Novel strategies to enforce an epithelial phenotype in mesenchymal cells.
Novel strategies to enforce an epithelial phenotype in mesenchymal cells.
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DOI：
10.1158/0008-5472.can-13-3231
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发表时间：
2014-07-15
期刊：
影响因子：
11.2
通讯作者：
Agaisse H
中科院分区：
文献类型：
--
作者：
Dragoi AM;Swiss R;Gao B;Agaisse H
E-钙粘附素在癌细胞中的下调与上皮向间充质转化(EMT)和转移能力有关，但其潜在的机制尚不完全清楚。在这项研究中，我们探讨了E-钙粘蛋白在细胞膜上的表达，作为一种功能分析来确定与E-钙粘蛋白下调相关的基因。该检测是基于细胞内单核细胞增多性李斯特菌的E-钙粘附素依赖的侵袭特性。在功能读数的基础上，使用自动显微镜和计算机辅助图像分析来筛选针对7000个人类基因的siRNA。该筛选的有效性得到了几个已知的E-钙粘蛋白表达调控因子的定义的支持，包括ZEB1、HDAC1和MMP14。我们发现了三个新的调控因子(Flash、CASP7和PCGF1)，它们的沉默足以恢复E-钙粘蛋白的高水平转录。此外，我们还发现了两个新的调控因子(FBXL5和CAV2)，它们的沉默足以在转录后水平上增加E-钙粘蛋白的表达。闪光沉默通过ZEB1转录后调控E-钙粘蛋白和其他ZEB1依赖基因的表达，但它也调节许多ZEB1非依赖基因的表达，这些基因的功能被预测有助于上皮表型的恢复。最后，我们还报告了通过模拟已知和假定的microRNAs的活性来鉴定能够有效恢复上皮表型的siRNA双链。我们的发现提示了通过阻断与EMT相关的侵袭和转移表型来加强上皮表型作为治疗癌症的一般策略的新方法。
E-cadherin downregulation in cancer cells is associated with epithelial-to-mesenchymal transition (EMT) and metastatic prowess, but the underlying mechanisms are incompletely characterized. In this study, we probed E-cadherin expression at the plasma membrane as a functional assay to identify genes involved in E-cadherin downregulation. The assay was based on the E-cadherin-dependent invasion properties of the intracellular pathogen Listeria monocytogenes. On the basis of a functional readout, automated microscopy and computer-assisted image analysis were used to screen siRNAs targeting 7,000 human genes. The validity of the screen was supported by its definion of several known regulators of E-cadherin expression, including ZEB1, HDAC1 and MMP14. We identified three new regulators (FLASH, CASP7 and PCGF1), the silencing of which was sufficient to restore high levels of E-cadherin transcription. Additionally, we identified two new regulators (FBXL5 and CAV2), the silencing of which was sufficient to increase E-cadherin expression at a post-transcriptional level. FLASH silencing regulated the expression of E-cadherin and other ZEB1-dependent genes, through post-transcriptional regulation of ZEB1, but it also regulated the expression of numerous ZEB1-independent genes with functions predicted to contribute to a restoration of the epithelial phenotype. Finally, we also report the identification of siRNA duplexes that potently restored the epithelial phenotype by mimicking the activity of known and putative microRNAs. Our findings suggest new ways to enforce epithelial phenotypes as a general strategy to treat cancer by blocking invasive and metastatic phenotypes associated with EMT.