen responsivenessthat carries forward into adulthood. Consistent with this idea,exposure of pre/peripubertal rats to an IF-rich diet reduces estro-gen-induced proliferative responses in the mammary gland ofovariectomized adults (33). The findings may support the notionthat dietary soy exposure initiated at puberty or premenarche isbeneficial for breast cancer prevention.

Mechanisms for these types of ER-modulating effects beyondsimple competitive interactions with E2 are unclear. Different IFshave greater affinity for ERb (34), which may reduce ERa-medi-ated transcription (35). Interestingly, soy resulted in lower ERamRNA after menarche and lower ERb mRNA before menarche.When considered alongside soy effects on GATA-3, which washigher in premenarche and lower in postmenarche, this pattern

supports the idea that a higher ratio of ERa:ERb activity early inpuberty may facilitate greater mammary gland differentiation.Our results indicate that the postmenarchal breast tissue of soy-fed and casein-fed animals expressed different set of genes andpathways, which could reflect induced differences in mammarygland differentiation. This finding, however, should be inter-preted with caution.

Recent evidence suggests that early-life exposure to exogenousestrogens may increase future breast cancer risk (36). Epigeneticchanges such as DNAmethylation may have an important role inmediating this type of latent effect (37). Promoter methylation isgenerally associated with transcriptional silencing although thereis a limited understanding of methylation-dependent regulationof ERs and ER-regulated genes, particularly in normal breastdevelopment. We examined the relationship between early-lifesoy exposure, DNAmethylation, and ER-mediated responses. Thesoy effect on ERa mRNA after menarche did not appear to bemediated by altered methylation within the CpG sites examined.We did observe several interesting methylation patterns based onpubertal development. The ESR1 gene has multiple promoters(38); we assessed CpG sites within promoter B, which is a CpG-rich region. CpG island promoters are mainly unmethylatedregardless of expression status of the gene, whereas low CpGpromoters are methylated during active or inactive states (39). Inbreast tumors, however, promoter B is often methylated (40).Here we found a generally low methylation level within thisregion across pubertal development with a significant increase

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Figure 5.GATA-3 protein in the pubertalmammary gland. Soy increasedGATA-3 before menarche (A), butdecreased it after menarche (B).Values are LSM for n¼ 11–17monkeys/group (bars ¼ SEM). Significantmain effect and interactions areindicated in each panel. Asterisks (�)indicate pairwise differences(P < 0.05, Tukey HSD).

Dewi et al.





in 2 of 4 CpG sites with maturity. This increase was subtle (�1%)and the biologic relevance of such amodest change is unclear. ForESR2, we assessed methylation of the region that corresponds tothe promoter 0N described in humans; this region is hypermethy-lated in most breast cancer cell lines (24). Similar to normalhuman breast epithelial cells, the macaque breast showed lowmethylation in this region. The promoter for PGR is also CpG-richwith a low overall methylation level. PGR expression in cancer is

epigenetically regulated via methylation of the alternative pro-moters A andB (41), but little is known regarding the regulation innormal breast. We found that specific CpG sites within PGRpromoter A also had subtle increases in methylation level aftermenarche regardless of diet. It is interesting to point out the tightregulation that maintains methylation level at each CpG site, asshown by sequential CpG sites having different levels of meth-ylation. Whether small methylation changes in certain sites of the

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Figure 6.mRNA levels for genes related toestrogen conjugation (A and B) andsynthesis/bioactivation (C–E) werenot affected by dietary treatment. STS(A) and SULT1E1 (B) decreased withmaturity, whereas HSD17B1 and CYP19increased (C and E), and HSD17B2 (D)did not differ with time. Values areLSM for n ¼ 11–17 monkeys/group(bars¼ SEM). Significant main effectsare indicated in each panel.






promoter could contribute to the decrease in ESR1 and PGRmRNA with maturity is unclear. It has been shown that the stateof only 2 CpG sites within the promoter region of oxytocinreceptor (OXTR) is crucial for the overall effect of promotermethylation on OXTR expression (42). Overall, there was a tightconstraint on shifting methylation at the regions examined; it ispossible that these regions are more vulnerable to shift at otherdevelopmental periods. For example, prenatal exposure to genis-tein has been shown to enhance DNA methylation and counterthe hypomethylating effect of bisphenol A (7, 43).

