Pyk2 on Cell Motility of Hepatocellular Carcinoma Figure 4. Effects of Pyk2 on the activation of STAT5b. (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. doi:10.1371/journal.pone.0018878.g004 transformation as reported by other investigators [17,24], it may be one of contributing factors to the Pyk2-mediated cell transformation in HCC. STAT5b and Twist have been reported to promote aggressiveness and EMT of cancer cells in HCC [16,35]. In this study, we demonstrated that Pyk2 also regulated the expression of STAT5b and Twist in HCC cells, suggesting a multiple regulatory roles of Pyk2 on EMT associated genes. In this study, forced overexpression of Pyk2 in epithelial cancer cell line Hep3B up-regulated the mesenchymal gene expression. However, some of the mesenchymal genes were not up-regulated in this study. This may be explained by the partial mesenchymal characteristics of the Hep3B cells, which resulted in an incomplete induction of EMT by Pyk2. In contrast, suppression of Pyk2 activation in MHCC97L cells resulted in the down- regulation of the expression a series of mesenchymal genes including STAT5b, Twist, N-cadherin and fibroectin. These results showed that Pyk2 regulated the expression of mesenchy- mal genes and promoted the migratory characteristics of HCC cells which can be attenuated by the forced expression of PRNK to suppress Pyk2 activation. For cells with a mesenchymal phenotype (MHCC97L cells), transfection of PRNK successfully transformed the cells to an epithelial phenotype, with the down- regulation of mesenchymal gene expression and up-regulation of epithelial gene expression. E-cadherin was up-regulated with the down-regulation of N-cadherin. The results showed that suppression of Pyk2 by PRNK transformed the cells from a mesenchymal phenotype, to an epithelial phenotype, by under- going mesenchymal to epithelial transition (MET). These results confirmed the involvement of Pyk2 in the regulation of EMT of HCC cells. Both EMT and MET are important processes in cancer progression. The induction of EMT in cancer cells may enhance their cell motility and invasiveness so that to facilitate the development of metastasis [13,36]. Subsequently, circulating tumor cells must undergo MET to initiate growth in a secondary site [37]. Failure of MET induction in bladder cancer cells resulted in micrometastasis, instead of secondary tumors, as shown by experimental models [9]. Novel therapeutic strategies targeting the MET process could be made to inhibit the development of cancer metastasis by preventing secondary tumor formation. In this study, we had shown that the process of both EMT and MET are regulated by the alternation of Pyk2 activation. Overexpression of Pyk2 in Hep3B cells resulted in the induction of EMT. On the other hand, transfection of PRNK in MHCC97L cells resulted in the induction of MET, suggesting that prevention of focal adhesion by targeting of Pyk2 may transform HCC cells from a mesenchymal phenotype to an epithelial phenotype. However, its underlying mechanism is still unclear and thus considered to be worthwhile for further study. Our previous study also demon- strated that the suppression of Pyk2 by PRNK domain in MHCC97L cells can suppress in vivo intrahepatic tumor growth and venous invasion as well as extrahepatic lung metastasis [15]. Together, our data suggested the possible role of Pyk2 in the regulation of EMT, MET and metastasis in HCC cells. Our study demonstrated the important role of Pyk2 on controlling cell motility of HCC cells through regulation of genes PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e18878