Abstract 807: 5-AZA/TSA reverses tamoxifen induced changes in chromatin remodeling enzymes in MCF-7/TAM-R cells
Authors: Pitta Chara A. 
Published: August 2012

Abstract
One major problem in breast cancer therapy is acquired resistance, necessitating the search for new approaches for overcoming this complication. The objective of this study is to overcome tamoxifen resistance in breast cancer cells using either a demethylating agent (such as 5-AZA) or a histone deacetylase inhibitor (such as trichostatin, TSA) or their combination. This is based on the observations that the promoters of ER-α and ER-α are rich in CpG islands, known to be associated with gene silencing. Methylated CpG clusters are expected to be especially susceptible to epigenetic modifications. Therefore, treatments with 5-AZA or TSA are expected, through chromatin remodelling, to activate the transcription of ER-α and ER-α m-RNA and produce functional proteins, reversing in this manner tamoxifen resistance. Using the MTT assay we determined the growth rates of MCF-7, MCF-7/TAM-R and MDA-MB-231 in the presence of 5-AZA, TSA and their combinations in the presence or absence of 4-OH tamoxifen. We identified the conditions (i.e order of inhibitor addition and concentration) that reverse tamoxifen resistance and those providing the maximum cell death of the MCF-7 and MCF-7/TAM-R. We found that pretreatment with 5-AZA (2μM) or TSA (0.1μM) enabled tamoxifen (10μM) to produce a considerable cell death (over 50%). Interestingly, MCF-7/TAM-R cells that normally do not express ER-α, after 24 hours of treatment with TSA and 5-AZA produced a marked increased in the expression of ER-α as determined by both Western Blot and fluorescence microscopy. Following 48 hours of treatment the levels of ER-α were further increased. Moreover, following 48 hours treatment with 5-AZA/TSA and tamoxifen the levels of p21 and p53 were substantially increased compared with the vehicle control. To determine if these changes are due to enzymes that modulate chromatin structure we evaluated the expression of HDAC1, 2, 3, 4, DNMT3a, and the levels of acetylated H3. We found that 4-OH-tamoxifen induced over a 10-fold increase in the HDAC3 expression levels compared to the untreated controls and this effect was reversed when 4-OH tamoxifen was provided in combination of 5-AZA and TSA. 4-OH tamoxifen also upregulated HDAC1 and DNMT3a protein levels which were accompanied by a dramatic increase in H3 acetylation. These results show clearly that: (a) the reversal of tamoxifen resistance can be achieved with relatively low concentrations of 5-AZA/TSA, (b) the upregulation of ER-α, p53 and p21 are essential components of the molecular pathway leading to this reversal, and c) these changes are accompanied by alterations in the expression levels of enzymes that control chromatin structure. These findings may find applications in clinical therapeutic protocols in the treatment of tamoxifen resistant breast cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 807. doi:1538-7445.AM2012-807