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Published OnlineFirst January 19, 2010; DOI: 10.1158/0008-5472.CAN-09-3068

Tumor and Stem Cell Biology
Cancer
Research
The G Protein–Coupled Receptor GPR30 Inhibits Proliferationof Estrogen Receptor–Positive Breast Cancer Cells
Eric A. Ariazi1, Eugen Brailoiu2, Smitha Yerrum1, Heather A. Shupp1, Michael J. Slifker1,Heather E. Cunliffe3, Michael A. Black4, Anne L. Donato1, Jeffrey B. Arterburn5,Tudor I. Oprea6, Eric R. Prossnitz7, Nae J. Dun2, and V. Craig Jordan1

Abstract

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The G protein–coupled receptor GPR30 binds 17β-estradiol (E2) yet differs from classic estrogen receptors(ERα and ERβ). GPR30 can mediate E2-induced nongenomic signaling, but its role in ERα-positive breastcancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcino-mas showed an association between increased GPR30 expression and ERα-positive status. We therefore ex-amined GPR30 in estrogenic activities in ER-positive MCF-7 breast cancer cells using G-1 and diethylstilbestrol(DES), ligands that selectively activate GPR30 and ER, respectively, and small interfering RNAs. In expressionstudies, E2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ERmediated. In Ca2+ mobilization studies, GPR30, but not ERα, mediated E2-induced Ca2+ responses because E2,4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca2+ increases not only inMCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E2-induced and G-1–induced Ca2+ mobilization, but ERα depletion did not. Interestingly, GPR30-coupled Ca2+

responses were sustained and inositol triphosphate receptor mediated in ER-positive MCF-7 cells but transi-tory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 de-pletion indicated that the role of GPR30 was to promote SKBr3 cell growth but reduce MCF-7 cell growth.Supporting this, G-1 profoundly inhibited MCF-7 cell growth, potentially via p53 and p21 induction. Further,flow cytometry showed that G-1 blocked MCF-7 cell cycle progression at the G1 phase. Thus, GPR30 antag-onizes growth of ERα-positive breast cancer and may represent a new target to combat this disease. Cancer Res;70(3); 1184–94. ©2010 AACR.

Introduction

The G protein–coupled receptor GPR30 is a seven-transmembrane domain protein identified as a novel 17β-estradiol (E2)–binding protein structurally distinct fromthe classic estrogen receptors α and β (ERα and ERβ).GPR30 can mediate rapid E2-induced nongenomic signalingevents, including stimulation of adenylyl cyclase, and,

ffiliations: 1Fox Chase Cancer Center; 2Department ofogy, Temple University School of Medicine, Philadelphia,nia; 3Computational Biology Division, The TranslationalResearch Institute, Phoenix, Arizona; 4Department ofry, University of Otago, Dunedin, New Zealand; 5Departmenttry and Biochemistry, New Mexico State University, Lasw Mexico; and Departments of 6Biochemistry and Moleculard 7Cell Biology and Physiology, University of New Mexicoedicine, Albuquerque, New Mexico

plementary data for this article are available at CancerOnline (http://cancerres.aacrjournals.org/).

dress for V.C. Jordan: Department of Oncology, Lombardinsive Cancer Center, Georgetown University School ofashington, District of Columbia.

ding Author: V. Craig Jordan, Department of Oncology, Lom-prehensive Cancer Center, Georgetown University Medical0 Reservoir Road Northwest, Washington, DC 20057. Phone:97; Fax: 202-687-6402; E-mail: [email protected].

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via transactivation of epidermal growth factor receptors, in-duces mobilization of intracellular calcium (Ca2+) storesand activation of mitogen-activated protein kinase (MAPK)and phosphoinositide 3-kinase (PI3K) signaling pathways (1,2). GPR30 also exhibits prognostic utility in endometrial (3),ovarian (4), and breast cancer (5, 6) and can modulategrowth of hormonally responsive cancer cells (7–11). There-fore, GPR30 likely plays important roles in modulating es-trogen responsiveness and in the development and/orprogression of hormonally responsive cancers. Moreover,GPR30 represents a promising new target for drug discoveryin hormonally responsive disease.In addition to E2, the selective ER modulator (SERM) ta-

moxifen, one of its active metabolites 4-hydroxytamoxifen(4OHT), and the complete antiestrogen fulvestrant all acti-vate GPR30 (12–14). GPR30 does not significantly bind thenonsteroidal full ER agonist diethylstilbestrol (DES; ref. 12).Drug discovery efforts have yielded two GPR30-selectivehigh-affinity ligands: an agonist termed G-1 (15) and, re-cently, an antagonist termed G-15 (16). G-1 and G-15 donot bind ERs at concentrations up to 10−6 mol/L (15, 16).Moreover, G-1 specificity for GPR30 was illustrated byshowing it does not significantly bind 25 other G protein–coupled receptors (17).GPR30 has been shown to mediate the proliferative effects

of E2 in thyroid (7), endometrial (8, 18), ovarian (9), and

1. © 2010 American Association for Cancer Research.

