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.