Abstract P1-07-11: ERa regulates epithelial-mesenchymal transition in breast cancers through suppressing Bmi1 expression
Authors: Zhang G-J , Wei X , Dou X 
Published: August 2014

Background: Bmi1, a polycomb-group protein, maintains stem cell self-renewal and is frequently overexpressed in cancers. Upregulation of Bmi1 by Twist1 can promote tumor-initiating capability and Epithelial-mesenchymal transition (EMT) by repressing E-cadherin expression. It has been demonstrated that Post-EMT breast cancer cells express cancer stem cell (CSC) markers including Bmi1, but display decreased ERα expression. ERα is a ligand-activated nuclear hormone receptor that regulates the transcription of E2-responsive genes in diverse target cells. It's reported that ligand-activated ERα could repress slug transcription by binding to the half-site ERE element in its promoter, and then regulate E-cadherin and EMT. Herein, we hypothesized that ERα signaling might regulate E-cadherin and EMT through Bmi1.

Methods: First, we determined Bmi1 and ERα expression at both mRNA and protein levels by quantitative RT-PCR and Western blot in various breast cancer cell lines. Further, we quantitatively analyzed mRNA and protein levels of Bmi1 as well as its down-stream genes including E-cadherin after silencing ERα in T47D cells with siRNA, or after overexpressing ERα in BT549 cells. The chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) were also performed to investigate whether ERα binds directly to Bmi1 promoter region. Luciferase assay with Bmi1 reporter was performed to ERα mediated regulation. To identify whether ERα regulates EMT, transwell and matrigel invasion, flow cytometry, mammosphere assay and wound healing assay were performed to invasion, metastases and characterize stem-cell properties of EMT. Finally, we analyzed the expression of ERα, Bmi1 and E-cadherin in human breast cancer specimens with immunohistochemistry.

Results: The study showed that Bmi1 inversely correlated with ERα expression in different subtypes of breast cancer cells. We further confirmed that Bmi1 was upregulated at both mRNA and protein levels and E-cadherin was downregulated with siRNA against ERα in T47D cells, and Bmi1 was downregulated and E-cadherin was upregulated when overexpressing ERα in BT549 cells with qRT-PCR and western blot analyses. The present study also demonstrated that ERα could directly binds to the half-ERE region of the Bmi1 gene and repressing Bmi1 expression transcriptionally with CHIP/EMSA and reporter analyses. Overexpression of ERα in BT549 cell significantly decreased levels of migration of transwell and wounding healing, invasiveness, CD44high/CD24low population, and the capabilities of mammosphere formation.

Conclusions: Our results demonstrated that ERα can suppress EMT through transcriptionally down-regulating Bmi-1 and its down-stream genes in human breast cancer cells. The inverse relationship between ERα and Bmi1 expression further supports the epithelial phenotype of ERα positive tumors or mesenchymal phenotype of ERα negative tumors. Our findings provide a novel mechanistic insight into how ERα regulates EMT, and is of valuable for developing biomarkers to predict prognosis and targeted therapies in breast cancers in the future.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-07-11.