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GPR30 Inhibits Growth of ER-Positive Breast Cancer


ER-negative SKBr3 breast cancer cell lines (9, 10) becauseGPR30 depletion, using antisense oligonucleotides or RNA in-terference (RNAi) methodologies, abrogated E2-stimulatedgrowth in these cells. However, GPR30-mediated growth ef-fects differ in ER-positive MCF-7 breast cancer cells. Aholaand colleagues (11) reported that transient overexpressionof GPR30 in MCF-7 cells inhibited bromodeoxyuridine incor-poration, an indicator of proliferation.We investigated GPR30 largely in ER-positive MCF-7 with

some comparisons to ER-negative SKBr3 breast cancer cells.First, a statistical association was sought between GPR30 andERα-positive status in publicly available breast carcinomamicroarray data sets. Next, the contribution of ERα andGPR30 in several E2-responsive activities, including regula-tion of GPR30 expression, intracellular Ca2+ mobilization, cel-lular growth, and cell cycle progression, was studied usingreceptor-specific ligands and small interfering RNA (siRNA)methodology.

Materials and Methods

Breast cancer microarray data mining. GPR30 mRNAlevels and ER status were extracted from gene expressionmicroarrays comprising 1,250 breast carcinomas across fivedistinct cohorts. The first cohort or the NKI cohort (n =295; Netherlands Cancer Institute, Amsterdam, the Nether-lands) was derived from van de Vijver and colleagues (19).8

NKI data were obtained using two-color 60-polymer oligo-nucleotide arrays. cRNA from one tumor was competitivelyhybridized against a pooled reference cRNA from all tu-mors. Expression values corresponded to normalized log2ratio intensity units. ER-positive status was supplied withthe microarray data (19). Pearson's correlation coefficientswere computed between GPR30 and all other genes usingthe R software package.9 Cohorts 2 to 5 were obtainedfrom Gene Expression Omnibus (GEO; ref. 20). These co-horts are termed the Uppsala cohort (GSE3494/GSE4922/GSE6532; samples collected in Uppsala County, Sweden),the Stockholm cohort (GSE1456; samples collected at theKarolinska Hospital in Stockholm, Sweden), the EMC co-hort (GSE2034/GSE5327; samples collected at the ErasmusMedical Center, Rotterdam, the Netherlands), and theTRANSBIG cohort (GSE7390; samples collected by thetranslational research network managed by the Breast In-ternational Group). The Uppsala and EMC cohortscontained samples processed at the same institution thatspan multiple GEO accession numbers. These four cohortsused Affymetrix microarray technology. Where available,raw data (in the form of CEL files) were downloaded; oth-erwise, MAS5.0 normalized data were downloaded (CELfiles were available for all studies except GSE2034 andGSE5327). All data preprocessing and MAS5.0 normaliza-tion were performed using R software and the justMASfunction in the simpleAffy library from Bioconductor (no

8 http://www.rii.com/publications/2002/nejm.html9 http://www.R-project.org

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background correction, target intensity of 600; ref. 21). Af-ter normalization, gene expression data were extracted forthe GPR30 probe 210640_s_at. ER status was provided viaSupplementary Data in GEO.Compounds and cell lines. E2, DES, and 2-aminoethyldi-

phenylborinate (2APB) were from Sigma-Aldrich. G-1 andfulvestrant (ICI 182,780, Faslodex) were from Tocris. Xestos-pongin C (XeC) and ryanodine (Ry) were from Calbiochem,EMD Biosciences. All agents were added to culture mediumat 1:10,000 to 1:1,000 (v/v). Fura-2 AM and all cell culture re-agents were from Invitrogen. MCF-7:WS8 human mammarycarcinoma cells were used in all experiments indicatingMCF-7 cells; they were clonally selected for sensitivity toE2-stimulated growth (22). SKBr3 cells were purchased fromthe American Type Culture Collection. Both cell lines weremaintained in estrogenized medium [RPMI-1640 plus 10%fetal bovine serum (FBS)]. MCF-7 cells were switched toestrogen-free medium (phenol red–free RPMI-1640 plus10% charcoal-stripped FBS) for 2 d before all experiments,except where noted.Real-time quantitative PCR assays. Quantitative PCR

