Epigenetics in nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) shows a remarkably distinctive ethnic and geographic distribution among the world suggesting that genetic susceptibility backgrounds cooperate with the environmental risk factors in etiology of NPCs. Among the environmental factors, much attention has been focused on a well-documented association with Epstein-Barr virus (EBV), which can be detected in 100% undifferentiated NPCs. During the passing decade, increasing evidence has shown that epigenetic changes play an important role at the early stage of carcinogenesis. Epigenetic changes could also serve as a surrogate for mutations or chromosomal alterations in inactivating tumor suppressor genes (TSGs) in the tumor development.

In the present work, we mainly focused on the study of methylation of NPC-related genes, including cellular TSGs and EBV-encoded gene, which may unravel the molecular basis of its tumorigenesis and thereby expand the prospects for the development of diagnostic and prognostic markers.

First, CDH13, which encodes cell adhesion molecule H-cadherin, was found to be methylated in 89.7% of NPC primary tumors, while only methylated in 10% normal nasopharyngeal epithelia (p<0.05). The potential of detecting methylation from nasopharyngeal swabs was then investigated. The high sensitivity (81%) and specificity (0% false positives) of detecting CDH13 methylation from nasopharyngeal swabs suggests it could be utilized as a tentative auxiliary tool for early diagnosis. We next explored another candidate TSG, RASSF2 which can bind directly to K-Ras and function as a negative effector of Ras protein. Promoter methylation of one of RASSF2 isoform, RASSF2A, could be detected in 80% NPC cell lines and 50.9% of primary tumors, but not in any of the normal epithelia. RASSF2A expression was found to be correlated with its promoter methylation. In addition, patients with methylated RASSF2A presented a higher frequency of lymph node metastasis (P<0.05). In characterizing the tumor suppressor function of RASSF2A in NPC, we found that RASSF2A could induce cell cycle arrest, and inhibit colony formation and cell migration, which provided further evidence for the tumor suppression function of RASSF2A.

The EBV-encoded latent membrane protein 1 (LMP1) is expressed in about 65% of NPC. In this study, how LMP1 expression is epigenetically regulated in NPC cells was investigated. Our results show that LMP1 promoters were heavily methylated in LMP1-silenced NPCs, while free of methylation in LMP1-expressing NPCs. Methyltransferase inhibitor 5-aza-dC could induce LMP1 mRNA and protein expression in NPC cell line C666-1. Although histone deacetylase inhibitor TSA relieved the repression by deacetylated histories at LMP1 promoter, LMP1 expression was not induced by treating with TSA alone. A synergistic effect on inducing LMP1 expression was observed after treating cells with 5-aza-dC plus TSA, suggesting both epigenetic mechanisms play a role in repressing LMP1 expression. Cytokine IL-4 failed to induce LMP1 expression in C666-1 cells in which LMP1 promoter is heavily methylated. A 15-fold induction on LMP1 expression was achieved by treating cells with 5aza-dC and IL4 together, compared with 5-aza-dC alone. It suggests that the presence of methylation on LMP1 promoter prevent the transctivation of LMP 1 by IL4.

Aiming at developing early diagnostic or prognostic tools for NPC, we developed a powerful technique "multiplex methylation specific-PCR (MMSP)". MMSP was designed to detect tumorspecific methylation status of several NPC-related genes. it is capable of acquiring multiplex information simultaneously by just single PCR reaction with the tiny tumor DNA derived from nasopharyngeal swabs. This MMSP assay is sensitive enough to detect multiplex information from as few as 10 cells. Among the sixty-nine samples (49 NPCs and 20 normal controls), the detection rate of NPC from NP swabs is 97.9%. The false positive rate of MMSP in detecting NPC is zero.