| Approximately one-fourth of human breast cancers do not express estrogen receptor alpha (ER). Loss of ER expression may be associated with epigenetic mechanisms such as DNA methylation and histone deacetylation. The goal of this study was to better understand the epigenetic mechanisms by which regulation of ER expression is achieved.; In order to determine the components of the protein complex present at the ER promoter, untreated MCF7 cells, untreated MDA-MB-231 cells or MDA-MB-231 cells treated with 5-azacytidine (Aza), Trichostatin A (TSA), or the combination of Aza and TSA were subjected to chromatin immunoprecipitation (ChIP) analyses. Silencing of ER was found to correlate with binding of specific methyl-binding proteins, DNA methyltransferases (DNMTs), and histone deacetylase (HDAC) proteins. Inhibition of HDAC activity by TSA resulted in the accumulation of hyperacetylated core histories. The activation of ER expression by Aza also involves the release of the repressor complex involving various methyl binding proteins, DNMTs, and HDACs.; Next, the effects of TSA or the novel compounds, LAQ824 and LBH589, on cell growth and ER status were studied. Treatment of MCF7 or MDA-MB-231 cells with these compounds resulted in accumulation of acetylated core histones at the ER promoter, release of HDACs from the ER promoter, and re-expression of ER and progesterone receptor (PR) mRNA in MDA-MB-231 cells, indicating that re-expressed ER is functional. Treatment of MDA-MB-231 cells with HDAC inhibitors sensitized the cells to concomitant treatment with the selective estrogen receptor modulator 4-hydroxy-tamoxifen, but not the selective estrogen receptor degrader fulvestrant.; Lastly, we wished to determine which HDAC proteins contribute to regulation of ER expression. RNA interference was used to selectively knock down expression of several members of the HDAC family alone or in combination, and the effects of RNAi knockdown on ER expression were assessed. The results of this study suggest that the class I and class II HDACs 1, 2, 3, 7 and 8 play a role in ER expression in human breast cancer cells.; Taken together, these data give us a better understanding of how ER is regulated and may lead to novel therapeutic interventions for the treatment of breast cancer. |
