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Esophageal squamous cell carcinoma (ESCC) is the sixth leading cause of cancer death among men in Taiwan, and the five-year survival rate is below 10%. The retinoic acid receptor beta2 (RAR-beta2) is frequently deleted or silenced at early stages in tumor progression. Most of RAR-beta2 repressions was associated with promoter hypermethylation in several epithelial carcinomas. The aim of this study is to investigate RAR-beta2 promoter methylation status in different stages of ESCC patients. Through methylation-specific PCR analysis, methylation rate of RAR-beta2 was 21.1% (15/71) in tumor cases, but unmethylated in all normal counterparts. In RAR-beta2 promoter hypermethylation cases, the methylation frequency of lymph nodes invasion and tumor metastasis was twofold increased. Moreover, RAR-beta2 promoter methylation frequency was increased with 14.7% and showed significant decrease survival rate in advanced stage. Esophageal cancer cell lines 81T, 81T 1-1 and 81T 1-4 were treated with 5-aza-dC (DNA methylation inhibitor), the level of RAR-beta2 methylation was decreased in all ESCC cell lines. Moreover, migration ability was inhibited in 81T cell line. The migration ability of 81T 1-1 and 81T 1-4 cell lines were decreased about 50%. Our findings indicate that RAR-beta2 hypermethylation in ESCC could be useful for prognosis marker. RAR-beta2 methylation may become one of the clinical therapy targets and invasion-migration markers. Citation Format: Ruei-Siang Wu, Chun-Chieh Yu, Yue-Yuah Li, Ming-Tsang Wu, Ruei-Nian Li. Study of the relationship between RAR-beta2 hypermethylation and invasive esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C5.