Department of Medical Biology, Research group in Molecular Oncology and Endocrinology, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada.
Cell Death Dis. 2014 Feb 6;5(2):e1044. doi: 10.1038/cddis.2014.7.
A growing body of evidence supports that the epithelial-to-mesenchymal transition (EMT), which occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Transforming growth factor-β (TGF-β) is known to induce EMT in a number of cancer cell types; however, the mechanism underlying this transition process is not fully understood. In this study we have demonstrated that TGF-β upregulates the expression of tumor suppressor protein Par-4 (prostate apoptosis response-4) concomitant with the induction of EMT. Mechanistic investigations revealed that exogenous treatment with each TGF-β isoform upregulates Par-4 mRNA and protein levels in parallel levels of phosphorylated Smad2 and IκB-α increase. Disruption of TGF-β signaling by using ALK5 inhibitor, neutralizing TGF-β antibody or phosphoinositide 3-kinase inhibitor reduces endogenous Par-4 levels, suggesting that both Smad and NF-κB pathways are involved in TGF-β-mediated Par-4 upregulation. NF-κB-binding sites in Par-4 promoter have previously been reported; however, using chromatin immunoprecipitation assay we showed that Par-4 promoter region also contains Smad4-binding site. Furthermore, TGF-β promotes nuclear localization of Par-4. Prolonged TGF-β3 treatment disrupts epithelial cell morphology, promotes cell motility and induces upregulation of Snail, vimentin, zinc-finger E-box binding homeobox 1 and N-Cadherin and downregulation of Claudin-1 and E-Cadherin. Forced expression of Par-4, results in the upregulation of vimentin and Snail expression together with increase in cell migration. In contrast, small interfering RNA-mediated silencing of Par-4 expression results in decrease of vimentin and Snail expression and prevents TGF-β-induced EMT. We have also uncovered a role of X-linked inhibitor of apoptosis protein in the regulation of endogenous Par-4 levels through inhibition of caspase-mediated cleavage. In conclusion, our findings suggest that Par-4 is a novel and essential downstream target of TGF-β signaling and acts as an important factor during TGF-β-induced EMT.
越来越多的证据表明，上皮间质转化（EMT）在癌症的发生和发展过程中发生，通过增强肿瘤细胞的迁移能力在转移中起关键作用。转化生长因子-β（TGF-β）已知可诱导多种癌细胞类型发生 EMT；然而，这种转化过程的机制尚不完全清楚。在这项研究中，我们已经证明 TGF-β上调肿瘤抑制蛋白 Par-4（前列腺凋亡反应蛋白-4）的表达，同时诱导 EMT。机制研究表明，外源性处理每种 TGF-β同工型都会平行上调 Par-4 mRNA 和蛋白水平，同时磷酸化 Smad2 和 IκB-α水平增加。使用 ALK5 抑制剂、中和 TGF-β 抗体或磷酸肌醇 3-激酶抑制剂破坏 TGF-β信号会降低内源性 Par-4 水平，表明 Smad 和 NF-κB 途径都参与了 TGF-β介导的 Par-4 上调。先前已经报道了 Par-4 启动子中的 NF-κB 结合位点；然而，通过染色质免疫沉淀分析，我们发现 Par-4 启动子区域还含有 Smad4 结合位点。此外，TGF-β促进 Par-4 的核定位。长时间 TGF-β3 处理会破坏上皮细胞形态，促进细胞迁移，并诱导 Snail、波形蛋白、锌指 E 盒结合同源框 1 和 N-钙黏蛋白的上调以及 Claudin-1 和 E-钙黏蛋白的下调。强制表达 Par-4 会导致波形蛋白和 Snail 表达上调，并伴随着细胞迁移增加。相比之下，通过小干扰 RNA 介导的 Par-4 表达沉默会导致波形蛋白和 Snail 表达下调，并阻止 TGF-β 诱导的 EMT。我们还发现 X 连锁凋亡抑制蛋白（XIAP）通过抑制半胱天冬酶介导的切割来调节内源性 Par-4 水平。总之，我们的研究结果表明 Par-4 是 TGF-β 信号的一个新的和必需的下游靶标，在 TGF-β 诱导的 EMT 中发挥重要作用。
