ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ERK signaling pathway

Abstract

Background: ER-α36, a novel ER-α66 variant, has been demonstrated to promote tamoxifen resistance in breast cancer cells. However, the role and mechanisms of ER-α36 in cisplatin resistance of breast cancer cells remain unclear. This study investigates the expression and role of ER-α36 in cisplatin resistance of breast cancer cells and elucidates its underlying mechanisms.

Methods: The expression of ER-α36 and the proteins involved in nongenomic estrogen signaling was evaluated by western blot analysis. Cisplatin sensitivity was explored by CCK-8 assay, monolayer colony formation assay and apoptosis assays, respectively. ER-α36 siRNAs/shRNAs and overexpression vector were transfected into cells to down-regulate or up-regulate ER-α36 expression. Loss-and gain-of function assays were performed to investigate the role of ER-α36 in cisplatin sensitivity. The interaction between ER-α36 and EGFR/HER-2 were detected using CoIP. A mouse xenograft model of breast cancer was established to verify the role of ER-α36 in vivo.

Results: ER-α36 is expressed at higher levels in cisplatin-resistant breast cancer cells compared to cisplatin sensitive cells. Cisplatin induced up-regulation of ER-α36 in a dose-dependent manner in breast cancer cells. Overexpression of ER-α36 leaded to cell resistant to cisplatin and knockdown of ER-α36 in cisplatin-resistant breast cancer cells restored cisplatin sensitivity. The up-regulation of ER-α36 resulted in increased activation of nongenomic estrogen signaling, which was responsible for cisplatin resistance. Disruption of ER-α36-mediated nongenomic estrogen signaling with kinase inhibitors significantly inhibited cisplatin-induced expression of ER-α36 and increased cisplatin sensitivity. The in vivo experiment also confirmed that up-regulation of ER-α36 attenuated cisplatin sensitivity in a mouse xenograft model of breast cancer.

Conclusions: The results for the first time demonstrated that ER-α36 mediates cisplatin resistance in breast cancer cells through nongenomic estrogen signaling, suggesting that ER-α36 may serve as a novel target for cisplatin resistance and a potential indicator of cisplatin sensitivity in breast cancer treatment.

Keywords: Breast cancer; Cisplatin resistance; EGFR; ER-α36; HER-2.

Fig. 1

Effect of cisplatin on proliferation and ER-α36 expression of breast cancer cells. a MCF-7 and MCF-7/DDP cells were treated with increasing concentrations of cisplatin (DDP) for 48 h, and then cell proliferation was measured using CCK-8 assay kit. b Cisplatin sensitivity of MCF-7 and MCF-7/DDP cells was examined by monolayer colony formation assay. c ER-α36 protein expression in MCF-7 and MCF-7/DDP cells was analyzed using western blot. d The quantitative analysis of ER-α36 expression of (c). e MCF-7 cells were treated with or without 5 μg/mL cisplatin for 48 h, cisplatin-induced expression of ER-α36 was measured by western blot. f The quantitative analysis of cisplatin-induced ER-α36 expression of (e). g MCF-7, BT474 and MDA-MB-231 cells were treated with cisplatin at the indicated concentrations for 48 h and then the protein levels of ER-α36 were analyzed by western blot. h The quantitative analysis of cisplatin-induced ER-α36 expression of (g). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 2

Overexpression of ER-α36 contributes to cisplatin resistance in breast cancer cells. a MCF-7 cells stably overexpressing ER-α36 were screened using G418 for 4 weeks and identified by western blot. b The quantitative analysis of ER-α36 expression of (a). c MCF-7/V and MCF-7/ER-α36 cells were treated with increasing concentrations of cisplatin (DDP) for 48 h, and then cell proliferation was measured with CCK-8 assay kit. d Cisplatin sensitivity of MCF-7/V and MCF-7/ER-α36 cells was examined by monolayer colony formation assay. e, f MCF-7/V and MCF-7/ER-α36 cells were treated with or without 5 μg/mL cisplatin for 48 h. The cell nucleus were stained with Hoechst 33258 and then observed under fluorescence microscope. The representative images were shown and the typical apoptotic bodies were marked with white arrows (e). The cells were stained with annexin V-FITC/PI. Then the percentage of apoptotic cells was calculated using flow cytometry (f). ^*P < 0.05, ^** P < 0.01, ^***P < 0.001