There was no change in the methylation of ERE within GREB1promoter despite the differential gene expression with diet andtime.GREB1has three consensusEREs spanning approximately 20kbupstreamof the TSS that are functional transcription enhancers.The region assessed in this study is the closest to the TSS (1.6 kbupstream), which has basal promoter activity, strongest ER recruit-ment, and repressed activity in the presence of ER antagonism(44). Our results show that the methylation status of CpG siteswithin this region does not appear to regulate GREB1 transcrip-tion. The TFF1 promoter has high level of methylation, consistentwith the fact that the region is low in CpG content. Althoughmethylation may not be the main mechanism regulating tran-scriptional activity in low CpG promoters, DNA methylationstatus is important for tissue-specific regulation of certain genesincluding TFF1 (39, 45). Dramatic change in methylation istypically attained in genes only under specific circumstances suchas cancer and neurodevelopmental disorders, but a study inhuman embryonic kidney carcinoma cell line derivative showedthat mild differences in methylation level may repress gene tran-scription(46).Here,we found that the change inmethylation levelaround the ERE in the TFF1promoter across pubertywas relativelymild (�5%–10%) but the CpG methylation was inversely corre-lated with TFF1 expression. Further investigation is needed toevaluate the biologic relevance of changes in TFF1 promotermethylation across puberty as a biomarker of ERa responsiveness.

GATA-3 is required for luminal differentiation, ESR1 promoteractivity, and ER-mediated transcription (47). We showed that inthe macaque breast, GATA-3 was expressed in ductal and alveolarluminal epithelial cells but not myoepithelial cells, as in mice(48). Interestingly, GATA-3 expression in the soy group was higharound the time of menarche, which could indicate a higherluminal differentiation at this stage as GATA-3 promotes thedifferentiation of lineage-restricted progenitor cells (23). Thisfinding is consistent with the large increase of lobuloalveolardifferentiation found in these animals (18), suggesting a soypromotional effect on breast differentiation through menarche.There was also a trend of lower GATA-3 with soy followingmenarche, which was consistent with the pattern of ERa andGREB1 mRNA and a study in ovariectomized mice that showeddownregulation of GATA-3 expression through ER modulationinduced by exogenous estrogen (49).

The breast is capable of synthesizing and activating estrogenlocally by the interaction of various enzymes. Here, we showed

that the immunoreactivity profile of these enzymes in themacaque breast is comparable with that in the normal humanbreast (50), supporting the translational potential of themacaquebreast model for studying agents that may alter estrogen metab-olism. No effects of soy IF exposure on steroidogenic enzymeexpression in the pubertal breast were observed.

Estrogen signaling during adolescence is a key driver of mam-mary gland development. We show for the first time that ERaexpression and response markers in the breast are higher early inthe pubertal transition, supporting the idea that this life stagerepresents a critical period for phenotypic modulation by ER-modulating compounds. Our findings suggest that adolescentintake of dietary soymaymodestly enhance breast differentiationearly in puberty anddampen estrogen responsiveness in thebreastlater in adulthood. Both of these phenotypes would be anticipat-ed to lower breast cancer risk. Additional experimental evidence isneeded to confirm these findings and to examine whether anysuch shifts in ER activity may reduce risk of breast cancer devel-opment later in life.

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

Authors' ContributionsConception and design: F.N. Dewi, C.E. Wood, T.C. Register, J.M. ClineDevelopment of methodology: F.N. Dewi, C.E. Wood, T.C. Register, Z. Huang,J.M. ClineAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F.N. Dewi, C.J. Willson, T.C. Register, C.J. Lees,S.K. Murphy, J.M. ClineAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F.N. Dewi, C.J. Willson, T.C. Register, T.D. Howard,J.A. Tooze, J.A. Chou, L.D. Miller, J.M. ClineWriting, review, and/or revision of the manuscript: F.N. Dewi, C.E. Wood,C.J. Willson, T.C. Register, C.J. Lees, T.D. Howard, S.K. Murphy, J.W. Chou,J.M. ClineAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. HuangStudy supervision: C.E. Wood, J.M. Cline

AcknowledgmentsThe authors thank Dr. Greg Hawkins for facilitating the pyrosequencing

experiment. The authors also thank Jean Gardin, Hermina Borgerink, LisaO'Donnell, Joseph Finley, CaroleGrenier, LouCraddock, andAbdoulayeDiallofor their outstanding technical contributions.

