Promoter hypermethylation of the EDNRB gene in nasopharyngeal carcinoma

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Abstract
To identify the epigenetic changes in nasopharyngeal carcinoma (NPC), we performed methylation‐sensitive restriction fingerprinting (MSRF) analysis on NPC cell lines and xenografts. A 190 bp sequence methylated in NPC tumors was isolated and showed high homology to the 5′ CpG island of the endothelin receptor B (EDNRB) gene. Since the EDNRB gene is commonly inactivated in prostate and bladder cancers, it may be a candidate target gene involved in NPC tumorigenesis. By bisulfite sequencing, we have confirmed that hypermethylation of the 5′ CpG island of EDNRB occurred in both xenografts and all 4 cell lines but not in 2 normal nasopharyngeal outgrowths. RT‐PCR demonstrated that only original EDNRB transcripts, but not the splicing transcripts, were expressed in normal nasopharyngeal epithelial cells. Loss of the original EDNRB expression was consistently found in 2 xenografts and 3 cell lines with dense methylation patterns. Treatment of these 3 cell lines with 5′‐aza‐2′‐deoxycytidine led to re‐expression of the EDNRB transcript and demethylation of its promoter regions. Our results demonstrate that silencing of EDNRB gene expression in NPC is associated with promoter hypermethylation. Using methylation‐specific PCR, we also detected methylation of the 5′ CpG island of EDNRB in 19/21 (90.5%) primary tumors, while no methylation was found in all 6 normal nasopharyngeal epithelia. The high frequencies of promoter hypermethylation suggest that repression of the EDNRB gene may play a role in the development of NPC.