RNAi‐Mediated Knockdown of DNMT3b in Hypermethylator Breast Cancer Cell Lines Leads to Normalization of Gene Expression Patterns

Abstract

A subset of breast cancer cell lines express a hypermethylation defect characterized by DNMT hyperactivity, overexpression of DNMT3b, and silencing of numerous genes. To investigate the role of DNMT3b in the hypermethylation defect, we examined the effect of RNAi‐mediated DNMT3b knockdown on expression of methylation‐sensitive genes in model hypermethylator cell lines: MDA‐MB‐453 and BT549. Western blot analysis confirmed reduction of DNMT3b in cells transfected with the DNMT3b RNAi targeting construct. RT‐PCR was used to analyze expression of six methylation‐sensitive genes (CEACAM6, CST6, ESR1, SCNN1A, GNA11, CDH1 and/or MUC1) that are methylated and exhibit diminished or no expression in parent cell lines compared to MCF12A. In MDA‐MB‐453 cells, all six genes were reexpressed or increased expression in response to DNMT3b knockdown. CEACAM6, CST6, ESR1, and GNA11 were expressed at normal levels, while MUC1 and SCNN1A were expressed at low but detectable levels. In BT549 cells, CEACAM6, CST6, ESR1, GNA11, and SCNN1A were reexpressed or increased expression in response to DNMT3b knockdown, whereas CDH1 was not expressed. These results strongly suggest that CEACAM6, CST6, ESR1, GNA11, SCNN1A, and MUC1 are direct methylation targets for DNMT3b. Further, these results suggest that DNMT3b overexpression governs the hypermethylator defect associated with some breast cancer cell lines.Support: NIH CA78343