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. 2014 Oct 2;5(10):e1428.
doi: 10.1038/cddis.2014.398.

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo

W Wei  1 , Z-J Chen  2 , K-S Zhang  3 , X-L Yang  4 , Y-M Wu  1 , X-H Chen  1 , H-B Huang  2 , H-L Liu  4 , S-H Cai  5 , J Du  1 , H-S Wang  1
Affiliations

The activation of G protein-coupled receptor 30 (GPR30) inhibits proliferation of estrogen receptor-negative breast cancer cells in vitro and in vivo

W Wei et al. Cell Death Dis. .

Erratum in

Abstract

There is an urgent clinical need for safe and effective treatment agents and therapy targets for estrogen receptor negative (ER-) breast cancer. G protein-coupled receptor 30 (GPR30), which mediates non-genomic signaling of estrogen to regulate cell growth, is highly expressed in ER--breast cancer cells. We here showed that activation of GPR30 by the receptor-specific agonist G-1 inhibited the growth of ER--breast cancer cells in vitro. Treatment of ER--breast cancer cells with G-1 resulted in G2/M-phase arrest, downregulation of G2-checkpoint regulator cyclin B, and induction of mitochondrial-related apoptosis. The G-1 treatment increased expression of p53 and its phosphorylation levels at Serine 15, promoted its nuclear translocation, and inhibited its ubiquitylation, which mediated the growth arrest effects on cell proliferation. Further, the G-1 induced sustained activation and nuclear translocation of ERK1/2, which was mediated by GPR30/epidermal growth factor receptor (EGFR) signals, also mediated its inhibition effects of G-1. With extensive use of siRNA-knockdown experiments and inhibitors, we found that upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was also involved in G-1-induced cell growth arrest. In vivo experiments showed that G-1 treatment significantly suppressed the growth of SkBr3 xenograft tumors and increased the survival rate, associated with proliferation suppression and upregulation of p53, p21 while downregulation of cyclin B. The discovery of multiple signal pathways mediated the suppression effects of G-1 makes it a promising candidate drug and lays the foundation for future development of GPR30-based therapies for ER- breast cancer treatment.

