The Mechanism Of Activation Of Fyn-BCKDK-BCAT1 Signaling Pathway To Promote Glioma Growth

Background: Glioblastoma(Glioblastoma,GBM)is a malignant tumor of the central nervous system with an extremely poor prognosis.The average overall survival of patients is 14.5-16.6 months,and the five-year survival rate is only 5%-7%.Although studies have found that GBM is associated with aberrant activation of multiple receptor tyrosine kinase(Receptor tyrosine kinase,RTK)pathways,these findings have not improved the treatment and overall survival of glioma patients.In addition to targeting RTKs themselves,another strategy for targeted therapy is to target the downstream RTKs that are druggable and valuable for tumor growth,such as targeting key pathways involved in tumor metabolic reprogramming.Branched-chain keto acid dehydrogenase kinase(Branched-chain keto acid dehydrogenase kinase,BCKDK),a key kinase in the catabolism of branched-chain amino acids,promotes cancer proliferation and metastasis and is considered an emerging kinase target for tumors.The role of BCKDK in the occurrence and development and whether it can be a target of glioma have not been reported.Objective: 1.To clarify the role of BCKDK in the occurrence and development of glioma.2.To find the direct upstream kinase and the downstream target of BCKDK and clarify the mechanism and biological effect.Methods: 1.The relationship between BCKDK expression levels and clinical prognosis was analyzed through bioinformatics,glioma clinical specimens and tissue microarrays 2.BCKDK or BCAT1 was silenced in glioma cell lines.Growth curves and colony formation assay were performed to elevate cell proliferation and colony formation.3.The immunoprecipitation assay and pull down assay were applied to demonstrate whether BCKDK can interact with Fyn or BCAT1.4.In vitro kinase assay,isotopic peptide experiments,mass spectrometry methods were combined to explore the upstream kinases,downstream substrates of BCKDK and specific phosphorylation sites.5.Prepare and purify a rabbit polyclonal antibody that can recognize the Y151 site of BCKDK.And this antibody was used to evaluated whether phosphorylation modification of BCKDK existed in the glioma cells and clinical tissues.6.Glioma cell lines stably expressing inactivating mutation,activating mutation of BCKDK Y151 site or BCAT1 S5,S9,T312 site were constructed.The effect of phosphorylation sites on protein stability was explored using cycloheximide assay and ubiquitination assay.The effect of phosphorylation sites on glioma growth was explored using mouse intracranial orthotopic glioma model.Results: 1.Both the m RNA and the protein level of BCKDK were positively correlated with the grade,and negatively correlated with the overall survival of glioma patients.2.Silencing BCKDK weakened the ability of cell proliferation and colony formation.Overexpression of BCKDK promoted the growth of intracranial glioma cells and shortened mouse progression-free survival.3.Silencing of BCKDK did not alter BCAT1 m RNA levels but reduced protein levels by enhancing ubiquitination of BCAT1.4.BCKDK bound to BCAT1 and phosphorylated the S5,S9,and T312 sites of BCAT1.The inactivating mutation of these three sites reduced the stability and activity of the BCAT1,and inhibited the growth of intracranial glioma cells and prolonged progression-free survival of mice.5.Fyn bound to BCKDK and phosphorylated the Y151 site of BCKDK.The inactivating mutation of Y151 site weakened its protein stability by enhancing the ubiquitination level of BCKDK,and inhibited the growth of intracranial glioma cells and lengthened progression-free survival of mice.6.The phosphorylation of Y151 site of BCKDK existed in glioma cells and clinical tissues,and was an important indicator of poor prognosis.Conclusion: 1.BCKDK promoted the growth of gliomas and was closely related to the poor prognosis of gliomas.2.BCKDK phosphorylated BCAT1 at S5,S9 and T312 sites,which enhanced the stability and activity of BCAT1,and promoted glioma growth.3.Fyn phosphorylated BCKDK at the Y151 site,which enhanced the stability of BCKDK and promoted glioma growth.