Epigenetic alterations disrupt, (or activate), cancer genes including TSGs, (or oncogenes), involved in early cancer initiation and progression, through the regulation of cell transformation and malignant outgrowth [2
]. Abnormal epigenetic modifications including mainly CpG methylation and histone modifications, which could be valuable biomarkers for the diagnosis and therapeutics of EBV-associated tumors.
3.1. CpG Methylation
CpG methylation is a well-studied epigenetic alteration associated with cancers. DNA methylation is reversible through active or passive processes. DNMTs, as master regulators of DNA methylation, are required for the maintenance of DNA methylation (5mC) and establishment of a new methylation pattern. Recent findings showed that 5-hydroxymethylcytosine (5hmC) is the sixth DNA base in mammalian genomic DNA [26
]. Ten-eleven translocation (TET) family enzymes and isocitrate dehydrogenases (IDHs) mediate the demethylation conversion of 5mC to 5hmC.
Using genome-wide techniques, epigenomes (CpG methylomes) of EBV-associated tumors have been established, with the discovery of novel and known methylated genes involved in EBV-associated tumorigenesis. NPC methylomes have been established using methylated DNA immunoprecipitation coupled with microarrays (MeDIP-chip) [27
], HumanMethylation450 (analyzing 485,000 CpG sites per genome) BeadChip [28
], and MethylCap-sequencing [30
]. EBVaGC methylomes have been profiled using Infinium HumanMethylation27 (analyzing 27,000 CpG sites per genome) and HumanMethylation450 BeadChips [31
]. NK-cell lymphoma methylomes have been characterized using methyl-sensitive cut counting (MSCC) and reduced representation bisulfite sequencing (RRBS) platforms [35
]. Epigenomes and transcriptomes of EBV-associated tumors display distinct biological patterns compared to their EBV-negative counterparts, with higher frequencies of gene methylation and >50% of gene expression and methylation affected by EBV infection [36
A list of known and novel cancer genes inactivated by CpG methylation, involved in various cell signaling pathways, have been identified in EBV-associated tumorigenesis. For examples, promoter methylation silencing of RASAL1
], and DOK1
] in Ras and Rho GTPase signaling; methylation silencing of PCDH10
, and SFRP5
], and ROR2
] in Wnt/β-Catenin signaling and epithelial-mesenchymal transition (EMT) regulation; methylation inactivation of DLEC1
] and PTPRK
] in STAT3 signaling; UCHL1
] and MGMT
] methylation linked to p53 and DNA repair signaling; ZNF382
], and PRDM5
methylation involved in chromatin and nuclear signaling; p16
] methylation in cell-cycle regulation; as well as ADAMTS18
], and DAPK1
] methylation related to cell apoptosis regulation. Specifically, p16
silencing by epigenetic modulation occurs widely in the early stage of EBV-associated tumors, to overcome senescence for further oncogenic transformation and malignant proliferation. EBV infection precedes E-cadherin
methylation, which was found in carcinoma tissues but not in dysplastic tissues [61
], supporting the view that early epigenetic alterations induced by EBV are involved in EBV-associated pathogenesis. Therefore, more investigations should be performed to identify methylated novel cancer genes in EBV-associated tumorigenesis, verify their expression and methylation in tumor samples, and to assess their relationship to clinical features, as well as their potential as biomarkers.
Promoter CpG methylation of cancer genes are ubiquitously present in all human cancers but less in precancerous lesions, thus makes them as ideal biomarkers for cancer prognosis and prevention. Compared with other molecular markers such as mRNA and proteins, CpG methylation has many advantages in diagnosis application, including being stable, easily amplifiable and detectable, highly frequent, and non-invasive (directly from body fluids). Moreover, it occurs at the early stage of tumorigenesis. In EBV-associated tumors, some methylation markers and signatures have been identified, such as methylation of PCDH10
], and DLEC1
] as early markers; ZNF382
] methylation as a metastasis marker; p16
] and WNT5A
] methylation as EBV-positive infection markers; PTPRK
] methylation as a prognosis marker for NKTCL. Further investigations are thus needed for the discovery of more epigenetic biomarkers, especially at the early stage of EBV-associated malignancies.
3.2. Histone Modifications
Histones modification, as one of the epigenetic features, is involved in the regulation of chromatin structure and gene transcription. Its deregulation leads to cellular transformation and cancer progression [66
]. Histone modifications include acetylation (-ac), methylation (-me), phosphorylation, ubiquitination, and sumoylation. Histone modifications regulate the accessibility of DNMTs, PcG complex proteins, and transcription factors, also as a link between DNA methylation and promoter activity. For example, histone H3 trimethylation of lysine 9 (H3K9me3) and histone H3 lysine 27 trimethylation (H3K27me3) are normally correlated with transcriptional repression, while H3K27ac and H3K4me3 are linked with active promoters.
Histone modifications regulate both EBV viral gene and host cell gene expression, to finely modulate EBV infection and EBV-induced tumorigenesis [67
]. Histone deacetylation is correlated with the transcriptional repression of LMP1, BZLF1, and EBNA3C, as well as EBNA2 silencing, to regulate EBV latency [69
]. LMP1 drives the expression of host cancer-promoting genes through activating poly(ADP-ribose) polymerase (PARP) and decreasing repressive H3K27me3 modification [70
]. Histone modification is thus critically involved in EBV-mediated epigenetic reprogramming, which could be a therapeutic target for EBV-associated tumors.