Gain-of-Function E76K-Mutant SHP2 Promotes Proliferation,Metastasis And Tumor Growth Of Glioblastoma Cells

Background: Glioblastoma,originated in neuroepithelial cells,is the most common primary intracranial tumors.Although giving operation,radion therapy,chemotherapy and other intergrated treatment,the prognosis is still poor.SHP2 is a non-receptor protein tyrosine phosphatase that regulates cell proliferation,migration and invasion through a variety of signaling pathways.Earlier studies reported SHP2 mutations in 35% of patients with juvenile myelomonocytic leukemia and followed research found the mutant SHP2 also existed in some solid tumors.SHP2 activation mutations are important for the development and prognosis of blood tumors and solid tumors.SHP2 mutants have been found in clinical glioblastoma patients,but their effects on glioblastomas and their mechanisms are rarely reported.ERK1/2(extracellular signal-regulated kinase 1/2)joined in the RAS/ERK signaling pathway is one of the most important signal transduction pathway in cells,widely involved in regulating basic life activities of cells,such as proliferation,survival and differentiation.The cAMP response element-binding protein(CREB)is a nuclear regulatory factor in eukaryotes and regulates the expression of a wide range of genes.Phosphorylation status of CREB enhances its basic activity for hundreds times.Studies have shown that CREB over-activation in leukemia,non-small cell lung cancer,pancreatic cancer and glioblastoma are closely related to the occurrence and development of these cancers.Based on the above research background,we asked the questions: What is the effect of activating mutant SHP2 on glioblastoma cells? Is ERK/CREB signaling pathway involved in regulating the process? Methods: 1.SHP2 vector,WT-SHP2 and MT-SHP2 stable cell lines were established in glioblastoma cell lines of A172 and U87.SHP2 expression and cell biological behavioral experiments were tested(Proliferation experiments: MTT,plate cloning,soft agar colony formation,flow cytometry;Migration and Invasion experiments: Transwell experiments);2.The expression of the ERK/CREB pathway and related proteins in the stable cell lines of A172 and U87 were detected by Western Blot;3.Nude mice were inoculated with normal,vector,wild-type and mutant SHP2 U87 cells respectively in armpit subcutaneously and were feeded in SPF animal laboratory.The growth of tumors was recorded regularly and analyzed statistically.4.After cultivating with U0126(the ERK inhibitor-specific inhibitor),the expression of ERK/CREB signaling pathway and cell biology behavior experiments were performed of the different groups.Result: 1.SHP2 null control,wild type and activating mutant stable cell lines were successfully established in glioblastoma cell lines of A172 and U87;2.Activating mutant SHP2 enhanced the ability of cell proliferation,migration and invasion in glioblastoma cells;3.Activating mutant SHP2 activated ERK/CREB signaling pathway and the expression of downstream related proteins such as cyclin D1 and MMP9 was significantly increased;4.The tumorigenicity of U87 cells in SHP2 activation mutation group was significantly enhanced than that in the control groups;5.The ERK/CREB signaling pathway of SHP2 mutation group was significantly inhibited by ERK inhibitor U0126,and the proliferation and migration ability were also decreased.Conclusion: 1.Activating mutant SHP2 enhanced the proliferation,migration,invasion and tumorigenicity of glioblastoma cells;2.The mutant SHP2 promotes the malignant biological behaviors of glioblastoma cells by activating the ERK/CREB signaling pathway.