Grant SupportThis work was supported by NIH R01 AT000639 (to J.M. Cline) and T32

OD010957 (to J.M. Cline and C.J. Willson). The Comprehensive Cancer Centerof Wake Forest University Microarray Shared Resource is supported by NCICCSG P30CA012197 (to B.C. Pasche).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 30, 2015; revised October 6, 2015; accepted November 6,2015; published OnlineFirst March 22, 2016.

References1. Yager JD, Davidson NE. Estrogen carcinogenesis in breast cancer. N Engl J

Med 2006;354:270–82.2. Arendt LM, Kuperwasser C. Form and function: how estrogen and proges-

teroneregulate the mammary epithelial hierarchy. J Mamm Gland BiolNeoplasia 2015;20:9–25.

3. Bjornstrom L, Sjoberg M. Mechanisms of estrogen receptor signaling:convergence of genomic and nongenomic actions on target genes. MolEndocrinol 2005;19:833–42.

4. Oseni T, Patel R, Pyle J, Jordan VC. Selective estrogen receptor modulatorsand phytoestrogens. Planta Med 2008;74:1656–65.

Dewi et al.





5. Nechuta SJ, Caan BJ, Chen WY, Lu W, Chen Z, Kwan ML, et al. Soy foodintake after diagnosis of breast cancer and survival: an in-depth analysis ofcombined evidence from cohort studies of US and Chinese women. Am JClin Nutr 2012;96:123–32.

6. Chen M, Rao Y, Zheng Y, Wei S, Li Y, Guo T, et al. Association between soyisoflavone intake and breast cancer risk for pre- and post-menopausalwomen: a meta-analysis of epidemiological studies. PLoS One 2014;9:e89288.

7. DolinoyDC,Weidman JR,WaterlandRA, Jirtle RL.Maternal genistein alterscoat color and protects Avymouse offspring from obesity bymodifying thefetal epigenome. Environ Health Perspect 2006;114:567–72.

8. Day JK, Bauer AM, DesBordes C, Zhuang Y, Kim BE, Newton LG, et al.Genistein altersmethylation patterns inmice. J Nutr 2002;132:2419S–23S.

9. Qin W, Zhu W, Shi H, Hewett JE, Ruhlen RL, MacDonald RS, et al. Soyisoflavones have an antiestrogenic effect and alter mammary promoterhypermethylation in healthy premenopausal women. Nutr Cancer2009;61:238–44.

10. King-BatoonA, Leszczynska JM, KleinCB.Modulation of genemethylationby genistein or lycopene in breast cancer cells. Environ Mol Mutagen2008;49:36–45.

11. Wei EK,Wolin KY, ColditzGA. Time course of risk factors in cancer etiologyand progression. J Clin Oncol 2010;28:4052–7.

12. Mahabir S. Association between diet during preadolescence and adoles-cence and risk for breast cancer during adulthood. J Adolesc Health2013;52:S30–5.

13. Lee SA, ShuXO, LiH, YangG,CaiH,WenW, et al. Adolescent and adult soyfood intake and breast cancer risk: results from the Shanghai Women'sHealth Study. Am J Clin Nutr 2009;89:1920–6.

14. Brown NM, Wang J, Cotroneo MS, Zhao YX, Lamartiniere CA. Prepubertalgenistein treatment modulates TGF-alpha, EGF and EGF-receptor mRNAsand proteins in the rat mammary gland. Mol Cell Endocrinol 1998;144:149–65.

