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 breast cancer remains unclear. Gene expression microarray data from five cohorts comprising 1,250 breast carcinomas showed an association between increased GPR30 expression and ERα-positive status. We therefore examined 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 expression studies, E2 and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. 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 in MCF-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 transitory and ryanodine receptor mediated in ER-negative SKBr3 cells. Proliferation studies involving GPR30 depletion 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 antagonizes growth of ERα-positive breast cancer and may represent a new target to combat this disease. Cancer Res; 70(3); 1184–94
The G protein–coupled receptor GPR30 is a seven-transmembrane domain protein identified as a novel 17β-estradiol (E2)–binding protein structurally distinct from the classic estrogen receptors α and β (ERα and ERβ). GPR30 can mediate rapid E2-induced nongenomic signaling events, including stimulation of adenylyl cyclase, and, via transactivation of epidermal growth factor receptors, induces mobilization of intracellular calcium (Ca2+) stores and 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 modulate growth of hormonally responsive cancer cells (7–11). Therefore, GPR30 likely plays important roles in modulating estrogen responsiveness and in the development and/or progression of hormonally responsive cancers. Moreover, GPR30 represents a promising new target for drug discovery in hormonally responsive disease.
In addition to E2, the selective ER modulator (SERM) tamoxifen, one of its active metabolites 4-hydroxytamoxifen (4OHT), and the complete antiestrogen fulvestrant all activate GPR30 (12–14). GPR30 does not significantly bind the nonsteroidal full ER agonist diethylstilbestrol (DES; ref. 12). Drug discovery efforts have yielded two GPR30-selective high-affinity ligands: an agonist termed G-1 (15) and, recently, an antagonist termed G-15 (16). G-1 and G-15 do not bind ERs at concentrations up to 10−6 mol/L (15, 16). Moreover, G-1 specificity for GPR30 was illustrated by showing 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 ER-negative SKBr3 breast cancer cell lines (9, 10) because GPR30 depletion, using antisense oligonucleotides or RNA interference (RNAi) methodologies, abrogated E2-stimulated growth in these cells. However, GPR30-mediated growth effects differ in ER-positive MCF-7 breast cancer cells. Ahola and colleagues (11) reported that transient overexpression of GPR30 in MCF-7 cells inhibited bromodeoxyuridine incorporation, 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 and ERα-positive status in publicly available breast carcinoma microarray data sets. Next, the contribution of ERα and GPR30 in several E2-responsive activities, including regulation of GPR30 expression, intracellular Ca2+ mobilization, cellular growth, and cell cycle progression, was studied using receptor-specific ligands and small interfering RNA (siRNA) methodology.
Materials and Methods
Breast cancer microarray data mining
GPR30 mRNA levels and ER status were extracted from gene expression microarrays comprising 1,250 breast carcinomas across five distinct cohorts. The first cohort or the NKI cohort (n = 295; Netherlands Cancer Institute, Amsterdam, the Netherlands) was derived from van de Vijver and colleagues (19).819). Pearson's correlation coefficients were computed between GPR30 and all other genes using the R software package.920). These cohorts are termed the Uppsala cohort (GSE3494/GSE4922/GSE6532; samples collected in Uppsala County, Sweden), the Stockholm cohort (GSE1456; samples collected at the Karolinska Hospital in Stockholm, Sweden), the EMC cohort (GSE2034/GSE5327; samples collected at the Erasmus Medical Center, Rotterdam, the Netherlands), and the TRANSBIG cohort (GSE7390; samples collected by the translational research network managed by the Breast International Group). The Uppsala and EMC cohorts contained samples processed at the same institution that span multiple GEO accession numbers. These four cohorts used Affymetrix microarray technology. Where available, raw data (in the form of CEL files) were downloaded; otherwise, MAS5.0 normalized data were downloaded (CEL files were available for all studies except GSE2034 and GSE5327). All data preprocessing and MAS5.0 normalization were performed using R software and the justMAS function in the simpleAffy library from Bioconductor (no background correction, target intensity of 600; ref. 21). After normalization, gene expression data were extracted for the GPR30 probe 210640_s_at. ER status was provided via Supplementary Data in GEO.
