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 time-dependent 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-beta, 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.

Figure 1

(a) Morphology and proliferation of SAEC, HBEC, and A549 cells exposed to CSC. Dose-dependent alterations in cell morphology seemed to coincide with growth inhibition after CSC exposure. CSC-mediated morphologic changes and growth inhibition in A549 lung cancer cells were considerably less than those observed in short-term SAEC or immortalized HBEC. (b) Upper panel: Western blot analysis of H4K16 acetylation and H4K20 trimethylation in HBEC after exposure to CSC. A dose-dependent decrease in H4K16 acetylation was observed within 4 h of CSC exposure; this phenomenon persisted after exposures of 24 and 48 h. No appreciable change in H4K20 trimethylation was observed after short-term CSC exposure. Lower panel: Immunoflourescence analysis of acetylated H4K16 and trimethylated H4K20 expression in HBEC exposed to CSC for 3 days. The results reveal a decrease in H4K16 acetylation in HBEC without significant reduction in H4K20 trimethylation in HBEC exposed to 1% CSC. These results are consistent with western blot data in upper panel. (c) Western blot analysis of H4K16Ac, and H4K20Me3 and total H4 levels in cultured NHBE and SAEC exposed to CSC for 72 h. Short-term CSC exposure seemed to decrease H4K16 acetylation, without altering H2K20Me3 in both types of normal respiratory epithelia. (d) Western blot analysis of histone alterations as well as DNMT and EZH2 expression in HBEC exposed to CSC. Densitometry analysis revealed time-dependent decreases in H4K16 acetylation and H4K20 trimethylation without appreciable alterations in total histone H4 levels. These alterations coincided with a relative increase in H3K27 trimethylation as well as a modest increase in EZH2, the polycomb protein mediating this repressive mark. These phenomena, which were evident after 5-month CSC exposure, coincided with a decrease in DNMT1:DNMT3b protein ratios.

Figure 2

Pyrosequencing analysis of methylation status in repetitive DNA sequences before and after CSC exposure. (a) Pyrosequencing analysis of NBL2, D4Z4, and LINE-1 methylation in untreated respiratory epithelia and A549 lung cancer cells. Relative to SAEC and NHBE, immortalized HBEC exhibited somewhat reduced methylation in these DNA repeats. This phenomenon was even more pronounced in A549 lung cancer cells. (b) Pyrosequencing analysis of NBL2, D4Z4, and LINE-1 sequences in HBEC exposed to CSC for 5 months. A dose-dependent decrease in DNA methylation was observed, suggestive of global DNA demethylation mediated by cigarette smoke. (c) Left panel: Quantitative RT–PCR and correlative RT–PCR analysis of imprinted and cancer-testis gene expression in HBEC after CSC exposure (upper and lower panels, respectively). The results of RT–PCR depicted as fold change relative to untreated HBEC. CSC dose-dependently increased expression of H19 and IGF-2 imprinted loci, as well as MAGE-A1 and MAGE-A3. No increase in NY-ESO-1 expression was observed. The results of RT–PCR analysis were consistent with qRT–PCR data. Right panel: Pyrosequencing analysis of genomic DNA and cDNA pertaining to IGF2 and H19 in control HBEC as well as HBEC exposed to CSC for 5 months. Genotyping revealed polymorphisms that could distinguish between maternal and paternal alleles of IGF2 and H19. After prolonged CSC exposure, only the maternally imprinted IGF2 and the paternally imprinted H19 loci were expressed. These results suggest that enhanced expression of these loci was attributable to mono-allelic up-regulation rather than loss of imprinting. (d) qRT–PCR with representative RT–PCR, and pyrosequencing analyses of tumor suppressor gene loci in HBEC after prolonged CSC exposure (upper and lower panels, respectively). A progressive, dose-dependent increase in promoter methylation was observed in RASSF1A and RAR-β promoter regions. No apparent hypermethylation was observed in several tumor suppressor genes such as p16, E-cadherin, DAP kinase, and MGMT, which are frequently silenced in tobacco-associated lung cancers by epigenetic mechanisms. DAP kinase expression in untreated and CSC-exposed HBEC was exceedingly low. Dkk-1 was not methylated, but was repressed, a phenomenon possibly attributable to polycomb repressor complexes (Hussain et al., 2009). Diminution of RASSF1A expression coincided with hypermethylation of the RASSF1A promoter. Hypermethylation of RAR-β seemed insufficient to silence this gene under exposure conditions used for these experiments.

