Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

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

Oncogene volume 29, pages 3650–3664 (2010)Cite this article

Subjects

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-β, 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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  • Agherbi H, Gaussmann-Wenger A, Verthuy C, Chasson L, Serrano M, Djabali M . (2009). Polycomb mediated epigenetic silencing and replication timing at the INK4a/ARF locus during senescence. PLoS One 4: e5622.

    Article  Google Scholar 

  • Barlesi F, Giaccone G, Gallegos-Ruiz MI, Loundou A, Span SW, Lefesvre P et al. (2007). Global histone modifications predict prognosis of resected non small-cell lung cancer. J Clin Oncol 25: 4358–4364.

    Article  Google Scholar 

  • Belinsky SA, Nikula KJ, Baylin SB, Issa JP . (1996). Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer. Proc Natl Acad Sci USA 93: 4045–4050.

    Article  CAS  Google Scholar 

  • Cho HJ, Caballero OL, Gnjatic S, Andrade VC, Colleoni GW, Vettore AL et al. (2006). Physical interaction of two cancer-testis antigens, MAGE-C1 (CT7) and NY-ESO-1 (CT6). Cancer Immun 6: 12.

    PubMed  Google Scholar 

  • D'Alessio AC, Szyf M . (2006). Epigenetic tête-à-tête: the bilateral relationship between chromatin modifications and DNA methylation. Biochem Cell Biol 84: 463–476.

    Article  CAS  Google Scholar 

  • Damiani LA, Yingling CM, Leng S, Romo PE, Nakamura J, Belinsky SA . (2008). Carcinogen-induced gene promoter hypermethylation is mediated by DNMT1 and causal for transformation of immortalized bronchial epithelial cells. Cancer Res 68: 9005–9014.

    Article  CAS  Google Scholar 

  • Dovey JS, Zacharek SJ, Kim CF, Lees JA . (2008). Bmi1 is critical for lung tumorigenesis and bronchioalveolar stem cell expansion. Proc Natl Acad Sci USA 105: 11857–11862.

    Article  CAS  Google Scholar 

  • Fields WR, Leonard RM, Odom PS, Nordskog BK, Ogden MW, Doolittle DJ . (2005). Gene expression in normal human bronchial epithelial (NHBE) cells following in vitro exposure to cigarette smoke condensate. Toxicol Sci 86: 84–91.

    Article  CAS  Google Scholar 

  • Fraga MF, Ballestar E, Villar-Garea A, Boix-Chornet M, Espada J, Schotta G et al. (2005). Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat Genet 37: 391–400.

    Article  CAS  Google Scholar 

  • Ho YS, Chen CH, Wang YJ, Pestell RG, Albanese C, Chen RJ et al. (2005). Tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces cell proliferation in normal human bronchial epithelial cells through NFkappaB activation and cyclin D1 up-regulation. Toxicol Appl Pharmacol 205: 133–148.

    Article  CAS  Google Scholar 

  • Hoogeboom D, Burgering BM . (2009). Should I stay or should I go: beta-catenin decides under stress. Biochim Biophys Acta 1796: 63–74.

    CAS  PubMed  Google Scholar 

  • Huang CL, Liu D, Nakano J, Ishikawa S, Kontani K, Yokomise H et al. (2005). Wnt5a expression is associated with the tumor proliferation and the stromal vascular endothelial growth factor—an expression in non-small-cell lung cancer. J Clin Oncol 23: 8765–8773.

    Article  Google Scholar 

  • Huang H, He X . (2008). Wnt/beta-catenin signaling: new (and old) players and new insights. Curr Opin Cell Biol 20: 119–125.

    Article  CAS  Google Scholar 

  • Hussain M, Rao M, Humphries AE, Hong JA, Liu F, Yang M et al. (2009). Tobacco smoke induces polycomb-mediated repression of Dickkopf-1 in lung cancer cells. Cancer Res 69: 3570–3578.

    Article  CAS  Google Scholar 

  • Jensen TJ, Wozniak RJ, Eblin KE, Wnek SM, Gandolfi AJ, Futscher BW . (2009). Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation. Toxicol Appl Pharmacol 235: 39–46.

    Article  CAS  Google Scholar 

  • Jin Q, Menter DG, Mao L, Hong WK, Lee HY . (2008). Survivin expression in normal human bronchial epithelial cells: an early and critical step in tumorigenesis induced by tobacco exposure. Carcinogenesis 29: 1614–1622.

    Article  CAS  Google Scholar 

  • Jorgensen ED, Dozmorov I, Frank MB, Centola M, Albino AP . (2004). Global gene expression analysis of human bronchial epithelial cells treated with tobacco condensates. Cell Cycle 3: 1154–1168.

    Article  CAS  Google Scholar 

  • Kang Y, Hong JA, Chen GA, Nguyen DM, Schrump DS . (2007). Dynamic transcriptional regulatory complexes including BORIS, CTCF and Sp1 modulate NY-ESO-1 expression in lung cancer cells. Oncogene 26: 4394–4403.