(qPCR) was conducted as previously described (23). TargetmRNA levels were normalized to PUM1 [pumilio homologue1 (Drosophila)] mRNA levels (24). See Supplementary Materi-als and Methods for primer sequences. Data were analyzedby comparison with a serial dilution series of MCF-7 cellcDNA. Values in each group were averaged from four biolog-ical replicates (unless otherwise indicated), and each biolog-ical replicate was averaged from four technical replicates.siRNA transfection. MCF-7 cells that had been maintained

in estrogenized medium were transfected with siRNAs for 6h in serum-free Opti-MEM (Invitrogen) using Dharmafect 1(Dharmacon RNAi Technologies) followed by overnight re-covery in estrogenized medium. The transfection was carriedout a second time, and then the cells were immediatelyswitched to estrogen-free medium and again allowed over-night recovery. siRNAs were transfected at 200 nmol/L finalconcentration, except in Ca2+ experiments in which siRNAswere transfected at 100 nmol/L and cotransfected with si-GLO Green, a 6-carboxyfluorescein–labeled inactive double-stranded RNA. See Supplementary Materials and Methodsfor ERα and GPR30 siRNA sequences (Dharmacon RNAiTechnologies).Ca2+ imaging. Cytoplasmic Ca2+concentrations were mea-

sured using Fura-2 AM and microscopy as previously de-scribed (25). Flat SKBr3 cells were imaged because theywere the major morphologic cell type, whereas roundedSKBr3 cells were not imaged. All compounds were adminis-tered at 1 min.Cellular proliferation. Cell growth was assessed using

Hoechst 33258 (Invitrogen) and compared with a standardcurve of serial-diluted calf thymus DNA as previously de-scribed (26, 27).Cell cycle analyses. Cell cycle distribution was determined

by propidium iodide staining and using a fluorescence-activated cell sorter (Becton Dickinson) as previously de-scribed (27). Datawere analyzed using FlowJo 7.2.5 forWindows(Tree Star).

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Immunoblot analyses. Immunoblotting was carried outusing 40 μg protein per lane as previously described (23).Membranes were probed using antibodies against ERα(AER 611; Lab Vision), p53 (DO-1; Calbiochem), p21 (F-5; San-ta Cruz Biotechnology), cyclin D1 (DCS-6; Santa Cruz Bio-technology), cyclin B1 (D-11; Santa Cruz Biotechnology),and β-actin (AC-15; Sigma-Aldrich). Membranes were visual-ized using the Odyssey Infrared Imaging System (LI-CORBiosciences).Statistical analyses. Mann-Whitney tests were performed

using Prism 4.03 for Windows (GraphPad Software). All otherstatistical analyses were performed using Excel 2003 forWindows (Microsoft). Error is represented by SEMs in Ca2+

mobilization experiments and by SDs in all other experi-ments. Also in the Ca2+ experiments, P values reflect one-way ANOVA comparing the maximum Ca2+ concentrationversus the concentration at the treatment start time, unlessotherwise stated.

Results

Increased GPR30 mRNA expression associates with ERα-positive status in 1,250 breast carcinomas. Evidence of arelationship between GPR30 and ERα expression was soughtby mining publicly available and well-annotated gene expres-sion microarray data sets across five independent cohortscomprising 1,250 breast carcinomas. In the NKI cohort (n =254), data were collected using two-color oligonucleotidemicroarrays (Fig. 1A). According to the nonparametricMann-Whitney rank sum test, GPR30 mRNA levels were sig-nificantly higher in ERα-positive versus ERα-negative tumors(P < 0.0001). The upper range of GPR30 expression was 7.7-foldhigher on a linear scale in the ERα-positive compared withERα-negative carcinomas. In addition, GPR30 and ERαmRNAlevels correlated as continuous variables (Pearson's coefficientρ = 0.30, adjusted P < 0.0001). The other four cohorts used one-color Affymetrix oligonucleotide microarrays (Fig. 1B). In eachof these four cohorts, GPR30 mRNA levels were significantlyhigher in the ERα-positive compared with the ERα-negativebreast cancers (Uppsala cohort, n = 244, P = 0.040; Stockholmcohort, n = 159, P = 0.0091; EMC cohort, n = 344, P = 0.0050;TRANSBIG cohort, n = 198, P = 0.0024).E2 downregulates GPR30 mRNA expression via ER and

