ErbB2 signaling epigenetically suppresses microRNA-205 transcription via Ras/Raf/MEK/ERK pathway in breast cancer

We previously reported that microRNA-205 (miR-205) is downregulated by overexpression of the receptor tyrosine kinase ErbB2 and that ectopic transfection of miR-205 precursor decreases ErbB2 tumorigenicity in soft agar. In this study, we further analyzed the regulatory mechanisms linking ErbB2 overexpression and miR-205 downregulation. In ErbB2-overexpressing breast epithelial cells, miR-205 expression was significantly increased by treatment with MEK inhibitor U0126 or PD98059, Raf-1 inhibitor ZM-336372, and ERK inhibitor SCH772984, but PI3K inhibitor LY294002 and p38 MAPK inhibitor SB203580 had no effect. We established breast epithelial cells overexpressing RafCAAX, a constitutively active form of Raf-1, and showed that overexpression of RafCAAX dramatically reduced miR-205 expression. In RafCAAX-overexpressing cells, miR-205 expression was also significantly increased by SCH772984. Moreover, miR-205 expression was significantly increased by treatment with DNA methyltransferase (DNMT) inhibitor 5-aza-2-deoxycytidine and expression of several DNMT family members was increased in both ErbB2- and RafCAAX-overexpressing cells. DNA methylation analysis by bisulfite sequencing revealed that the putative miR-205 promoters were predominantly hypermethylated in both ErbB2- and RafCAAX-overexpressing cells. Reporter activity of the putative miR-205 promoters was reduced in both ErbB2-overexpressing and RafCAAX-overexpressing cells. Together, these findings indicate that ErbB2 signaling epigenetically suppresses miR-205 transcription via the Ras/Raf/MEK/ERK pathway.