Trichostatin A and 5-aza-2' deoxycytidine decrease ER mRNA stability in ER positive MCF7 cells through decreased HuR concentrations. Free

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Pharmacologic inhibition of epigenetic mechanisms including DNA methylation and histone acetylation results in the re-expression of functional estrogen receptor alpha (ER) mRNA and protein in ER negative breast cancer cell lines. Treatment of ER positive cell lines with the same pharmacologic agents, 5-aza 2’ deoxycytidine (AZA) and Trichostatin A (TSA), however, results in decreased ER mRNA and protein expression. Inhibition of nascent RNA synthesis with actinomycin D treatment suggests that this decrease in ER mRNA is due, at least in part, to a decrease in ER half-life from 4 to 1.5 hours. Similar studies in ER negative MDA-MB-231 cells treated with AZA/TSA indicate that while ER is re-expressed following treatment of these cells, the half-life of re-expressed ER mRNA is only 2.5 - 3 hours, less than that of untreated MCF7 cells, suggesting that inhibiting epigenetic mechanisms may not fully restore all proteins controlling ER mRNA stability in the cell. Further, this suggests that a protein(s) controlling ER mRNA stability is expressed in MCF7 but not expressed, or is not expressed to the same extent, in MDA-MB-231 cells. Control of ER mRNA stability is mediated through RNA binding protein interaction with the 3’UTR. Several RNA binding proteins including HuR are key mediators in the control of mRNA stability and have been shown to alter cyclin E, c-Yes and COX2 mRNA stability in breast cancer cell lines. Recent studies have shown that both the overall and the cytoplasmic-to-nuclear ratio of HuR expression has been shown to be increased in MCF7 cells compared with that of non-tumorigenic, normal mammary epithelial MCF10A cells. This suggests that HuR may be a key contributor to tumorigencity in breast cancer. Higher HuR protein concentrations are also detected in both whole cell lysates and cytoplasmic extracts of MCF7 compared with MDA-MB-231 cells. HuR cytoplasmic protein concentration decreased following AZA/TSA treatment in both cell lines, suggesting that a potential mechanism for AZA/TSA mediated ER mRNA decrease could be through altered interaction of HuR with the ER 3’UTR. Immunoprecipitation of HuR in MCF7 and AZA/TSA treated MDA-MB-231 cells followed by Real Time RT-PCR indicates that HuR does associate with ER mRNA, further suggesting a role for HuR in control of ER mRNA stability. Taken together, our data suggest that the epigenetic modulators, AZA and TSA, play a larger role in the determination of mRNA concentrations in the cell. This not only includes epigenetic activation of gene expression, but also involves control of mRNA stability or decay. Studies are ongoing to decipher the role of these epigenetic modulators in control of mRNA decay and determination of mRNA concentrations in the cell.