Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Carvalho RH

PROVIDER: S-EPMC3407794 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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

Carvalho Rejane Hughes RH Haberle Vanja V Hou Jun J van Gent Teus T Thongjuea Supat S van Ijcken Wilfred W Kockx Christel C Brouwer Rutger R Rijkers Erikjan E Sieuwerts Anieta A Foekens John J van Vroonhoven Mirjam M Aerts Joachim J Grosveld Frank F Lenhard Boris B Philipsen Sjaak S

Epigenetics & chromatin 20120622 1

<h4>Background</h4>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 ...[more]

PMID: 22726460

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Project description:Purpose: Generate genome-wide methylation profiles of non-small cell lung carcinomas (NSCLC) and their matching lung tissues for detection of hypermethylated and hypomethylated regions present in the tumors. Methods: MethylCapture followed by next-generation sequencing (Illumina GAIIx) of 7 nsclc tumor samples and paired lung tissues in replicated, plus one cell line, 2 fully artificially methylated and 2 fully artificially unmethylated controls. Normalization of methylation reads based on CpG coupling factor–method. Relative methylation scores (rms) in 500bp non-overlapping windows. 90th percentile of rpm (reads per million) values for all 500bp genome-wide windows, with rpm <1.33 were considered. Distributions of 10bp bins rms values within each 500bp genomic region were compared using both one-sided Student’s t-test and one-sided Wilcoxon rank-sum test. Testing was done separately for hypo- and hypermethylation and p-value threshold of 10-18. Results: MethylCap-seq data revealed strong positive correlation between replicate experiments and between paired tumor/lung samples. 14472 differentially methylated regions (DMR) with non-overlapping 500 bp windows were found. 57 DMRs were present in all NSCLC tumors. 287 were unique for squamous-cell carcinomas and 26 unique for adenocarcinomas. 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. Conclusion: 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. The DMRs can be in further studies to develop sensitive biological markers for NSCLC, which may enable non-invasive diagnosis and early detection of the disease, and potentially allow histological classification.

Project description:BackgroundEukaryotic histone methyltransferases 2 (EHMT2 or G9A) has been regarded as a potential target for non-small cell lung cancer (NSCLC) therapy. This study investigated the regulatory roles of G9A in tumorigenesis and stemness in NSCLC. We isolated and enriched tumor-initiating cells (TIC) from surgically resected NSCLC tissues by FACS and sphere formation assays. We then knocked down G9A using shRNA and carried out genome-wide 850K methylation array and RNA sequencing analyses. We carried out in vivo tumorigenecity asssay using mice xenografts and examined G9A interactions with its novel target using chromatin Immunoprecipitation (ChIP).ResultsWe identified 67 genes hypomethylated and 143 genes upregulated following G9A knockdown of which 43 genes were both hypomethylated and upregulated. We selected six genes (CDYL2, DPP4, SP5, FOXP1, STAMBPL1, and ROBO1) for validation. In addition, G9A expression was higher in TICs and targeting G9a by shRNA knockdown or by selective inhibitor UNC0642 significantly inhibited the expression of cancer stem cell markers and sphere forming capacity, in vitro proliferation, and in vivo growth. Further, transient overexpression of FOXP1, a protein may promote normal stem cell differentiation, in TICs resulted in downregulation of stem cell markers and sphere forming capacity and cell proliferation in vitro indicating that the genes we identified are directly regulated by G9A through aberrant DNA methylation and subsequent expression. Similarly, ChIP assay has shown that G9a interacts with its target genes through H3K9me2 and downregulation of H3K9me2 following G9a knockdown disrupts its interaction with its target genes.ConclusionsThese data suggest that G9A is involved in lung cancer stemness through epigenetic mechanisms of maintaining DNA methylation of multiple lung cancer stem cell genes and their expression. Further, targeting G9A or its downstream genes could be a novel therapeutic approach in treating NSCLC patients.

Project description:Chordomas are an aggressive rare type of malignant bone tumors arising from the remnant of the notochord. Chordomas occur mainly in vertebral bones and account for 1-4% of malignant bone tumors. Management and treatment of chordomas are difficult as they are resistant to conventional chemotherapy; therefore, they are mainly treated with surgery and radiation therapy. In this study, we performed DNA methylation profiling of 26 chordomas and normal nucleus pulposus samples plus UCH-1 chordoma cell line using the Illumina Infinium HumanMethylation450 BeadChips. Combined bisulfite restriction analysis and bisulfite sequencing was used to confirm the methylation data. Gene expression was analyzed using RT-PCR before and after 5-aza-2'-deoxycytidine (5-azaDC) treatment of chordoma cell lines. Analysis of the HumanMethylation450 BeadChip data led to the identification of 8,819 loci (2.9%) that were significantly differentially methylated (>0.2 average ?-value difference) between chordomas and nucleus pulposus samples (adjusted P < 0.05). Among these, 5,868 probes (66.5%) were hypomethylated, compared to 2,951 (33.5%) loci that were hypermethylated in chordomas compared to controls. From the 2,951 differentially hypermethylated probes, 33.3% were localized in the promoter region (982 probes) and, among these, 104 probes showed cancer-specific hypermethylation. Ingenuity Pathway Analysis indicates that the cancer-specific differentially methylated loci are involved in various networks including cancer disease, nervous system development and function, cell death and survival, cellular growth, cellular development, and proliferation. Furthermore, we identified a subset of probes that were differentially methylated between recurrent and non-recurrent chordomas. BeadChip methylation data was confirmed for these genes and gene expression was shown to be upregulated in methylated chordoma cell lines after treatment with 5-azaDC. Understanding epigenetic changes in chordomas may provide insights into chordoma tumorigenesis and development of epigenetic biomarkers.

Dataset Information

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

Publications

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

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