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

• Published in: Clinical cancer research 2004-03, Vol.10 (5), p.1733-1742
• Main Authors: YOUSSEF, Emile M, LOTAN, Dafna, HONG, Waun K, LOTAN, Reuben, ISSA, Jean-Pierre, WAKASA, Kenichi, FAN, You-Hong, LI MAO, HASSAN, Khaled, LEI FENG, LEE, J. Jack, LIPPMAN, Scott M
• Format: Article
• Language: English
• Subjects: Antineoplastic agents; Biological and medical sciences; Medical sciences; Pharmacology. Drug treatments; Tumors
• ISSN: 1078-0432; 1557-3265
• DOI: 10.1158/1078-0432.CCR-0989-3

Purpose: Retinoic acid receptor-β 2 (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-β 2 gene expression in such lesions can be silenced by promoter methylation. Experimental Design: RAR-β 2 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-β 2 promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-β 2 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-β 2 methylation in the cell lines was correlated with loss of RAR-β 2 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-β 2 inducibility by all- trans -retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-β 2 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-β 2 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.