Investigation of Epstein-Barr virus impact on epigenetic regulation of host genes and characterization of a host chromosome 14q candidate gene, MIPOL1, in the development of nasopharyngeal carcinoma

Abstract

Epstein-Barr virus (EBV) infection and genetic susceptibility are two risk factors contributing to nasopharyngeal carcinoma (NPC) tumorigenesis. At present, the involvement of EBV in epigenetic regulation has been widely reported. It is generally believed that inactivation of key tumor suppressor genes (TSGs), including Ras Association Domain Family member 1 (RASSF1A) and p16, are early events of NPC development that occur even before EBV infection. Therefore, the current studies aim to elucidate the mechanism of EBV to regulate expression of host cells via epigenetic modification in nasopharyngeal epithelial (NPE) cells in order to decipher its role in NPC development and functional validation of a TGS candidate gene, Mirror-image polydactyly 1 (MIPOL1), in NPC. In this study, EBV infection of epithelial cells was shown to be associated with aberrant modification of histone bivalent switches to reduce a transcriptional-activation histone mark (H3K4me3) and enhance a suppressive mark (H3K27me3) at the promoter regions of DNA damage repair family members. Sixteen DNA damage repair family members from the base excision repair (BER), homologous recombination (HR), non-homologous end-joining (NHEJ), and mismatch repair (MMR) pathways showed aberrant modifications after EBV infection. Involved in MMR, MLH1 is a candidate gene among the panel of DNA damage repair members demonstrated to be down-regulated in EBV-infected NPE cells, EBV-positive NPC cell lines, and NPC tumor specimens. The down-regulation is associated with aberrant histone bivalent switch in a promoter hypermethylation-independent manner. The expression of MLH1 was functionally correlated with cisplatin sensitivity in both in vitro and in vivo assays. Moreover, a pathway analysis showed that totally seven BER members with aberrant histone bivalent switches in the EBV-positive NPE cell lines were significantly enriched (adjusted p-value<0.1). This observation is further validated by their down-regulation in EBV-infected NPE cells in a promoter hypermethylation-independent manner. The in vitro comet and AP site assays further confirmed a reduced DNA damage repair responsiveness in the EBV-infected NPE cells. On the other hand, by using two truncation constructs of MIPOL1 as negative controls, re-expression of wild type MIPOL1 in NPC cell lines suppressed the expression of angiogenic factors. Moreover, the in vitro and in vivo functional assays further showed that cell migration, invasion, and metastasis were significantly inhibited, when wild type MIPOL1 was re-expressed. In conclusion, the findings of this study highlight the importance of EBV-associated aberrant histone bivalent switch in epithelial cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner. It also indicated the critical role of MIPOL1 in suppression of cell migration, invasion, and metastasis in NPC.