    Article  CAS  Google Scholar 

  • Kaplan R, Luettich K, Heguy A, Hackett NR, Harvey BG, Crystal RG . (2003). Monoallelic up-regulation of the imprinted H19 gene in airway epithelium of phenotypically normal cigarette smokers. Cancer Res 63: 1475–1482.

    CAS  PubMed  Google Scholar 

  • Katoh M, Katoh M . (2007). WNT signaling pathway and stem cell signaling network. Clin Cancer Res 13: 4042–4045.

    Article  CAS  Google Scholar 

  • Keshet I, Schlesinger Y, Farkash S, Rand E, Hecht M, Segal E et al. (2006). Evidence for an instructive mechanism of de novo methylation in cancer cells. Nat Genet 38: 149–153.

    Article  CAS  Google Scholar 

  • Killian JK, Bilke S, Davis S, Walker RL, Killian MS, Jaeger EB et al. (2009). Large-scale profiling of archival lymph nodes reveals pervasive remodeling of the follicular lymphoma methylome. Cancer Res 69: 758–764.

    Article  CAS  Google Scholar 

  • Kim H, Kwon YM, Kim JS, Han J, Shim YM, Park J et al. (2006). Elevated mRNA levels of DNA methyltransferase-1 as an independent prognostic factor in primary nonsmall cell lung cancer. Cancer 107: 1042–1049.

    Article  CAS  Google Scholar 

  • Le FN, Rivat C, De WO, Bruyneel E, Mareel M, Dale T et al. (2005). The proinvasive activity of Wnt-2 is mediated through a noncanonical Wnt pathway coupled to GSK-3beta and c-Jun/AP-1 signaling. FASEB J 19: 144–146.

    Article  Google Scholar 

  • Lee SJ, Jeon HS, Jang JS, Park SH, Lee GY, Lee BH et al. (2005). DNMT3B polymorphisms and risk of primary lung cancer. Carcinogenesis 26: 403–409.

    Article  CAS  Google Scholar 

  • Licchesi JD, Westra WH, Hooker CM, Machida EO, Baylin SB, Herman JG . (2008). Epigenetic alteration of Wnt pathway antagonists in progressive glandular neoplasia of the lung. Carcinogenesis 29: 895–904.

    Article  CAS  Google Scholar 

  • Lin RK, Hsu HS, Chang JW, Chen CY, Chen JT, Wang YC . (2007). Alteration of DNA methyltransferases contributes to 5′CpG methylation and poor prognosis in lung cancer. Lung Cancer 55: 205–213.

    Article  Google Scholar 

  • Malusecka E, Krzyzowska-Gruca S, Gawrychowski J, Fiszer-Kierzkowska A, Kolosza Z, Krawczyk Z . (2008). Stress proteins HSP27 and HSP70i predict survival in non-small cell lung carcinoma. Anticancer Res 28: 501–506.

    PubMed  Google Scholar 

  • Mathews LA, Crea F, Farrar WL . (2009). Epigenetic gene regulation in stem cells and correlation to cancer. Differentiation 78: 1–17.

    Article  CAS  Google Scholar 

  • McCabe MT, Lee EK, Vertino PM . (2009). A multifactorial signature of DNA sequence and polycomb binding predicts aberrant CpG island methylation. Cancer Res 69: 282–291.

    Article  CAS  Google Scholar 

  • Monte M, Simonatto M, Peche LY, Bublik DR, Gobessi S, Pierotti MA et al. (2006). MAGE-A tumor antigens target p53 transactivation function through histone deacetylase recruitment and confer resistance to chemotherapeutic agents. Proc Natl Acad Sci USA 103: 11160–11165.

    Article  CAS  Google Scholar 

  • Narayan S, Jaiswal AS, Kang D, Srivastava P, Das GM, Gairola CG . (2004). Cigarette smoke condensate-induced transformation of normal human breast epithelial cells in vitro. Oncogene 23: 5880–5889.

    Article  CAS  Google Scholar 

  • Richter AM, Pfeifer GP, Dammann RH . (2009). The RASSF proteins in cancer; from epigenetic silencing to functional characterization. Biochim Biophys Acta 1796: 114–128.

    CAS  PubMed  Google Scholar 

  • Schrump DS, Hong JA, Nguyen DM . (2007). Utilization of chromatin remodeling agents for lung cancer therapy. Cancer J 13: 56–64.

    Article  CAS  Google Scholar 

  • Seligson DB, Horvath S, McBrian MA, Mah V, Yu H, Tze S et al. (2009). Global levels of histone modifications predict prognosis in different cancers. Am J Pathol 174: 1619–1628.

