Proline-Rich Tyrosine Kinase 2 (Pyk2) Promotes Cell Motility of Hepatocellular Carcinoma through Induction of Epithelial to Mesenchymal Transition

Aims: Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells. Methodology/Principal Findings: We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization. Conclusion: These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT. © 2011 Sun et al.published_or_final_versio

Effects of Pyk2 on the activation of STAT5b.

<p>(A) Pyk2 over-expression did not increase activation of STAT5b in Hep3B cells (green, white arrow). (B) Western blot analyses of protein level of phosphorylated STAT5b and Twist between Hep3B-vector and Hep3B-Pyk2 cells. (C) Suppression of Pyk2 down-regulated the nuclear localization of STAT5b (green, white arrow) in MHCC97L cells. (D) Suppression of Pyk2 down-regulated the phosphorylated STAT5b and other mesenchymal genes including Twist, N-cadherin and fibronectin analyzed by Western blot.</p

Effects of Pyk2 on epithelial genes cytokeratin and E-cadherin.

<p>(A) Effects of Pyk2 over-expression on the localization of E-cadherin in Hep3B cells. Positive staining of E-cadherin (green, white arrow) was present in the cytoplasm of the transfectants. (B) Over-expression of Pyk2 down-regulated the expression of cytokeratin and E-cadherin in Hep3B cells as shown by Western Blotting. (C) Forced expression of PRNK in MHCC97L cells restored the expression of E-cadherin (green, white arrow) on the cell membrane and cytoplasm. (D) The expression of E-cadherin and cytokeratin was increased upon suppression of Pyk2 in MHCC97L cells.</p