not GPR30. GPR30 regulation in response to E2 was inves-tigated. MCF-7 cells were treated with E2 or without E2(control, vehicle only) over a 96-hour time course followedby determination of ERα and GPR30 mRNA levels by qPCR.As expected, E2 steadily downregulated ERα mRNA levelsby 59% over 96 hours (Fig. 2A). E2 also downregulatedGPR30 but with faster kinetics than with ERα (Fig. 2B);GPR30 mRNA levels were decreased by 37% at 2 hours(P = 0.0013) and by 79% at 24 hours (P < 0.0001). Afterwards,GPR30 mRNA levels rebounded. Additionally, GPR30 mRNAexpression decreased in a concentration-dependent mannerfrom 10−12 mol/L E2 to 10−10 mol/L E2 (Fig. 2C). The GPR30-specific agonist G-1 did not alter GPR30 mRNA expression,but the ER-specific agonist DES did repress GPR30 expres-

Cancer Res; 70(3) February 1, 2010


sion relative to control treatment by 54% P = 0.0009), whichwas very similar to the effect of E2 (Fig. 2D). Fulvestrantcompletely blocked E2 and DES effects. Therefore, E2 likelyacted via ER and not GPR30 to transiently downregulateGPR30 mRNA expression.GPR30 and not ERαmediates E2-induced Ca2+ mobiliza-

tion responses. To begin to delineate whether endogenousERα and/or GPR30 mediates E2-induced Ca2+ responses inbreast cancer cells, changes in intracellular Ca2+ concentra-tions [Ca2+]i were measured in ER-positive MCF-7 and ER-negative SKBr3 breast cancer cells at the single-cell levelusing Fura-2 AM (Fig. 3). In ER-positive MCF-7 cells(Fig. 3A), E2 induced [Ca2+]i by 112 ± 1.6 nmol/L (n = 47 cells,P = 0.0063), G-1 by 511 ± 3.4 nmol/L (n = 58 cells, P = 0.0007),and 4OHT by 234 ± 3.4 nmol/L (n = 31 cells, P = 0.0017),whereas DES did not significantly increase the [Ca2+]i(change = 41 ± 0.8 nmol/L, n = 23 cells, P = 0.66). In ER-neg-ative SKBr3 cells (Fig. 3B), the rank order of ligand-inducedcytosolic Ca2+ increases was the same as in MCF-7 cells, butthe magnitude of the increases was much greater and the re-sponses were transitory instead of sustained. In ER-negativeSKBr3 cells, E2 induced oscillating increases in [Ca2+]i withan average maximum of 294 ± 1.6 nmol/L (n = 36 cells, P =0.0037). G-1 and 4OHT induced transitory increases in [Ca2+]iof 1,517 ± 10.3 nmol/L (n = 79 cells, P = 0.0001) and of 558 ±2.7 nmol/L (n = 37 cells, P = 0.0013), respectively, whereasDES did not ([Ca2+]i change = 51 ± 1.3 nmol/L, n = 21 cells,P = 0.26). Therefore, because G-1 and two ER ligands thatalso bind GPR30, E2 and 4OHT, but not ER-selective DES, eli-cited Ca2+ responses in both ER-positive MCF-7 cells and ER-negative SKBr3 cells, they likely did so via GPR30.Two of the major Ca2+ channels, inositol triphosphate re-

ceptors (IP3R) and Ry receptors (RyR; ref. 28), were tested forwhether they mediated G-1–induced Ca2+ mobilization. Thepharmacologic probes 2APB and XeC, both of which inhibitIP3Rs, and Ry, which at high concentrations blocks RyRs,were used. Cells were pretreated for 30 min before inducingCa2+ responses with G-1. In MCF-7 cells, both 2APB and XeCblocked G-1–induced [Ca2+]i increases by 78% (both 2APB +G-1 versus G-1 alone and XeC + G-1 versus G-1 alone, P =0.0018) but Ry did not. In contrast, in SKBr3 cells, Ry blockedG-1–induced [Ca2+]i increases by 80% (Ry + G-1 versus G-1alone, P = 0.0006), whereas XeC did not. In addition, 2APBallowed G-1 to almost fully induce [Ca2+]i increases, althoughthe response was significantly lower by 17% versus G-1 alone(P = 0.0094), but this was likely due to blockade of store-operated Ca2+ entry, another activity of 2APB. Therefore,GPR30 was coupled to IP3Rs in ER-positive MCF-7 cells butto RyRs in ER-negative SKBr3 cells.To confirm that GPR30 and not ERα mediates Ca2+

mobilization in response to E2 in MCF-7 cells, cells weretransfected with siRNAs targeting these receptors (character-ization of siRNAs in Supplementary Materials and Methods)and a nontargeting siRNA pool as a control. First, the GPR30siRNA was validated by showing that it led to an almostcomplete blockade of G-1–induced Ca2+ responses ([Ca2+]iincrease = 58 ± 1.3 nmol/L, n = 19 cells, P = 0.62; Fig. 4A). Next,E2-induced Ca2+ mobilization responses were investigated