Figure 3

(a) The results of Wnt superarray analysis showing marked up-regulation of a variety of Wnt ligands and down-regulation of antagonists of Wnt signaling including SFRP1 and Dkk-1. (b) The results of superarrays showing CSC-mediated changes in expression levels of numerous genes regulating cellular response to oxidative stress.

Figure 4

(a, b) Clonogenic potential of HBECs after prolonged CSC exposure. Soft-agar assay showing a time-dependent increase in clonogenic potential of HBEC after prolonged CSC treatment. (c) Pyrosequencing analysis of DNA methylation within repetitive DNA sequences and tumor suppressor genes in HBEC after prolonged CSC exposure. A progressive time-dependent increase in RASSF1A and RARβ promoter methylation was observed without an apparent increase in methylation of p16, DAPK, E-cadherin, or CDH13. Increased clonogenic potential also coincided with a time-dependent decrease in methylation status within DNA repeats, particularly NBL2 and D4Z4. (d) Pyrosequencing analysis of DNA methylation in soft-agar clones emerging spontaneously from untreated HBEC and representative soft-agar clones derived from HBEC exposed to CSC for 9 months. The CSC-exposed clones showed markedly diminished methylation status within NBL2 and D4Z4, findings which were consistent with results observed in bulk cultures. Once again, there was no change in methylation status within p16, DAPK, E-cadherin, H-cadherin, or MGMT. However, clones derived from CSC-exposed HBEC exhibited markedly increased methylation status of RASSF1A and RARβ. These data suggest that CSC induced alterations, which contributed to enhanced clonogenicity rather than selecting for outgrowth of cells exhibiting aberrant methylation profiles in bulk untreated HBEC.

Figure 5

(a) Illumina array analysis of DNA methylation in control bulk HBEC, control spontaneous soft-agar clones, and soft-agar clones derived from HBEC exposed to CSC for 9 months. Treatment groups were as follows—1: control untreated HBEC; 2: control spontaneous soft-agar clones; 3: CSC-derived soft-agar clones expanded in the absence of CSC; 4: CSC-derived soft-agar clones expanded in the presence of CSC; 5: pooled HBEC exposed to CSC for 9 months. Each lane pertains to an individual clone or pooled sample. Spontaneous clones exhibited methylation profiles remarkably similar to those observed in untreated HBEC cells. In contrast, CSC-exposed clones showed considerable alterations in methylation status across numerous gene loci. Additional short-term CSC exposure did not seem to further alter methylation profiles in CSC-derived soft-agar colonies. Heat map represents a differential score >25 for genes hypo- or hypermethylated by CSC treatment. These data are summarized in Table 1. (b) Quantitative RT–PCR analysis of HSPB1 expression in untreated control and CSC-treated HBEC, as well as spontaneous and CSC-derived soft-agar clones. Data from two independent experiments revealed that CSC exposure diminished HSPB1 expression in HBEC. Interestingly, HSPB1 expression in spontaneous soft-agar clones was decreased relative to untreated controls, albeit to a lesser extent than CSC-treated HBEC. (c) Pyrosequencing analysis of HSPB1 promoter methylation in untreated and pooled CSC-treated HBEC, as well as spontaneous and CSC-derived soft-agar clones. CSC exposure mediated a progressive increase in HSPB1 promoter methylation. No apparent increase in HSPB1 promoter methylation was observed despite diminished HSPB1 expression in spontaneous clones relative to untreated HBEC controls. (d) ChIP analysis showing decreased H3K9Ac (activation mark) with increased H3K27Me3 (repression mark) within the HSPB1 promoter in HBEC exposed to CSC. These findings are consistent with CSC-mediated down-regulation of HSPB1. (e) Quantitative RT–PCR analysis of HSPB1 expression in a panel of immortalized respiratory epithelia and lung cancer lines. The results are depicted as fold relative to SAEC. HSPB1 expression in immortalized respiratory epithelia (HBEC and BEAS) and numerous lung cancer lines was lower than that observed in SAEC.

Figure 6

(a) Illumina array analysis of gene expression in untreated HBEC, and spontaneous clones from control HBEC emerging from soft agar, as well as clones exposed to CSC and then expanded with or without additional tobacco smoke exposure. Gene expression profiles in untreated controls as well as spontaneous control clones were remarkably similar. Interestingly, array analysis revealed that gene expression profiles in clones derived from CSC-treated HBEC that were subsequently expanded without further CSC exposure were markedly altered; additional CSC exposure further modulated gene expression in HBEC, reflecting acute effects of CSC. (b) Ingenuity analysis of Illumina gene expression arrays revealing major pathways modulated in CSC-derived soft-agar clones expanded in the absence (CSC−) or presence (CSC+) of CSC.