Inactivation of RASSF1A, RARβ2 and DAP-kinase by promoter methylation correlates with lymph node metastasis in nasopharyngeal carcinoma

Epigenetic modification is one of the mechanisms leading to gene silencing in neoplastic cells. By methylation-specific PCR, we analyzed the promoter methylation of three cancer-related genes: Ras Association domain Family 1A (RASSF1A), Death Associated Protein kinase (DAP-kinase) and Retinoic Acid Receptor beta 2 (RAR beta 2) in two NPC xenografts (C15 and C17), 68 primary NPC tumors, and 9 normal nasopharyngeal epithelia. We showed that C15 and C17 displayed a complete promoter methylation of RASSF1A, RAR beta 2 and DAP-kinase genes. In primary NPC tumors, the incidence of promoter methylation was very high for all three tested genes: 91% for RASSF1A, 88% for both RAR beta 2 and DAP-kinase whereas all normal nasopharyngeal epithelia were unmethylated. Interestingly, our study revealed that aberrant promoter methylation of the three genes were statistically associated with the lymph node involvement (p < 0.0001). In addition, hypermethylation of RASSF1A was correlated with age at diagnosis (p = 0.047) and T stage (p = 0.037) while the RAR beta 2 hypermethylation was associated with histological type (p = 0.011). Taken together, our results demonstrate that silencing of RASSF1A and RAR beta 2 expression by promoter hypermethylation is associated with highly differentiated tumors, advanced tumor stage and the presence of lymph node metastasis. To assess the functional significance of the epigenetic silencing of RAR beta 2 and DAP-kinase in NPC, we analysed the expression of two downstream target genes COX-2 and p53 by reverse PCR (RT-PCR) and immunohistochemistry (IHC). We revealed a significant association between expression of COX-2 and loss of RAR beta 2 through aberrant methylation (p = 0.003) in NPC biopsies. We concluded that the inactivation of RASSF1A, RAR beta 2 and DAP-Kinase by hypermethylation is a key step in NPC tumorigenesis and progression.