Advertisement

Tumor Biology

, Volume 33, Issue 2, pp 287–296 | Cite as

DNA methylation biomarkers for lung cancer

  • Tibor A. Rauch
  • Zunde Wang
  • Xiwei Wu
  • Kemp H. Kernstine
  • Arthur D. Riggs
  • Gerd P. Pfeifer
Research Article

Abstract

Changes in DNA methylation patterns are an important characteristic of human cancer including lung cancer. In particular, hypermethylation of CpG islands is a signature of malignant progression. Methylated CpG islands are promising diagnostic markers for the early detection of cancer. However, the full extent and sequence context of DNA hypermethylation in lung cancer has remained unknown. We have used the methylated CpG island recovery assay and high-resolution microarray analysis to find hypermethylated CpG islands in squamous cell carcinomas (SCC) and adenocarcinomas of the lung. Each tumor contained several hundred hypermethylated CpG islands. In an initial microarray screen, 36 CpG islands were methylated in five of five (=100%) of the SCC tumors tested and 52 CpG islands were methylated in at least 75% of the adenocarcinomas tested (n = 8). Using sodium-bisulfite-based approaches, 12 CpG islands (associated with the BARHL2, EVX2, IRX2, MEIS1, MSX1, NR2E1, OC2, OSR1, OTX1, PAX6, TFAP2A, and ZNF577 genes) were confirmed to be methylated in 85% to 100% of the squamous cell carcinomas and 11 CpG islands (associated with the CHAD, DLX4, GRIK2, KCNG3, NR2E1, OSR1, OTX1, OTX2, PROX1, RUNX1, and VAX1 genes) were methylated in >80% of the adenocarcinomas. From the list of genes that were methylated in lung adenocarcinomas, we identified the gene FAT4 and found that this gene was methylated in 39% of the tumors. FAT4 is the closest mammalian homologue of the Drosophila tumor suppressor Fat which is an important component of the Hippo growth control pathway. Many of these newly discovered methylated CpG islands hold promise for becoming biomarkers for the early detection of lung cancer.

Keywords

DNA methylation CpG islands Methylation marker Lung cancer FAT4 

Notes

Acknowledgments

This work was supported by NIH grant CA084469 and a grant from the University of California Tobacco Related Disease Research Program (TRDRP) (to G.P.P.)

Conflicts of interest

Under a licensing agreement between City of Hope and Active Motif (Carlsbad, CA), the methylated CpG island recovery assay (MIRA) technique was licensed to Active Motif, and the authors T.A.R. and G.P.P. are entitled to a share of the royalties received by City of Hope from sales of the licensed technology.