    Article  CAS  Google Scholar 

  • Seng TJ, Currey N, Cooper WA, Lee CS, Chan C, Horvath L et al. (2008). DLEC1 and MLH1 promoter methylation are associated with poor prognosis in non-small cell lung carcinoma. Br J Cancer 99: 375–382.

    Article  CAS  Google Scholar 

  • Sexton K, Balharry D, BeruBe KA . (2008). Genomic biomarkers of pulmonary exposure to tobacco smoke components. Pharmacogenet Genomics 18: 853–860.

    Article  CAS  Google Scholar 

  • Shin HJ, Sohn HO, Han JH, Park CH, Lee HS, Lee DW et al. (2009). Effect of cigarette filters on the chemical composition and in vitro biological activity of cigarette mainstream smoke. Food Chem Toxicol 47: 192–197.

    Article  CAS  Google Scholar 

  • Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ . (2005). Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 5: 615–625.

    Article  CAS  Google Scholar 

  • Smith CJ, Perfetti TA, Garg R, Hansch C . (2003). IARC carcinogens reported in cigarette mainstream smoke and their calculated log P values. Food Chem Toxicol 41: 807–817.

    Article  CAS  Google Scholar 

  • Toyooka S, Maruyama R, Toyooka KO, McLerran D, Feng Z, Fukuyama Y et al. (2003). Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer. Int J Cancer 103: 153–160.

    Article  CAS  Google Scholar 

  • Toyooka S, Tokumo M, Shigematsu H, Matsuo K, Asano H, Tomii K et al. (2006). Mutational and epigenetic evidence for independent pathways for lung adenocarcinomas arising in smokers and never smokers. Cancer Res 66: 1371–1375.

    Article  CAS  Google Scholar 

  • Van Den BA, Brambilla E, Moro-Sibilot D, Lantuejoul S, Brambilla C, Eymin B et al. (2008). Loss of histone H4K20 trimethylation occurs in preneoplasia and influences prognosis of non-small cell lung cancer. Clin Cancer Res 14: 7237–7245.

    Article  Google Scholar 

  • Vrzalikova K, Skarda J, Ehrmann J, Murray PG, Fridman E, Kopolovic J et al. (2008). Prognostic value of Bmi-1 oncoprotein expression in NSCLC patients: a tissue microarray study. J Cancer Res Clin Oncol 134: 1037–1042.

    Article  CAS  Google Scholar 

  • Watanabe H, Soejima K, Yasuda H, Kawada I, Nakachi I, Yoda S et al. (2008). Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells. Cancer Cell Int 8: 15.

    Article  Google Scholar 

  • West KA, Brognard J, Clark AS, Linnoila IR, Yang X, Swain SM et al. (2003). Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells. J Clin Invest 111: 81–90.

    Article  CAS  Google Scholar 

  • Winn RA, Van SM, Hammond M, Rodriguez K, Crossno Jr JT, Heasley LE et al. (2006). Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferator-activated receptor gamma. J Biol Chem 281: 26943–26950.

    Article  CAS  Google Scholar 

  • Wu JP, Chang LW, Yao HT, Chang H, Tsai HT, Tsai MH et al. (2009). Involvement of oxidative stress and activation of aryl hydrocarbon receptor in elevation of CYP1A1 expression and activity in lung cells and tissues by arsenic: an in vitro and in vivo study. Toxicol Sci 107: 385–393.

    Article  CAS  Google Scholar 

  • Xing J, Stewart DJ, Gu J, Lu C, Spitz MR, Wu X . (2008). Expression of methylation-related genes is associated with overall survival in patients with non-small cell lung cancer. Br J Cancer 98: 1716–1722.

    Article  CAS  Google Scholar 

  • Yang B, O′Herrin SM, Wu J, Reagan-Shaw S, Ma Y, Bhat KM et al. (2007). MAGE-A, mMage-b, and MAGE-C proteins form complexes with KAP1 and suppress p53-dependent apoptosis in MAGE-positive cell lines. Cancer Res 67: 9954–9962.

    Article  CAS  Google Scholar 

  • Zhou X, Li Q, Arita A, Sun H, Costa M . (2009). Effects of nickel, chromate, and arsenite on histone 3 lysine methylation. Toxicol Appl Pharmacol 236: 78–84.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Thoracic Oncology Section, Surgery Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA

    F Liu, M Yang, J A Hong, M Zhang, Y Zhang, M Hussain, S Xi, M Rao & D S Schrump

  2. Genetics Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    J K Killian, R L Walker, S Davis & P A Meltzer

Authors
  1. F Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J K Killian
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. R L Walker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J A Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. S Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Y Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. M Hussain
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. S Xi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. M Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. P A Meltzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. D S Schrump
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D S Schrump.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, F., Killian, J., Yang, M. et al. Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate. Oncogene 29, 3650–3664 (2010). https://doi.org/10.1038/onc.2010.129

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2010.129

Keywords

This article is cited by

Search

Advanced search

Quick links