Silencing of caspase-8 in murine hepatocellular carcinomas is mediated via methylation of an essential promoter element
Silencing of caspase-8 in murine hepatocellular carcinomas is mediated via methylation of an essential promoter element
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DOI：
10.1053/j.gastro.2005.08.007
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发表时间：
2005-11-01
期刊：
影响因子：
29.4
通讯作者：
Trautwein, C
中科院分区：
文献类型：
--
作者：
Liedtke, C;Zschemisch, NH;Trautwein, C
背景和目的：Caspase-8 是触发 Fas 诱导细胞凋亡所必需的顶端 caspase。在本研究中，我们使用最近描述的 HCC 小鼠模型（c-myc 和 IgEGF 转基因）研究了肝细胞癌 (HCC) 中 caspase-8 的表达。方法：从 c-myc 和 IgEGF 转基因动物中分离 HCC。通过逆转录聚合酶链反应监测 caspase-8 的表达。通过荧光素酶报告基因分析对小鼠 caspase-8 启动子进行表征，并通过亚硫酸氢盐基因组测序进行启动子甲基化分析。结果：在研究的 HCC 中，我们经常发现 caspase-8 信使 RNA 表达缺失。 caspase-8 位点的基因组缺失不会导致 caspase-8 沉默。我们检查了肿瘤来源的启动子序列，发现不同的 CpG 位点有显着的高甲基化。与此同时，我们对小鼠 caspase-8 启动子进行了表征，并鉴定了一个对于基础启动子活性必不可少的 30 bp 启动子元件。该最小启动子元件包含与 CpG 位点共定位的 SP1 结合基序，并在肿瘤衍生的启动子序列中甲基化。电泳迁移率变动分析表明这些 SPI 位点的甲基化足以阻止 SP1 复合物的形成。为了支持我们的数据，我们使用 CpG 甲基化酶在体外模拟了肿瘤来源的 caspase-8 启动子的甲基化模式，发现启动子活性大幅降低。结论：我们表明 HCC 经常与 caspase-8 表达沉默相关，并提供数据表明 caspase-8 沉默是抑制 SP1 依赖性反式激活的直接结果，SP1 依赖性反式激活是由启动子区域的 CpG 甲基化引起的。我们的数据支持抑制细胞凋亡会引发肝癌的假设。
Background& Aims: Caspase-8 is the apical caspase essential for triggering Fas-induced apoptosis. In this study, we investigated caspase-8 expression in hepatocellular carcinomas (HCCs) using recently described HCC mouse models (c-myc and IgEGF transgenes).Methods: HCCs were isolated from c-myc and IgEGF transgenic animals. Expression of caspase-8 was monitored by reverse-transcription polymerase chain reaction. The murine caspase-8 promoter was characterized by luciferase-reporter analysis and the analysis of promoter methylation was performed by bisulfite genomic sequencing.Results: In HCCs investigated, we frequently found a lack of caspase-8 messenger RNA expression. Genomic deletions at the caspase-8 locus did not contribute to caspase-8 silencing. We examined tumor-derived promoter sequences and found significant hypermethylation at distinct CpG sites. In parallel, we characterized the murine caspase-8 promoter and identified a 30-bp promoter element that is indispensable for basal promoter activity. This minimal promoter element contained SP1 binding motifs that are colocalized with CpG sites and were methylated in tumor-derived promoter sequences. Electrophoretic mobility shift assay analysis showed that methylation of these SPI sites is sufficient to prevent SP1 complex formation. To support our data, we mimicked the methylation pattern of a tumor-derived caspase-8 promoter in vitro using CpG methylase and found a strong reduction of promoter activity.Conclusion : We show that HCCs are correlated frequently with silencing of caspase-8 expression and provide data suggesting that caspase-8 silencing is a direct consequence of inhibiting SP1-dependent transactivation caused by CpG methylation at its essential binding sites in the promoter region. Our data support the hypothesis that inhibition of apoptosis triggers hepatocarcinogenesis.