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(Fig. 4B). In nontargeting siRNA–transfected cells, E2 inducedan [Ca2+]i increase of 159 ± 1.6 nmol/L (n = 14 cells, P =0.0075). However, in ERα siRNA–transfected cells, E2 causedalmost a 2-fold further rise in [Ca2+]i (314 ± 3.2 nmol/L,n = 17 cells; P = 0.0006). In GPR30 siRNA–transfectedcells, the E2-induced Ca2+ response was blocked ([Ca2+]iincrease = 52 ± 0.8 nmol/L, n = 16 cells, P = 0.35). ERαand GPR30 expression were depleted in the appropriatesiRNA-transfected cells (Fig. 4C). However, GPR30 mRNA



levels were increased by 73% in ERα-depleted cells, a find-ing consistent with the prior observation that E2 and DESrepressed GPR30 expression (Fig. 2B–D). Hence, the 2-foldpotentiation of E2-induced [Ca2+]i in ERα-depleted cellslikely reflected, at least in part, the increased GPR30expression.GPR30 functions to promote growth of ER-negative

SKBr3 cells but to inhibit growth of ER-positive MCF-7cells. The role of GPR30 in cellular proliferation was examined

Figure 1. GPR30 mRNA expression shows an association with ERα-positive status in human breast carcinomas. A, GPR30 mRNA levels in the NKI cohortderived from two-color arrays. Expression values are normalized log2 ratio intensity units corresponding to a single tumor cRNA hybridized against apooled reference cRNA from all tumors. B, GPR30 mRNA levels in the Uppsala, Stockholm, EMC, and TRANSBIG cohorts all derived from one-color arrays.Expression values are MAS5.0 normalized intensity units. A and B, sample sizes of ERα-positive (ER+) and ER-negative (ER−) cancers are shown, andbars indicate the 75th, 50th (median), and 25th percentiles. Significance was assessed using the nonparametric Mann-Whitney rank test.




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by transfecting cells with nontargeting and GPR30 siRNAs andthen measuring cellular DNA mass after 5 days of growth.First, SKBr3 cells were evaluated (Fig. 5A). The number of cellsseeded in each group was similar as indicated by a lack of dif-ference in DNA masses at day 0. After 5 days of growth, DNAmass was 45% lower in GPR30 siRNA compared with nontar-geting siRNA–transfected cells (P < 0.0001). Thus, the functionof GPR30 was to promote growth of SKBr3 cells, in accordancewith other reports (9, 10). Next, MCF-7 cells were similarlyevaluated (Fig. 5B). Again, equivalent numbers of nontargetingand GPR30 siRNA–transfected cells were seeded as indicatedby DNAmasses at day 0. After 5 days, GPR30 depletion did notaffect basal growth (control treatment). However, GPR30 de-



pletion did potentiate E2-stimulated growth by 2.1-fold (non-targeting versus GPR30 siRNA–transfected cells, P < 0.0001).Analysis of progesterone receptor (PgR) and TFF1 (pS2)mRNA levels by qPCR indicated no significant differencesin their induction by E2 between nontargeting and GPR30siRNA–transfected cells (data not shown). Therefore, in con-trast to SKBr3 cells, the function of GPR30 in MCF-7 cells wasto inhibit growth.The role of GPR30 in MCF-7 cell proliferation was further

evaluated by examining effects of G-1 on E2-stimulated (Fig.5C) and DES-stimulated (Fig. 5D) growth over 6 days. G-1blocked the concentration-dependent growth stimulatory re-sponse of E2 (all E2 treatment groups versus paired E2 + G-1

Figure 2. E2 represses ERα and GPR30 mRNA levels via ER and not GPR30 in MCF-7 cells. E2 regulation of ERα (A) and GPR30 mRNA (B) levels across atime course. MCF-7 cells were treated with 10−9 mol/L E2 or with the vehicle ethanol alone for 2, 6, 12, 24, 48, 72, and 96 h. C, GPR30 mRNA levelsin response to 24-h treatment with a serial dilution series of E2. D, ERα and GPR30 mRNA levels in response to 48-h treatment with ER and GPR30 ligandsas determined by qPCR. Each data point represents the average of six (A and B) or four (C and D) biological replicates. FUL, fulvestrant.