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Figures

Figure 1
Figure 1
The activation of GPR30 inhibited the proliferation of ER− breast cancer cells. (a) SkBr3 and MDA-MB-231 cells were treated with various concentrations (10−8 to 10−5 M) of G-1 for 48 h, and then cell viability was assessed by CCK-8 kit. (b) SkBr3 and MDA-MB-231 cells were treated with 1 μM G-1 for 24, 36, and 48 h, respectively, and then cell viability was assessed by CCK-8 kit. (c) After 24 h pre-transfection with si-NC or si-GPR30 siRNAs, the protein levels of GPR30 were analyzed by western blotting. (d) After 24 h pre-transfection with si-NC or si-GPR30 siRNAs, SkBr3 or MDA-MB-231 cells were further treated with 1 μM G-1 for 48 h, and then cell viability was assessed by CCK-8 kit. Data were presented as means±S.D. of three independent experiments (ten independent experiments for cell viability). *P<0.05 compared with control; **P<0.01 compared with control
Figure 2
Figure 2
The activation of GPR30 induced G2/M cell-cycle arrest. (a) SkBr3 cells were synchronized at the G1/S transition by a double TdR block, and then treated with 1 μM G-1 for the indicated times. The cycle cycles were analyzed by FCM; (b) SkBr3 cells were treated with 1 μM G-1 for 24 h, and then the mRNA levels of cyclins were measured by qRT-PCR; SkBr3 (c) and MDA-MB-231 (d) cells were treated with 1 μM G-1 for 72 h, and then protein levels of cyclins were analyzed by western blotting; (e) SkBr3 and MDA-MB-231 cells were treated with 1 μM G-1 for the indicated times, the protein levels of cyclin B were measured by western blotting. Data were presented as means±S.D. of three independent experiments. *P<0.05 compared with control
Figure 3
Figure 3
The activation of GPR30 induced mitochondrial-related apoptosis. (a) SkBr3 and MDA-MB-231 cells were treated with increasing concentrations of G-1 for 48 h, stained with annexin V-FITC and PI, and then analyzed by flow cytometry for cell apoptosis. (b) SkBr3 cells were treated with G-1 as the indicated concentrations for 24 h, and then JC-1, the mitochondria-specific dye, was added to measure the membrane polarity (ΔΨm) and cell apoptosis. Apoptotic cells mainly show green fluorescence (FITC), while healthy cells show red fluorescence (PE). (c) SkBr3 cells were treated with various concentrations of G-1 for 4 h, and then loaded with CM-H2DCFDA. The fluorescence intensity was measured by FCM. (d) SkBr3 cells were treated with G-1 as the indicated concentrations for 48 h, and then Bcl-2, Bax, Bim, and caspase-3 protein expression levels were analyzed by western blotting. Data were presented as means±S.D. of three independent experiments
Figure 4
Figure 4
p53 mediated growth arrest of G-1 in ER− breast cancer cells. SkBr3 cells were treated with 1 μM G-1 for the indicated time periods, and then mRNA levels of p53 (a) and MDM2 (b) were quantified by real-time PCR, the protein levels of p53 (c) were detected by western blotting. SkBr3 cells transfected with si-p53 or si-NC for 24 h, and the mRNA and protein expression of p-53 were measured by qRT-PCR and western blotting (d), respectively. The transfected cells were then stimulated with or without G-1 (1 μM) for another 24 h, the cell viability was assessed by CCK-8 kit (e). (f) After treatment with G-1 for 24 h, nuclear and cytoplasmic cellular fractions were isolated by differential lysis. The levels of p53 in nuclear and cytoplasmic cellular fractions were detected by western blotting. (g) SkBr3 cells were treated with or without G-1 (1 μM) for 24 h. After fixation, the cellular location of p53 (red) was examined by immunofluorescence staining and nuclei were stained with Hoechst (blue). (h) SkBr3 cells treated with 1 μM G-1 for the indicated time periods. After p53 was immunoprecipitated from equal amount of lysates, the ubiquitination of p53 was examined by Western blotting. (i) SkBr3 cells were treated with 1 μM G-1 for the indicated time periods, and then p-Ser15-p53 and p53 were measured by western blotting. Data were presented as means±S.D. of three independent experiments. *P<0.05 compared with control
Figure 5
Figure 5
Activation of ERK by GPR30/EGFR mediated the growth arrest effects of G-1. SkBr3 (a) and MDA-MB-231 (b) cells were treated with 1 μM G-1 for the indicated time periods, and then the phosphorylation and total protein levels of ERK1/2, JNK, and p-38 were detected by western blotting. (c) SkBr3 cells were treated with or without G-1 (1 μM) for 24 h. After fixation, the cellular location of p-ERK1/2 (green) was examined by immunofluorescence staining and nuclei were stained with DAPI (blue). SkBr3 cells treated with 10 μM MEK inhibitor PD98059 (PD), PI3K inhibitor LY294002 (LY), p38 MAPK inhibitor SB203580 (SB), or EGFR inhibitor AG1478 (AG) for 24 h, and then treated with 1 μM G-1 for further 48 h, the protein levels of cyclin B, cyclin A, cyclin E, p-ERK1/2, and p21 were detected by western blotting (d), and the cell proliferation was measured by CCK-8 kit (e)
Figure 6
Figure 6
Upregulation of p21 by the cross-talk of GPR30/EGFR and p53 was involved in G-1-induced ER− breast cancer cell growth arrest. SkBr3 cells transfected with si-p21 or si-NC for 24 h, and then protein expression of p-21 was measured by western blotting (a). The transfected cells were then stimulated with or without G-1 (1 μM) for another 24 h, the cell viability was assessed by CCK-8 kit (b). SkBr3 cells treated with 10 μM MEK inhibitor PD98059 (PD), PI3K inhibitor LY294002 (LY), p38 MAPK inhibitor SB203580 (SB), or EGFR inhibitor AG1478 (AG) for 24 h, and then treated with 1 μM G-1 for further 24 h (c) or 12 h (d), the protein levels of p21, p-Ser15-p53, and p53 were detected by western blotting
Figure 7
Figure 7
Activation of GPR30 by G-1 inhibited the ER− breast tumor xenograft growth in vivo. (a) After treatment with or without 1 μM G-1 for 24 h, MDA-MB-231 cells injection subcutaneously into the fourth right mammary fat pad at the base of the nipple of nude mice with 50% Matrigel. Tumor size was measured at the indicated time intervals. (b) The survival rate of mice treated during the experiment. The survival rate of mice in G-1 treated group was significantly (P<0.05) higher than that of the control group from 27 days on after tumor implantation (indicated by the black arrow). (c) The proteins related to the growth inhibition effects of G-1 were determined by western blotting analysis in the tumor lysates from the control and G-1 treated group. *P<0.05 compared with control; **P<0.01 compared with control
Figure 8
Figure 8
A proposed model to illustrate the mechanism of GPR30 mediated growth arrest and apoptosis of ER− breast cancer cells
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