The G protein-coupled receptor GPR30 inhibits proliferation of estrogen receptor-positive breast cancer cells

Abstract

The G protein-coupled receptor GPR30 binds 17beta-estradiol (E(2)) yet differs from classic estrogen receptors (ERalpha and ERbeta). GPR30 can mediate E(2)-induced nongenomic signaling, but its role in ERalpha-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 ERalpha-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, E(2) and DES, but not G-1, transiently downregulated both ER and GPR30, indicating that this was ER mediated. In Ca(2+) mobilization studies, GPR30, but not ERalpha, mediated E(2)-induced Ca(2+) responses because E(2), 4-hydroxytamoxifen (activates GPR30), and G-1, but not DES, elicited cytosolic Ca(2+) increases not only in MCF-7 cells but also in ER-negative SKBr3 cells. Additionally, in MCF-7 cells, GPR30 depletion blocked E(2)-induced and G-1-induced Ca(2+) mobilization, but ERalpha depletion did not. Interestingly, GPR30-coupled Ca(2+) 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 G(1) phase. Thus, GPR30 antagonizes growth of ERalpha-positive breast cancer and may represent a new target to combat this disease.

Figure 1. GPR30 mRNA expression shows an association with ERα-positive status in human breast carcinomas

(A) GPR30 mRNA levels in the NKI cohort derived from 2-color arrays. Expression values are normalized log₂ ratio intensity units corresponding to a single tumor cRNA hybridized against a pooled reference cRNA from all tumors. B) GPR30 mRNA levels in the Uppsala, Stockholm, EMC, and TRANSBIG cohorts all derived from 1-color arrays. Expression values are MAS5.0 normalized intensity units. A-B, Sample sizes of ERα-positive (ER+) and ER-negative (ER-) cancers are shown, and bars indicate the 75^th, 50^th (median), and 25^th percentiles. Significance was assessed using the non-parametric Mann-Whitney rank test.

Figure 2. E₂ represses ERα and GPR30 mRNA levels via ER and not GPR30 in MCF-7 cells

E₂ regulation of (A) ERα and (B) GPR30 mRNA levels across a time course. MCF-7 cells were treated with 10^-9 M E₂ or with the vehicle ethanol alone for 2, 6, 12, 24, 48, 72, and 96 h. (C) GPR30 mRNA levels in 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 ligands as determined by qPCR. Each data point represents the average of 6 (A-B), or 4 (C-D) biological replicates.

Figure 3. ER ligands that also activate GPR30 induce Ca²⁺ mobilization responses in both ER-positive MCF-7 and ER-negative SKBr3 cells

Ligand-induced Ca²⁺ responses (A-B), and blockade of G-1-induced responses using Ca²⁺ channel inhibitors (C-D) in MCF-7 and SKBr3 cells. Cells were loaded with Fura-2AM and intracellular Ca²⁺ concentrations [Ca²⁺]_i were determined in individual cells vs. time using fluorescence microscopy. Cells were perfused with all ligands at 10^-6 M starting at 1 m. 2APB was used at 10^-4 M, xestospongin C (XeC) at 10^-5 M, and ryanodine (Ry) at 10^-5 M. SKBr3 cells with flat, not rounded, morphology were imaged. G-1-induced Ca²⁺ traces in (A, B) were redrawn in (C, D), respectively.

Figure 4. GPR30 and not ERα mediates E₂-induced Ca²⁺ mobilization in MCF-7 cells

(A) G-1-induced and (B) E₂-induced Ca²⁺ responses. Cells were transfected with non-targeting pool, ERα, and GPR30 siRNAs. Transfected cells were labeled using siGLO Green and appear green. Ca²⁺ imaging was performed 48 h following the transfection as in Fig. 3. Low levels of basal [Ca²⁺]_i are visualized as blue and then green, while higher levels of [Ca²⁺]_i are seen as red and then white. (C) ERα protein levels were measured by immunobloting and GPR30 mRNA levels by qPCR in siRNA-transfected cells 48 h following transfection.

Figure 5. GPR30 promotes growth of ER-negative SKBr3 but inhibits growth of ER-positive MCF-7 cells

Proliferation of (A) SKBr3 and (B) MCF-7 cells transfected with non-targeting pool and GPR30 siRNAs. Cells were transfected and seeded at 15,000 cells per well in 24-well dishes. Media was replenished the day after seeding on day 0, and every other day thereafter. Cells were collected on days 0 and 5. SKBr3 cells were cultivated in their passage media, and MCF-7 cells in estrogen-free media supplemented with 10^-9 M E₂ (E2) or without E₂ (Control, C). Proliferation was assessed as cellular DNA mass (μg/well) using 24 replicate wells. GPR30 mRNA levels were determined by qPCR 48 h following the transfection in both cell lines, and in MCF-7 cells, after 24 h of 10^-9 M E₂ or control treatment. (C-D) Proliferation of MCF-7 cells over 6 days treated with (C) a serial dilution series of E₂, or with (D) 10^-9 M DES, in the absence and presence of 10^-6 M G-1. Twelve replicate wells were used per group.

Figure 6. G-1 inhibits cell cycle progression in E₂-stimulated MCF-7 cells by producing a block at G(1)-phase

(A) Cell cycle distribution as determined by propidium iodide staining of DNA content and flow cytometry. Cells were synchronized by 3 days cultivation in estrogen-free media, and then treated as indicated for 24 h. Thirty-thousand cells per sample and 3 replicates per group were collected. Representative histograms are shown. Immunoblot analyses of (B) p53 and p21, and of (C) cyclins D1 and B1 protein levels. MCF-7 cells were control (C), 10^-9 M E₂ (E) and 10^-6 M G-1 (G) -treated as indicated. Quantitated protein levels normalized to β-actin are indicated.