15. Cabanes A, Wang M, Olivo S, DeAssis S, Gustafsson JA, Khan G, et al.Prepubertal estradiol and genistein exposures up-regulate BRCA1 mRNAand reduce mammary tumorigenesis. Carcinogenesis 2004;25:741–8.

16. Cline JM, Wood CE. The mammary glands of macaques. Toxicol Pathol2008;36:130S–41S.

17. Weinbauer GF, Niehoff M, Niehaus M, Srivastav S, Fuchs A, Van Esch E,et al. Physiology and endocrinology of the ovarian cycle in macaques.Toxicol Pathol 2008;36:7S–23S.

18. Dewi FN,WoodCE, Lees CJ,WillsonCJ, Register TC, Tooze JA, et al. Dietarysoy effects onmammary gland development during the pubertal transitionin nonhuman primates. Cancer Prev Res 2013;6:832–42.

19. Wood CE, Register TC, Lees CJ, Chen H, Kimrey S, Cline JM. Effects ofestradiol with micronized progesterone or medroxyprogesterone acetateon risk markers for breast cancer in postmenopausal monkeys. BreastCancer Res Treat 2007;101:125–34.

20. Franke AA, Halm BM, Kakazu K, Li X, Custer LJ. Phytoestrogenic isoflavo-noids in epidemiologic and clinical research.Drug Test Anal 2009;1:14–21.

21. Stute P, Sielker S, Wood CE, Register TC, Lees CJ, Dewi FN, et al. Life stagedifferences in mammary gland gene expression profile in non-humanprimates. Breast Cancer Res Treat 2012;133:617–34.

22. Theodorou V, Stark R,Menon S, Carroll JS. GATA3 acts upstreamof FOXA1inmediating ESR1 binding by shaping enhancer accessibility. Genome Res2013;23:12–22.

23. Kouros-MehrH, Slorach EM, SternlichtMD,Werb Z.GATA-3maintains thedifferentiation of the luminal cell fate in the mammary gland. Cell2006;127:1041–55.

24. Zhao C, Lam EW, Sunters A, Enmark E, De Bella MT, Coombes RC, et al.Expressionof estrogen receptorbeta isoforms innormal breast epithelial cellsand breast cancer: regulation by methylation. Oncogene 2003;22:7600–6.

25. Smyth GK.Linear models and empirical bayes methods for assessingdifferential expression in microarray experiments. Stat Appl Genet MolBiol 2004;3:Article3.

26. SubramanianA, TamayoP,Mootha VK,Mukherjee S, Ebert BL,GilletteMA,et al. Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles. Proc Natl Acad Sci US A2005;102:15545–50.

27. Liberzon A, Subramanian A, Pinchback R, Thorvaldsdottir H, Tamayo P,Mesirov JP. Molecular signatures database (MSigDB) 3.0. Bioinformatics2011;27:1739–40.

28. Russo J, Russo IH. Development of the human breast. Maturitas 2004;49:2–15.

29. Keeling JW, Ozer E, King G, Walker F. Oestrogen receptor alpha in femalefetal, infant, and child mammary tissue. J Pathol 2000;191:449–51.

30. Boyd M, Hildebrandt RH, Bartow SA. Expression of the estrogen receptorgene in developing and adult human breast. Breast Cancer Res Treat1996;37:243–51.

31. Wood CE, Kaplan JR, Stute P, Cline JM. Effects of soy on the mammaryglands of premenopausal female monkeys. Fertil Steril 2006;85Suppl1:1179–86.

32. Wood CE, Register TC, Franke AA, Anthony MS, Cline JM. Dietary soyisoflavones inhibit estrogen effects in the postmenopausal breast. CancerRes 2006;66:1241–9.

33. Molzberger AF, Soukup ST, Kulling SE, Diel P. Proliferative and estrogenicsensitivity of the mammary gland are modulated by isoflavones duringdistinct periods of adolescence. Arch Toxicol 2013;87:1129–40.

34. Hwang CS, Kwak HS, Lim HJ, Lee SH, Kang YS, Choe TB, et al. Isoflavonemetabolites and their in vitro dual functions: they can act as an estrogenicagonist or antagonist depending on the estrogen concentration. J SteroidBiochem Mol Biol 2006;101:246–53.

