Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.
J Cell Physiol. 2012 Nov;227(11):3621-8. doi: 10.1002/jcp.24068.
High levels of plasminogen activator inhibitor-1 (PAI-1), which is produced by stromal, endothelial, and cancer cells and has multiple complex effects on cancers, correlate with poor cancer prognosis. To more definitively study the role of endogenously produced PAI-1 in human pancreatic adenocarcinoma (PAC) PANC-1 cell line biology, we used anti-PAI-1 shRNA to create stable PAI-1 deficient cells (PD-PANC-1s). PD-PANC-1s exhibited a heterogeneous morphology. While the majority of cells exhibited a cuboidal shape similar to the parental PANC-1 or the vector-infected control cells, numerous large cells with long filopodia and a neuronal-like appearance were observed. Although both Vector-control cells and PD-PANC-1s expressed mRNAs that are characteristic of mesenchymal, neural, and epithelial phenotypes, epithelial marker RNAs were up-regulated (e.g., E-cadherin, 32-fold) whereas mesenchymal marker RNAs were down-regulated (e.g., Thy1, ninefold) in PD-PANC-1s, suggesting mesenchymal-to-epithelial transition. Neural markers exhibited both up- and down-regulation. Immunocytochemistry indicated that epithelial-like PD-PANC-1s expressed E-cadherin and β-catenin in significantly more cells, while neural-like cells exhibited robust expression of organized β-3-tubulin. PAI-1 and E-cadherin were rarely co-expressed in the same cells. Indeed, examination of PAI-1 and E-cadherin mRNAs expression in additional cell lines yielded clear inverse correlation. Indeed, infection of Colo357 PAC cells (that exhibit high expression of E-cadherin) with PAI-1-expressing adenovirus led to a marked decrease in E-cadherin expression and to enhanced migration of cells from clusters. Our results suggest that endogenous PAI-1 suppresses expression of E-cadherin and differentiation in PAC cells in vitro, supporting its negative impact on tumor prognosis.
高水平的纤溶酶原激活物抑制剂-1（PAI-1）由基质细胞、内皮细胞和癌细胞产生，对癌症有多种复杂影响，与癌症预后不良相关。为了更明确地研究内源性产生的 PAI-1 在人胰腺腺癌（PAC）PANC-1 细胞系生物学中的作用，我们使用抗 PAI-1 shRNA 构建了稳定缺乏 PAI-1 的细胞（PD-PANC-1s）。PD-PANC-1s 表现出异质形态。虽然大多数细胞呈类似亲本 PANC-1 或载体感染对照细胞的立方体形，但观察到许多具有长丝状伪足和神经元样外观的大细胞。尽管 Vector-control 细胞和 PD-PANC-1s 都表达了具有间充质、神经和上皮表型特征的 mRNAs，但上皮标志物 RNA 上调（例如，E-钙粘蛋白上调 32 倍），而间充质标志物 RNA 下调（例如，Thy1 下调 9 倍）在 PD-PANC-1s 中，提示间充质向上皮转化。神经标志物既有上调也有下调。免疫细胞化学表明，上皮样 PD-PANC-1s 在更多细胞中表达 E-钙粘蛋白和β-连环蛋白，而神经样细胞则表现出强烈的组织化β-3-微管蛋白表达。PAI-1 和 E-钙粘蛋白很少在同一细胞中共同表达。事实上，在其他细胞系中检查 PAI-1 和 E-钙粘蛋白 mRNAs 的表达也得到了明确的反比关系。事实上，用表达 PAI-1 的腺病毒感染 Colo357 PAC 细胞（E-钙粘蛋白表达较高）会导致 E-钙粘蛋白表达明显下降，并增强细胞从簇中的迁移。我们的结果表明，内源性 PAI-1 在体外抑制 PAC 细胞中 E-钙粘蛋白的表达和分化，支持其对肿瘤预后的负面影响。
