Protective Effects of Prepubertal Genistein …...by agarose gel electrophoresis. The B004/B005 primer combination generates a 450-bp product that corresponds to the wild-type Brca1,

Research Article

Protective Effects of Prepubertal Genistein Exposure on MammaryTumorigenesis Are Dependent on BRCA1 Expression

Sonia de Assis1, Anni Warri1, Carlos Benitez1, William Helferich2, and Leena Hilakivi-Clarke1

AbstractThis study investigated whether prepubertal dietary exposure to genistein reduces mammary tumori-

genesis by upregulating Brca1 expression in mice. Heterozygous Brca1þ/� mice and their wild-type (WT)

littermates were fed control AIN93G diet or 500 ppm genistein–supplemented AIN93G diet from

postnatal day (PND) 15 to PND30 and then switched to AIN93G diet. Prepubertal dietary exposure to

genistein reduced 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary incidence (P ¼ 0.029)

and aggressiveness of the tumors (P < 0.001) in the WT mice and upregulated the expression of Brca1 in

their mammary glands (P ¼ 0.04). In contrast, prepubertal genistein diet neither significantly reduced

mammary tumorigenesis or tumor aggressivity nor increased Brca1 mRNA expression in the Brca1þ/�

mice. These results may be related to the opposing effects of prepubertal genistein diet on the expression

of Rankl and CK5/CK18 ratio (marker of luminal epithelial cell differentiation) in the mammary gland

and estrogen receptor (ER-a) and progesterone receptor (PgR) protein levels in the mammary tumor:

these all were reduced in the WT mice or increased in Brca1þ/� mice. Both the WT and Brca1þ/� mice

exhibited reduced levels of amphiregulin, CK5, and CK18, delayed ductal elongation and a reduction

in terminal end bud number in the normal mammary gland, and reduced HER-2 protein levels in

the mammary tumors; however, these effects were not sufficient to significantly reduce mammary

tumorigenesis in Brca1þ/� mice. Our results show that upregulation of Brca1 may be required for

prepubertal dietary genistein exposure to reduce later mammary tumorigenesis, perhaps because in the

absence of this upregulation, mice do not exhibit genistein-induced downregulation of ER-a, PgR, andRankl. Cancer Prev Res; 4(9); 1436–48. �2011 AACR.

Introduction

Soy consumption during childhood and adolescencehas been consistently linked to a marked reduction inbreast cancer risk in Asians, American Asians, and Cau-casians (1–5). Genistein, a soy-derived isoflavone, isthought to play a key role in the cancer-preventive activityof pubertal soy intake. In agreement with this view,pubertal genistein exposure reduces mammary cancer riskin rodent models (6). The protective effect of soy/genis-tein may be limited to puberty, as an adult exposure doesnot alter the risk in mice or rats (6). Consumption of soylimited to adult life may not affect breast cancer risk inhumans either (7).

The mechanisms mediating the protective effect of pub-ertal genistein exposure on mammary tumorigenesisremain to be established. Genistein is a weak estrogenreceptor a (ER-a) agonist, but it binds relatively stronglyto ER-b (8). However, with the exception of very low doses,genistein activates both estrogen receptors equally, simi-larly to estradiol (9). Previous studies indicate that genis-teinmodifies the expression of ER-a in vitro and in vivo (10–13) and increases progesterone receptor (PgR) in humanbreast tumors (14), human breast cancer cells in a mousemodel (15), and normal mammary tissue in animal mod-els (16). Genistein also targets many other signaling path-ways including tyrosine kinases ErbB2/HER-2 (17). It hasbeen shown that genistein downregulates HER-2 in humanbreast cancer cells (18), and following pubertal genisteinexposure, in rat mammary tumors (19). Prepubertal gen-istein exposure also affects mammary glandmorphology. Itreduces the number of targets formalignant transformation(terminal end buds, TEB), and this is proposed to explainthe cancer-protective effects of genistein (6).

Because genistein targets several biological pathways, it isnot clear which one of them may contribute to its breastcancer risk–reducing effects. Our earlier study showed thatprepubertal genistein exposure, similarly to prepubertalestradiol exposure, upregulates the tumor suppressor gene

Authors' Affiliations: 1Lombardi Comprehensive Cancer Center andDepartment of Oncology, Georgetown University, Washington, Districtof Columbia; and 2Department of Food Science and Human Nutrition,University of Illinois, Urbana-Champaign, Illinois

Corresponding Author: Leena Hilakivi-Clarke, Georgetown University,3970 Reservoir Road, NW, The Research Building, RoomW405, Washing-ton, DC 20057. Phone: 202-687-7237; Fax: 202-687-7505; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-10-0346

�2011 American Association for Cancer Research.

