title: The role of c-Src in the invasion and metastasis of hepatocellular carcinoma cells induced by association of cell surface GRP78 with activated α2M.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Central laboratory, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121001, China. Zhaosong517@163.com.
authors.forename: Song
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Central laboratory, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121001, China. Lihongdan101@126.com.
authors.forename: Hongdan
authors.initials: H
authors.lastname: Li
authors.affiliation: Oncology Department, the First Affiliated Hospital of Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121001, China. qingjunwang666@163.com.
authors.forename: Qingjun
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Veterinary Medicine Department, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121001, China. changsu80@163.com.
authors.forename: Chang
authors.initials: C
authors.lastname: Su
authors.affiliation: Central laboratory, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121001, China. Wang591716708@163.com.
authors.forename: Guan
authors.initials: G
authors.lastname: Wang
authors.affiliation: Central laboratory, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121001, China. shj-shj@tom.com.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Song
authors.affiliation: Pharmacy Department, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121000, China. liangzhao79@163.com.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhao
authors.affiliation: Development Department, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121000, China. zhdluanqwe@hotmail.com.
authors.forename: Zhidong
authors.initials: Z
authors.lastname: Luan
authors.affiliation: Central laboratory, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121001, China. rongjiansu@hotmail.com.|Cell Biology AND Genetic Department, Liaoning Medical College, No 40 Songpo Road, Jinzhou, 121000, China. rongjiansu@hotmail.com.
authors.forename: Rongjian
authors.initials: R
authors.lastname: Su
mesh_terms: D000081247:CSK Tyrosine-Protein Kinase; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D000091342:Endoplasmic Reticulum Chaperone BiP; D066246:ErbB Receptors / Q000378:metabolism; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction; D000511:alpha-Macroglobulins / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D000511:alpha-Macroglobulins; D066246:ErbB Receptors; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human