Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan.
Oncol Rep. 2014 Feb;31(2):693-700. doi: 10.3892/or.2013.2901. Epub 2013 Dec 5.
Esophageal cancer ranks sixth in cancer mortality worldwide and patients with esophageal squamous cell carcinoma (ESCC) have a poor prognosis with a 5-year survival rate of less than 10%. Elucidation of the mechanisms of carcinogenesis and tumor progression in esophageal cancer is urgently required to develop targets for therapy and prognostic biomarkers. In the present study, the expression and regulatory mechanism of the differentially expressed in normal and neoplastic cells domain containing 2D (DENND2D), which is a regulator of Rab GTPases, were investigated to explore its potential as a tumor suppressor gene for ESCC. The level of DENND2D mRNA expression in ESCC cell lines and surgical specimens was determined using a quantitative real-time reverse transcription-polymerase chain reaction assay, and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanism of DENND2D expression in ESCC. The level of DENND2D mRNA expression was reduced in 8/9 ESCC cell lines and in 59/65 surgical specimens, and the mean expression levels were significantly lower in cancerous tissues compared to corresponding normal tissues (p<0.001). The expression pattern of DENND2D protein and mRNA was consistent. Downregulation of DENND2D mRNA in ESCC tissues was identified as an independent prognostic factor in multivariate analysis (hazard ratio, 2.194; p=0.039). The DENND2D promoter was methylated in 5/9 ESCC cell lines, and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in ESCC tissues (64.6%) and was significantly associated with downregulation of DENND2D mRNA expression (P=0.008). Taken together, our data suggest that DENND2D is a candidate tumor suppressor gene that was inactivated by promoter hypermethylation in patients with ESCC and may serve as a novel biomarker of ESCC.
食管癌在全球癌症死亡率中排名第六，食管鳞状细胞癌（ESCC）患者预后较差，5 年生存率低于 10%。阐明食管癌的致癌机制和肿瘤进展机制，对于开发治疗靶点和预后生物标志物至关重要。本研究旨在探讨 Rab GTPases 调节因子差异表达在正常和肿瘤细胞域 2D（DENND2D）的表达和调控机制，探索其作为 ESCC 肿瘤抑制基因的潜力。采用实时定量逆转录聚合酶链反应检测 ESCC 细胞系和手术标本中 DENND2D mRNA 的表达水平，并评估 DENND2D mRNA 表达水平与临床病理因素的关系。采用免疫组织化学法检测 DENND2D 的表达和分布。进行 DNA 甲基化分析以确定 DENND2D 在 ESCC 中的表达调控机制。结果显示，8/9 种 ESCC 细胞系和 59/65 例手术标本中 DENND2D mRNA 表达降低，癌组织中 DENND2D mRNA 的平均表达水平明显低于相应的正常组织（p<0.001）。DENND2D 蛋白和 mRNA 的表达模式一致。多因素分析显示，ESCC 组织中 DENND2D mRNA 下调是独立的预后因素（危险比，2.194；p=0.039）。DENND2D 启动子在 5/9 种 ESCC 细胞系中发生甲基化，DNA 去甲基化可重新激活 DENND2D mRNA 的表达。ESCC 组织中 DENND2D 高甲基化发生率较高（64.6%），与 DENND2D mRNA 表达下调显著相关（P=0.008）。综上所述，本研究数据表明，DENND2D 是一种候选肿瘤抑制基因，在 ESCC 患者中因启动子甲基化而失活，可能作为 ESCC 的新型生物标志物。