References

  1. 1.
    Anglim PP, Alonzo TA, Laird-Offringa IA. DNA methylation-based biomarkers for early detection of non-small cell lung cancer: an update. Mol Cancer. 2008;7:81.PubMedCrossRefGoogle Scholar
  2. 2.
    Baylin SB, Hoppener JW, de Bustros A, Steenbergh PH, Lips CJ, Nelkin BD. DNA methylation patterns of the calcitonin gene in human lung cancers and lymphomas. Cancer Res. 1986;46:2917–22.PubMedGoogle Scholar
  3. 3.
    Belinsky SA. Gene-promoter hypermethylation as a biomarker in lung cancer. Nat Rev Cancer. 2004;4:707–17.PubMedCrossRefGoogle Scholar
  4. 4.
    Berndt A, Cario CL, Silva KA, Kennedy VE, Harrison DE, Paigen B, Sundberg JP. Identification of fat4 and tsc22d1 as novel candidate genes for spontaneous pulmonary adenomas. Cancer Res. 2011;71:5779–91.PubMedCrossRefGoogle Scholar
  5. 5.
    Burbee DG, Forgacs E, Zochbauer-Muller S, Shivakumar L, Fong K, Gao B, Randle D, Kondo M, Virmani A, Bader S, Sekido Y, Latif F, Milchgrub S, Toyooka S, Gazdar AF, Lerman MI, Zabarovsky E, White M, Minna JD. Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst. 2001;93:691–9.PubMedCrossRefGoogle Scholar
  6. 6.
    Cho E, Feng Y, Rauskolb C, Maitra S, Fehon R, Irvine KD. Delineation of a Fat tumor suppressor pathway. Nat Genet. 2006;38:1142–50.PubMedCrossRefGoogle Scholar
  7. 7.
    Costello JF, Fruhwald MC, Smiraglia DJ, Rush LJ, Robertson GP, Gao X, Wright FA, Feramisco JD, Peltomaki P, Lang JC, Schuller DE, Yu L, Bloomfield CD, Caligiuri MA, Yates A, Nishikawa R, Su Huang H, Petrelli NJ, Zhang X, O’Dorisio MS, et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat Genet. 2000;24:132–8.PubMedCrossRefGoogle Scholar
  8. 8.
    Costello JF, Plass C. Methylation matters. J Med Genet. 2001;38:285–303.PubMedCrossRefGoogle Scholar
  9. 9.
    Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP. Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet. 2000;25:315–9.PubMedCrossRefGoogle Scholar
  10. 10.
    Dammann R, Takahashi T, Pfeifer GP. The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas. Oncogene. 2001;20:3563–7.PubMedCrossRefGoogle Scholar
  11. 11.
    Dammann R, Strunnikova M, Schagdarsurengin U, Rastetter M, Papritz M, Hattenhorst UE, Hofmann HS, Silber RE, Burdach S, Hansen G. CpG island methylation and expression of tumour-associated genes in lung carcinoma. Eur J Cancer. 2005;41:1223–36.PubMedCrossRefGoogle Scholar
  12. 12.
    Esteller M, Corn PG, Baylin SB, Herman JG. A gene hypermethylation profile of human cancer. Cancer Res. 2001;61:3225–9.PubMedGoogle Scholar
  13. 13.
    Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet. 2007;8:286–98.PubMedCrossRefGoogle Scholar
  14. 14.
    Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983;301:89–92.PubMedCrossRefGoogle Scholar
  15. 15.
    Feng Q, Hawes SE, Stern JE, Wiens L, Lu H, Dong ZM, Jordan CD, Kiviat NB, Vesselle H. DNA methylation in tumor and matched normal tissues from non-small cell lung cancer patients. Cancer Epidemiol Biomarkers Prev. 2008;17:645–54.PubMedCrossRefGoogle Scholar
  16. 16.
    Gama-Sosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW, Ehrlich M. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res. 1983;11:6883–94.PubMedCrossRefGoogle Scholar
  17. 17.
    Gonzalez-Zulueta M, Bender CM, Yang AS, Nguyen T, Beart RW, Van Tornout JM, Jones PA. Methylation of the 5′ CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res. 1995;55:4531–5.PubMedGoogle Scholar
  18. 18.
