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, 5 (3), 146-160

GPER Mediates Estrogen-Induced Signaling and Proliferation in Human Breast Epithelial Cells and Normal and Malignant Breast

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GPER Mediates Estrogen-Induced Signaling and Proliferation in Human Breast Epithelial Cells and Normal and Malignant Breast

Allison L Scaling et al. Horm Cancer.

Abstract

17β-Estradiol (estrogen), through receptor binding and activation, is required for mammary gland development. Estrogen stimulates epithelial proliferation in the mammary gland, promoting ductal elongation and morphogenesis. In addition to a developmental role, estrogen promotes proliferation in tumorigenic settings, particularly breast cancer. The proliferative effects of estrogen in the normal breast and breast tumors are attributed to estrogen receptor α. Although in vitro studies have demonstrated that the G protein-coupled estrogen receptor (GPER, previously called GPR30) can modulate proliferation in breast cancer cells both positively and negatively depending on cellular context, its role in proliferation in the intact normal or malignant breast remains unclear. Estrogen-induced GPER-dependent proliferation was assessed in the immortalized nontumorigenic human breast epithelial cell line, MCF10A, and an ex vivo organ culture model employing human breast tissue from reduction mammoplasty or tumor resections. Stimulation by estrogen and the GPER-selective agonist G-1 increased the mitotic index in MCF10A cells and proportion of cells in the cell cycle in human breast and breast cancer explants, suggesting increased proliferation. Inhibition of candidate signaling pathways that may link GPER activation to proliferation revealed a dependence on Src, epidermal growth factor receptor transactivation by heparin-bound EGF and subsequent ERK phosphorylation. Proliferation was not dependent on matrix metalloproteinase cleavage of membrane-bound pro-HB-EGF. The contribution of GPER to estrogen-induced proliferation in MCF10A cells and breast tissue was confirmed by the ability of GPER-selective antagonist G36 to abrogate estrogen- and G-1-induced proliferation, and the ability of siRNA knockdown of GPER to reduce estrogen- and G-1-induced proliferation in MCF10A cells. This is the first study to demonstrate GPER-dependent proliferation in primary normal and malignant human tissue, revealing a role for GPER in estrogen-induced breast physiology and pathology.

