Abstract
BackgroundEmerging 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.MethodsThe 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.ResultsIn 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.Conclusionc-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.
Highlights
Emerging data have suggested that cell surface glucose-regulated protein 78 (GRP78) is a multifunctional receptor and has been linked to proliferative and antiapoptotic signaling cascades
To investigate whether cell surface GRP78 is the surrogate of α2M* in this process, serum starved QGY-7703 and PLC cells were pretreated with the antibody directed against the NH2-termnial domain (NTD), COOHterminal domain (CTD) of GRP78 or isotype IgG for 1 h followed by α2M* stimulation [25, 26]
We found that blockade of cell surface GRP78 with the NTD antibody decreased the binding ability of tumor cells to fibronectin in QGY-7703 cells (Fig. 1h)
Summary
Emerging data have suggested that cell surface GRP78 is a multifunctional receptor and has been linked to proliferative and antiapoptotic signaling cascades. Association of cell surface GRP78 with α2M* is involved in the regulation of cell proliferation, survival and apoptosis in human cancers. Α2M binds directly with corresponding cell surface receptors and functions as a regulator of many signaling pathways and plays a growth factor-like role in many human cancers. GRP78 is regarded as an endoplasmic reticulum chaperone, whose major function is to fold and process the unfolded or malfolded proteins [6] It is presented on the cell surface under stress condition [7]. Ligation of cell surface GRP78 with α2M* activates the NF-kappaB signaling pathway, decreases p53 level and plays a stimulatory role in the proliferation and viability of prostate cancer cells [11, 12]
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