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Published OnlineFirst June 16, 2011; DOI: 10.1158/1940-6207.CAPR-10-0346

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BRCA1 (breast cancer susceptibility gene 1; ref. 12). Othershave reported that genistein upregulates BRCA1 in vitro inhuman breast and prostate cancer cells (20) and in vivo inthe mammary gland of ovariectomized rats (21). BRCA1expression in the mammary gland is also elevated duringpregnancy when estrogen and progesterone levels are high(22) and is reduced by ovariectomy (23). BRCA1 physicallyinteracts with ER-a and PgR to inhibit their transcriptionalactivity (24). The link between BRCA1 and these steroidreceptors as well as the role of BRCA1 in inducing differ-entiation of stem/progenitor cells to luminal cells (25)mayexplain why this tumor suppressor reduces breast cancerrisk.To further explore the role of BRCA1 as a mechanism

mediating the effects of prepubertal genistein exposure onthe mammary gland, we used Brca1þ/� mice. BRCA1þ/�

mutations are the main known cause for familial breastcancer (26), and women who have inherited a germ lineBRCA1 mutation have a 66% to 85% risk of developingbreast cancer by the age of 70 years (27). The BRCA1-induced changes were also investigated, focusing on ER-aand PgR and their target genes amphiregulin and receptoractivator of NF-kB ligand (Rankl), respectively. Amphire-gulin is an epithelial growth factor receptor (EGFR) ligand(28, 29). It is transcriptionally regulated by ER-a (30) andBRCA1 (31), and it mediates the proliferative actions ofestradiol by activating the EGFR (30). Amphiregulin isrequired for mammary gland ductal morphogenesis atpuberty (28), and its absence prevents duct-limited mam-mary progenitor cell proliferation but has no effect onlobule-limited mammary progenitor cells (32). It is notknown whether genistein alters amphiregulin expression.RANKL is a member of the TNF family and mediatesprogesterone-induced mammary cell proliferation down-stream of PgR (33). Genistein downregulates RANKL in thebone (34) and prostate cancer cells (35), but the effects onthe mammary gland have not been explored.We found that prepubertal dietary genistein exposure

reduced mammary tumor incidence in the WT mice butfailed to do so in the Brca1þ/�mice. In contrast toWTmice,prepubertal genistein diet did not increase Brca1 or reduceER-a, PgR, or Rankl expression in the mammary gland ortumor in these mice. However, prepubertal genistein expo-sure elevated CK5/CK18 ratio in the mammary gland of theBrca1þ/� mice, indicative of reduced luminal differentia-tion (36). Our results show that upregulation of Brca1maybe required for prepubertal dietary genistein exposure toreduce later mammary tumorigenesis.

Materials and Methods

AnimalsBrca1tm1Cxd heterozygous (þ/�) frozen mouse embryos

at 129Sv/C57BL/6 background were obtained from theNational Cancer Institute mouse repository (Bethesda,MD). Frozen embryos were implanted in ICR female miceand a colony was established at the Georgetown Uni-versity’s Animal Facility. Mice were fed a semipurified

AIN93G (the American Institute of Nutrition) diet uponarrival. Animals were housed in a temperature- and humid-ity-controlled room under a 12-hour light–dark cycle. Allanimal procedures were approved by the GeorgetownUniversity Animal Care and Use Committee, and theexperiments were carried out following the NIH guidelinesfor the proper and humane use of animals in biomedicalresearch.

GenotypingWild-type (WT) and heterozygous Brca1 (þ/�) mice

were genotyped using DNA extracted from the tail clipsusing the NaOH method. PCR amplification was carriedout using forward and reverse primers to obtain a 450- and550-bp bands for wild-type and knockout alleles, respec-tively. Primer sequences were: forward primer (Brca1 004):50-CTGGGTAGTTTGTAAGCATGC30 and reverse primer(Brca1 005): 50-CAATAAACTGCTGGTCTCAGG-30 and(Brca1 007): 50-ATCGCCTTCTATCGCCTTCTTGACGAGT-TC-30. After PCR amplification, products were analyzedby agarose gel electrophoresis. The B004/B005 primercombination generates a 450-bp product that correspondsto the wild-type Brca1, whereas the B004/B007 primercombination generates a 550-bp product that correspondsto the Neo cassette.

Prepubertal dietary exposuresOn postnatal day 15 (PND15), WT and Brca1þ/� female

mice were divided into 2 dietary groups (n ¼ 30 mice pergenotype per group): AIN93G diet (control) or genistein(500 ppm)-supplemented AIN93G diet obtained fromHarlan-Teklad. The mice were kept on these diets untilPND30, and all mice were then switched to the non-supplemented AIN93G diet.

