Abstract 111: Regulation of alcohol dehydrogenase 1B by DNA methylation and histone modification in epithelial breast cell lines

Abstract

Homeostasis of normal breast epithelium requires maintenance of a gene expression profile that is, in part, dependent on DNA methylation and histone modification. Tumorgenesis is marked by a disturbance of this profile, as the bulk of the tumor genome is hypomethylated with the exception of tumor suppressor genes that are typically hypermethylated. The hypermethylation interacts with histone modifications to suppress gene expression. Our previous research demonstrated that alcohol dehydrogenase (ADH) isozymes are expressed in normal breast tissue and down-regulated in breast cancer. In this study, qRT-PCR analysis of RNA extracted from normal breast epithelium reveals that ADH1B accounts for 95.8% of total ADH isozyme expression. Expression of ADH1B in RNA extracts from breast tumor is significantly down-regulated to 5.4% of normal expression (p < 0.0001). Similar findings have been identified in lung and colon epithelium. The consistent down-regulation of ADH1B in epithelial cancers suggests that it plays an important role in epithelial maintenance and may act as a tumor suppressor. In addition to its role in oxidation of alcohols, ADH1B participates in a variety of major biochemical processes important to tissue homeostasis such as lipid peroxidation and the oxidation of retinol. Epigenetic modifications that may participate in the observed abrogation of ADH1B expression in breast cancer were investigated. Using qRT-PCR, the expression profile of ADH1B was determined for 3 epithelial breast cell lines: MCF-10A, MCF-10AT, and HS-578T. Neither MCF-10A nor MCF-10AT express ADH1B. HS-578T cells express less than 1% of the ADH1B found in RNA extracted from normal breast epithelium. These results are consistent with observed ADH1B expression in breast tumor samples. Chromatin immunoprecipitation assays were performed on cell lysates from untreated cells and cells treated with 5-Aza-2′-Deoxycytidine (5-aza-2dC) or Trichostatin A (TSA) alone or in combination. 5-aza-2dC inhibits methylation by forming a covalent bond with DNA methyltransferase and TSA is a reversible histone deacetylase inhibitor. Interaction between ADH1B and RNA Pol II increased six fold in cells treated with TSA (p < 0.05). ADH1B expression significantly increased in these cells and further increased in cells treated with a combination of 5-aza-2dC and TSA (p < 0.0001). However, no significant difference was found in cells treated with 5-aza-2dC alone. These results suggest that histone deacetylation plays an important role in the down-regulation of ADH1B expression in these breast cell lines. The methylation state of the promoter region may also interact with histone acetylation to down-regulate ADH1B expression. The down-regulation of ADH1B observed in breast cancer may result from similar changes in epigenetic modifications and the availability of key transcription factors during carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 111. doi:10.1158/1538-7445.AM2011-111