Fig. 3

Knockdown of ER-α36 restores cisplatin sensitivity in cisplatin-resistant breast cancer cells. a, b MCF-7/DDP cells were transfected with siER-α36 and negative control siRNA (siNC) for 48 h. The cells were collected and analyzed for ER-α36 protein expression using western blot (a). The transfected MCF-7/DDP cells were treated with 5 μg/mL cisplatin (DDP) for 48 h, and cell proliferation was measured with CCK-8 assay kit (b). c ER-α36 expression in MCF-7/DDP cells expressing ER-α36 shRNA and the control cells analyzed using western blot. d Cisplatin sensitivity of MCF-7/DDP cells expressing ER-α36 shRNA and the control cells was examined by monolayer colony formation assay. e, f MCF-7/ER-α36 were treated as in (a, b), then the peotein level of ER-α36 was detected by western blot (e). The proliferation of the transfected MCF-7/ER-α36 cells was evaluated using CCK-8 assay kit (f). g ER-α36 expression in MCF-7/ER-α36 cells expressing ER-α36 shRNA and the control cells analyzed using western blot. h Cisplatin sensitivity of MCF-7/ER-α36 cells expressing ER-α36 shRNA and the control cells was examined by monolayer colony formation assay. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 4

Up-regulation of ER-α36 leads to increased activation of nongenomic estrogen signaling. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h. Then the protein levels of EGFR, HER-2, ER-α36, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. b, c The quantitative analysis of cisplatin-induced expression of ER-α36, EGFR, HER-2 and P-ERK/ERK of (a). d MCF-7, BT474 and MDA-MB-231 cells were treated with cisplatin at the indicated concentrations for 48 h and then the protein levels of EGFR, HER-2, ER-α36, ERK and P-ERK were analyzed by western blot. e The quantitative analysis of cisplatin-induced expression of P-ERK/ERK of (d). f MCF-7 cells were treated as in (a). The cell lysates were immunoprecipitated with anti-HER-2 or anti-EGFR antibodies. Then the immunoprecipitates were separated by SDS-PAGE and probed with anti-ER-α36 antibodies. Immunoprecipitation of IgG was used as a negative control

Fig. 5

Increased activation of ER-α36-mediated nongenomic estrogen signaling is responsible for cisplatin resistance. a, b MCF-7 and MCF-7/DDP cells (a) or MCF-7/V and MCF-7/ER-α36 cells (b) were harvested and the protein levels of ER-α36, EGFR, HER-2, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. c, d MCF-7/DDP (c) and MCF-7/ER-α36 (d) cells were transfected with siER-α36 and negative control siRNA (siNC) for 48 h. Then the cells were collected and the levels of ER-α36, EGFR, HER-2, P-ERK, ERK were analyzed by western blot

Fig. 6

Disruption of ER-α36-mediated nongenomic estrogen signaling increases cisplatin sensitivity in breast cancer cells. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the levels of ER-α36, total EGFR (EGFR) and phosphorylated EGFR (P-EGFR), total HER-2 (HER-2) and phosphorylated HER-2 (P-HER-2), total ERK (ERK) and phosphorylated ERK (P-ERK) were evaluated using western blot. b MCF-7 cells were treated as in (a), and then the cell proliferation was measured with CCK-8 assay kit. c MCF-7/ER-α36 cells were treated with 5 μg/mL cisplatin for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the total ERK (ERK) and phosphorylated ERK (P-ERK) was detected by western blot. d MCF-7/ER-α36 cells were treated as in (c), and then the cell proliferation was examined using CCK-8 assay kit. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 7

Up-regulation of ER-α36 attenuates cisplatin sensitivity in a nude mouse xenograft model. a, b The nude mice bearing MCF-7/V cell-derived and MCF-7/ER-α36 cell-derived subcutaneous tumors were treated intraperitoneally with or without cisplatin (DDP) for 2 weeks, the xenograft tumors were harvested (a) and the tumor volume was calculated as described in Methods (b). c ER-α36 protein levels in each group were evaluated by western blot. d MCF-7/V tumors treated with or without cisplatin were analyzed for ER-α36 protein levels using western blotting. ^*P < 0.05, ^** P < 0.01, ^***P < 0.001