Effects on mammary tumorigenesisBrca1þ/� mice do not develop spontaneous mammary

tumors, and therefore, mammary tumors were induced byadministration of a subcutaneous injection of 15 mg in100 mL of medroxyprogesterone acetate (MPA; DepoPro-vera, Pfizer) to mice at 6 weeks of age, followed byadministration of 1 mg 7,12-dimethylbenz(a)anthracene(DMBA; Sigma) weekly for 4 weeks (weeks 7–10). Thecarcinogen was dissolved in corn oil and administeredby oral gavage in a volume of 0.1 mL. This study included22 WT mice fed the control diet, 21 WT mice fedprepubertally genistein diet, 25 Brca1þ/� mice fed thecontrol diet, and 26 Brca1þ/� mice fed genistein diet.Animals were examined for mammary tumors by palpa-tion once per week. The endpoints for data analysis were(i) latency to tumor appearance, (ii) the number ofanimals with tumors (tumor incidence), and (iii) thenumber of tumors per animal (tumor multiplicity). Dur-ing the follow-up, animals in which tumor burdenapproximated 10% of total body weight were euthanized,as required by the ethical guidelines of our institution.All surviving animals were sacrificed 20 weeks after thelast dose of DMBA administration.

Genistein, BRCA1, and Breast Cancer

www.aacrjournals.org Cancer Prev Res; 4(9) September 2011 1437




Hyperplastic alveolar nodulesWhole mounts of the mammary glands were obtained

from mice 20 weeks after DMBA exposure, processed asdescribed below. Whole mounts were evaluated under themicroscope to determine the number of hyperplastic alveo-lar nodules (HAN) in each gland. Whole mounts of thefourth abdominal mammary glands were obtained frommice 20 weeks after DMBA exposure and processed aspreviously described (37). Whole mounts were evaluatedunder the microscope to determine the number of HANs ineach gland.

Histopathologic evaluationMammary tumors were classified according to its histo-

pathology by a veterinarian pathologist, through ARUPveterinarian services.

Effects on mammary gland morphology andcytokeratin (CK) 5 and CK18 mRNA expression

To assess changes in mammary gland morphology,whole mounts of the fourth abdominal glands obtainedon PND50. The total number of TEBs was countedblindly by 2 independent investigators using an Olym-pus dissecting microscope. Identification of TEBswas based on the guidelines established by Russo andRusso (38). Ductal elongation (epithelial outgrowth)was measured as the growth, in centimeters, fromthe end of the lymph node to the end of the epithelialtree.

We also determined whether the mRNA expression ofCK5 (committed progenitor cells) or CK18 (luminal cells;ref. 36) or CK5/CK18 ratio were different between the WTand Brca1þ/� mice and whether prepubertal genisteinexposure affected the expression of these cytokeratins.The mRNA expression was determined as described belowunder the section Mammary gland amphiregulin, Rankl,CK5, and CK18 mRNA levels.

Mammary gland and tumor ER-a and HER-2 proteinlevels

Protein levels of ER-a and HER-2 were assessed byWestern blotting in the mammary glands and tumors.Briefly, mammary glands and tumors were homogenized,centrifuged, and the protein extract collected from thesupernatant. A total of 50 mg of protein extract was loadedonto a NuPAGE 12% Bis-Tris gel (Invitrogen Life Technol-ogies), and gels were run at 150 V. Membranes were thenwashed with TBS with Tween (TBST) and blocked in 5%milk in TBST for 30 minutes at room temperature. Afterblocking, membranes were incubated with antibodiesagainst ER-a (1:100 dilution; Vector Laboratories) andHER-2 (1:1,000 dilution; Lab Vision Corp) overnight at4�C. Next, membranes were incubated with secondary anti-rabbit IgG or mouse IgG horseradish peroxidase antibodies(anti-mouse, 1:5,000 dilution; Amersham Pharmacia Bio-tech) and developed using Super Signal (Pierce). Folddifferences were calculated by normalization againstb-actin.

Mammary gland and tumor PgR protein levelsUsing immunohistochemistry, PgR expression was

assessed in mammary glands and tumors collected at theend of the follow-up period. Briefly, tissues were fixed in10% buffered formalin, embedded in paraffin, and sec-tioned (5 mm). Sections were deparaffinized in xylene,hydrated through graded alcohols, and incubated withH2O2 to block endogenous peroxidases. Antigen retrievalwas carried out in a Target Retrieval solution (pH¼ 9; DakoS2368) in a pressure cooker for 20 minutes followed by 2hours of cool down. Tissue sections were incubated over-night with the primary antibody against PgR at a 1:400dilution (polyclonal rabbit anti-human PgR; A0098,Dako). After several washes, sections were treated withsecondary antibody (anti-rabbit) and developed usingthe Dako EnVisionþ Dual Link System-HRP, DABþ

(K4065) as instructed by the manufacturer. The PgR statusof each sample was evaluated according to a modifiedversion of the scoring system proposed by Allred andcolleagues (39). The total score for each sample was calcu-lated as the sum of the estimated proportion of positive-staining cells (0–7) plus the estimated intensity of positive-staining cells (0–4).

