DNA methylation of genes linked to retinoid signaling in squamous cell carcinoma of the esophagus: DNA methylation of CRBP1 and TIG1 is associated with tumor stage

Abstract

Hypermethylation of CpG islands is associated with the silencing of various tumor suppressor genes. Retinoic acid receptor-beta (RAR-beta), cellular retinol-binding protein 1 (CRBP1), and tazarotene-induced gene 1 (TIG1) have been linked to retinoic acid signaling. Little is known about the involvement of these three genes in esophageal squamous cell carcinoma (ESCC). In this study, we investigated the methylation status of these genes and analyzed the role of methylation of their DNA in ESCC. Methylation-specific polymerase chain reaction (PCR) was performed to study the methylation of CpG islands in 28 ESCC (stages I, II, and III) and 10 samples of corresponding non-neoplastic mucosa. The mRNA expression levels of the three genes were measured by quantitative reverse transcription-PCR. DNA hypermethylation of RAR-beta was found in seven (25.0%) of the 28 ESCC, of CRBP1 in five (17.9%), and of TIG1 in five (17.9%). DNA methylation of RAR-beta was identified in one of 10 samples of corresponding non-neoplastic mucosa (10.0%), whereas no DNA methylation of CRBP1 or TIG1 was detected. In total, at least one of the three genes was hypermethylated in 12 (42.9%) ESCC. Reduced expression of RAR-beta, CRBP1, and TIG1 was found in 14 (50.0%), 15 (53.6%), and 13 (46.4%) ESCC, respectively. DNA methylation of each gene was significantly associated with reduced expression of the respective mRNA. No correlation was found between the DNA methylation status of RAR-beta and clinicopathological factors such as depth of invasion, lymph node metastasis, or tumor stage. In contrast, DNA methylation of both CRBP1 and TIG1 was observed only in stage III ESCC. These results show that inactivation of the retinoic acid signaling-associated genes RAR-beta, CRBP1, and TIG1 by DNA methylation occurs frequently in ESCC.