Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate

Abstract

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose-and timedependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT-PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-b, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces 'cancer-associated' epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

Cigarette smoking induces small airway epithelial epigenetic changes with corresponding modulation of gene expression

“…Consistent with our observations in the SAE, several studies have demonstrated that cigarette smoke in vivo and in vitro can mediate differences in the methylation status of individual genes in lung cells as well as non-lung cells. For example, immortalized human bronchial epithelial cells (HBECs) exposed to cigarette smoke extract display hypermethylation in association with tumor suppressor genes such as (RASSF1A) and retinoic acid receptor b2 (RARb2) in a dose-dependent manner (14). For RASSF1A, the increase in DNA methylation correlated with a decrease in gene expression.…”

Oxidative Stress and Chromatin Remodeling in Chronic Obstructive Pulmonary Disease and Smoking-Related Diseases

“…In vitro exposure of airway epithelial cells or immortalized bronchial epithelial cells to CS condensate/ extract (oxidants) reduced H4K16ac and H4K20me3, but increased H3K27me3. This may have implications in tumor development in several cancer types associated with tobacco smoking (77). It is well known that COPD is associated with a significantly increased incidence of lung cancer and other comorbidities in susceptible chronic smokers.…”

Wif1 Hypermethylation as Unfavorable Prognosis of Non-Small Cell Lung Cancers with EGFR Mutation

“…Transcriptional silencing of genes by CpG island (CGI) methylation is now recognized as a critical component in initiation and progression of lung cancer (Kerr et al, 2007;Kim et al, 2007;Na et al, 2010;Toyooka et al, 2001). In addition, variation in methylation status has been associated with tobacco smoking (Liu et al, 2010).…”