Mammary gland amphiregulin, Rankl, CK5, and CK18mRNA levels

Frozen tissue samples were weighed and homogenizedusing a PowerGen 35 handheld homogenizer (Fisher Scien-tific) with RNase-free disposable OMNI-Tips (Fisher Scien-tific). RNA extraction was then carried out according to theQiagen’s RNeasy Lipid Tissue Kit (Qiagen Inc.) instruc-tions. The quantity and quality of RNA were measured bycomparing the optical density ratios (OD260/OD280)obtained using a Nanodrop (ND-1000) Spectrophot-ometer (Thermo Scientific). RNA samples were stored at�80�C until use.

cDNA synthesis and quantitative real-time PCRanalysis

A total of 200 ng of total RNA per sample was used asa template for random primed cDNA synthesis with arecombinant Moloney murine leukemia virus reversetranscriptase (TaqMan MultiScribe Reverse Transcriptaseand RT-PCR Reagents, Applied Biosystems), accordingto manufacturer’s instructions. A reverse transcriptaseenzyme minus control reaction was also included. ThecDNA samples were then used as templates for quanti-tative real-time PCR (qRT-PCR) analysis with previouslydescribed specific primers for the target genes Brca1 (40),amphiregulin (30), Rankl (41), and CK5 and CK18 (42)using QuantiTect SYBR green PCR kit (Qiagen Inc.)and an ABI Prism 7900 Sequence Detection System. Eachsample was run in triplicate, and qPCR run was repeated1 to 2 times. Absolute gene expression levels were deter-mined using SDS2.3 software from Applied Biosystemsand the standard curve method. Concentration ofeach sample was normalized to the reference gene 18SrRNA (42).

de Assis et al.

Cancer Prev Res; 4(9) September 2011 Cancer Prevention Research1438




Statistical analysesData obtained on (i) mammary gland morphology

(total number of TEBs and ductal elongation), (ii) mRNAlevels, (iii) mammary gland and tumor HER-2 and ER-aprotein levels, (iv) PgR staining, and (v) number of HANswere analyzed by 2-way ANOVA using genotype (WT orknockout) and treatment (control or genistein) as vari-ables. Kaplan–Meier curves were used to compare differ-ences in tumor incidence, followed by the log-rank test.Normalized qRT-PCR results were analyzed with Kruskal–Wallis 1-way ANOVA on Ranks and 1-way ANOVA withHolm–Sidak method for pairwise multiple comparisonprocedures. All tests were carried out using the SPSSSigmaStat Software, and differences were considered sig-nificant if the value of P was less than 0.05. All prob-abilities are 2 tailed.

Results

Mammary tumorigenesisPrepubertal genistein exposure significantly reduced

mammary tumor incidence in the WT mice (P ¼ 0.029;Fig. 1A). Among the Brca1þ/�mice, however, the differencein mammary tumor incidence failed to reach statisticalsignificance (P ¼ 0.26; Fig. 1B). The number of HANs(Fig. 1C), tumor multiplicity (the total number of tumorsper animal; ranged between 1.2 and 1.6 in the WT andBrca1þ/� mice), or tumor latency (time to tumor appear-ance; ranged between 10.1 and 11.9 weeks) were notaffected by prepubertal genistein diet in either the WT orBrca1þ/� mice (Table 1).

There was no evidence that the incidence of mammarytumors was higher in the Brca1þ/� mice; in fact, a slightly

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Figure 1.Mammary tumorigenesis and Brca1 expression. Effect of prepubertal dietary exposure to 500 ppm genistein on (A) tumor incidence in wild-typemice(n ¼ 21–22 mice per group) and (B) in Brca1þ/� mice (n ¼ 25–26 mice per group), (C) HANs in these mice, and (D) Brca1 mRNA levels (n ¼ 4–6 miceper group) on PND50. Means � SEM of Brca1 mRNA are shown; bars with different letters are significantly different from each other, P < 0.05. Prepubertalgenistein exposure significantly reduced mammary tumorigenesis in WT mice (P < 0.029) but not in Brca1þ/� mice (P < 0.26).






lower number of these mice (14 of 25, 56%) developedmammary tumors compared with WT mice (15 of 22,68%). The number of premalignant lesions, that is, HANs,was significantly higher in the Brca1þ/� mice than in theWT mice (P ¼ 0.047; Fig. 1C).

Histopathologic analysis indicated that most mammarytumors were malignant carcinomas (Table 2) in all 4groups. The incidence of benign adenomas was 10% and8% in the WT and Brca1þ/� mice fed the control diet,respectively, and 20% in both groups which were fedgenistein-containing diet before puberty. The malignanttumors consisted of adenocarcinomas and other carcino-mas. These malignant carcinomas were divided into low-and high-grade tumors, and the ratio of low- to high-gradetumors was similar in the WT and Brca1þ/� mice (Table 2).In the WT mice, the incidence of high-grade malignanttumors dropped from 60% in the control diet group to20% in the genistein group (P < 0.001) but only from 67%to 50% in the Brca1þ/� mice (nonsignificant). The tumormitotic index, another indicator of tumor growth andaggressiveness, was reduced by prepubertal exposure togenistein in the WT mice (P < 0.011), but genistein hadno effect on the Brca1þ/� mice (P ¼ 0.93; Table 2).

