Genome-wide DNA methylation profiling of non-small cell lung carcinomas

Epigenetics & Chromatin

Sjaak Philipsen⁷  Boris Lenhard⁴  Frank Grosveld¹  Joachim Aerts⁸  Mirjam van Vroonhoven³  John Foekens⁶  Anieta Sieuwerts⁶  Erikjan Rijkers⁵  Rutger Brouwer⁵  Christel Kockx⁵  Wilfred van IJcken⁵  Supat Thongjuea⁴  Teus van Gent⁷  Jun Hou⁷  Vanja Haberle⁴  Rejane Hughes Carvalho² 

[1] Center for Biomedical Genetics, ErasmusMC, Rotterdam, The Netherlands;Center for Cancer Genomics, ErasmusMC, Rotterdam, The Netherlands;Department of Bioinformatics, ErasmusMC, Rotterdam, The Netherlands;Department of Biology and Computational Biology Unit, Uni BCCS, University of Bergen, Hoyteknologisenteret, Thormohlensgate 55, Bergen, N-5008, Norway;Center for Biomics, ErasmusMC, Rotterdam, The Netherlands;Internal Oncology, ErasmusMC, Rotterdam, The Netherlands;Dutch Consortium for Systems Biology, ErasmusMC, Rotterdam, The Netherlands;Department of Pulmonology, ErasmusMC, Rotterdam, The Netherlands

关键词: Non-small cell lung Cancer;    Next generation sequencing;    MethylCap;    Epigenetics;    DNA Methylation;   

Others  :  813790
DOI  :  10.1186/1756-8935-5-9

 received in 2012-04-03, accepted in 2012-06-22,  发布年份 2012

【 摘 要 】

Background

Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions.

Results

Analysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs) present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas.

Conclusions

Collectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC.

