The Inhibition And Molecular Mechanism Of Dual Targeting Src And PLK1 On EGFRvⅢ Positive Glioma Stem Cells

Background:EGFRvⅢ(epidermal growth factor receptor type Ⅲ mutant)is the most common EGFR(epidermal growth factor receptor)mutant in gliomas.EGFRvⅢ is closely related to the malignant degree of glioma and the poor prognosis of patients.The clinical treatment of EGFRvⅢ-positive gliomas has been a difficult problem.At present,some studies have shown that the expression of EGFRvⅢ is closely related to the malignant behavior of glioma stem cells.This may be one of the main reasons for the poor prognosis of glioma patients with positive EGFRvⅢ expression.In our previous studies,we found that the expression of Src and PLK1 proteins was higher in EGFRvⅢ-positive glioma stem cells,suggesting that EGFRvⅢ is significantly related to the activation of Src and PLK1-related signaling pathways.Therefore,we speculated that EGFRvⅢ may promote the malignant behavior of glioma stem cells by activating Src and PLK1 signaling pathways,but the molecular mechanism of its effect is not clear.Purpose:The aim was to explore the relationship between EGFRvⅢ and glioma stem cells.At the same time,combined with the results of our previous research,further study the molecular mechanism of EGFRvⅢ regulating the phenotypic characteristics and tumorigenicity of glioma stem cells by activating Src and PLK1 expression.To provide a theoretical basis for the combined targeting of Src and PLK1 in the treatment of EGFRvⅢpositive gliomas.Methods:1.The bioinformatics method was used to analyze the mutation rate of EGFR in glioma tissue and its correlation with the prognosis of glioma patients.At the same time,we used immunohistochemistry,western-blot and other methods to analyze the co-expression of EGFRvⅢ and glioma stem cell markers(CD133,Nestin)in glioma tissues.2.EGFRvⅢ positive glioma stem cells were established and examined the effect of EGFRvⅢ on the expression of glioma stem cell markers by immunofluorescence and Western-blot methods.The effect of EGFRvⅢ on the self-renewal ability of glioma stem cells was tested by secondary spheroidization and limiting dilution method.At the same time,we constructed a model of intracranial xenograft tumors in nude mice,and analyzed the effects of EGFRvⅢ on the expression of related signaling pathway proteins by immunohistochemical methods.3.The immunofluorescence,western-blot,limiting dilution,protein immuno-precipitation and other methods were used to detect the effects of down-regulation or inhibition of Src expression on the "stemness" characteristics of EGFRvⅢ-positive glioma stem cells and its molecular mechanism.4.We used immunofluorescence,western-blot,limiting dilution,Co-IP(co-immunoprecipitation),and other methods to detect the effects and molecular mechanisms of down-regulating or inhibiting the expression of PLK1 on the "stemness"characteristics of EGFRvⅢ-positive glioma stem cells.5.The apoptosis of EGFRvⅢ glioma stem cells were analyzed in vitro and in vivo using the Src inhibitor Saracatinib and the PLK1 inhibitor BI2536 by flow cytometry,Western-blot,intracranial transplantation tumor model,and immunohistochemistry.Results:1.The results showed that the abnormal expression of EGFR in glioma tissue was not only closely related to the malignant degree of glioma,but also could be used as one of the indicators of poor prognosis of glioma patients.The clinical examination of glioma tissues showed that the expression rate of EGFRvⅢ increased with the increase of glioma grade,indicating that the expression level of EGFRvⅢ is related to the malignancy of glioma.At the same time,the expression levels of CD133 and Nestin protein were higher in EGFRvⅢ positive glioma tissues.2.Compared with EGFRvⅢ-negative glioma stem cells,EGFRvⅢ-positive glioma stem cells have higher expression levels of glioma stem cell surface markers(CD 133 and Nestin)and a faster rate of secondary spheroid formation.These data indicate that EGFRvⅢ can promote the "stem" characteristics and tumorigenicity of glioma stem cells.For the possible molecular mechanism,we found that EGFRvⅢ is likely to play a role by activating Src and PLK1 signaling pathways3.Compared with EGFRvⅢ-negative glioma stem cells,EGFRvⅢ-positive glioma stem cells have higher activation expression of Src.In vitro experiments,the Src inhibitor Saracatinib can inhibit the self-renewal ability of EGFRvⅢ-positive glioma stem cells,indicating that EGFRvⅢ activation of Src regulation promotes the self-renewal ability of glioma stem cells.At the same time,the molecular mechanism research results show that EGFRvⅢ activated Src promotes the maintenance of glioma stem cell characteristics by regulating the Notch 1-SOX2 signaling pathway.4.Compared to EGFRvⅢ-positive glioma stem cells,PLK1 protein expression is higher than that of EGFRvⅢ-negative glioma stem cells.PLK1 inhibitor BI2536 was found to inhibit the self-renewal ability of EGFRvⅢ-positive glioma stem cells in vitro.At the same time,further research shows that EGFRvⅢ promotes the expression of PLK1,and also affects the biological characteristics of glioma stem cells mainly by activating Notch1-SOX2 signaling pathway.5.Compared with the single use of an inhibitor,Saracatinib combined with BI2536 induced a higher apoptosis rate of EGFRvⅢ-positive glioma stem cells,and combined treatment in vivo also had a more efficient killing effect on EGFRvⅢ-positive glioma stem cellsConclusions:This study focused on EGFRvⅢ-positive glioma stem cells,and explored possible molecular mechanisms for EGFRvⅢ-positive gliomas with higher malignancy and worse prognosis.We showed that EGFRvⅢ promotes malignant progression of EGFRvⅢ-positive gliomas by activating the expression of Src and PLK1,and then regulating the Notch1-SOX2 signaling pathway.It provides a sufficient scientific basis for the clinical use of Src inhibitors and PLK1 inhibitors in the treatment of EGFRv III positive gliomas.