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treatment groups, P = 0.0001, one-way ANOVA); in particular,G-1 inhibited 10−10 mol/L E2-stimulated growth by 77% rela-tive to E2 alone (P < 0.0001, t test). G-1 also blocked DES-stimulated growth by 72% (DES versus DES + G-1, P <0.0001). Additionally, in both the E2 and DES experiments,G-1 inhibited basal (control treatment) growth by 32% (P <0.0001) and 47% (P < 0.0001), respectively. Therefore, G-1–activated GPR30 blocked growth of ER-positive breast cancercells but did so independently of ligand-activated ER.G-1–activated GPR30 blocks cell cycle progression

at G1 phase. The effect of G-1 on cell cycle progressionwas investigated. MCF-7 cells were synchronized by estro-gen withdrawal and then treated with E2 and G-1 for 24hours followed by propidium iodide staining and flow cyto-metric analysis (Fig. 6A). Treatment with G-1 alone signifi-cantly decreased the proportion of S-phase cells from 19.8%(control) to 14.7% (G-1; P < 0.0001). Importantly, the addi-tion of G-1 to E2 led to retention of an additional 11.6% ofthe cells in G1 phase (42.7% in E2-treated cells versus 54.4%in E2 + G-1–treated cells, P < 0.0001) and prevented 13.2%of cells from entering S phase (37.7% in E2-treated cells ver-sus 24.5% in E2 + G-1–treated cells, P < 0.0001). Therefore, G-1blocked E2-stimulated cells from cell cycle progression at theG1 phase.The G-1–induced cell cycle block was further investigated

by measuring protein expression of the tumor suppressor



p53, the cyclin-dependent kinase inhibitor (CDK-I) p21 (Fig.6B), the G1-phase–specific cyclin D1, and the G2/M-phase–specific cyclin B1 (Fig. 6C). MCF-7 cells were treated withE2 and G-1 and then collected at 24, 48, and 72 hours for im-munoblot analysis. Both p53 and p21 proteins were upregu-lated in G-1 and E2 + G-1–treated cells across all time pointscompared with control-treated cells (Fig. 6B). As expected, E2upregulated both cyclins D1 and B1 across the time coursecompared with control treatment, whereas G-1 alone did not(Fig. 6C). However, the addition of G-1 to E2 potentiated theupregulation of cyclin D1 while nearly completely preventingcyclin B1 accumulation compared with E2 alone across thetime points. Because cyclin D1 is induced during G1 phaseand degraded in S phase (29, 30), whereas cyclin B1 accumu-lates during G2-phase and degrades on M-phase entry (31),these data are consistent with G-1 blocking cell cycle pro-gression in G1 phase before cyclin D1 degradation occurredand before cyclin B1 accumulated.

Discussion

Filardo and colleagues (5) and Kuo and colleagues (6)have previously shown in breast carcinomas a positive as-sociation between GPR30 and ERα expression by immu-nohistochemistry and qPCR, respectively. We confirmedand extended this finding by examining gene expression

Figure 3. ER ligands that alsoactivate GPR30 induce Ca2+

mobilization responses in bothER-positive MCF-7 andER-negative SKBr3 cells.Ligand-induced Ca2+ responses(A and B) and blockade ofG-1–induced responses usingCa2+ channel inhibitors (C and D) inMCF-7 and SKBr3 cells. Cellswere loaded with Fura-2 AM, andintracellular Ca2+ concentrations[Ca2+]i were determined inindividual cells versus time usingfluorescence microscopy. Cellswere perfused with all ligands at10−6 mol/L starting at 1 min. 2APBwas used at 10−4 mol/L, XeC at10−5 mol/L, and Ry at 10−5 mol/L.SKBr3 cells with flat, notrounded, morphology wereimaged. G-1–induced Ca2+ tracesin A and B were redrawn in Cand D, respectively.




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microarray data sets of five independent patient cohortscomprising 1,250 breast cancers. In all cohorts, highGPR30 mRNA levels showed an association with ERα pos-itivity (Fig. 1). It is unknown why high GPR30 levelswould be selected for in ERα-positive breast carcinomasgiven GPR30 attenuates growth of ER-positive breast can-



cer, but some GPR30-dependent functions may be neces-sary for tumorigenesis and cell survival, such as activationof adenylyl cyclase, PI3K, and MAPK (1, 2). Additionalroles of GPR30 may be needed for disease progression,such as in cell migration (15, 32), which may then pro-mote metastasis (5).