Mammary gland Brca1 mRNA levelsThe expression levels of Brca1 mRNA in the mammary

gland were determined on PND50 (Fig. 1C). As expected,qRT-PCR showed lower levels of Brca1 mRNA in Brca1þ/�

mice compared with WTmice (P < 0.001), regardless of the

diet. Higher levels of Brca1 mRNA were observed in themammary glands of WT mice fed genistein-containing dietcompared with those fed AIN93G control diet (P ¼ 0.04).No effects by genistein diet on Brca1 levels were observed inBrca1þ/� mice (Fig. 2).

Mammary gland morphologyMammary gland morphology and the expression of CK5

and CK18 were assessed at PND50. CK5 levels were sig-nificantly higher in the Brca1þ/� mice fed the control dietthan in the WT controls (P < 0.016; Fig. 2A), but CK18levels were not altered (Fig. 2B). Regardless of the genotype,the mice fed genistein during prepuberty exhibited signifi-cantly reduced levels of both cytokeratines compared withthe mice fed the control diet (CK5: P < 0.002; CK18: P <0.003). The ratio between CK5 and CK18 was higher in theBrca1þ/� mice than in the WT mice (P < 0.022). Thedifference was particularly apparent in mice fed genisteinduring prepuberty because genistein reduced it in the WTmice and significantly increased the ratio in the Brca1þ/�

mice (P < 0.04; Pinteraction ¼ 0.079; Fig. 2C).The reductions in both CK5 and CK18 by genistein

reflected changes in the mammary gland morphology. Asseen in Figure 3A, mice fed genistein during prepubertyexhibited significantly smaller mammary epithelium thanmice fed the control diet. No significant differences in thenumber of TEBs or ductal elongation were seen betweenthe WT and Brca1þ/� mice, when determined at PND50.However, prepubertal genistein exposure reduced the

Table 1. Mammary tumor multiplicity and latency in DMBA-exposed WT and Brca1þ/� mice fed a controlor genistein-supplemented AIN93G diet during prepuberty

Group Number oftumors

Tumor multiplicity(number of tumors/tumor-bearing mice)

Tumor latency,wk

WT control 10 1.2 � 0.4 10.1 � 0.7WT genistein 5 1.6 � 0.3 11.6 � 1.2Brca1þ/� control 12 1.2 � 0.3 11.9 � 1.0Brca1þ/� genistein 10 1.2 � 0.2 10.8 � 1.1

NOTE: Mean � SEM are shown.

Table 2. Mammary tumor histopathology in DMBA-exposed WT and Brca1þ/� mice fed a control orgenistein-supplemented AIN93G diet during prepuberty

Group Benign Malignant Tumor grade Mitotic indexMean � SEM

Adenoma Adeno carcinoma Carcinoma (other) Low High

WT control 1 (10%) 6 (60%) 3 (30%) 3 (33%) 6 (67%)a 5.0 � 0.8a

WT genistein 1 (20%) 1 (20%) 3 (60%) 3 (75%) 1 (25%)b 1.4 � 1.1b

Brca1þ/� control 1 (8%) 9 (75%) 2 (17%) 3 (27%) 8 (63%)a 2.1 � 0.7b

Brca1þ/� genistein 2 (20%) 4 (40%) 4 (40%) 3 (37%) 5 (63%)a 2.0 � 0.8b

NOTE: Values within a column which are marked with a different letter are significantly different from each other, P < 0.05.

de Assis et al.





number of TEBs (P < 0.001) and inhibited ductal elonga-tion (P ¼ 0.007) in both the WT and Brca1þ/� mice(Fig. 3B).

Mammary gland and mammary tumor proteinexpression levels of ER-a, ER-b, PgR, and HER-2, andtheir downstream targets amphiregulin and RANKLBecause genistein acts by binding to ER-a and ER-b

(8, 9) and upregulation of PgR is indicative of ER-aactivation, we determined the expression of these recep-tors. We also tested pathways downstream of ER-a and

PgR, that is, the expression of amphiregulin and RANKL.In addition, genistein is a tyrosine kinase inhibitor (17)and therefore HER-2 levels were assessed. ER-a and HER-2 protein levels were detected by Western blotting; ER-b,amphiregulin, and Rankl mRNA levels by RT-qPCR; andPgR by qRT-PCR and immunohistochemistry.