Compounds and cell lines
E2, DES, and 2-aminoethyldiphenylborinate (2APB) were from Sigma-Aldrich. G-1 and fulvestrant (ICI 182,780, Faslodex) were from Tocris. Xestospongin C (XeC) and ryanodine (Ry) were from Calbiochem, EMD Biosciences. All agents were added to culture medium at 1:10,000 to 1:1,000 (v/v). Fura-2 AM and all cell culture reagents were from Invitrogen. MCF-7:WS8 human mammary carcinoma cells were used in all experiments indicating MCF-7 cells; they were clonally selected for sensitivity to E2-stimulated growth (22). SKBr3 cells were purchased from the American Type Culture Collection. Both cell lines were maintained in estrogenized medium [RPMI-1640 plus 10% fetal bovine serum (FBS)]. MCF-7 cells were switched to estrogen-free medium (phenol red–free RPMI-1640 plus 10% 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). Target mRNA levels were normalized to PUM1 [pumilio homologue 1 (Drosophila)] mRNA levels (24). See Supplementary Materials and Methods for primer sequences. Data were analyzed by comparison with a serial dilution series of MCF-7 cell cDNA. Values in each group were averaged from four biological replicates (unless otherwise indicated), and each biological replicate was averaged from four technical replicates.
MCF-7 cells that had been maintained in estrogenized medium were transfected with siRNAs for 6 h in serum-free Opti-MEM (Invitrogen) using Dharmafect 1 (Dharmacon RNAi Technologies) followed by overnight recovery in estrogenized medium. The transfection was carried out a second time, and then the cells were immediately switched to estrogen-free medium and again allowed overnight recovery. siRNAs were transfected at 200 nmol/L final concentration, except in Ca2+ experiments in which siRNAs were transfected at 100 nmol/L and cotransfected with siGLO Green, a 6-carboxyfluorescein–labeled inactive double-stranded RNA. See Supplementary Materials and Methods for ERα and GPR30 siRNA sequences (Dharmacon RNAi Technologies).
Cytoplasmic Ca2+concentrations were measured using Fura-2 AM and microscopy as previously described (25). Flat SKBr3 cells were imaged because they were the major morphologic cell type, whereas rounded SKBr3 cells were not imaged. All compounds were administered at 1 min.
Cell growth was assessed using Hoechst 33258 (Invitrogen) and compared with a standard curve of serial-diluted calf thymus DNA as previously described (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 described (27). Data were analyzed using FlowJo 7.2.5 for Windows (Tree Star).
Immunoblotting was carried out using 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; Santa Cruz Biotechnology), cyclin D1 (DCS-6; Santa Cruz Biotechnology), cyclin B1 (D-11; Santa Cruz Biotechnology), and β-actin (AC-15; Sigma-Aldrich). Membranes were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences).
Mann-Whitney tests were performed using Prism 4.03 for Windows (GraphPad Software). All other statistical analyses were performed using Excel 2003 for Windows (Microsoft). Error is represented by SEMs in Ca2+ mobilization experiments and by SDs in all other experiments. Also in the Ca2+ experiments, P values reflect one-way ANOVA comparing the maximum Ca2+ concentration versus the concentration at the treatment start time, unless otherwise stated.
Increased GPR30 mRNA expression associates with ERα-positive status in 1,250 breast carcinomas
Evidence of a relationship between GPR30 and ERα expression was sought by mining publicly available and well-annotated gene expression microarray data sets across five independent cohorts comprising 1,250 breast carcinomas. In the NKI cohort (n = 254), data were collected using two-color oligonucleotide microarrays (Fig. 1A). According to the nonparametric Mann-Whitney rank sum test, GPR30 mRNA levels were significantly higher in ERα-positive versus ERα-negative tumors (P < 0.0001). The upper range of GPR30 expression was 7.7-fold higher on a linear scale in the ERα-positive compared with ERα-negative carcinomas. In addition, GPR30 and ERα mRNA levels correlated as continuous variables (Pearson's coefficient ρ = 0.30, adjusted P < 0.0001). The other four cohorts used one-color Affymetrix oligonucleotide microarrays (Fig. 1B). In each of these four cohorts, GPR30 mRNA levels were significantly higher in the ERα-positive compared with the ERα-negative breast cancers (Uppsala cohort, n = 244, P = 0.040; Stockholm cohort, 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 investigated. MCF-7 cells were treated with E2 or without E2 (control, vehicle only) over a 96-hour time course followed by determination of ERα and GPR30 mRNA levels by qPCR. As expected, E2 steadily downregulated ERα mRNA levels by 59% over 96 hours (Fig. 2A). E2 also downregulated GPR30 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 mRNA expression decreased in a concentration-dependent manner from 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 expression relative to control treatment by 54% P = 0.0009), which was very similar to the effect of E2 (Fig. 2D). Fulvestrant completely blocked E2 and DES effects. Therefore, E2 likely acted via ER and not GPR30 to transiently downregulate GPR30 mRNA expression.
