The role of c-Src in the invasion and metastasis of hepatocellular carcinoma cells induced by association of cell surface GRP78 with activated α2M

Song Zhao, Hongdan Li, Qingjun Wang, Chang Su, Guan Wang, Huijuan Song, Liang Zhao, Zhidong Luan, Rongjian Su

Emerging data have suggested that cell surface GRP78 is a multifunctional receptor and has been linked to proliferative and antiapoptotic signaling cascades. Activated α2-macroglobin (α2M*) is a natural circulating ligand of cell surface GRP78. Association of cell surface GRP78 with α2M* is involved in the regulation of cell proliferation, survival and apoptosis in human cancers. the invasion and metastasis of HCC cells were examined using transwell and wound healing assay; Cell surface expression of GRP78 was detected by in cell western assay. Translocation of GRP78 from cytosol to cell surface was observed by transfection of GRP78-EGFP plus TRIRC-WGA staining. The levels of Src, phosphor-Src, FAK, phospho-FAK, EGFR, phospho-EGFR, phospho-Cortactin, phospho-Paxillin were determined by western blot. Cell surface expression of GRP78 in HCC tissue samples was observed by immunofluorescence. The distribution of Paxillin and Cortactin in HCC cells was also observed by immunofluorescence. The interaction between GRP78 and Src were detected by far-western blot, co-immunoprecipitation and GST pulldown. GRP78 mRNA was detected by RT-PCR. In the current study, we showed that association of cell surface GRP78 with α2M* stimulated the invasion and metastasis of HCC. Cell surface GRP78 could interact directly with c-Src, promoted the phosphorylation of c-Src at Y416. Inhibition of the tyrosine kinase activity of c-Src with PP2 reverted the stimulatory effect caused by association of cell surface GRP78 with α2M*. Moreover, association of cell surface GRP78 with α2M* facilitates the interaction between EGFR and c-Src and consequently phosphorylated EGFR at Y1101 and Y845, promoting the invasion and metastasis of HCCs. However, inhibition of the tyrosine kinase of c-Src do not affect the interaction between EGFR and Src. c-Src plays a critical role in the invasion and metastasis of HCC induced by association of cell surface GRP78 with α2M*. Cell surface GRP78 directly binds and phosphorylates c-Src. As a consequence, c-Src phosphorylated EGFR, promoting the invasion and metastasis of HCCs.