A unique CpG-island methylator phenotype associated with Epstein-Barr virus (EBV) and CCCTC-binding factor (CTCF) in nasopharyngeal carcinoma

Abstract

Background: Nasopharyngeal carcinoma (NPC), an endemic cancer in Southeast Asia, is distinct from the other cancer types in terms of its relatively low somatic mutation rate, wider mutational diversity and its intrinsic association with Epstein-Barr Virus (EBV). EBV is reported to be an epigenetic driver in lymphoma and EBV-positive gastric cancer. Recent Illumina Human methylation450 array-based studies targeting the CpG-rich regions revealed the frequent DNA hypermethylation in NPC and emphasized the importance of methylation alterations in NPC development. Method: The aim of this study is to comprehensively profile the global methylation changes in NPC by whole-genome bisulfite sequencing (WGBS). Sixteen EBV-positive NPC tumor tissues and nine non-tumor adjacent tissues were included in the study. Results: Two methylome subgroups of NPCs were identified. Moderate to high level of global methylation was found in the majority of the cases, while global hypomethylation was observed in a subset of NPC, only accounting for 20% of the total cases. Group comparisons were performed to identify the differentially methylated regions (DMRs) between non-tumor and tumor tissues in these two subgroups, respectively. Surprisingly, both subgroups had predominant hypermethylation which is mostly enriched in CpG islands (OR=34.1), indicating that NPC has a CpG island methylator phenotype (CIMP). Hypermethylation in both groups is also enriched in the gene-regulatory regions, including promoters (OR=6.02), enhancers (OR=5.39), and 5’untranslated regions (5’UTR) (OR=5.66). The binding sites of EZH2 are highly enriched for hypermethylation (OR=80.1). As an important member of the polycomb repressive complex 2, EZH2 imposes the repressive H3K27me3 mark on the target genes. Interestingly, CTCF (OR=5.67) binding sites are associated with hypermethylation as well. This result was further confirmed by the motif analysis for the sequences in the DMRs. CTCF plays an essential role to regulate the EBV latent gene expression and loops the cellular chromatin to define topologically associating domains that regulate cellular gene expression and gene-gene interaction. We expanded our analysis to the methylome data in The Cancer Genome Atlas (TCGA) study for EBV-positive and -negative gastric cancer. Unsupervised clustering using a subset of CpG sites from the NPC DMRs significantly enriched for the CTCF binding motif clearly separate the EBV-positive and -negative groups (Fisher’s exact test, p<2.2x10-16), indicating that EBV and CTCF may function together to contribute to this phenotype. Conclusions:This study reveals a NPC-specific CIMP. As hypermethylation is enriched in active-regulatory regions and presumably associated with EBV and CTCF, it may be a key driver for NPC development.