Overexpression and Tyr421-phosphorylation of cortactin is induced by three-dimensional spheroid culturing and contributes to migration and invasion of pancreatic ductal adenocarcinoma (PDAC) cells

Katharina Stock,Marcel Trautmann,Jan-Henrik Mikesch,Jan Sperveslage,Eva Wardelmann,Jan Rehkämper,Konrad Steinestel,Rebekka Borrink,Anna Hansmeier,Wolfgang Hartmann

doi:10.1186/s12935-019-0798-x

Abstract

BackgroundThe 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).MethodsCortactin 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.ResultsImmunohistochemically, 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.ConclusionOur 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.

Highlights

The nucleation-promoting factor cortactin is expressed and promotes tumor progression and metas‐ tasis in various cancers
Enhanced cortactin expression level and phosphorylation correlates with tumor metastasis The expression and the activation status of cortactin was determined by immunohistochemical staining of pancreatic ductal adenocarcinoma (PDAC) specimens
PDAC metastases revealed a significant upregulation of cortactin (p = 0.0049 Chi square) and phospho-cortactin (p = 0.0184 Chi square) compared to those from primary tumors (Fig. 1a + b)

Summary

Introduction

The nucleation-promoting factor cortactin is expressed and promotes tumor progression and metas‐ tasis in various cancers. Little is known about the biological role of cortactin in the progression of pancreatic ductal adenocarcinoma (PDAC). By the time of diagnosis, in as many as 80% of patients the tumor has already metastasized, leading to a 5-year survival rate of less than 5% [2]. In this context, understanding metastatic progression and the highly invasive potential of PDAC cells has become increasingly important. Irrespective of a single-cell or cohesive cell mode of invasion, many of the steps involved in metastatic progression require the remodeling of the actin cytoskeleton. These functions can lead to deregulated cell migration and invasion [9, 10]

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Conclusion