Valproic acid, in combination with all-trans retinoic acid and 5-aza-2′-deoxycytidine, restores expression of silenced RARβ2 in breast cancer cells

Epigenetic silencing of tumor suppressor genes has been established as an important process of carcinogenesis. The retinoic acid (RA) receptor 2 (RAR beta 2) gene is one such tumor suppressor gene often silenced during carcinogenesis. The combined use of histone deacetylase and DNA methyltransferase inhibitors has been shown to reverse the epigenetic silencing of numerous growth regulatory genes. Valproic acid (VPA), which has long been used in the treatment of epilepsy, was shown recently to be an effective histone deacetylase inhibitor that can induce differentiation of neoplastically transformed cells. In this study, we show for the first time that VPA, in combination with RA and the DNA methyltransferase inhibitor 5-aza-2 ' deoxycytidine (Aza-dC), can overcome the epigenetic barriers to transcription of a prototypical silenced tumor suppressor gene, RAR beta 2, in human breast cancer cells. Chromatin immunoprecipitation assays show that the combination of VPA, RA, and Aza-dC increases histone acetylation at the silenced RAR beta 2 promoter of MCF-7 breast cancer cells. Furthermore, reverse transcription-PCR analyses reveal cell type-specific effects in the actions of VPA on RAR beta 2 expression in cultured human breast cancer cells. Finally, we show that VPA, in combination with RA and Aza-dC, inhibits the proliferation of both estrogen receptor alpha-positive (MCF-7) and estrogen receptor alpha-negative (MDA-MB-231) breast cancer cell lines. These data suggest that VPA may ultimately be useful in combination therapies in the treatment of human breast cancers.