Comparative methylome analysis in solid tumors reveals aberrant methylation at chromosome 6p in nasopharyngeal carcinoma

Abstract

Altered patterns of DNA methylation are key features of cancer. Nasopharyngeal carcinoma (NPC) has the highest incidence in Southern China. Aberrant methylation at the promoter region of tumor suppressors is frequently reported in NPC; however, genome-wide methylation changes have not been comprehensively investigated. Therefore, we systematically analyzed methylome data in 25 primary NPC tumors and nontumor counterparts using a high-throughput approach with the Illumina HumanMethylation450 BeadChip. Comparatively, we examined the methylome data of 11 types of solid tumors collected by The Cancer Genome Atlas (TCGA). In NPC, the hypermethylation pattern was more dominant than hypomethylation and the majority of de novo methylated loci were within or close to CpG islands in tumors. The comparative methylome analysis reveals hypermethylation at chromosome 6p21.3 frequently occurred in NPC (false discovery rate; FDR=1.33 × 10(-9) ), but was less obvious in other types of solid tumors except for prostate and Epstein-Barr virus (EBV)-positive gastric cancer (FDR<10(-3) ). Bisulfite pyrosequencing results further confirmed the aberrant methylation at 6p in an additional patient cohort. Evident enrichment of the repressive mark H3K27me3 and active mark H3K4me3 derived from human embryonic stem cells were found at these regions, indicating both DNA methylation and histone modification function together, leading to epigenetic deregulation in NPC. Our study highlights the importance of epigenetic deregulation in NPC. Polycomb Complex 2 (PRC2), responsible for H3K27 trimethylation, is a promising therapeutic target. A key genomic region on 6p with aberrant methylation was identified. This region contains several important genes having potential use as biomarkers for NPC detection.

Keywords: Chromosome 6p; EBV; Illumina HumanMethylation450; methylome; nasopharyngeal carcinoma.

Figure 1

Aberrant methylation in NPC and other types of solid tumors. (A) Percentage of hypermethylated and hypomethylated CpG sites identified by LIMMA analysis with FDR < 0.001 in NPC and other types of solid tumors. hypo: hypomethylated CpG sites, hyper: hypermethylated CpG sites. (B) Categories of the genomic locations of de novo methylated CpG sites in NPC. Island: CpG island, N_Shelf: north shelf region of CGI, N_shore: north shore region of CGI, S_Shelf: south shelf region of CGI, N_Shelf: north shelf region of CGI. (C) Verification of the aberrant methylation of six selected loci identified in NPC by bisulfite pyrosequencing. 100% methylation control; 0% no methylation control; N: normal adjacent tissues, T: NPC tumor tissues. *Methylation level in tumor is significantly higher than matched nontumor tissue examined by Mann–Whitney U-test with P < 0.05. NPC, nasopharyngeal carcinoma; FDR, false discovery rate; CGI, CpG island.

Figure 2

Enrichment of bivalent marks at the de novo methylated loci and overexpression of polycomb complex 2 genes in NPC. (A) The de novo methylated loci were more likely to be EZH2 and SUZ12 targets, and often had active H3K4 trimethylation marks and repressive H3K27 trimethylation marks derived from hESCs. (B) Significant overlap between the de novo methylated loci with H3K27me3 and H3K4me3. (C) Genes with bivalent marks and associated with de novo methylated loci were downregulated in NPC. Top: Unsupervised hierarchical clustering using expression data of the selected genes with bivalent marks and aberrantly methylated in NPC. The expression profiles of these genes can separate the majority of the normal controls from NPC tissues; Bottom: Median expression of bivalent genes in normal controls and NPC tissues. (D) Overexpression of EZH2, SUZ12, EED, and RBBP7 in NPC. All the expression data were obtained from GEO data set GSE12452. NPC, nasopharyngeal carcinoma.

Figure 3

Aberrantly methylated regions identified in NPC and other types of solid tumors. (A) Aberrantly methylated regions across genome in NPC. Enrichment of the genes from 6p21.3 was identified by DAVID analysis. (B) Aberrantly methylated regions on 6p22.1-6p21.3 in NPC. Gray dots: the regions not reaching the significance level. Yellow dots: the regions reaching the significance level and which were unmethylated in normal adjacent tissues. The genes close to these regions are highlighted on the graph. Black dots: the regions reaching the significance level, but were methylated in normal adjacent tissues. (C) Aberrantly methylated regions in EBV-positive gastric cancer compared to EBV-negative gastric cancer. Enrichment of the genes from 6p21.3 was identified by DAVID analysis. (D–F) Aberrantly methylated regions in colon, kidney, and pancreatic cancers. No enrichment of the genes from 6p21.3 was observed. NPC, nasopharyngeal carcinoma; EBV, Epstein–Barr virus.

Figure 4

Methylation and expression of the genes from chromosome 6p in NPC. (A) Methylation level measured by Illumina Infinium Assay in normal tissues (control) and NPC tissues at the genes associated with de novo methylated regions from chromosome 6p. (B) We identified the genes aberrantly methylated in NPC from chromosome 6p and examined the expression level of these genes in normal tissues (control) and NPC tissues. There was downregulation of expression at genes B3GALT4, TNXB, TRIM31, LY6G5C, GNL1, IER3, NKAPL, and SERPINB6 in NPC (Mann–Whitney U-test P < 0.05), presumably through epigenetic regulation. Genes HLA-L/HCG20, PRRT1, and HIST1HF4 were not present on the Affymetrix Human U133Plus2 array, and thus, were excluded in this analysis. The P value was estimated by Mann–Whitney U-test. NPC, nasopharyngeal carcinoma.

Figure 5

Bisulfite pyrosequencing of the selected loci from 6p21.3 in the validation set. Aberrant methylation at the selected loci from 6p21.3 was further examined by bisulfite pyrosequencing in an additional NPC patient cohort. The P value was estimated using the Mann–Whitney U-test. *P < 0.05; ***P < 0.001; ****P < 0.0001. NPC, nasopharyngeal carcinoma.