Suppression of WIF-1 through promoter hypermethylation causes accelerated proliferation of the aryl hydrocarbon receptor (AHR) overexpressing MCF10AT1 breast cancer cells

The cause for increased cell proliferation in AHR overexpressing breast cancer cells still remains unknown. Here we studied the molecular basis of aggressive cell proliferation of an AHR overexpressing and ERα functionally down-regulated MCF10AT1 cell line, designated as P20E, in comparison to a matched sub-line, P20C with normal AHR expression and ERα function. We found that a 4-day treatment of P20E cells with 5-aza-2′-deoxycytidine (AZ) caused a significant suppression of cell proliferation. Such an effect of AZ was accompanied with the significant recovery of ERα function. Among diagnostic markers of AZ-induced cellular changes we found conspicuous up-regulation of mRNA expression of Wnt inhibitory factor-1 (WIF-1), particularly in P20E. The possibility of AZ-induced demethylation on the promoter of WIF-1 gene was confirmed through methylation specific PCR assay. Such AZ-induced changes in P20E cells were also accompanied with the decrease in the binding of nuclear proteins to the 32P labeled TRE (TCF response element) and the reduced accumulation of β-catenin protein in the cell nucleus, indicating the importance of Wnt/β-catenin pathway in maintaining the increased cell proliferation in P20E line over P20C line. The importance of WIF-1 in this regard has been validated by transfecting cells with siRNA against WIF-1, which caused an increase in cell proliferation. Moreover, artificial overexpression of ERα in both P20E as well as MDA-MB-231 cells increased the mRNA expression of WIF-1. Together these results support our main conclusion that the aggressive cell proliferation of this AHR-overexpressing sub-type of transformed mammary epithelial cells is assisted by the epigenetic suppression of WIF-1 gene, which is caused by the loss of ERα.

► 5-Aza-2′-deoxycytidine (AZ) causes proliferation suppression and ERα recovery. ► AZ down-regulates Wnt/β-catenin pathway mainly by increasing WIF-1 expression. ► Both ERα and AhR have some effects on DNA methylation in breast cancer cells. ► Artificial overexpression of ERα in ER negative cells increases WIF-1 expression. ► WIF-1 promoter hypermethylation is one of the major causes for accelerated proliferation.