Proline-Rich Tyrosine Kinase 2 (Pyk2) Promotes Cell Motility of Hepatocellular Carcinoma through Induction of Epithelial to Mesenchymal Transition Chris K. Sun1., Kevin T. Ng1., Zophia X. Lim1, Qiao Cheng 1 , Chung Mau Lo1, Ronnie T. Poon 1 , Kwan Man1*, Nathalie Wong2, Sheung Tat Fan1 1 Department of Surgery, LKS Faculty of Medicine, Centre for Cancer Research, The University of Hong Kong, Pokfulam, Hong Kong, China, 2 Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Shatin, Hong Kong Abstract 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. Citation: Sun CK, Ng KT, Lim ZX, Cheng Q, Lo CM, et al. (2011) Proline-Rich Tyrosine Kinase 2 (Pyk2) Promotes Cell Motility of Hepatocellular Carcinoma through Induction of Epithelial to Mesenchymal Transition. PLoS ONE 6(4): e18878. doi:10.1371/journal.pone.0018878 Editor: Paul Dent, Virginia Commonwealth University, United States of America Received January 27, 2011; Accepted March 11, 2011; Published April 20, 2011 Copyright: 2011 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the General Research Fund (7574/06M) and Collaborative Research Fund (HKU5/CRF/08) of the Research Grant Council Hong Kong. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kwanman@hku.hk . These authors contributed equally to this work. Introduction Hepatocellular carcinoma (HCC) is the primary malignancy of the liver. It is fifth in popularity and third in cancer-related deaths worldwide [1]. Prognosis and treatment of HCC remains unsatisfactory due to tumor recurrence, metastasis of the primary tumor and poor therapeutic response to chemotherapy and radiotherapy [2,3]. Metastasis is not only a complex process but also the major cause of cancer-related deaths [4]. Transformation of cells to a fibroblastic phenotype is important for the cancer cells to successfully metastasize [5]. Several lines of evidences suggested that the induction of epithelial to mesenchymal transition (EMT) plays an important role in cancer cell transformation [6,7]. It contributes significantly to metazoan embryogenesis and patho- genesis such as tissue fibrosis and cancer progression [8]. On the other hand, the process of mesenchymal to epithelial transition (MET) may promote the growth of the metastatic cancer cells in secondary sites [9]. The critical hallmarks of EMT include the down-regulation of E-cadherin which is considered to be a tumor suppressor gene [10], activation of Rho small GTPases such as Rac1/RhoA which may increase cell motility by up-regulating actin turnover and formation of focal adhesion [11], cytoskeletal rearrangement and nuclear translocation of several transcription factors such as Snail and Twist [12,13]. Understanding the mechanism of HCC cell migration and metastasis may have great value to develop effective diagnostic and therapeutic strategies for treatment of HCC patients. Proline-rich tyrosine kinase 2 (Pyk2) is a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family. Our previous study had shown that up-regulation of Pyk2 in tumor tissues of HCC patients is significantly associated to poor prognosis [14]. Moreover, forced overexpression of Pyk2 in HCC cells promotes cell proliferation, invasion and migration via the activation of the c-Src and ERK/MAPK pathways which can be attenuated by forced overexpression of its C-terminal non-kinase region (PRNK)[15]. Furthermore, Pyk2 up-regulates the formation of PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e18878