Abstract 5194: MiR-200c expression and methylation status determines epithelial characteristics of NSCLC

Abstract

Abstract Background: MicroRNA (miR)-200 family members are frequently silenced in advanced cancer and have been implicated in the process of epithelial-to-mesenchymal transition (EMT) and tumor invasion. In this study, we investigated the roles of miR-200c in non-small cell lung cancer (NSCLC). Materials and Methods: We analyzed miR-200c promoter methylation status by methylation specific PCR and expression by quantitative RT-PCR in 34 NSCLC cell lines and 10 NSCLC clinical samples. To eliminate the effects of contamination of normal lung epithelial cells and mesenchymal cells in clinical samples, we used primary cultured NSCLC cells obtained by surgical resection. We assessed the association of miR-200c status with histology, EMT-related protein expression, and genetic alteration. Results: Mir-200c was methylated in 10 (30%) NSCLC cell lines and partially methylated in 7 (21%) cell lines. Expression of miR-200c was silenced in all methylated cell lines and restored with 5-aza-2′-deoxycytidine treatment. In cell lines with miR-200c methylation and silenced, protein expression of epithelial markers was reduced and mesenchymal markers were increased. Introduction of miR-200c into highly invasive cells with miR-200c silencing inhibited cell invasion. Interestingly, miR-200c was unmethylated and not silenced in many EGFR mutated NSCLC cell lines and clinical samples. In HEK293T cells stably transfected with wild-type EGFR, mutant EGFR, wild-type KRAS, or mutant KRAS, miR-200c expression was up-regulated only in mutant EGFR transfectant. Conclusions: Mir-200c expression was regulated by promoter methylation in NSCLC, and related to epithelial characteristics and cell invasion. In EGFR mutated NSCLC cells, miR-200c was generally unmethylated and expressed, suggesting the existence of mechanism that retain EGFR mutated NSCLC cells to the epithelial state through the miR-200c expression. Citation Format: Kazuhiko Shien, Shinichi Toyooka, Junichi Soh, Hiromasa Yamamoto, Shinsuke Hashida, Ken Suzawa, Tomoaki Otsuka, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi. MiR-200c expression and methylation status determines epithelial characteristics of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5194. doi:10.1158/1538-7445.AM2014-5194