Cancer Research Institute, Seoul National University College of Medicine, South Korea.
Lab Invest. 2012 Jul;92(7):1033-44. doi: 10.1038/labinvest.2012.61. Epub 2012 Apr 16.
Epiregulin (EREG) induces cell growth by binding to the epidermal growth factor receptor (EGFR). Expression of EREG affects sensitivity to cetuximab a chimeric monoclonal antibody that inhibits the EGFR signaling pathway. The mechanism through which EREG is regulated is largely unknown, but a methyl-array study previously performed by our group revealed that EREG is methylated in gastric cancer cells. In this study, we found that EREG gene expression was low in 7 out of 11 gastric cancer cells and this downregulation was mediated by aberrant CpG methylation of the EREG promoter. Treatment with 5-aza-CdR restored EREG expression and demethylated CpG sites in the EREG promoter. Compared with DNA methyltransferase 1 (DNMT1), knock-down of DNA methyltransferase 3b (DNMT3b) significantly increased the expression of EREG and led to the demethylation of specific CpG sites in the EREG promoter, suggesting that DNMT3b primarily regulates CpG methylation and silencing of the EREG gene. EREG methylation was observed in 30% (4/13) of human primary gastric tumor tissues we evaluated. In addition to DNA methylation, results from a chromatin immunoprecipitation assay demonstrated that transcriptional levels of EREG were associated with the enrichment of active histone marks (H3K4me3 and AcH3) and of a repressive mark (H3K27me2). Treatment with 5-aza-CdR dynamically increased the low occupancy of H3K4me3 and AcH3, while decreasing the high enrichment of H3K27me2, indicating that dynamic histone modifications contribute to EREG regulation in addition to DNA methylation. Finally, the combination of 5-aza-CdR and cetuximab exerted a synergistic anti-proliferative effect on gastric cancer cells. Taken together, the results of our study showed for the first time that EREG is epigenetically silenced in gastric cancer cells by aberrant DNA methylation and histone modification.
表皮调节素 (EREG) 通过与表皮生长因子受体 (EGFR) 结合诱导细胞生长。EREG 的表达会影响西妥昔单抗的敏感性，西妥昔单抗是一种抑制 EGFR 信号通路的嵌合单克隆抗体。EREG 的调控机制在很大程度上尚不清楚，但我们小组之前进行的甲基化芯片研究表明，表皮调节素在胃癌细胞中被甲基化。在这项研究中，我们发现 11 个胃癌细胞中有 7 个 EREG 基因表达较低，这种下调是由 EREG 启动子的异常 CpG 甲基化介导的。用 5-氮杂-2'-脱氧胞苷 (5-aza-CdR) 处理可恢复 EREG 表达并使 EREG 启动子上的 CpG 去甲基化。与 DNA 甲基转移酶 1 (DNMT1) 相比，敲低 DNA 甲基转移酶 3b (DNMT3b) 显著增加了 EREG 的表达，并导致 EREG 启动子上特定 CpG 位点的去甲基化，表明 DNMT3b 主要调节 EREG 基因的 CpG 甲基化和沉默。我们评估的 13 个人类原发性胃癌组织中有 30%（4/13）观察到 EREG 甲基化。除了 DNA 甲基化，染色质免疫沉淀分析的结果表明，EREG 的转录水平与活性组蛋白标记（H3K4me3 和 AcH3）和抑制性标记（H3K27me2）的富集有关。用 5-氮杂-2'-脱氧胞苷 (5-aza-CdR) 处理可动态增加 H3K4me3 和 AcH3 的低占有率，同时降低 H3K27me2 的高占有率，表明除了 DNA 甲基化外，动态组蛋白修饰也有助于 EREG 的调节。最后，5-aza-CdR 和西妥昔单抗联合应用对胃癌细胞表现出协同的抗增殖作用。综上所述，我们的研究结果首次表明，异常的 DNA 甲基化和组蛋白修饰使 EREG 在胃癌细胞中被表观沉默。