Figures

Fig. 1
Fig. 1. 17β-Estradiol stimulates proliferation in MCF10A cells
Mitotic index was assessed as a surrogate for proliferation by immunofluorescence using an anti-Histone H3 (phospho-ser10) (pH3) antibody in MCF10A cells cultured in the presence of vehicle control or the indicated concentrations of E2 for 24 hr (A). Data represents the average of three independent experiments. Results are expressed as mean ± SEM and statistical significance (p ≤ .05) was assessed by one-way ANOVA followed by a Dunnett’s test (*, significantly different relative to control). GPER expression was assessed in MCF10A cells by immunofluorescence (B; scale bar = 75 μM) and western immunoblotting (C), probing with an anti-human GPER C-terminal peptide antibody. Reduced GPER protein and RNA expression following siRNA knockdown was also confirmed (representative experiment shown in C). Cells transfected with non-specific (scrambled) control siRNA express normal levels of protein (C). (D), Densitometric quantitation of three independent GPER immunoblots following no transfection (NT), or 72 hr following transfection with control siRNA or GPER-specific siRNA. Quantitation is normalized to β-actin immunodetection. Results are expressed as mean ± SEM and statistical significance (p = .0176) was assessed by one-way ANOVA followed by a Dunnett’s test (*, statistically significant relative to non-transfected cells).
Fig. 2
Fig. 2. E2 and G-1-induced proliferation is dependent on GPER in MCF10A cells
Mitotic index as a surrogate for proliferation was assessed in MCF10A cells grown on glass coverslips in the presence of indicated concentrations of GPER agonists (E2, G-1), antagonist (G36), or combinations, for 24 hr (A, B). Proliferation was also assessed after GPER siRNA or control siRNA transfection followed by 24-hr stimulation with E2 or G-1 (C). Mitotic index was quantified by immunofluorescence using an anti-pH3 antibody. GPER knockdown was confirmed by Western immunoblotting (D). Data is representative of a minimum of three independent experiments. Results are expressed as mean ± SEM and statistical significance (P ≤ .05) was assessed by one-way ANOVA followed by a Dunnett’s test (*, significantly different relative to control; #, significantly different relative to E2 or G-1; ns, not significant).
Fig. 3
Fig. 3. GPER activation induces activation of the MAPK signaling cascade
MCF10A cells were stimulated with indicated concentrations of E2 or G-1 alone or in combination with GPER antagonist G36, for 15 min (A). Lysates were prepared and immunoblotted with antibodies specific to phospho-ERK (pERK). Equal protein loading was confirmed by β-actin immunoblotting. Histograms represent fold change (pERK relative to actin) in pERK protein expression, relative to control-treated cells. pERK was also assayed in cells transfected with control or GPER siRNA-treated cells 72 hr after transfection, and then stimulated with E2 or G-1 for 15 min (B). Data are representative of three independent experiments. Results are expressed as mean ± SEM and statistical significance (P ≤ .05) was assessed by one-way ANOVA followed by a Dunnett’s test (*, significantly different relative to control; #, significantly different relative to E2 or G-1).
Fig. 4
Fig. 4. GPER-dependent activation of MAPK (ERK1 and ERK2) is dependent on Src activation but not MMP activation in MCF10A cells
Signal transduction inhibitors were tested for their ability to block GPER-dependent ERK activation in MCF10A cells. Cells were pre-incubated for 30 min with either control, AG1478 (A; 250 nM, inhibitor of EGFR), U0126, (A; 10 M, inhibitor of MEK), PP2 (A; 10 nM, inhibitor of Src), GM6001 (B, 25 M, inhibitor of MMPs), CRM-197 (B, 0.2 mg/mL, inhibitor of HB-EGF or HB-EGF neutralizing antibody (B, 6 ng/mL), then stimulated with 10 nM EGF, 10 nM E2 or 100 nM G-1 for 15 min. Lysates were immunoblotted with anti-phospho-ERK antibody. Histograms represent fold change in pERK protein expression relative to -actin loading control. Data are representative of three independent experiments. Results are expressed as mean ± SEM and statistical significance (P ≤ .05) was assessed by one-way ANOVA followed by a Dunnett’s test. (* significantly different relative to control)
Fig. 5
Fig. 5. GPER-dependent proliferation requires transactivation of EGFR
Signal transduction inhibitors were tested for their ability to block GPER-dependent proliferation in MCF10A cells. Cells were pre-incubated for 30 min with either vehicle (control), AG1478 (A; 250 nM, EGFR inhibitor), U0126 (A; 10 M, MEK inhibitor), LY294002 (A; 10 M, PI3K inhibitor), PP2 (B; 10 nM, Src inhibitor), GM6001 (B; 25 M, MMP inhibitor), CRM197 (B; 0.2 mg/mL HB-EGF release inhibitor) or HB-EGF neutralizing antibody (B; 6 ng/mL) and then stimulated with 10 nM EGF, 10 nM E2 or 100 nM G-1 for 24 hr. Mitotic index as a surrogate for proliferation was quantified by immunofluorescence using an anti-pH3 antibody. Data are representative of a minimum of three independent experiments. Results are expressed as mean ± SEM and statistical significance (P ≤ .05) was assessed by one-way ANOVA followed by a Dunnett’s test (* significantly different relative to control).
Fig. 6
Fig. 6. Estrogen-induced GPER activation stimulates proliferation in a 3D model of breast morphogenesis
MCF10A cells were grown in 3D on Matrigel™ basement membrane in the presence of 10 nM E2 or 100 nM G-1 for six days. Mitotic index as a surrogate for proliferation (B) was quantified by immunofluorescence using an anti-pH3 antibody. A representative spheroid immunolabeled with anti-pH3 (green) and anti-gamma tubulin (red) is shown (A; arrow indicates anti-pH3 immunolabeled chromatin; arrowhead indicates mitotic spindle). Total cell number per spheroid was quantified for each treatment group (C). Data are representative of three independent experiments (scale bar = 25 μM). Results are expressed as mean ± SEM and statistical significance (P ≤ .05) was assessed by one-way ANOVA followed by a Dunnett’s test (*, significantly different relative to control).
Fig. 7
Fig. 7. E2 and G-1 promote proliferation in normal human breast tissue
(A) GPER protein expression was detected in epithelia and stroma by immunohistochemistry on tissue sections. (B) ERα protein expression was detected in nuclei of breast epithelia. Breast epithelial proliferation was quantified by immunofluorescence using anti-Ki67 antibody in the presence of GPER agonists E2 and G-1 (C, D) and antagonist G36 (D) in alveolar structures within normal human breast tissue explants. Each treatment group consisted of tissue samples from a minimum of five different patients (scale bars = 50 μM). Results are expressed as mean ± SEM, and statistical significance (P ≤ 0.05) was assessed by one-way ANOVA followed by a Dunnett’s t-test (* significantly different relative to control, # significantly different relative to E2 or G-1).
Fig. 8
Fig. 8. E2 and G-1 promote proliferation in tumorigenic human breast tissue
(A) GPER protein expression was detected in breast tumor cells by immunohistochemistry on tissue sections. (B) Tumor cell proliferation was quantified by immunofluorescence using anti-Ki67 antibody in the presence of GPER agonists E2 and G-1 and antagonist G36. Each treatment group consisted of tissue samples from a minimum of five different patients (scale bars = 50 μM). Results are expressed as mean ± SEM, and statistical significance (P ≤ 0.05) was assessed by one-way ANOVA followed by a Dunnett’s t-test (* significantly different relative to control, # significantly different relative to E2 or G-1).

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