Methylation of the claudin-3 promoter predicts the prognosis of advanced gastric adenocarcinoma

Abstract

Claudin-3 expression is associated with gastric cancer progression, but the role of epigenetic modifcations remains unclear. We investigated methylation of the claudin-3 promoter and expression profles in gastric adenocarcinoma and their associations with clinicopathological characteristics and prognosis of the patients. A total of 122 patients with advanced gastric cancer [stage IIB-IV, with lymph node (LN) metastasis] were enrolled. Each patient provided 4 tissue samples: normal gastric epithelium, intestinal metaplasia, primary tumor and metastatic LN. Claudin-3 protein expression was examined by immunohistochemistry. Claudin-3 promoter methylation was determined by methylation-specifc PCR and verified by bisulfite sequencing PCR. Claudin-3 mRNA expression was measured by real-time PCR in a subset of cases, and its correlation with protein expression was analyzed using Spearman correlation. Kaplan-Meier survival analysis was performed (log-rank test). Factors associated with survival were identifed by Cox regression. The strong expression rate of claudin-3 in intestinal metaplasia, primary tumor, metastatic LN and normal gastric epithelium was 91.8, 58.2, 30.3 and 13.9%, respectively. The promoter hypermethylation rate in intestinal metaplasia, primary tumor, normal gastric epithelium and metastatic LN was 5.7, 27.9, 36.9 and 49.2%, respectively. Claudin-3 mRNA and protein expression were positively correlated (P<0.001) with normal gastric epithelium (rs=0.745), intestinal metaplasia (rs=0.876), primary gastric adenocarcinoma (rs=0.915) and metastatic LN (rs=0.819). Claudin-3 mRNA expression was negatively correlated with claudin-3 promoter methylation. Median patient survival was 38, 22 and 11 months in the hypomethylated, partially methylated and hypermethylated groups, respectively (P<0.001). Claudin-3 promoter methylation status (HR: 5.67; 95% CI: 2.27-14.17) but not claudin-3 expression was an independent predictor of survival. Claudin-3 promoter hypermethylation reduces claudin-3 expression and independently predicts poor prognosis.