Changes in mammary glandsThe assays revealed that mammary glands and tumors

were positive for ER-a, ER-b, PgR, and HER-2 expression.Protein levels of ER-a (Fig. 4A,mammary gland) andHER-2

CK5on PND50 in mice fed genistein during prepuberty

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Figure 2. CK5 and CK18 expression and CK5/CK18 ratio. Effect of prepubertal dietary exposure to 500 ppm genistein on CK5 and CK18 expression andCK5/CK18 ratio in 50-day-old wild-type and Brca1þ/� mice. Mean � SEM of 4 to 6 mice per group are shown; bars marked with different letters aresignificantly different from each other, P < 0.05. Prepubertal genistein exposure did not have a significantly different effect on CK5/CK18 ratio in the wild-typecompared with Brca1þ/� mice (Pinteraction < 0.079).






(Fig. 4B) in mammary gland tissues were not differentbetween the genotypes or dietary groups. PgR protein levelswere higher in the mammary glands of Brca1þ/� mice thanin theWTmice (P < 0.033; Fig. 4C). Genistein decreased thePgR protein levels in the WTmice (P < 0.030). ER-b proteinlevelswerenotdifferentamongthegroups(datanotshown).

Changes in mammary tumorsAssessment of expression of the different receptors in

the mammary tumors revealed that ER-a protein levelswere not different in the mammary tumors between WTand Brca1þ/� mice fed the control diet. The expression ofER-a was reduced in the tumors by prepubertal genistein

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Figure 3. Mammary gland morphology. A, histologic depiction of whole mounts of the fourth abdominal mammary gland (arrow indicates where theepithelial tree ends); (B) number of TEBs; and (C) ductal elongation (distance, in cm, from the end of the lymph node to the end of the epithelial tree) onPND50 in wild-type and Brca1þ/� mice fed 500 ppm genistein during prepuberty. Mean� SEM of 4 to 6 mice per group are shown; bars marked with differentletters are significantly different from each other, P < 0.05.

de Assis et al.





exposure in the WT but not in the Brca1þ/� mice(Pinteraction < 0.04; Fig. 5A, mammary tumor).In contrast to normal mammary gland tissue, the levels

of PgR were significantly lower in the mammary tumors ofthe Brca1þ/�mice than in theWTmice (P¼ 0.007; Fig. 5C).Furthermore, although theWTmice exposed to genistein atprepuberty exhibited a significant reduction in PgR expres-sion, no reduction was seen in the in mammary tumors ofthe Brca1þ/� mice (Pinteraction < 0.034).Mammary tumors of Brca1þ/�� mice expressed higher

levels of HER-2 than the tumors in the WT mice, regardless

of diet (P < 0.03; Fig. 5B). Prepubertal exposure to genisteindiet decreased the expression levels of HER-2 in mammarytumors of WT and Brca1þ/� mice compared with theirrespective controls (P < 0.002).

Amphiregulin and Rankl expression in the mammarygland

Althoughmammary gland ER-a levels were similar in theWT and Brca1þ/� mice, amphiregulin mRNA levels werelower in the mammary glands of Brca1þ/� mice than WTmice (P < 0.011; Fig. 6A). Prepubertal genistein exposure

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Figure 4. Mammary gland and tumor protein levels of ER-a, PgR, and HER-2. Protein levels of (A) ER-a, (B) HER-2, and (C) PgR determined on PND50. Allvalues are expressed as the mean � SEM of 4 to 6 mice per group. ER-a and HER-2 levels were determined by Western blotting, and PR levels weredetermined by immunohistochemistry. Bars marked with different letters are significantly different from each other, P < 0.05. C, control; G, genistein; KO,knockout.






significantly reduced the levels in both groups (P < 0.001),and consequently, no differences in amphiregulin levelsbetween the two genotypes were seen.

Downstream target of PgR, Rankl, was significantlyhigher in the control diet–fed WT mice than in theBrca1þ/� mice on PND50 (P < 0.018), but the differencewas lost in mice fed genistein during prepuberty (Fig. 6B).This is because among the WT mice genistein reducedRankl, but among the Brca1þ/� mice, genistein exposureincreased Rankl mRNA (Pinteraction < 0.026).

Discussion

Effect of prepubertal genistein diet on mammarytumorigenesis

We found that in the WT mice, prepubertal dietaryexposure to 500 ppm genistein significantly reduced carci-

nogen-induced mammary tumor incidence, in accordancewith data previously reported in rats (6). Because the dosewe used generates blood genistein levels comparable tothose seen in the Asians (43), our study adds to theconvincing evidence obtained in human studies that child-hood soy intake protects against the development of breastcancer later in life (1–5).

However, no significant reduction in mammarytumorigenesis was seen in the Brca1þ/� mice fed thegenistein diet before puberty onset. Furthermore, incontrast to the WT mice, these mice did not exhibit anincrease in Brca1 expression, possibly because theremaining Brca1 allele was already maximally expressedin the heterozygous Brca1þ/� mice. The increase in Brca1expression in the WT mice is in agreement with theearlier findings obtained in both in vitro and in vivostudies indicating that genistein upregulates BRCA1

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Figure 5. Mammary tumor protein levels of ER-a, PR, and HER-2. Protein levels of (A) ER-a, (B) HER-2, and (C) PgR were determined in the DMBA-inducedadenocarcinomas. All values are expressed as the mean� SEM of 5 to 7mice per group. Bars marked with different letters are significantly different from eachother, P < 0.05. Genistein had opposing effect on ER-a (Pinteraction < 0.04) and PgR expression (Pinteraction < 0.034) in wild-type and Brca1þ/� mice. IHC,immunohistochemistry.

de Assis et al.





