Oncogene (2010) 29, 3650–3664 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate F Liu1, JK Killian2, M Yang1, RL Walker2, JA Hong1, M Zhang1, S Davis2, Y Zhang1, M Hussain1,SXi1, M Rao1, PA Meltzer2 and DS Schrump1 1Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA and 2Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Limited information is available regarding epigenomic events Introduction mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to Mounting evidence implicates aberrant expression/ examine epigenomic effects of cigarette smoke in respiratory activity of epigenetic regulators of gene expression in epithelia. Normal human small airway epithelial cells and the initiation and progression of lung cancers, the cdk-4/hTERT-immortalized human bronchial epithelial cells majority of which are directly attributable to cigarette (HBEC) were cultured in normal media with or without smoking (Lee et al., 2005; D’Alessio and Szyf, 2006; cigarette smoke condensate (CSC) for up to 9 months under Lin et al., 2007; Schrump et al., 2007). For example, potentially relevant exposure conditions. Western blot increased DNA methyltransferase (DNMT) expression analysis showed that CSC mediated dose- and time- coincides with progression to malignancy in murine dependent diminution of H4K16Ac and H4K20Me3, while pulmonary adenomas induced by tobacco carcinogens increasing relative levels of H3K27Me3; these histone (Belinsky et al., 1996). Over-expression of DNMT1 and alterations coincided with decreased DNA methyltransferase DNMT3b, as well as methyl-binding domain 2, correlates 1 (DNMT1) and increased DNMT3b expression. Pyrose- with hypermethylation of tumor suppressor genes in quencing and quantitative RT–PCR experiments revealed tobacco-induced lung cancers, and diminished survival of time-dependent hypomethylation of D4Z4, NBL2, and patients with these neoplasms (Kim et al., 2006; Lin et al., LINE-1 repetitive DNA sequences; up-regulation of H19, 2007; Xing et al., 2008). Aberrant activities of histone IGF2, MAGE-A1, and MAGE-A3; activation of Wnt lysine methyl transferases and histone deacetylases signaling; and hypermethylation of tumor suppressor genes enhance oncogenic transformation of bronchial epithelial such as RASSF1A and RAR-b, which are frequently cells (Watanabe et al., 2008), and induce global alterations silenced in human lung cancers. Array-based DNA methyla- in the histone code, which correlate with advanced stage tion profiling identified additional novel DNA methylation of disease and poor prognosis in lung cancer patients targets in soft-agar clones derived from CSC-exposed (Barlesi et al., 2007; Van Den et al., 2008; Seligson et al., HBEC; a CSC gene expression signature was also identified 2009). Over-expression of polycomb proteins such as Bmi in these cells. Progressive genomic hypomethylation and 1 and Ezh2 facilitates epigenetic silencing of tumor locoregional DNA hypermethylation induced by CSC suppressor genes, and enhances stem cell signaling in coincided with a dramatic increase in soft-agar clonogeni- lung cancer cells (Dovey et al., 2008; Vrzalikova et al., city. Collectively, these data indicate that cigarette smoke 2008; Hussain et al., 2009; McCabe et al.,2009). induces ‘cancer-associated’ epigenomic alterations in cul- Epigenetic alterations during malignant transforma- tured respiratory epithelia. This in vitro model may prove tion seem analogous to those regulating gene expression useful for delineating early epigenetic mechanisms regulating during gametogenesis and stem cell development (Simp- gene expression during pulmonary carcinogenesis. son et al., 2005; Mathews et al., 2009). For example, Oncogene (2010) 29, 3650–3664; doi:10.1038/onc.2010.129; tumor suppressor genes such as p16 and p19/ARF, published online 3 May 2010 which mediate replicative senescence and apoptosis in response to oncogene signaling (Agherbi et al., 2009), Keywords: tobacco smoke; lung cancer; epigenetics; are frequently observed to be silenced in cancer cells respiratory epithelial cells exhibiting coordinate de-repression of germ-line re- stricted genes such as NY-ESO-1 and MAGE family members, several of which physically interact and suppress p53-mediated apoptosis (Cho et al., 2006; Correspondence: Dr DS Schrump, Thoracic Oncology Section, Monte et al., 2006; Yang et al., 2007). These observa- Surgery Branch, Center for Cancer Research, National Cancer tions suggest that epigenetic perturbations in lung Institute, 10 Center Drive, Building 10, Room 4-3940, Bethesda, MD cancer cells are not merely manifestations of random 