Department of Medicine, Division of Gastroenterology, University of California, San Francisco, CA, USA.
Mol Cancer. 2011 Jul 27;10:91. doi: 10.1186/1476-4598-10-91.
We have previously reported that RAS-MEK (Cancer Res. 2003 May 1;63(9):2088-95) and TGF-β (Cancer Res. 2006 Feb 1;66(3):1648-57) signaling negatively regulate coxsackie virus and adenovirus receptor (CAR) cell-surface expression and adenovirus uptake. In the case of TGF-β, down-regulation of CAR occurred in context of epithelial-to-mesenchymal transition (EMT), a process associated with transcriptional repression of E-cadherin by, for instance, the E2 box-binding factors Snail, Slug, SIP1 or ZEB1. While EMT is crucial in embryonic development, it has been proposed to contribute to the formation of invasive and metastatic carcinomas by reducing cell-cell contacts and increasing cell migration.
Here, we show that ZEB1 represses CAR expression in both PANC-1 (pancreatic) and MDA-MB-231 (breast) human cancer cells. We demonstrate that ZEB1 physically associates with at least one of two closely spaced and conserved E2 boxes within the minimal CAR promoter here defined as genomic region -291 to -1 relative to the translational start ATG. In agreement with ZEB1's established role as a negative regulator of the epithelial phenotype, silencing its expression in MDA-MB-231 cells induced a partial Mesenchymal-to-Epithelial Transition (MET) characterized by increased levels of E-cadherin and CAR, and decreased expression of fibronectin. Conversely, knockdown of ZEB1 in PANC-1 cells antagonized both the TGF-β-induced down-regulation of E-cadherin and CAR and the reduction of adenovirus uptake. Interestingly, even though ZEB1 clearly contributes to the TGF-β-induced mesenchymal phenotype of PANC-1 cells, TGF-β did not seem to affect ZEB1's protein levels or subcellular localization. These findings suggest that TGF-β may inhibit CAR expression by regulating factor(s) that cooperate with ZEB1 to repress the CAR promoter, rather than by regulating ZEB1 expression levels. In addition to the negative E2 box-mediated regulation the minimal CAR promoter is positively regulated through conserved ETS and CRE elements.
This report provides evidence that inhibition of ZEB1 may improve adenovirus uptake of cancer cells that have undergone EMT and for which ZEB1 is necessary to maintain the mesenchymal phenotype. Targeting of ZEB1 may reverse some aspects of EMT including the down-regulation of CAR.
我们之前曾报道 RAS-MEK（Cancer Res. 2003 May 1;63(9):2088-95）和 TGF-β（Cancer Res. 2006 Feb 1;66(3):1648-57）信号通路负调控柯萨奇病毒和腺病毒受体（CAR）细胞表面表达和腺病毒摄取。在 TGF-β的情况下，CAR 的下调发生在上皮-间充质转化（EMT）的情况下，这是一种与 E-钙粘蛋白转录抑制有关的过程，例如，通过 E2 盒结合因子 Snail、Slug、SIP1 或 ZEB1 进行。虽然 EMT 在胚胎发育中至关重要，但它已被提议通过减少细胞-细胞接触和增加细胞迁移来促进侵袭性和转移性癌的形成。
在这里，我们表明 ZEB1 在 PANC-1（胰腺）和 MDA-MB-231（乳腺）人类癌细胞中均抑制 CAR 表达。我们证明 ZEB1 与此处定义的最小 CAR 启动子内的两个紧密间隔且保守的 E2 盒中的至少一个物理结合，该启动子相对于翻译起始 ATG 的基因组区域为-291 至-1。与 ZEB1 作为上皮表型的负调节剂的既定作用一致，沉默 MDA-MB-231 细胞中的表达诱导了部分间充质-上皮转化（MET），其特征是 E-钙粘蛋白和 CAR 水平增加，纤连蛋白表达减少。相反，在 PANC-1 细胞中敲低 ZEB1 拮抗 TGF-β 诱导的 E-钙粘蛋白和 CAR 下调以及腺病毒摄取减少。有趣的是，尽管 ZEB1 显然有助于 PANC-1 细胞的 TGF-β 诱导的间充质表型，但 TGF-β 似乎不影响 ZEB1 的蛋白水平或亚细胞定位。这些发现表明 TGF-β 可能通过调节与 ZEB1 合作抑制 CAR 启动子的因子而不是通过调节 ZEB1 表达水平来抑制 CAR 表达。除了负 E2 盒介导的调节外，最小的 CAR 启动子还通过保守的 ETS 和 CRE 元件被正向调节。
本报告提供的证据表明，抑制 ZEB1 可能会提高已经经历 EMT 的癌症细胞的腺病毒摄取，并且 ZEB1 对于维持间充质表型是必需的。ZEB1 的靶向可能会逆转 EMT 的某些方面，包括 CAR 的下调。