    Guo C, Tommasi S, Liu L, Yee JK, Dammann R, Pfeifer GP. RASSF1A is part of a complex similar to the Drosophila Hippo/Salvador/Lats tumor-suppressor network. Curr Biol. 2007;17:700–5.PubMedCrossRefGoogle Scholar
  19. 19.
    Guo C, Zhang X, Pfeifer GP. The tumor suppressor RASSF1A prevents dephosphorylation of the mammalian STE20-like kinases MST1 and MST2. J Biol Chem. 2011;286:6253–61.PubMedCrossRefGoogle Scholar
  20. 20.
    Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, Sidransky D, Baylin SB. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 1995;55:4525–30.PubMedGoogle Scholar
  21. 21.
    Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–92.PubMedCrossRefGoogle Scholar
  22. 22.
    Kalari S, Pfeifer GP. Identification of driver and passenger DNA methylation in cancer by epigenomic analysis. Adv Genet. 2010;70:277–308.PubMedCrossRefGoogle Scholar
  23. 23.
    Kane MF, Loda M, Gaida GM, Lipman J, Mishra R, Goldman H, Jessup JM, Kolodner R. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 1997;57:808–11.PubMedGoogle Scholar
  24. 24.
    Lahtz C, Pfeifer GP. Epigenetic changes of DNA repair genes in cancer. J Mol Cell Biol. 2011;3:51–8.PubMedCrossRefGoogle Scholar
  25. 25.
    Laird PW. The power and the promise of DNA methylation markers. Nat Rev Cancer. 2003;3:253–66.PubMedCrossRefGoogle Scholar
  26. 26.
    Lee KP, Lee JH, Kim TS, Kim TH, Park HD, Byun JS, Kim MC, Jeong WI, Calvisi DF, Kim JM, Lim DS. The Hippo–Salvador pathway restrains hepatic oval cell proliferation, liver size, and liver tumorigenesis. Proc Natl Acad Sci U S A. 2010;107:8248–53.PubMedCrossRefGoogle Scholar
  27. 27.
    Merlo A, Herman JG, Mao L, Lee DJ, Gabrielson E, Burger PC, Baylin SB, Sidransky D. 5′ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med. 1995;1:686–92.PubMedCrossRefGoogle Scholar
  28. 28.
    Ohm JE, McGarvey KM, Yu X, Cheng L, Schuebel KE, Cope L, Mohammad HP, Chen W, Daniel VC, Yu W, Berman DM, Jenuwein T, Pruitt K, Sharkis SJ, Watkins DN, Herman JG, Baylin SB. A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA hypermethylation and heritable silencing. Nat Genet. 2007;39:237–42.PubMedCrossRefGoogle Scholar
  29. 29.
    Pan D. The hippo signaling pathway in development and cancer. Dev Cell. 2010;19:491–505.PubMedCrossRefGoogle Scholar
  30. 30.
    Pfeifer GP, Dammann R, Tommasi S. RASSF proteins. Curr Biol. 2010;20:R344–5.PubMedCrossRefGoogle Scholar
  31. 31.
    Rauch T, Pfeifer GP. Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-CpG islands in cancer. Lab Investig. 2005;85:1172–80.PubMedCrossRefGoogle Scholar
  32. 32.
    Rauch T, Li H, Wu X, Pfeifer GP. MIRA-assisted microarray analysis, a new technology for the determination of DNA methylation patterns, identifies frequent methylation of homeodomain-containing genes in lung cancer cells. Cancer Res. 2006;66:7939–47.PubMedCrossRefGoogle Scholar
  33. 33.
    Rauch T, Wang Z, Zhang X, Zhong X, Wu X, Lau SK, Kernstine KH, Riggs AD, Pfeifer GP. Homeobox gene methylation in lung cancer studied by genome-wide analysis with a microarray-based methylated CpG island recovery assay. Proc Natl Acad Sci U S A. 2007;104:5527–32.PubMedCrossRefGoogle Scholar
  34. 34.
    Rauch TA, Zhong X, Wu X, Wang M, Kernstine KH, Wang Z, Riggs AD, Pfeifer GP. High-resolution mapping of DNA hypermethylation and hypomethylation in lung cancer. Proc Natl Acad Sci U S A. 2008;105:252–7.PubMedCrossRefGoogle Scholar
  35. 35.
    Rauch TA, Pfeifer GP. DNA methylation profiling using the methylated-CpG island recovery assay (MIRA). Methods. 2010;52:213–7.PubMedCrossRefGoogle Scholar