(12, 20, 21). Our results thus suggest that the genistein-induced upregulation of Brca1 may be required for pre-pubertal genistein exposure to reduce breast cancer risk.

Mammary tumorigenesis in the Brca1þ/� mice: role ofPgR signalingPalpable mammary tumor incidence was not different

between the genotypes, although Brca1þ/� mice developedmore premalignant HAN lesions than WT mice did. Thesefindings suggest that the Brca1þ/� mice have a higherpotential to develop mammary tumors than the WT micedo but most of these HANs do not progress to malignancyin the Brca1þ/� mice. Because Brca1þ/� mice expressedhigher levels of PgR than those of the WT mice; a findingwhich is consistent with the data by Poole and colleagues(44) in conditional Brca1�/�/p53�/�mice, these geneticallymodified mice should be at least as sensitive as the wild-type mice to the MPA exposure, which is required in bothwild-type and various genetically modified mouse modelsfor DMBA to induce mammary tumors (45). This wasconfirmed by determining that MPA similarly increasedRankl expression and TEB number in the WT and Brca1þ/�

mice (data not shown).

Differences in the response to prepubertal genisteindiet between the genotypesPrepubertal genistein diet affected some endpoints in

the mammary glands and tumors differentially, and somesimilarly in the WT and Brca1þ/� mice. This diet had anopposite effect on the following endpoints in the 2genotypes: Rankl expression and CK5/CK18 ratio in the

mammary gland, PgR expression in the mammary glandand tumor, ER-a expression in the tumor, and histo-pathology of the tumor. In the WT mice, prepubertalgenistein exposure reduced the expression of Rankl inthe mammary gland and PgR both in the mammary glandand tumor. The effect on the PgR is in agreement with thestudy by Pei and colleagues (46) showing that prepuber-tal genistein exposure reduced PgR expression in the (rat)mammary gland. Thus, consumption of genistein duringprepuberty may induce persistent downregulation of PgR.Downregulation of Rankl by genistein is consistent withprevious data in bone and prostate (34, 35). Becausereduced expression of PgR and Rankl may be associatedwith a reduction in the mammary progenitor cell prolif-eration (47), one of the mechanisms by which prepu-bertal genistein exposure reduces breast cancer risk maybe through increased mammary luminal cell differentia-tion. Changes in these three endpoints could haveoccurred through upregulation of BRCA1, which isknown to suppress progesterone signaling (24) andinduce luminal-specific differentiation of mammary pro-genitor cells (25).

Prepubertal genistein diet affected Rankl and CK5/CK18ratio differently in the Brca1þ/� mice. Genistein-treatedBrca1þ/� mice expressed more Rankl and higher CK5/CK18 ratio than the control diet–fed mice. Progenitor cells,including committed myoepithelial and some ductal lumi-nal progenitor cells, express CK5, and the expression is lostwhen the cells differentiate to mature myoepithelial andluminal cells (36). Because high CK5/CK18 ratio is indi-cative of increased presence of undifferentiated luminal

Amphiregulinon PND50 in mice fed genistein during prepuberty

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Figure 6. Expression of amphiregulin and Rankl in the mammary gland. mRNA levels of (A) amphiregulin and (B) Rankl in the mammary gland on PND50,determined using qRT-PCR. All values are expressed as the mean � SEM of 4 to 6 mice per group. Bars marked with different letters are significantlydifferent from each other, P < 0.05. Genistein reduced amphiregulin expression in both the wild-type and Brca1þ/� mice but had opposing effect onRankl expression in the 2 genotypes (Pinteraction < 0.026).






cells (36) as is high Rankl expression (47), prepubertalgenistein exposure may have led to reduced luminal celldifferentiation in the Brca1þ/� mice.

Prepubertal genistein diet reduced mammary tumorgrade and tumor mitotic index, both of which are indica-tors of aggressiveness of a tumor, in the wild-type but notBrca1þ/� mice. In addition, tumors in the prepubertallygenistein-fed wild-type mice expressed significantly lowerlevels of the ER-a, PgR, and HER-2 than tumors in thecontrol diet–fed wild-type mice. Again, mammary tumorsin Brca1þ/� mice failed to show similar changes, with theexception of downregulation of HER-2 in the genistein-fedmice. These findings indicate that genistein failed to reduceaggressiveness of the mammary tumors in the mice lacking1 Brca1 allele.

