Environmental-relevant bisphenol A exposure promotes ovarian cancer stemness by regulating microRNA biogenesis

Abstract

Bisphenol A (BPA) is a ubiquitous environmental xenobiotic impacting millions of people worldwide. BPA has long been proposed to promote ovarian carcinogenesis, but the detrimental mechanistic target remains unclear. Cancer stem cells (CSCs) are considered as the trigger of tumour initiation and progression. Here, we show for the first time that nanomolar (environmentally relevant) concentration of BPA can markedly increase the formation and expansion of ovarian CSCs concomitant. This effect is observed in both oestrogen receptor (ER)-positive and ER-defective ovarian cancer cells, suggesting that is independent of the classical ERs. Rather, the signal is mediated through alternative ER G-protein-coupled receptor 30 (GPR30), but not oestrogen-related receptor α and γ. Moreover, we report a novel role of BPA in the regulation of Exportin-5 that led to dysregulation of microRNA biogenesis through miR-21. The use of GPR30 siRNA or antagonist to inhibit GPR30 expression or activity, respectively, resulted in significant inhibition of ovarian CSCs. Similarly, the CSCs phenotype can be reversed by expression of Exportin-5 siRNA. These results identify for the first time non-classical ER and microRNA dysregulation as novel mediators of low, physiological levels of BPA function in CSCs that may underlie its significant tumour-promoting properties in ovarian cancer.

Keywords: bisphenol A; cancer stem cells; microRNA; oestrogen receptors; ovarian cancer.

FIGURE 1

BPA stimulates MCS formation at low concentration. (A) HEYA8 and (B) SKOV‐3 were cultured with BPA at 0, 10, 50 and 100 nM in non‐adherent culture dish for 72 h. The number of tumour spheres generated was photographed, quantified by MTT assay and counted. (C) HEYA8 and (D) SKOV‐3 were cultured with BPA at 0 or 10 nM in non‐adherent culture dish for 0, 24, 72 and 120 h. The number of tumour spheres generated was photographed and counted. Bar = 100 μm. Experiments were repeated three times and results are presented as the mean ± SD and were analysed using unpaired student's t test.

FIGURE 2

BPA induces cancer stem cell phenotypes. (A) HEYA8 and (B) SKOV‐3 were cultured with BPA at 0 and 10 nM in non‐adherent culture dish for 72 h. Total RNA was extracted and reverse transcription‐PCR was performed using BMI‐1, Nanog, and c‐Kit sequence‐specific primers. The signal intensities were quantified by densitometric analysis and the amount was normalized for the amount of β‐Actin. (C) HEYA8 and (D) SKOV‐3 were cultured with BPA at 0 or 10 nM in non‐adherent culture dish for 72 h. After 72 h, tumour spheres were recovered, dissociated and incubated for another 72 h for secondary sphere formation. The number of tumour spheres generated was photographed, quantified by MTT assay and counted. Bar = 100 μm. Experiments were repeated three times and results are presented as the mean ± SD and were analysed using unpaired student's t test.

FIGURE 3

BPA induces MCS formation via GPR30. (A) HEYA8 and (B) SKOV‐3 transfected with nonspecific (NS) siRNA, ERRα siRNA ERRγ siRNA or GPR30 siRNA were cultured with BPA at 0 or 10 nM in non‐adherent culture dish for 72 h. The number of tumour spheres generated was photographed, quantified by MTT assay and counted. Total RNA was extracted and reverse transcription‐PCR was performed using ERα, ERβ, ERRα, ERRβ, ERRγ and GPR30 sequence‐specific primers. Bar = 100 μm. Experiments were repeated three times and results are presented as the mean ± SD and were analysed using unpaired student's t test.

FIGURE 4

BPA induces Exportin‐5 and miR‐21 expression. (A) HEYA8 and (B) SKOV‐3 were cultured with BPA at 0 and 10 nM in non‐adherent culture dish for 72 h. Total RNA was extracted and reverse transcription‐PCR was performed using Drosha, Dicer, and Exportin‐5 sequence‐specific primers. The signal intensities were quantified by densitometric analysis and the amount was normalized for the amount of β‐Actin. (C) HEYA8 and (D) SKOV‐3 were cultured with BPA at 0 and 10 nM in non‐adherent culture dish for 72 h. Total RNA was extracted and reverse transcription‐PCR was performed using miR‐21 sequence‐specific primer. The signal intensities were quantified by densitometric analysis and the amount was normalized for the amount of U6. Experiments were repeated three times and results are presented as the mean ± SD and were analysed using unpaired student's t test.

FIGURE 5

Silencing of Exportin‐5 abolishes BPA‐induced MCS formation. (A) HEYA8 and (B) SKOV‐3 were cultured with BPA at 0, 10, 50 and 100 nM in non‐adherent culture dish for 72 h. The number of tumour spheres generated was photographed, quantified by MTT assay and counted. (C) HEYA8 and (D) SKOV‐3 transfected with nonspecific (NS) siRNA or Exportin‐5 siRNA were cultured in non‐adherent culture dish for 72 h. Total RNA was extracted and reverse transcription‐PCR was performed using GPR30, Exportin‐5 and miR‐21 sequence‐specific primers. Experiments were repeated three times and results are presented as the mean ± SD and were analysed using two‐way anova.

FIGURE 6

The use of GPR30 antagonist G15 abolishes BPA‐induced MCS formation. (A) HEYA8 and (B) SKOV‐3 were cultured with BPA at 0 and 10 nM in non‐adherent culture dish for 72 h with G15 or DMSO as the control. The number of tumour spheres generated was photographed, quantified by MTT assay and counted. (C) HEYA8 and (D) SKOV‐3 were cultured with BPA at 0 and 10 nM in non‐adherent culture dish for 72 h with G15 or DMSO as the control. Total RNA was extracted and reverse transcription‐PCR was performed using Exportin‐5 and miR‐21 sequence‐specific primers. The signal intensities were quantified by densitometric analysis and the amount was normalized for the amount of β‐Actin or U6 respectively. Bar = 100 μm. Experiments were repeated three times and results are presented as the mean ± SD and were analysed using two‐way anova.

FIGURE 7

Anti‐miR21 inhibits CSC viability and spheroid formation ability. (A) HEYA8 and (B) SKOV3 cells were treated with anti‐miR21 to a working concentration of 20 nM for 72 h with NS miR as control. The number of tumour spheres generated quantified by MTT assay and counted. Experiments were repeated three times and results are presented as the mean ± SD and were analysed using unpaired student's t test.