【 授权许可】

   
2012 Hughes Carvalho et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Jemal A, Siegel R, Xu J, Ward E: Cancer statistics, 2010. CA Cancer J Clin 2010, 60:277-300.
• [2]Little AG: No nodes is good nodes. Ann Thorac Surg 2006, 82:4-5.
• [3]Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA: Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 2008, 83:584-594.
• [4]Hou J, Aerts J, den Hamer B, van Ijcken W, den Bakker M, Riegman P, van der Leest C, van der Spek P, Foekens JA, Hoogsteden HC, Grosveld F, Philipsen S, et al.: Gene expression-based classification of non-small cell lung carcinomas and survival prediction. PLoS One 2010, 5:e10312.
• [5]Larsen JE, Pavey SJ, Passmore LH, Bowman R, Clarke BE, Hayward NK, Fong KM: Expression profiling defines a recurrence signature in lung squamous cell carcinoma. Carcinogenesis 2007, 28:760-766.
• [6]Liang P, Pardee AB: Analysing differential gene expression in cancer. Nat Rev Cancer 2003, 3:869-876.
• [7]Kameyama K, Huang CL, Liu D, Masuya D, Nakashima T, Sumitomo S, Takami Y, Kinoshita M, Yokomise H: Reduced ING1b gene expression plays an important role in carcinogenesis of non-small cell lung cancer patients. Clin Cancer Res 2003, 9:4926-4934.
• [8]Petty RD, Nicolson MC, Kerr KM, Collie-Duguid E, Murray GI: Gene expression profiling in non-small cell lung cancer: from molecular mechanisms to clinical application. Clin Cancer Res 2004, 10:3237-3248.
• [9]Ding L, Mardis ER, Li S, Larson DE, Chen K, Wallis JW, Harris CC, McLellan MD, Fulton RS, Fulton LL, Abbott RM, Hoog J, Dooling DJ, Koboldt DC, Schmidt H, Kalicki J, Zhang Q, Chen L, Lin L, Wendl MC, McMichael JF, Magrini VJ, Cook L, McGrath SD, Vickery TL, Appelbaum E, Deschryver K, Davies S, Guintoli T, Lin L, et al.: Genome remodelling in a basal-like breast cancer metastasis and xenograft. Nature 2010, 464:999-1005.
• [10]Lister R, Pelizzola M, Dowen RH, Hawkins RD, Hon G, Tonti-Filippini J, Nery JR, Lee L, Ye Z, Ngo QM, Edsall L, Antosiewicz-Bourget J, Stewart R, Ruotti V, Millar AH, Thomson JA, Ren B, Ecker JR: Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 2009, 462:315-322.
• [11]Weber M, Davies JJ, Wittig D, Oakeley EJ, Haase M, Lam WL, Schubeler D: Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 2005, 37:853-862.
• [12]Bird A: DNA methylation patterns and epigenetic memory. Genes Dev 2002, 16:6-21.
• [13]Bird AP, Wolffe AP: Methylation-induced repression–belts, braces, and chromatin. Cell 1999, 99:451-454.
• [14]Leonhardt H, Page AW, Weier HU, Bestor TH: A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 1992, 71:865-873.
• [15]Agathanggelou A, Honorio S, Macartney DP, Martinez A, Dallol A, Rader J, Fullwood P, Chauhan A, Walker R, Shaw JA, Hosoe S, Lerman MI, Minna JD, Maher ER, Latif F: Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours. Oncogene 2001, 20:1509-1518.
• [16]Kohonen-Corish MR, Sigglekow ND, Susanto J, Chapuis PH, Bokey EL, Dent OF, Chan C, Lin BP, Seng TJ, Laird PW, Young J, Leggett BA, Jass JR, Sutherland RL: Promoter methylation of the mutated in colorectal cancer gene is a frequent early event in colorectal cancer. Oncogene 2007, 26:4435-4441.
• [17]Liu L, Broaddus RR, Yao JC, Xie S, White JA, Wu TT, Hamilton SR, Rashid A: Epigenetic alterations in neuroendocrine tumors: methylation of RAS-association domain family 1, isoform A and p16 genes are associated with metastasis. Mod Pathol 2005, 18:1632-1640.
• [18]Pfeifer GP, Yoon JH, Liu L, Tommasi S, Wilczynski SP, Dammann R: Methylation of the RASSF1A gene in human cancers. Biol Chem 2002, 383:907-914.
• [19]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, 17:645-654.
• [20]Hsu HS, Chen TP, Hung CH, Wen CK, Lin RK, Lee HC, Wang YC: Characterization of a multiple epigenetic marker panel for lung cancer detection and risk assessment in plasma. Cancer 2007, 110:2019-2026.
• [21]Vaissiere T, Hung RJ, Zaridze D, Moukeria A, Cuenin C, Fasolo V, Ferro G, Paliwal A, Hainaut P, Brennan P, Tost J, Boffetta P, Herceg Z: Quantitative analysis of DNA methylation profiles in lung cancer identifies aberrant DNA methylation of specific genes and its association with gender and cancer risk factors. Cancer Res 2009, 69:243-252.
• [22]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, 25:315-319.
• [23]Clark SJ: Action at a distance: epigenetic silencing of large chromosomal regions in carcinogenesis. Hum Mol Genet 2007, 16 Spec No 1:R88-R95.
• [24]Mayor R, Casadome L, Azuara D, Moreno V, Clark SJ, Capella G, Peinado MA: Long-range epigenetic silencing at 2q14.2 affects most human colorectal cancers and may have application as a non-invasive biomarker of disease. Br J Cancer 2009, 100:1534-1539.
• [25]Brinkman AB, Simmer F, Ma K, Kaan A, Zhu J, Stunnenberg HG: Whole-genome DNA methylation profiling using MethylCap-seq. Methods 2010, 52:232-236.
• [26]Teodoridis JM, Hardie C, Brown R: CpG island methylator phenotype (CIMP) in cancer: causes and implications. Cancer Lett 2008, 268:177-186.
• [27]Keshet I, Schlesinger Y, Farkash S, Rand E, Hecht M, Segal E, Pikarski E, Young RA, Niveleau A, Cedar H, Simon I: Evidence for an instructive mechanism of de novo methylation in cancer cells. Nat Genet 2006, 38:149-153.
• [28]Rauch TA, Pfeifer GP: DNA methylation profiling using the methylated-CpG island recovery assay (MIRA). Methods 2010, 52:213-217.