Similarities in the response to prepubertal genisteindiet in the two genotypes

Genistein had some effects on the Brca1þ/� mice whichwere similar to those seen in the WT mice; that is, itreduced the number of targets for malignant transforma-tion (TEB), inhibited ductal elongation, downregulatedamphiregulin expression in the mammary gland, andreduced HER-2 expression in the tumors. Some of thesechanges have been previously reported to occur by gen-istein (6, 17, 48), but we are not aware of any studiesshowing that genistein delays ductal elongation or inhi-bits amphiregulin expression.

Activation of ER-a increases amphiregulin expressionwhich then binds to the EGFR/ErbB1/HER-1 and inducescell proliferation (30). ErbB2/HER-2 does not have anyknown ligands, but its activity and possibly expressioncan be modified through heterodimerization withamphiregulin-bound EGFR (49, 50). Amphiregulinexpression was lower in the control diet–fed Brca1þ/�

mice than in the WT controls, suggesting that ER-asignaling may be reduced in the Brca1þ/� mice, althoughno differences in the ER-a expression in the normalmammary gland were seen between the genotypes. Ourresults also indicate that both in the Brca1þ/� and WTmice, prepubertal genistein diet induced a persistentreduction in mammary amphiregulin levels. Lamberand colleagues have reported that BRCA1 suppressesamphiregulin (31), and therefore, the reduction in theWTmice may be due to genistein-induced upregulation ofBrca1. However, because amphiregulin was reduced alsoin the genistein-exposed Brca1þ/� mice, but genisteinfailed to upregulate their Brca1 expression, other mechan-isms than genistein-induced upregulation of Brca1 sup-pressed their amphiregulin expression.

Amphiregulin was recently found to mediate self-renewal of stem/progenitor cells in the mammary duct(32); its inhibition by siRNA reduced mammosphere for-mation by preventing the expansion of ductal progenitorcells. Reduction in amphiregulin expression by prepubertalgenistein diet could thus explain the significant delay inductal elongation observed here in the postpubertal mam-mary gland in both WT and Brca1þ/� mice. Because

amphiregulin also is expressed in the TEBs (51), genis-tein-induced reduction in TEB number could perhaps beexplained by a reduction in amphiregulin expression.Mammary gland morphology on PND50 in the controldiet–fed WT and Brca1þ/� mice was similar; that is, loss ofone Brca1 allele did not affect gross mammary glandmorphology. In humans, mammographic density also issimilar in germ line BRCA1 mutation carriers and controls(52, 53). Animal studies have generated conflicting dataand reported that conditional or mammary-specific BRCA1mutations impair mammary ductal development (16, 54–56) or induce ductal elongation (57).

Conclusions

In this study, we showed that a prepubertal dietaryexposure to genistein inmice reduces the risk of developingmammary tumors later in life and upregulates Brca1mRNAexpression in an adult mammary gland. We also showedthat the protective effect was not seen in mice with a germline Brca1 mutation and neither was the remaining Brca1allele upregulated by genistein in these mice. WT mice fedgenistein before puberty onset exhibited reduced expres-sion of PgR and Rankl; changes in the opposite directionwere seen in the Brca1þ/� mice, and they also exhibitedincreased CK5/CK18 ratio. These findings are indicative ofreduced progenitor cell proliferation and increased luminaldifferentiation in the genistein-exposed WT mice butreduced differentiation in the genistein-exposed Brca1þ/�

mice (36, 47).Genistein downregulated amphiregulin expression and

inhibited ductal elongation and reduced TEBs in the mam-mary glands of both the WT and Brca1þ/� mice and alsoreduced the expression of Her-2 in the mammary tumors,suggesting that prepubertal dietary genistein exposureinduces some breast cancer risk–protective effects in themammary gland, regardless of the level of Brca1 expression.However, these effects do not offer full protection toBrca1þ/� mice, indicating that upregulation of Brca1 isrequired for prepubertal genistein to prevent cancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Salim Shah for advising in establishing Brca1þ/�

mouse colony, LCCC Animal Shared Resource for help in feeding miceand in tumorigenesis experiments, and Histopathology Shared Resource fortissue processing.

Grant Support

The work was supported by the National Cancer Institute (U54 CA100970)and American Cancer Society (postdoctoral fellowship 116602-PF-09-018-01-CNE).

Received November 23, 2010; revised April 20, 2011; accepted May 23,2011; published OnlineFirst June 16, 2011.

de Assis et al.





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2011;4:1436-1448. Published OnlineFirst June 16, 2011.Cancer Prev Res Sonia de Assis, Anni Warri, Carlos Benitez, et al.

ExpressionBRCA1Tumorigenesis Are Dependent on Protective Effects of Prepubertal Genistein Exposure on Mammary

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Protective Effects of Prepubertal Genistein …...by agarose gel electrophoresis. The B004/B005 primer combination generates a 450-bp product that corresponds to the wild-type Brca1,