Abstract
Rationale: Whereas asbestos burden has been linked to cytogenetic alterations in malignant pleural mesotheliomas, epigenetic aberrations induced by these fibers have not been fully delineated. Objectives: The objective of this study was to establish an in vitro model to characterize early epigenetic events potentially contributing to malignant pleural mesothelioma. Methods: Normal human mesothelial cells (LP9 and LP3) were cultured with or without crocidolite asbestos fibers (1 or 2 μg/cm(2)) for up to 10 days. Messenger RNA, microRNA, and protein levels were assessed by quantitative reverse transcriptase–polymerase chain reaction and immunoblot techniques. Methylation-specific polymerase chain reaction, pyrosequencing, and quantitative chromatin immunoprecipitation techniques were used to correlate changes in gene expression with epigenetic alterations in the respective promoters. Results: Asbestos mediated time- and dose-dependent repression of Ras association domain–containing protein 1 (RASSF1A), p16 kinase 4a inhibitor (p16INK4a), and p14 alternative reading frame (p14ARF) in normal mesothelial cells; this phenomenon coincided with upregulation of DNA methyltransferase 1 (DNMT1) as well as increased expression of enhancer of zeste homolog 2 (EZH2). Upregulation of EZH2 coincided with repression of microRNA 26A and microRNA 101, which target the 3′ untranslated region of the EZH2 transcript. Silencing of RASSF1A, p16INK4a, and p14ARF coincided with recruitment of EZH2 and concomitant increases in the PRC-2–mediated repressive histone mark, histone H3 lysine 27 trimethylation (H3K27me3), and decreased levels of histone H3 lysine 27 acetylation (H3K27ac3) (histone activation mark) within the respective promoters. Asbestos induced de novo DNA methylation in the promoter of RASSF1A, but not within the INK4a/ARF locus. 5-Aza-2′-deoxycytidine (0–0.5 μM for 72 h) induced dose-dependent activation of RASSF1A (but not p16/p14) in malignant pleural mesothelioma cells; this phenomenon coincided with DNA demethylation and decreased occupancy of DNA methyltransferase 3B within the RASSF1A promoter. Conclusions: Asbestos mediates rapid inactivation of Hippo, Retinoblastoma, and p53 tumor suppressor pathways in normal human mesothelial cells via epigenetic repression of RASSF1A, p16INK4A, and p14ARF, respectively. This in vitro model may prove useful for delineating the sequence of epigenetic events contributing to malignant pleural mesothelioma, and the development of novel strategies for treatment and possible prevention of these neoplasms.