Pyk2 on Cell Motility of Hepatocellular Carcinoma Figure 3. Effects of Pyk2 on the phosphorylation and activation of Hic-5. (A) Pyk2 over-expression promoted the membrane localization of Hic-5. Positive staining of Hic-5 (green, white arrow) was up-regulated in Pyk2 transfected Hep3B cells. (B) Effect of Pyk2 over-expression on the phosphorylation of Hic-5 in Hep3B cells. (C) Suppression of Pyk2 by PRNK down-regulated the localization of Hic-5 (green, white arrow) on cell membrane and (D) level of activated Hic-5 in MHCC97L cells. doi:10.1371/journal.pone.0018878.g003 expression in MHCC97L-PRNK cells compared to that in MHCC97L-vector cells (Fig. 4D). In MHCC97L cells, suppression of Pyk2 activation by PRNK resulted in down-regulation of other mesenchymal genes such as Twist, N-cadherin and fibronectin (Fig. 4D). Discussion We previously demonstrated that the focal adhesion localization is a major determinant for Pyk2 to carry out its function in HCC cells [15]. In this study, the role and underlying molecular mechanism of Pyk2 on transformation of HCC cells to undergo EMT were investigated. Forced overexpression of Pyk2 in Hep3B cells transformed the cells from an epithelial phenotype to a migratory phenotype which is commonly acquired by cancer cells to induce EMT leading to the increase of metastatic potential [28]. Enhanced formation of membrane ruffles and migration ability in Pyk2-overexpressing Hep3B cells suggested the promoting effect of Pyk2 on cell motility of HCC cells. Increased activation of Rac1/ RhoA in Hep3B-Pyk2 cells indicated that Pyk2 might promote cell motility of HCC cells via regulation of Rac1/RhoA activity. Suppression of Pyk2 in a metastatic HCC cell line MHCC97L showing a repression of EMT and cell migratory ability of the cells also indicated the importance of Pyk2 in regulation of cell motility of HCC cells. The constitutive expression of E-cadherin in both normal and cancer cells may help to maintain adherence junctions and subsequently decrease the cell’s capacity to invade or migrate through the extracellular matrix [29,30]. E-cadherin is frequently down-regulated during tumor progression. Loss of E-cadherin is associated with increased tumorigenecity and metastasis of cancer cells, providing a close correlation between metastasis and EMT [31]. Down-regulation of E-cadherin is usually coupled with an up-regulation of N-cadherin [32]. In this study, overexpression of Pyk2 in Hep3B cells resulted in a down-regulation of E-cadherin expression while suppression of Pyk2 by PRNK in MHCC97L cells significantly up-regulated the expression of E-cadherin, suggesting the important mechanism of Pyk2 on the regulation of adhesiveness of HCC cell via regulation of E-cadherin expression. The transcription factor, Hic-5, plays important roles in tumorigenesis and metastasis of human cancers including prostate cancer [33] and breast cancer [34]. Hic-5 is also a crucial EMT regulator of cancer cells [20,24]. Our results demonstrated that overexpression of Pyk2 in Hep3B cells up-regulated the activation of Hic-5 and its focal adhesion localization. Moreover, suppression of Pyk2 activation in MHCC97L cells down-regulated the membrane localization of Hic-5. Hic-5 is a Pyk2-binding protein which is phosphorylated by Pyk2 at tyrosine residue 60 upon physical interaction [22,23]. Given the role of Hic-5 on cell PLoS ONE | www.plosone.org 5 April 2011 | Volume 6 | Issue 4 | e18878