Stock Katharina, Borrink Rebekka, Mikesch Jan-Henrik, Hansmeier Anna, Rehkämper Jan, Trautmann Marcel, Wardelmann Eva, Hartmann Wolfgang, Sperveslage Jan, Steinestel Konrad
1Gerhard-Domagk-Institute of Pathology, University Hospital Münster, Münster, Germany.
2Department of Medicine A, University Hospital Münster, Münster, Germany.
Cancer Cell Int. 2019 Mar 29;19:77. doi: 10.1186/s12935-019-0798-x. eCollection 2019.
The nucleation-promoting factor cortactin is expressed and promotes tumor progression and metastasis in various cancers. However, little is known about the biological role of cortactin in the progression of pancreatic ductal adenocarcinoma (PDAC).
Cortactin and phosphorylated cortactin (Y421) were investigated immunohistochemically in 66 PDAC tumor specimens. To examine the functional role of cortactin in PDAC, we modulated cortactin expression by establishing two cortactin knockout cell lines (Panc-1 and BxPC-3) with CRISPR/Cas9 technique. Cortactin knockout was verified by immunoblotting and immunofluorescence microscopy and functional effects were determined by cell migration and invasion assays. A proteomic screening approach was performed to elucidate potential binding partners of cortactin.
Immunohistochemically, we observed higher cortactin expression and Tyr421-phosphorylation in PDAC metastases compared to primary tumor tissues. In PDAC cell lines Panc-1 and BxPC-3, knockdown of cortactin impaired migration and invasion, while cell proliferation was not affected. Three-dimensional spheroid culturing as a model for collective cell migration enhanced cortactin expression and Tyr421-phosphorylation. The activation of cortactin as well as the migratory capacity of PDAC cells could significantly be reduced by dasatinib, a Src family kinase inhibitor. Finally, we identified gelsolin as a novel protein interaction partner of cortactin in PDAC.
Our data provides evidence that cohesive cell migration induces cortactin expression and phosphorylation as a prerequisite for the gain of an invasive, pro-migratory phenotype in PDAC that can effectively be targeted with dasatinib.
成核促进因子皮层肌动蛋白在多种癌症中表达并促进肿瘤进展和转移。然而，关于皮层肌动蛋白在胰腺导管腺癌（PDAC）进展中的生物学作用知之甚少。
对66例PDAC肿瘤标本进行免疫组织化学研究，检测皮层肌动蛋白和磷酸化皮层肌动蛋白（Y421）。为了研究皮层肌动蛋白在PDAC中的功能作用，我们利用CRISPR/Cas9技术建立了两个皮层肌动蛋白敲除细胞系（Panc-1和BxPC-3）来调节皮层肌动蛋白的表达。通过免疫印迹和免疫荧光显微镜验证皮层肌动蛋白敲除，并通过细胞迁移和侵袭试验确定功能效应。采用蛋白质组学筛选方法阐明皮层肌动蛋白的潜在结合伙伴。
免疫组织化学分析显示，与原发性肿瘤组织相比，PDAC转移灶中皮层肌动蛋白表达和Tyr421磷酸化水平更高。在PDAC细胞系Panc-1和BxPC-3中，皮层肌动蛋白的敲低会损害细胞迁移和侵袭能力，而细胞增殖不受影响。三维球体培养作为集体细胞迁移的模型，增强了皮层肌动蛋白的表达和Tyr421磷酸化。Src家族激酶抑制剂达沙替尼可显著降低皮层肌动蛋白的激活以及PDAC细胞的迁移能力。最后，我们确定凝溶胶蛋白是PDAC中皮层肌动蛋白的一种新的蛋白质相互作用伙伴。
我们的数据表明，凝聚性细胞迁移诱导皮层肌动蛋白表达和磷酸化，这是PDAC获得侵袭性、促迁移表型的先决条件，而达沙替尼可有效靶向该表型。