Figure 4. GPR30 and not ERα mediates E2-induced Ca2+ mobilization in MCF-7 cells. G-1–induced (A) and E2-induced (B) Ca2+ responses. Cells weretransfected with nontargeting pool, ERα, and GPR30 siRNAs. Transfected cells were labeled using siGLO Green and appear green. Ca2+ imaging wasperformed 48 h following the transfection as in Fig. 3. Low levels of basal [Ca2+]i are visualized as blue and then green, whereas higher levels of [Ca2+]i areseen as red and then white. C, ERα protein levels were measured by immunoblotting and GPR30 mRNA levels by qPCR in siRNA-transfected cells48 h following transfection.

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The interplay between GPR30 and ERα was further inves-tigated using MCF-7 breast cancer cells. E2 repressed GPR30expression in a time- and concentration-dependent manner(Fig. 2B and C). In addition, DES but not G-1 downregulated



GPR30; therefore, ER mediated this effect (Fig. 2D). Theinverse functional relationship between ERα and GPR30was also shown by depleting ERα, which led to derepressionof GPR30 mRNA expression and consequently potentiated

Figure 5. GPR30 promotes growth ofER-negative SKBr3 but inhibits growthof ER-positive MCF-7 cells. Proliferationof SKBr3 (A) and MCF-7 (B) cellstransfected with the nontargeting pooland GPR30 siRNAs. Cells weretransfected and then seeded at 15,000per well in 24-well dishes. Medium wasreplenished the day after seeding onday 0 and every other day thereafter.Cells were collected on days 0 and5. SKBr3 cells were cultivated in theirpassage medium, and MCF-7 cellsin estrogen-free medium supplementedwith 10−9 mol/L E2 or without E2

[control (C)]. Proliferation was assessedas cellular DNA mass (μg/well) using24 replicate wells. GPR30 mRNA levelswere determined by qPCR 48 hfollowing the transfection in both celllines, and in MCF-7 cells, after 24 h of10−9 mol/L E2 or control treatment. Cand D, proliferation of MCF-7 cells over6 d treated with a serial dilution seriesof E2 (C) or with 10−9 mol/L DES (D)in the presence and absence of10−6 mol/L G-1. Twelve replicate wellswere used per group.




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E2-induced Ca2+ mobilization responses (Fig. 4). E2 is knownto downregulate ERα expression in MCF-7 cells as a negativefeedback regulatory loop to prevent overresponsiveness (26).Likewise, GPR30 may also be negatively regulated by E2 via ERto prevent excessive GPR30-dependent activity, such as aber-rantly high [Ca2+]i. Interestingly, the maximum increases in[Ca2+]i were much larger in SKBr3 cells than in MCF-7 cells(Fig. 3B versus Fig. 3A). It is possible that this was due tothe lack of ERs in SKBr3 cells, which translated into a lackof negative feedback regulation.



GPR30 depletion decreased growth of SKBr3 cells (Fig. 5A)but potentiated E2-stimulated growth in MCF-7 cells(Fig. 5B), indicating that GPR30 functions to promote SKBr3but to inhibit MCF-7 cellular proliferation. Also in MCF-7cells, G-1 profoundly inhibited E2-stimulated (Fig. 5C) andDES-stimulated growth (Fig. 5D) as well as decreased thepercentage of cells entering S phase (Fig. 6A). However,these G-1 effects occurred in both the presence and theabsence of E2. These findings in MCF-7 cells complementthose of Ahola and colleagues (11) who reported that transient

Figure 6. G-1 inhibits cell cycle progression in E2-stimulated MCF-7 cells by producing a block at G1 phase. A, cell cycle distribution as determined bypropidium iodide staining of DNA content and flow cytometry. Cells were synchronized by 3-d cultivation in estrogen-free medium and then treated asindicated for 24 h. Thirty-thousand cells per sample and three replicates per group were collected. Representative histograms are shown. Immunoblotanalyses of p53 and p21 (B) and of cyclin D1 and cyclin B1 (C) protein levels. MCF-7 cells were control (C)-, 10−9 mol/L E2 (E)-, and 10−6 mol/L G-1(G)–treated as indicated. Quantitated protein levels normalized to β-actin are indicated.