  36. 36.
    Riggs AD, Jones PA. 5-Methylcytosine, gene regulation, and cancer. Adv Cancer Res. 1983;40:1–30.PubMedCrossRefGoogle Scholar
  37. 37.
    Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J, Eden E, Yakhini Z, Ben-Shushan E, Reubinoff BE, Bergman Y, Simon I, Cedar H. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer. Nat Genet. 2007;39:232–6.PubMedCrossRefGoogle Scholar
  38. 38.
    Seidel C, Schagdarsurengin U, Blumke K, Wurl P, Pfeifer GP, Hauptmann S, Taubert H, Dammann R. Frequent hypermethylation of MST1 and MST2 in soft tissue sarcoma. Mol Carcinog. 2007;46:865–71.PubMedCrossRefGoogle Scholar
  39. 39.
    Sudol M, Harvey KF. Modularity in the Hippo signaling pathway. Trends Biochem Sci. 2010;35:627–33.PubMedCrossRefGoogle Scholar
  40. 40.
    Tommasi S, Dammann R, Zhang Z, Wang Y, Liu L, Tsark WM, Wilczynski SP, Li J, You M, Pfeifer GP. Tumor susceptibility of Rassf1a knockout mice. Cancer Res. 2005;65:92–8.PubMedGoogle Scholar
  41. 41.
    Tommasi S, Karm DL, Wu X, Yen Y, Pfeifer GP. Methylation of homeobox genes is a frequent and early epigenetic event in breast cancer. Breast Cancer Res. 2009;11:R14.PubMedCrossRefGoogle Scholar
  42. 42.
    Topaloglu O, Hoque MO, Tokumaru Y, Lee J, Ratovitski E, Sidransky D, Moon CS. Detection of promoter hypermethylation of multiple genes in the tumor and bronchoalveolar lavage of patients with lung cancer. Clin Cancer Res. 2004;10:2284–8.PubMedCrossRefGoogle Scholar
  43. 43.
    Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer. 2005;5:223–31.PubMedCrossRefGoogle Scholar
  44. 44.
    Widschwendter M, Fiegl H, Egle D, Mueller-Holzner E, Spizzo G, Marth C, Weisenberger DJ, Campan M, Young J, Jacobs I, Laird PW. Epigenetic stem cell signature in cancer. Nat Genet. 2007;39:157–8.PubMedCrossRefGoogle Scholar
  45. 45.
    Wu X, Rauch TA, Zhong X, Bennett WP, Latif F, Krex D, Pfeifer GP. CpG island hypermethylation in human astrocytomas. Cancer Res. 2010;70:2718–27.PubMedCrossRefGoogle Scholar
  46. 46.
    Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 1997;25:2532–4.PubMedCrossRefGoogle Scholar
  47. 47.
    Yanagawa N, Tamura G, Oizumi H, Takahashi N, Shimazaki Y, Motoyama T. Promoter hypermethylation of tumor suppressor and tumor-related genes in non-small cell lung cancers. Cancer Sci. 2003;94:589–92.PubMedCrossRefGoogle Scholar
  48. 48.
    Zhou D, Conrad C, Xia F, Park JS, Payer B, Yin Y, Lauwers GY, Thasler W, Lee JT, Avruch J, Bardeesy N. Mst1 and Mst2 maintain hepatocyte quiescence and suppress hepatocellular carcinoma development through inactivation of the Yap1 oncogene. Cancer Cell. 2009;16:425–38.PubMedCrossRefGoogle Scholar
  49. 49.
    Zochbauer-Muller S, Fong KM, Virmani AK, Geradts J, Gazdar AF, Minna JD. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res. 2001;61:249–55.PubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2011

Authors and Affiliations

  • Tibor A. Rauch
    • 1
    • 4
  • Zunde Wang
    • 1
  • Xiwei Wu
    • 2
  • Kemp H. Kernstine
    • 3
  • Arthur D. Riggs
    • 1
  • Gerd P. Pfeifer
    • 1
  1. 1.Division of BiologyBeckman Research Institute of the City of HopeDuarteUSA
  2. 2.Department of Information SciencesBeckman Research Institute of the City of HopeDuarteUSA
  3. 3.Division of Thoracic SurgeryUniversity of Texas, Southwestern Medical CenterDallasUSA
  4. 4.Section of Molecular Medicine, Department of Orthopedic SurgeryRush University Medical CenterChicagoUSA

Personalised recommendations