• [29]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, 104:5527-5532.
• [30]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, 105:252-257.
• [31]Schmidt B, Liebenberg V, Dietrich D, Schlegel T, Kneip C, Seegebarth A, Flemming N, Seemann S, Distler J, Lewin J, Tetzner R, Weickmann S, Wille U, Liloglou T, Raji O, Walshaw M, Fleischhacker M, Witt C, Field JK: SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer based on bronchial aspirates. BMC Cancer 2010, 10:600.
• [32]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, 61:249-255.
• [33]Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 1996, 93:9821-9826.
• [34]Bibikova M, Lin Z, Zhou L, Chudin E, Garcia EW, Wu B, Doucet D, Thomas NJ, Wang Y, Vollmer E, Goldmann T, Seifart C, Jiang W, Barker DL, Chee MS, Floros J, Fan JB: High-throughput DNA methylation profiling using universal bead arrays. Genome Res 2006, 16:383-393.
• [35]Sano A, Kage H, Sugimoto K, Kitagawa H, Aki N, Goto A, Fukayama M, Nakajima J, Takamoto S, Nagase T, Yatomi Y, Ohishi N, Takai D: A second-generation profiling system for quantitative methylation analysis of multiple gene promoters: application to lung cancer. Oncogene 2007, 26:6518-6525.
• [36]Shames DS, Girard L, Gao B, Sato M, Lewis CM, Shivapurkar N, Jiang A, Perou CM, Kim YH, Pollack JR, Fong KM, Lam CL, Wong M, Shyr Y, Nanda R, Olopade OI, Gerald W, Euhus DM, Shay JW, Gazdar AF, Minna JD: A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies. PLoS Med 2006, 3:486.
• [37]Zhang Y, Wang R, Song H, Huang G, Yi J, Zheng Y, Wang J, Chen L: Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer. Cancer Letters 2011, 303:21-28.
• [38]Kwon YJ, Lee SJ, Koh JS, Kim SH, Lee HW, Kang MC, Bae JB, Kim YJ, Park JH: Genome-wide analysis of DNA methylation and the gene expression change in lung cancer. J Thorac Oncol 2012, 7:20-33.
• [39]Chavez L, Jozefczuk J, Grimm C, Dietrich J, Timmermann B, Lehrach H, Herwig R, Adjaye J: Computational analysis of genome-wide DNA methylation during the differentiation of human embryonic stem cells along the endodermal lineage. Genome Res 2010, 20:1441-1450.
• [40]Chung JH, Lee HJ, Kim BH, Cho NY, Kang GH: DNA methylation profile during multistage progression of pulmonary adenocarcinomas. Virchows Arch 2011, 459:201-211.
• [41]Esteller M: Epigenetic gene silencing in cancer: the DNA hypermethylome. Hum Mol Genet 2007, 16 Spec No 1:R50-R59.
• [42]Liu Y, Pang JC, Dong S, Mao B, Poon WS, Ng HK: Aberrant CpG island hypermethylation profile is associated with atypical and anaplastic meningiomas. Hum Pathol 2005, 36:416-425.
• [43]Marsit CJ, Houseman EA, Christensen BC, Eddy K, Bueno R, Sugarbaker DJ, Nelson HH, Karagas MR, Kelsey KT: Examination of a CpG island methylator phenotype and implications of methylation profiles in solid tumors. Cancer Res 2006, 66:10621-10629.
• [44]Ting DT, Lipson D, Paul S, Brannigan BW, Akhavanfard S, Coffman EJ, Contino G, Deshpande V, Iafrate AJ, Letovsky S, Rivera MN, Bardeesy N, Maheswaran S, Haber DA: Aberrant overexpression of satellite repeats in pancreatic and other epithelial cancers. Science 2011, 331:593-596.
• [45]Pfeifer GP, Rauch TA: DNA methylation patterns in lung carcinomas. Semin Cancer Biol 2009, 19:181-187.
• [46]Hou J, Lambers M, den Hamer B, den Bakker M, Hoogsteden MC, Grosveld F, Hegmans J, Aerts J, Philipsen S: Expression profiling-based subtyping identifies novel non-small cell lung cancer subgroups and implicates putative resistance to pemetrexed therapy. J Thorac Oncol 2012, 7:105-114.
• [47]Chatagnon A, Ballestar E, Esteller M, Dante R: A role for methyl-CpG binding domain protein 2 in the modulation of the estrogen response of pS2/TFF1 gene. PLoS One 2010, 5:e9665.
• [48]Clouaire T, de Las Heras JI, Merusi C, Stancheva I: Recruitment of MBD1 to target genes requires sequence-specific interaction of the MBD domain with methylated DNA. Nucleic Acids Res 2010, 38:4620-4634.
• [49]Helman E, Naxerova K, Kohane IS: DNA hypermethylation in lung cancer is targeted at differentiation-associated genes. Oncogene 2011, 31:1181-1188.
• [50]Karlen Y, McNair A, Perseguers S, Mazza C, Mermod N: Statistical significance of quantitative PCR. BMC Bioinformatics 2007, 8:131.
• [51]R Development Core Team: R: A language and environment for statistical computing. Reference index version: 2.11.1. R Foundation for Statistical Computing, Vienna, Austria; 2010. http://www.R-project.org webcite
• [52]Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, et al.: Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004, 5:R80.
• [53]Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D: The human genome browser at UCSC. Genome Res 2002, 12:996-1006.
• [54]Down TA, Rakyan VK, Turner DJ, Flicek P, Li H, Kulesha E, Graf S, Johnson N, Herrero J, Tomazou EM, Thorne NP, Bäckdahl L, Herberth M, Howe KL, Jackson DK, Miretti MM, Marioni JC, Birney E, Hubbard TJ, Durbin R, Tavaré S, Beck S: A Bayesian deconvolution strategy for immunoprecipitation-based DNA methylome analysis. Nat Biotechnol 2008, 26:779-785.
• [55]Pelizzola M, Koga Y, Urban AE, Krauthammer M, Weissman S, Halaban R, Molinaro AM: MEDME: an experimental and analytical methodology for the estimation of DNA methylation levels based on microarray derived MeDIP-enrichment. Genome Res 2008, 18:1652-1659.