Hypermethylation of the Retinoic Acid Receptor-β₂ Gene in Head and Neck Carcinogenesis

Purpose: Retinole acid receptor-β₂ (RAR-β ₂) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-β₂ gene expression in such lesions can be silenced by promoter methylation. Experimental Design: RAR-β₂ methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-_β2 promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-β₂ hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates. Results: Significantly higher RAR-β ₂ hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-β₂ methylation in the cell lines was correlated with loss of RAR-β₂ expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) restored RAR-β₂ inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-β₂ promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA. Conclusions: RAR-β ₂ silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-β₂ inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.