20892-1201, USA. E-mail: [email protected] stochastic events, but instead reflect strong selective Received 14 September 2009; revised 3 February 2010; accepted 17 pressure to reactivate/maintain stem cell gene expression March 2010; published online 3 May 2010 during multistage pulmonary carcinogenesis. Epigenetics of tobacco smoke exposure FLiuet al 3651 Despite the unequivocal association between cigarette CSC (1%) mediated time-dependent decreases in smoking and lung cancer, the epigenetic mechanisms by H4K16Ac, as well as H4K20me3 levels; reduced levels which tobacco smoke initiates and promotes pulmonary of H4K16Ac—but not H4K20Me3—were observed in carcinogenesis have not been fully delineated. In HBEC 5 days after initiation of CSC exposure. particular, epigenetic events associated with initiation Decreased H4K20Me3 levels were evident in HBEC 1 of tobacco-induced lung cancers have not as yet been month after initiation of CSC exposure. More pro- elucidated. This study was undertaken to characterize longed exposure to 1% CSC virtually abolished epigenomic alterations in cultured human respiratory H4K16Ac, and dramatically reduced H4K20Me3 levels epithelial cells mediated by cigarette smoke. without significantly diminishing total H4 levels in HBEC; these histone alterations coincided with a 2.6- fold relative increase in H3K27Me3, an 80% reduction in DNMT1, as well as B2–3-fold increase in EZH2 and Results DNMT3b levels, respectively. Growth inhibitory effects of cigarette smoke condensate in cultured cells DNA methylation changes mediated by CSC Preliminary experiments were initiated to examine the Additional experiments were performed to ascertain effects of cigarette smoke condensate (CSC) in cultured whether CSC exposure altered global DNA methylation respiratory epithelia and lung cancer cells to define status in HBEC. Initial pyrosequencing experiments appropriate exposure conditions for subsequent studies. focused on NBL2-seq3 and D4Z4 DNA repeats, as well As shown in Figure 1a, CSC mediated dose-dependent as LINE-1 elements, as these have been shown to be growth inhibition, which coincided with morphologic demethylated in cancer cells exhibiting decreased levels changes in cultured respiratory epithelia; after CSC of H4K16Ac and H4K20Me3 (Fraga et al., 2005). exposure, cdk-4/h-Tert immortalized human bronchial Preliminary analysis showed modest demethylation of epithelial cells (HBEC), and to a lesser extent normal these sequences in untreated immortalized HBEC human small airway epithelial cells (SAEC) became less relative to primary cultures of SAEC or NHBE cells, polygonal and appeared more elongated and rectangu- although not to the extent observed in A549 lung cancer lar in shape. Interestingly, under these exposure condi- cells (Figure 2a). Subsequent experiments revealed a tions, the effects of CSC were less apparent in fully dose-dependent demethylation of NBL2 and D4Z4 transformed A549 lung cancer cells. subtelomeric DNA repeats, and to a lesser extent, LINE-1 elements in HBEC after 5-month continuous ‘Cancer-associated’ histone alterations mediated by CSC CSC exposure (Figure 2b). Malignant transformation is associated with global loss Quantitative RT–PCR experiments were performed of monoacetylated H4K16 as well as decreased to ascertain whether hypomethylation of the aforemen- H4K20me3 levels (Fraga et al., 2005; Van Den et al., tioned repetitive DNA sequences coincided with activa- 2008). As such, western blot experiments were per- tion of imprinted loci, as well as cancer-testis–X formed to ascertain whether CSC exposure could induce chromosome (CT-X) genes that are frequently de- these histone changes in respiratory epithelial cells. repressed in primary lung cancers through epigenetic Briefly, SAEC and HBEC were cultured in normal mechanisms. As shown in Figure 2c (left panel), CSC media with or without CSC at various concentrations mediated dose-dependent up-regulation of IGF2 and and exposure durations. As shown in Figure 1b (upper H19 imprinted loci, as well as several CT-X genes, panel), within 24 h of exposure, CSC mediated a dose- including MAGE-A1 and MAGE-A3. Interestingly, dependent decrease in H4K16Ac levels, without NY-ESO-1 (cancer-testis antigen 1), a CT-X gene appreciably changing total H4 expression in HBEC. A frequently de-repressed in lung cancer cells (Schrump similar phenomenon was observed after 24 or 48 h CSC et al., 2007), did not seem to be activated in HBEC by exposure. Densitometry analysis revealed no appreci- CSC under these exposure conditions. able change in H4K20Me3 levels under these exposure Additional