The Molecular Mechanism Of C-Kit-G?i/Gab1 Signal Complex In The Proliferation And Survival Of Osteosarcoma Cells

Background and Objective:Osteosarcoma(OS)is the most common primary malignant bone tumors,most in 10 to 20 years old teenagers.OS high malignant degree,can be transferred in a short period.Since the 1970 s,chemotherapy drugs(cisplatin,methotrexate and adriamycin)began treatment applied to the OS which makes the OS total 5 years survival rate has reached more than 60~70%.But some OS chemotherapy in patients with poor response,prone to local recurrence or distant metastasis,the long-term survival of patients did not improve.Studies confirm that multiple oncogenes excessive expression and activation is the main reason for the chemotherapy resistance,invasion and metastasis of OS.Therefore it is particularly important to explore the main molecular mechanism of OS and to find new intervention strategy.Abnormal expression and activation of receptor tyrosine kinases(RTK)play an important role in the development and progression of OS.Herein,c-Kit is a type III RTK,and its ligand is a stem cell factor(SCF).After specific binding to SCF,c-Kit dimerization or oligomerization activates multiple downstream signaling pathways,including PI3K-Akt-mTOR pathway and ERK-MAPK pathway,ultimately promoting cell survival,proliferation,apoptosis resistance and so on.In recent years,studies have confirmed that G?i protein and Gab1 protein-coupled activation of tyrosine kinase receptors(Tyrosine Kinase Receptors,RTKs)can mediate some growth factors such as: EGFR,FGFR,PDFGR,activation of downstream signaling pathway.However,the mechanism of expression of c-Kit receptor in osteosarcoma and activation of downstream PI3K-Akt-mTOR pathway and ERK-MAPK pathway remains unclear.Therefore,this study was to explore the effect of SCF on osteosarcoma cells,and to investigate the mechanism of c-Kit-G?i / Gab1 complex in the activation of SCF signaling pathway in osteosarcoma cells.Methods:1.The expression of c-Kit protein in MC3T3-E1 osteoblasts,OB-6 osteoblasts and osteosarcoma cell lines MG-63 and U-2OS were compared by western blot.2.MG-63 and U-2OS cells were treated with different concentrations of SCF for 24 hours and 48 hours.The cell clone formation was detected by crystal violet staining.The proliferation of osteoblasts was detected by MTT assay and Brdu method.3.To compare the expression of G?i1/3 and Gab1 proteins in MC3T3-E1 osteoblasts,OB-6 osteoblasts and osteosarcoma cell lines MG-63 and U-2OS,we used western blot.4.The expression of c-Kit,G?i,Gab1 in osteosarcoma tissue and peripheral normal bone tissues were compared by immunocytoprecipitation(Co-IP)assay,and the expression of c-Kit-G?i-Gab1 in osteosarcoma cell lines MG-63 and the mouse embryonic fibroblasts(MEFs)with SCF stimulated were compared.5.In order to further explore the expression of AKT-mTORC and ERK-MAPK signaling pathway in SCF stimulated MG-63 cells,and how G?i protein and Gab1 protein play roles in SCF / c-Kit activation of AKT-mTORC and ERK-MAPK signaling pathway respectively We detected the expression and activity of Akt(Thr308 and Ser473),mTORC1,S6,GSK,Gab1,c-Kit,S6 K and ERK protein in MG-63 cells and MEFs-related cells respectively by western blot.Results:1.The expression of c-Kit in cultured OS cell lines(MG-63,U-2OS)was significantly higher than that in MC3T3-E1 osteoblasts and OB-6 osteoblasts(p <0.05).2.SCF can promote the proliferation and clone formation of osteosarcoma cells(MG-63,U-2OS)(p <0.05).3.The expression levels of c-Kit,G?i1/3 and Gab1 were significantly higher in cultured OS cell lines(MG-63,U-2OS)than those in osteoblasts and osteocytes.4.Immunoprecipitation(Co-IP)results showed that the expression of c-Kit,G?i1/3 and Gab1 in osteosarcoma was significantly higher than that in peripheral normal bone tissue.In addition,SCF could induce c-Kit-G?i-Gab1 protein to form a coupled complex in MG-63 cells and mouse embryonic fibroblasts(MEFs)in vitro.5.SCF can activate AKT-mTORC and ERK-MAPK signaling pathways in osteosarcoma cells,and this activation is mediated by G?i protein in mouse embryonic fibroblasts(MEFs).6.Gab1 can mediate SCF activation of Akt-mTORC and ERK-MAPK signaling pathway in osteosarcoma cells(MG-63)and mouse fibroblasts(MEFs).7.In MEFs,knockdown of G?i can inhibit SCF-induced phosphorylation of Gab1(Tyr627 and Tyr307).Conclusions:1.SCF can promote the survival and proliferation of osteosarcoma cells(MG-63,U-2OS),and the expression of c-Kit in osteosarcoma tissues and cells is also higher than that of normal tissues and osteoblasts.2.G?i-Gab1 signaling complexes act as c-Kit junction proteins,which promote the survival and proliferation of OS cells by mediating the downstream signals(PI3K-Akt-mTOR,ERK-MAPK).This study will provide a theoretical basis for G?i-Gab1 signaling complexes as new biomarkers and therapeutic targets for OS.Targeting to this complex,it is expected to find a new way to effectively inhibit osteosarcoma by interfering with the signal pathway.