Carcinogenesis Advance Access originally published online on September 1, 2008
Carcinogenesis 2008 29(11):2096-2105; doi:10.1093/carcin/bgn203
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Proline-rich tyrosine kinase 2 (Pyk2) promotes proliferation and invasiveness of hepatocellular carcinoma cells through c-Src/ERK activation
Department of Surgery and Centre for Cancer Research, The University of Hong Kong, Pokfulam, Hong Kong, China
* To whom correspondence should be addressed. Department of Surgery and Centre for Cancer Research, The University of Hong Kong, L9-55, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China. Tel: +852 28199646; Fax: +852 28199634; Email: kwanman{at}hkucc.hku.hk Correspondence may also be addressed to Ronnie T.Poon. Tel: +852 28553641; Fax: +852 28175475; Email: poontp{at}hkucc.hku.hk
The aim of the current study is to elucidate the mechanism of proline-rich tyrosine kinase 2 (Pyk2)-mediated cell proliferation and invasiveness in hepatocellular carcinoma (HCC) cells. Human HCC cell lines PLC and MHCC97L were stably transfected with either full-length Pyk2 or C-terminal non-kinase region of Pyk2 (PRNK). Functional studies on cell proliferation and invasion were conducted in vitro by colony formation assay, adhesion assay, migration assay and wound-healing assay. For the in vivo study, an orthotopic nude mice liver tumor model was applied to investigate the effects of Pyk2 overexpression on tumor growth and metastasis. Overexpression of Pyk2 in PLC cells resulted in an upregulation of colony formation (P = 0.021) and adhesion toward laminin (P = 0.018). Pyk2 promoted wound recovery by stimulation of actin stress fiber polymerization. In the in vivo study, transfection of PRNK in MHCC97L cells significantly decreased tumor volume (P = 0.001) and the incidence of lung metastasis (P = 0.014). Overexpression of Pyk2 promoted the activation of c-Src, formation of Pyk2/c-Src complex and activated the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-signaling pathway. Pyk2 upregulated the activation of ERK1/2 that is insensitive to MAPK/ERK kinase (MEK)1/2 inhibition. On the contrary, PRNK overexpression downregulated the activation of c-Src and ERK/MAPK-signaling pathways. Immunofluorescence staining showed that the focal adhesion localization of Pyk2 is a major determinant for c-Src and ERK/MAPK activation. In conclusion, our results showed that Pyk2 promoted cell proliferation and invasiveness by upregulation of the c-Src and ERK/MAPK-signaling pathways.
Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FAT, focal adhesion targeting; FBS, fetal bovine serum; HCC, hepatocellular carcinoma; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; PRNK, C-terminal non-kinase region of Pyk2; Pyk2, proline-rich tyrosine kinase 2
Received April 26, 2008; revised July 29, 2008; accepted August 23, 2008.
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