Synergistic activation of functional estrogen receptor (ER)-alpha by DNA methyltransferase and histone deacetylase inhibition in human ER-alpha-negative breast cancer cells

Formation of transcriptional repression complexes such as DNA methyltransfe rase (DNMT) 1/histone deacetylase (HDAC) or methyl-CpG binding protein/HDAC is emerging as an important mechanism in silencing a variety of methylated tissue-specific and imprinted genes. Our previous studies showed that trea tment of estrogen receptor (ER)-alpha -negative human breast cancer cells w ith the DNMT inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) led to ER mRNA and protein re-expression. Also, the HDAC inhibitor trichostatin A (TSA) could induce ER transcript about 5-fold. Here we show that 5-aza-dC alone induce d ER transcript about 30-40-fold, and the addition of TSA elevated ER mRNA expression about 10-fold more in the human ER-negative breast cancer cell l ines MDA-MB-231 and MDA-MB-435. Overall, the combination of 5-aza-dC and TS A induced a 300-400-fold increase in ER transcript. Restoration of estrogen responsiveness was demonstrated by the ability of the induced ER protein t o elicit estrogen response clement-regulated reporter activity from an exog enous plasmid as well as induce expression of the ER target gene, progester one receptor. The synergistic activation of ER occurs concomitantly with ma rkedly reduced soluble DNMT1 expression and activity, partial demethylation of the ER CpG island, and increased acetylation of histones H-3 and H-4. T hese data suggest that the activities of both DNMT1 and HDAC are key regula tors of methylation-mediated ER gene silencing.