δEF1 associates with DNMT1 and maintains DNA methylation of the E-cadherin promoter in breast cancer cells

Abnormal DNA methylation at the C-5 position of cytosine (5mC) of CpG dinucleotides is a well-known epigenetic feature of cancer. Levels of E-cadherin, which is regularly expressed in epithelial tissues, are frequently reduced in epithelial tumors due to transcriptional repression, sometimes accompanied by hypermethylation of the promoter region. delta EF1 family proteins (delta EF1/ZEB1 and SIP1/ZEB2), key regulators of the epithelial-mesenchymal transition (EMT), suppress E-cadherin expression at the transcriptional level. We recently showed that levels of mRNAs encoding delta EF1 proteins are regulated reciprocally with E-cadherin level in breast cancer cells. Here, we examined the mechanism underlying downregulation of E-cadherin expression in three basal-type breast cancer cells in which the E-cadherin promoter region is hypermethylated (Hs578T) or moderately methylated (BT549 and MDA-MB-231). Regardless of methylation status, treatment with 5-aza-2'-deoxycytidine (5-aza), which inhibits DNA methyltransferases, had no effect on E-cadherin expression. Knockdown of delta EF1 and SIP1 resulted in recovery of E-cadherin expression in cells lacking hypermethylation, whereas combined treatment with 5-aza synergistically restored E-cadherin expression, especially when the E-cadherin promoter was hypermethylated. Moreover, delta EF1 interacted with DNA methyltransferase 1 (DNMT1) through the Smad-binding domain. Sustained knockdown of delta EF1 family proteins reduced the number of 5mC sites in the E-cadherin promoter region, suggesting that these proteins maintain 5mC through interaction with DNMT1 in breast cancer cells. Thus, delta EF1 family proteins appear to repress expression of E-cadherin during cancer progression, both directly at the transcriptional level and indirectly at the epigenetic level.