Abstract 4250: Cigarette smoke induces expression of the putative stem cell marker ABCG2 in cultured esophageal adenocarcinoma cells

Abstract

Abstract Limited information is available regarding epigenetic mechanisms by which cigarette smoke mediates initiation and progression of esophageal cancer. In order to examine this issue, esophageal adenocarcinoma cell (EsC) lines (NCI-EsC1 and NCI-EsC2) were cultured in normal media (NM) with or without cigarette smoke condensate (CSC) under clinically relevant exposure conditions. Microarray analysis revealed that CSC exposure significantly upregulated expression of ABCG2, which encodes a xenobiotic pump protein believed to be highly expressed in cancer stem cells, and recently shown to correlate with aggressive phenotype of primary esophageal squamous cell carcinomas. Analysis revealed an average of 11 fold upregulation of ABCG2 as well as a 4 fold upregulation of another ABC transporter, ABCC3. Quantitative reverse transcription-PCR (RT-PCR), western blot, and immunohistochemistry experiments confirmed upregulation of ABCG2 in esophageal cancer cells exposed to CSC. Interestingly, basal as well as CSC-induced levels of ABCG2 were considerably higher in NCI-EsC2 derived from a smoker, relative to NCI-EsC1, which was established from a never-smoker. Time course experiments revealed that induction of ABCG2 commenced approximately 8 hours and peaked 48 hours following initiation of CSC exposure; thereafter, ABCG2 expression was sustained at these levels with continuous CSC exposure. HDAC inhibitors such as TSA and depsipeptide repressed basal levels of ABCG2 expression, and reduced CSC-mediated induction of ABCG2 in EsC cells. HDAC inhibitors in combination with verapamil (a ABCC3 inhibitor) and Depsipeptide virtually abolished basal, as well as CSC-induced ABCG2 expression in these cells. Preliminary pyrosequencing and chromatin immunoprecipitation (ChIP) experiments revealed that CSC-mediated ABCG2 induction was independent of promoter DNA demethylation, and coincided with hyperacetylation of H3K9 within the ABCG2 promoter. Flow cytometry analysis utilizing hoescht dye with and without fumitremorgin C, an ABCG2 inhibitor, revealed an increased side population in CSC-treated ESC cells relative to untreated controls. Experiments are in progress to further delineate the mechanisms by which CSC induces ABCG2 expression, and to ascertain if ABCG2 induction enhances the malignant phenotype of esophageal adenocarcinoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4250.