Discovery of epigenetically masked tumor suppressor genes in gyneoobgical cancer

认识到许多肿瘤抑制基因被表观遗传机制沉默，刺激了新的肿瘤抑制基因的发现。我们使用多种研究工具来寻找在人类子宫内膜癌中被表观遗传沉默的基因。分析了去甲基化剂5-aza-2′-脱氧胞苷（5-Aza-CdR）联合组蛋白去乙酰化酶抑制剂亚酰苯胺双氢酰胺（SAHA）治疗子宫内膜癌细胞系Ishikawa后基因表达的变化。通过筛选超过22,000个基因，确定了候选肿瘤抑制基因。对正常和癌性子宫内膜样本的微阵列分析和实时RTPCR，以及对CpG岛的搜索进一步完善了该列表。选择Tigl和C/ebpa进行进一步研究。这两个基因在子宫内膜癌细胞系和临床样本中表达较低，但在正常子宫内膜组织中表达较高。亚硫酸氢盐测序和限制性内切分析显示，在所有6种子宫内膜癌细胞系中，Tigl基因的CpG岛甲基化异常，而C/ebpa启动子在子宫内膜癌中保持未甲基化。染色质免疫沉淀显示，在5-Aza-CdR和/或SAHA治疗后，Tigl和C/ebpa基因结合的乙酰化组蛋白H3增加。强迫表达TIG1或C/EBPa均可导致Ishikawa细胞的生长显著降低。免疫沉淀和报告基因实验表明，C/EBPa结合并抑制E2F1的转录活性，E2F1是一个关键的细胞周期调节因子。我们的数据表明，C/ebpa和Tigl在子宫内膜癌中具有肿瘤抑制蛋白的功能，它们的再表达可能是治疗靶点。

Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated discovery of novel tumor suppressor genes. We used a variety of research tools to search for genes that are epigenetically silenced in human endometrial cancers. Changes in global gene expression of the endometrial cancer cell line Ishikawa was analyzed after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) combined with the histone deacetylase inhibitor, suberoyl anilide bishydroxamide (SAHA). By screening over 22,000 genes, candidate tumor suppressor genes were identified. Additional microarray analysis and real-time RTPCR of normal and cancerous endometrial samples, and search for CpG islands further refined the list. Tigl and C/ebpa were chosen for further study. Expression of both genes was low in endometrial cancer cell lines and clinical samples, but high in normal endometrial tissues. Bisulfite sequencing and restriction analysis revealed aberrant methylation of the CpG island in the Tigl gene of all 6 endometrial cancer cell lines examined, whereas the C/ebpa promoter remained unmethylated in endometrial cancers Chromatin immunoprecipitation showed increased acetylated histones H3 bound to both Tigl and C/ebpa genes after treatment with 5-Aza-CdR and/or SAHA. Forced expression of either TIG1 or C/EBPa led to significant growth reduction of Ishikawa cells. Immunoprecipitation and reporter gene assays demonstrated that C/EBPa bound to and suppressed transcriptional activity of E2F1, a key cell cycle regulator. Our data suggest that C/ebpa and Tigl function as tumor suppressor proteins in endometrial cancers and that their reexpression may be a therapeutic target.