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GPR30 overexpression decreased the percentage of proliferat-ing MCF-7 cells independent of E2. Indeed, GPR30 likely doesnot directly regulate ER transcriptional activity becausethere were no significant differences in E2-induced mRNAexpression of well-established ER target genes PgR andTFF1 between GPR30 siRNA–transfected and nontargetingsiRNA–transfected cells (data not shown).Rather, we propose that GPR30 antagonizes growth of

MCF-7 cells by inducing sustained increases in cytosolicCa2+ concentrations (Figs. 3A and 4), in contrast to transitoryincreases in SKBr3 cells where GPR30 promotes growth. Ab-errant sustained increases in intracellular Ca2+ levels canlead to inhibition of proliferation and induce apoptosis(33). For example, the plasma membrane Ca2+-ATPase(PMCA) pumps Ca2+ across the plasma membrane out ofthe cell to lower cytosolic Ca2+ levels after Ca2+ increases.Partial inhibition of PMCA in MCF-7 cells causes a moderateincrease in intracellular Ca2+ levels, which leads to inhibitionof proliferation by altering cell cycle kinetics (34). Addition-ally, the mechanism of action of numerous antitumor agentsinvolves increases in [Ca2+]i (35).It is possible that differences in GPR30-coupled Ca2+ sig-

naling, which mediate sustained versus transitory responses,associate with ER status. In support of this hypothesis,GPR30 was coupled to differing Ca2+ channels: to IP3Rs inER-positive MCF-7 cells but to RyRs in ER-negative SKBr3cells. Alternatively, sustained versus transitory Ca2+ re-sponses could have been due to potential alterations in fac-tors that participate in lowering cytosolic Ca2+, such asplasma membrane or sarcoplasmic/endoplasmic reticulumCa2+-ATPase pumps. We intend to explore these possibilitiesinvolving differing Ca2+ responses in future studies.As shown by propidium iodide staining and flow cytome-

try, G-1 induced a cell cycle block at the G1 phase (Fig. 6A).Consistent with a G1-phase arrest, G-1 increased accumula-tion of the tumor suppressor p53, the CDK-I p21, and theG1-phase–specific cyclin D1 but prevented E2-induced accu-mulation of the G2/M-phase–specific cyclin B1 (Fig. 6B andC). Ca2+ signaling has been shown to induce p53 via activa-tion of cyclic AMP–responsive element binding protein (28).In MCF-7 cells, p53 induction by E2 is Ca

2+ and calmodulinkinase IV dependent (36). In addition, aberrant Ca2+ mobili-zation in response to anticancer/cytotoxic agents correlates



with p53 induction (35). Thus, G-1 could lead to p53 induc-tion via Ca2+ mobilization in MCF-7 cells. Then, p53 couldinduce p21 via a p53 response element to mediate arrest inG1 phase of the cell cycle (28).As a SERM, tamoxifen acts as an antiestrogen in ER-posi-

tive breast cancer but as an estrogen in the endometriumand bone (37). Similarly, G-1 inhibits growth of ER-positiveMCF-7 breast cancer cells but promotes growth of the endo-metrium (16) and plays an important role in promoting bonegrowth in vivo (38, 39). Thus, the tissue-specific proliferativeeffects of G-1 may parallel those of tamoxifen. It is interestingto speculate that 4OHT-induced Ca2+ mobilization (Fig. 3Aand B) may be involved in some of the tissue-specific effectsof tamoxifen.Taken together, GPR30 inhibits growth of ERα-positive

breast cancer. Our studies also indicate that pharmacologicactivation of GPR30 shows promise in combating ER-positivebreast cancer. G-1 would also probably be well tolerated be-cause it, like E2, exerts beneficial effects against an animalmodel of multiple sclerosis but without E2-associated side ef-fects (17, 40). Thus, G-1 may represent the first in a new classof therapeutically relevant agents for use alone or in conjunc-tion with conventional antihormonal therapeutics in breastcancer.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

Department of Defense grant BC050277 Center of Excellence (V.C. Jordan;views and opinions of, and endorsements by the author(s) do not reflect thoseof the US Army or the Department of Defense), and Weg and Genuardis funds(V.C. Jordan) and NIH grants P30CA006927 (Fox Chase Cancer Center),HL090804 and HL090804-01A2S109 (E. Brailoiu), CA127731 (E.R. Prossnitz,T.I. Oprea, and J.B. Arterburn), CA116662 (E.R. Prossnitz), U54MH084690 andCA118100 (University of New Mexico Cancer Center), and NS18710 andHL51314 (N.J. Dun).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8/19/09; revised 10/28/09; accepted 11/24/09; publishedOnlineFirst 1/19/10.

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Cancer Research



2010;70:1184-1194. Published OnlineFirst January 19, 2010.Cancer Res Eric A. Ariazi, Eugen Brailoiu, Smitha Yerrum, et al.

Positive Breast Cancer Cells−of Estrogen Receptor Coupled Receptor GPR30 Inhibits Proliferation−The G Protein

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