Abstract LB-358: Carcinogen-induced epigenetic silencing of miR-205 and miR-200 family promotes EMT and transformation of lung epithelial cells

Abstract

Abstract An in vitro carcinogen-induced transformation model was developed using immortalized bronchial epithelial cells (HBECs) to identify key molecular changes in preneoplastic lung epithelial cells that occur during the development of lung cancer in smokers. HBECs were treated for twelve weeks (one exposure per week) with low-doses of tobacco carcinogens (MNU and BPDE) that resulted in transformation from genotoxic stress (DNA damage). The HBECs displayed a morphological change consistent with an epithelial-to-mesenchymal transition (EMT) after 4 weeks of carcinogen treatment that persisted in colonies that were recovered and expanded from soft agar (transformed cell line). Expression of mesenchymal markers vimentin, fibronectin, and N-cadherin increased significantly, while the epithelial marker E-cadherin was significantly decreased. The most notable changes associated with EMT included 4 - 55 fold reduced expression of miR-200b, -200c, and -205 that correlated with 2-1500 fold increased expression of E-box binding transcription factors (SNAI1, ZEB1, ZEB2) and basic helix-loop-helix transcription factor (TWIST1). Restoring the expression of miR-200b, -200c or -205 in carcinogen transformed cells prevented growth in soft agar; meanwhile the cells maintained a fibroblast-like mesenchymal appearance. Levels of DNA methyltransferase 1 (DNMT1) protein also increased significantly during carcinogen treatment and stable knockdown of DNMT1, but not DNMT3A and DNMT3B prior to carcinogen prevented EMT and transformation. ChIP for H3K4me2 (indicative of active/open chromatin) revealed a progressive decrease of this mark at the promoters of miR-200b, -200c, and -205 over 12 weeks of carcinogen treatment and during transformation. A modest enrichment for H3K9me2 (inactive/closed chromatin mark) was observed at all time points. In contrast, H3K27me3 (indicative of inactive/closed chromatin) was enriched at the miR promoters after 4 weeks of carcinogen treatment and enrichment declined over time. Bisulfite sequencing revealed CpG DNA methylation increased from 3% to 42% in the miR-200 family and miR-205 promoters during transformation. To examine the clinical relevance of miR-200b, -200c, and -205 their expression was analyzed in lung tumors relative to distant normal lung tissue from 24 patients. Reduced expression of at least one miR was observed in 50% of lung adenocarcinomas. These findings implicate a major role for epigenetic regulation of EMT that extends beyond that of cancer metastasis to causality for development of premalignant lung cancer. Supported by ES008801. 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 LB-358.