35. Matthews J, Gustafsson JA. Estrogen signaling: a subtle balance between ERalpha and ER beta. Mol Interv 2003;3:281–92.

36. Hilakivi-Clarke L. Maternal exposure to diethylstilbestrol during pregnan-cy and increased breast cancer risk in daughters. Breast Cancer Res2014;16:208.

37. FoleyDL, Craig JM,Morley R,Olsson CA,Dwyer T, Smith K, et al. Prospectsfor epigenetic epidemiology. Am J Epidemiol 2009;169:389–400.

38. Kos M, Reid G, Denger S, Gannon F. Minireview: genomic organization ofthe human ERalpha gene promoter region. Mol Endocrinol 2001;15:2057–63.

39. Weber M, Hellmann I, Stadler MB, Ramos L, Paabo S, Rebhan M, et al.Distribution, silencing potential and evolutionary impact of promoterDNA methylation in the human genome. Nat Genet 2007;39:457–66.

40. Gaudet MM, Campan M, Figueroa JD, Yang XR, Lissowska J, Peplonska B,et al. DNA hypermethylation of ESR1 and PGR in breast cancer: pathologicand epidemiologic associations. Cancer Epidemiol Biomarkers Prev2009;18:3036–43.

41. Pathiraja TN, Shetty PB, Jelinek J, He R, Hartmaier R, Margossian AL, et al.Progesterone receptor isoform-specific promoter methylation: associationof PRA promoter methylation with worse outcome in breast cancerpatients. Clin Cancer Res 2011;17:4177–86.

42. Mamrut S, Harony H, Sood R, Shahar-Gold H, Gainer H, Shi YJ, et al. DNAmethylation of specific CpG sites in the promoter region regulates thetranscription of the mouse oxytocin receptor. PLoS One 2013;8:e56869.

43. Dolinoy DC, Huang D, Jirtle RL. Maternal nutrient supplementationcounteracts bisphenol A-induced DNA hypomethylation in early devel-opment. Proc Natl Acad Sci USA 2007;104:13056–61.

44. Deschenes J, Bourdeau V, White JH, Mader S. Regulation of GREB1transcription by estrogen receptor alpha through a multipartite enhancerspread over 20 kb of upstream flanking sequences. J Biol Chem 2007;282:17335–9.

45. Fujii Y, Shimada T, Koike T, Hosaka K, Tabei K, Namatame T, et al. Reviewarticle: regulation of TFF1 (pS2) expression in gastric epithelial cells.Aliment Pharmacol Ther 2006;24:285–91.

46. Hsieh CL. Dependence of transcriptional repression on CpG methylationdensity. Mol Cell Biol 1994;14:5487–94.

47. Eeckhoute J, Keeton EK, Lupien M, Krum SA, Carroll JS, BrownM. Positivecross-regulatory loop ties GATA-3 to estrogen receptor alpha expression inbreast cancer. Cancer Res 2007;67:6477–83.

48. Asselin-Labat ML, Sutherland KD, Barker H, Thomas R, Shackleton M,Forrest NC, et al. Gata-3 is an essential regulator of mammary-glandmorphogenesis and luminal-cell differentiation. Nat Cell Biol 2007;9:201–9.

49. Calvo E, Luu-The V, Belleau P, Martel C, Labrie F. Specific transcriptionalresponse of four blockers of estrogen receptors on estradiol-modulatedgenes in the mouse mammary gland. Breast Cancer Res Treat 2012;134:625–47.

50. Sasaki Y, Miki Y, Hirakawa H, Onodera Y, Takagi K, Akahira J, et al.Immunolocalization of estrogen-producing and metabolizing enzymesin benign breast disease: comparison with normal breast and breastcarcinoma. Cancer Sci 2010;101:2286–92.






Published OnlineFirst March 22, 2016.Cancer Prev Res Fitriya N. Dewi, Charles E. Wood, Cynthia J. Willson, et al. Receptor Activity in the Breast of Cynomolgus MacaquesEffects of Pubertal Exposure to Dietary Soy on Estrogen

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