GPR30 and not ERα mediates E2-induced Ca2+ mobilization responses
To begin to delineate whether endogenous ERα and/or GPR30 mediates E2-induced Ca2+ responses in breast cancer cells, changes in intracellular Ca2+ concentrations [Ca2+]i were measured in ER-positive MCF-7 and ER-negative SKBr3 breast cancer cells at the single-cell level using 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-negative SKBr3 cells (Fig. 3B), the rank order of ligand-induced cytosolic Ca2+ increases was the same as in MCF-7 cells, but the magnitude of the increases was much greater and the responses were transitory instead of sustained. In ER-negative SKBr3 cells, E2 induced oscillating increases in [Ca2+]i with an average maximum of 294 ± 1.6 nmol/L (n = 36 cells, P = 0.0037). G-1 and 4OHT induced transitory increases in [Ca2+]i of 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, whereas DES did not ([Ca2+]i change = 51 ± 1.3 nmol/L, n = 21 cells, P = 0.26). Therefore, because G-1 and two ER ligands that also bind GPR30, E2 and 4OHT, but not ER-selective DES, elicited 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 receptors (IP3R) and Ry receptors (RyR; ref. 28), were tested for whether they mediated G-1–induced Ca2+ mobilization. The pharmacologic probes 2APB and XeC, both of which inhibit IP3Rs, and Ry, which at high concentrations blocks RyRs, were used. Cells were pretreated for 30 min before inducing Ca2+ responses with G-1. In MCF-7 cells, both 2APB and XeC blocked 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 blocked G-1–induced [Ca2+]i increases by 80% (Ry + G-1 versus G-1 alone, P = 0.0006), whereas XeC did not. In addition, 2APB allowed G-1 to almost fully induce [Ca2+]i increases, although the 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 but to 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 were transfected with siRNAs targeting these receptors (characterization of siRNAs in Supplementary Materials and Methods) and a nontargeting siRNA pool as a control. First, the GPR30 siRNA was validated by showing that it led to an almost complete blockade of G-1–induced Ca2+ responses ([Ca2+]i increase = 58 ± 1.3 nmol/L, n = 19 cells, P = 0.62; Fig. 4A). Next, E2-induced Ca2+ mobilization responses were investigated (Fig. 4B). In nontargeting siRNA–transfected cells, E2 induced an [Ca2+]i increase of 159 ± 1.6 nmol/L (n = 14 cells, P = 0.0075). However, in ERα siRNA–transfected cells, E2 caused almost a 2-fold further rise in [Ca2+]i (314 ± 3.2 nmol/L, n = 17 cells; P = 0.0006). In GPR30 siRNA–transfected cells, the E2-induced Ca2+ response was blocked ([Ca2+]i increase = 52 ± 0.8 nmol/L, n = 16 cells, P = 0.35). ERα and GPR30 expression were depleted in the appropriate siRNA-transfected cells (Fig. 4C). However, GPR30 mRNA levels were increased by 73% in ERα-depleted cells, a finding consistent with the prior observation that E2 and DES repressed GPR30 expression (Fig. 2B–D). Hence, the 2-fold potentiation of E2-induced [Ca2+]i in ERα-depleted cells likely reflected, at least in part, the increased GPR30 expression.
