Eliminating epigenetic barriers induces transient hormone-regulated gene expression in estrogen receptor negative breast cancer cells

In breast cancer, approximately one-third of tumors express neither the estrogen receptor ( ER alpha) nor estrogen-regulated genes such as the progesterone receptor gene ( PR). Our study provides new insights into the mechanism allowing hormone-activated expression of ER alpha target genes silenced in ER alpha-negative mammary tumor cells. In cell lines derived from ER alpha-negative MDA-MB231 cells, stable expression of different levels of ER alpha from a transgene did not result in transcription of PR. A quantitative comparative analysis demonstrates that inhibiting DNA methyltransferases using 5-aza-2 '-deoxycytidine or specific disruption of DNMT1 by small interfering RNAs and treatment with the histone-deacetylase inhibitor trichostatin A enabled ER alpha-mediated hormone-dependent expression of endogenous PR. We show that demethylation of a CpG island located in the first exon of PR was a prerequisite for ER alpha binding to these regulatorysequences. Although not a general requirement, DNA demethylation is also necessary for derepression of a subset of ER alpha target genes involved in tumorigenesis. PR transcription did not subsist 4 days after removal of the DNA methyltransferase blocking agents, suggesting that hormone-induced expression of ER alpha target genes in ER alpha-negative tumor cells is transient. Our observations support a model where an epigenetic mark confers stable silencing by precluding ER alpha access to promoters.