GPR30 functions to promote growth of ER-negative SKBr3 cells but to inhibit growth of ER-positive MCF-7 cells
The role of GPR30 in cellular proliferation was examined by transfecting cells with nontargeting and GPR30 siRNAs and then measuring cellular DNA mass after 5 days of growth. First, SKBr3 cells were evaluated (Fig. 5A). The number of cells seeded in each group was similar as indicated by a lack of difference in DNA masses at day 0. After 5 days of growth, DNA mass was 45% lower in GPR30 siRNA compared with nontargeting siRNA–transfected cells (P < 0.0001). Thus, the function of GPR30 was to promote growth of SKBr3 cells, in accordance with other reports (9, 10). Next, MCF-7 cells were similarly evaluated (Fig. 5B). Again, equivalent numbers of nontargeting and GPR30 siRNA–transfected cells were seeded as indicated by DNA masses at day 0. After 5 days, GPR30 depletion did not affect basal growth (control treatment). However, GPR30 depletion did potentiate E2-stimulated growth by 2.1-fold (nontargeting versus GPR30 siRNA–transfected cells, P < 0.0001). Analysis of progesterone receptor (PgR) and TFF1 (pS2) mRNA levels by qPCR indicated no significant differences in their induction by E2 between nontargeting and GPR30 siRNA–transfected cells (data not shown). Therefore, in contrast to SKBr3 cells, the function of GPR30 in MCF-7 cells was to 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-1 blocked the concentration-dependent growth stimulatory response of E2 (all E2 treatment groups versus paired E2 + G-1 treatment groups, P = 0.0001, one-way ANOVA); in particular, G-1 inhibited 10−10 mol/L E2-stimulated growth by 77% relative 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 cancer cells 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 progression was investigated. MCF-7 cells were synchronized by estrogen withdrawal and then treated with E2 and G-1 for 24 hours followed by propidium iodide staining and flow cytometric analysis (Fig. 6A). Treatment with G-1 alone significantly decreased the proportion of S-phase cells from 19.8% (control) to 14.7% (G-1; P < 0.0001). Importantly, the addition of G-1 to E2 led to retention of an additional 11.6% of the 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 versus 24.5% in E2 + G-1–treated cells, P < 0.0001). Therefore, G-1 blocked E2-stimulated cells from cell cycle progression at the G1 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 with E2 and G-1 and then collected at 24, 48, and 72 hours for immunoblot analysis. Both p53 and p21 proteins were upregulated in G-1 and E2 + G-1–treated cells across all time points compared with control-treated cells (Fig. 6B). As expected, E2 upregulated both cyclins D1 and B1 across the time course compared with control treatment, whereas G-1 alone did not (Fig. 6C). However, the addition of G-1 to E2 potentiated the upregulation of cyclin D1 while nearly completely preventing cyclin B1 accumulation compared with E2 alone across the time points. Because cyclin D1 is induced during G1 phase and degraded in S phase (29, 30), whereas cyclin B1 accumulates during G2-phase and degrades on M-phase entry (31), these data are consistent with G-1 blocking cell cycle progression in G1 phase before cyclin D1 degradation occurred and before cyclin B1 accumulated.
Filardo and colleagues (5) and Kuo and colleagues (6) have previously shown in breast carcinomas a positive association between GPR30 and ERα expression by immunohistochemistry and qPCR, respectively. We confirmed and extended this finding by examining gene expression microarray data sets of five independent patient cohorts comprising 1,250 breast cancers. In all cohorts, high GPR30 mRNA levels showed an association with ERα positivity (Fig. 1). It is unknown why high GPR30 levels would be selected for in ERα-positive breast carcinomas given GPR30 attenuates growth of ER-positive breast cancer, but some GPR30-dependent functions may be necessary for tumorigenesis and cell survival, such as activation of adenylyl cyclase, PI3K, and MAPK (1, 2). Additional roles of GPR30 may be needed for disease progression, such as in cell migration (15, 32), which may then promote metastasis (5).
The interplay between GPR30 and ERα was further investigated using MCF-7 breast cancer cells. E2 repressed GPR30 expression 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). The inverse functional relationship between ERα and GPR30 was also shown by depleting ERα, which led to derepression of GPR30 mRNA expression and consequently potentiated E2-induced Ca2+ mobilization responses (Fig. 4). E2 is known to downregulate ERα expression in MCF-7 cells as a negative feedback regulatory loop to prevent overresponsiveness (26). Likewise, GPR30 may also be negatively regulated by E2 via ER to prevent excessive GPR30-dependent activity, such as aberrantly 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 to the lack of ERs in SKBr3 cells, which translated into a lack of 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 SKBr3 but to inhibit MCF-7 cellular proliferation. Also in MCF-7 cells, G-1 profoundly inhibited E2-stimulated (Fig. 5C) and DES-stimulated growth (Fig. 5D) as well as decreased the percentage of cells entering S phase (Fig. 6A). However, these G-1 effects occurred in both the presence and the absence of E2. These findings in MCF-7 cells complement those of Ahola and colleagues (11) who reported that transient GPR30 overexpression decreased the percentage of proliferating MCF-7 cells independent of E2. Indeed, GPR30 likely does not directly regulate ER transcriptional activity because there were no significant differences in E2-induced mRNA expression of well-established ER target genes PgR and TFF1 between GPR30 siRNA–transfected and nontargeting siRNA–transfected cells (data not shown).
Rather, we propose that GPR30 antagonizes growth of MCF-7 cells by inducing sustained increases in cytosolic Ca2+ concentrations (Figs. 3A and 4), in contrast to transitory increases in SKBr3 cells where GPR30 promotes growth. Aberrant sustained increases in intracellular Ca2+ levels can lead to inhibition of proliferation and induce apoptosis (33). For example, the plasma membrane Ca2+-ATPase (PMCA) pumps Ca2+ across the plasma membrane out of the cell to lower cytosolic Ca2+ levels after Ca2+ increases. Partial inhibition of PMCA in MCF-7 cells causes a moderate increase in intracellular Ca2+ levels, which leads to inhibition of proliferation by altering cell cycle kinetics (34). Additionally, the mechanism of action of numerous antitumor agents involves increases in [Ca2+]i (35).
It is possible that differences in GPR30-coupled Ca2+ signaling, which mediate sustained versus transitory responses, associate with ER status. In support of this hypothesis, GPR30 was coupled to differing Ca2+ channels: to IP3Rs in ER-positive MCF-7 cells but to RyRs in ER-negative SKBr3 cells. Alternatively, sustained versus transitory Ca2+ responses could have been due to potential alterations in factors that participate in lowering cytosolic Ca2+, such as plasma membrane or sarcoplasmic/endoplasmic reticulum Ca2+-ATPase pumps. We intend to explore these possibilities involving differing Ca2+ responses in future studies.
As shown by propidium iodide staining and flow cytometry, G-1 induced a cell cycle block at the G1 phase (Fig. 6A). Consistent with a G1-phase arrest, G-1 increased accumulation of the tumor suppressor p53, the CDK-I p21, and the G1-phase–specific cyclin D1 but prevented E2-induced accumulation of the G2/M-phase–specific cyclin B1 (Fig. 6B and C). Ca2+ signaling has been shown to induce p53 via activation of cyclic AMP–responsive element binding protein (28). In MCF-7 cells, p53 induction by E2 is Ca2+ and calmodulin kinase IV dependent (36). In addition, aberrant Ca2+ mobilization in response to anticancer/cytotoxic agents correlates with p53 induction (35). Thus, G-1 could lead to p53 induction via Ca2+ mobilization in MCF-7 cells. Then, p53 could induce p21 via a p53 response element to mediate arrest in G1 phase of the cell cycle (28).
As a SERM, tamoxifen acts as an antiestrogen in ER-positive breast cancer but as an estrogen in the endometrium and bone (37). Similarly, G-1 inhibits growth of ER-positive MCF-7 breast cancer cells but promotes growth of the endometrium (16) and plays an important role in promoting bone growth in vivo (38, 39). Thus, the tissue-specific proliferative effects of G-1 may parallel those of tamoxifen. It is interesting to speculate that 4OHT-induced Ca2+ mobilization (Fig. 3A and B) may be involved in some of the tissue-specific effects of tamoxifen.
Taken together, GPR30 inhibits growth of ERα-positive breast cancer. Our studies also indicate that pharmacologic activation of GPR30 shows promise in combating ER-positive breast cancer. G-1 would also probably be well tolerated because it, like E2, exerts beneficial effects against an animal model of multiple sclerosis but without E2-associated side effects (17, 40). Thus, G-1 may represent the first in a new class of therapeutically relevant agents for use alone or in conjunction with conventional antihormonal therapeutics in breast cancer.
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 those of 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 and CA118100 (University of New Mexico Cancer Center), and NS18710 and HL51314 (N.J. Dun).
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