LHPP Inhibits Energy Metabolism And Malignant Progression By Inducing Degradation Of PKM2 In Glioblastoma

Background:Phospholysine phosphorhistidine inorganic pyrophosphate phos-phatase(LHPP)was a protein encoding gene which was first discovered in pig brain.Recent studies uncovered that LHPP inhibits oncogenesis and tumor progression in various cacers.However,the expression characteristic and clinical relevance of LHPP in human glioblastoma,and its biological function on the malignant progression of glioblastoma and relative molecular mechanisms are still unclear.Purpose:To investigate the expression characteristic and clinical relevance of LHPP in glioblastoma,as well as the biological function on the malignant progression of glioblastoma and relative molecular mechanisms.Methods:Bioinformatic analysis based on thress open databases,including The Human Protein Atlas,The Cancer Genome Atlas,and Chinese Glioma Genome Atlas,integrated with immunohistochemical staining,and western blot assays were conducted for studying the expression profile and clinical revelance of LHPP in glioblastoma.Cell growth assay in vitro and tumor formation assay in nude mice brain were performed to evaluate the biological function of LHPP in the malignant progression of glioblastoma.Glucose uptake capacity and metabolites measuring.as well as the stress tests of glycolysis and oxidative respiration in mitochondria were performed to evaluate the effect of LHPP in energy metabolism of glioblastoma.Mass spectrometry,chromatin immuno precipitation sequencing,genome transcriptome sequencing,real-time fluorescence quantitative PCR,western blotting,Co-immunoprecipitation,half-life of protein degradation testing,and ubiquitination in vivo testing were conducted for investigating the underlying mechanism of LHPP in suppressing the energy metabolic process and malignant progression in glioblastoma.Phenotypic rescue assay and mechanism rescue assay were performed to verify above-mentioned mechanism.Results:The level of LHPP mRNA or protein level was significantly down-regulated,even absent in glioblastoma,and it's negatively correlated with the median survival,which the median survival in patients with low LHPP expression level was noteworthily shortened.Overexpression of LHPP by plasmid transfection or viral infection in glioblastoma cells significantly inhibited the tumor cells growth in vitro,suppressed the tumorigenicity of gliobastoma cells transplanted in nude mice brain,and improved the median survival of the tumor-bearing mice.As to the molecular mechanism,LHPP can interacts with pyruvate kinase M2(PKM2),and the interaction would interfere the protein stability of PKM2,and then induce protein degradation of PKM2 via ubiquitin-mediated pathway.The down-regulated protein level PKM2 caused decreasing protein level of dimeric and tetrameric PKKM2,as well as the down-regulated kinase activity of PKM2 in glioblastoma cells,which caused restrained level of the glycolysis and oxidative respiration,and leading to reduced production of ATP,thus inhibits the glioblastoma progression.In the rescue assays,expression restoration of PKM2 could effectively reverse the inhibition of energy metabolism and malignant progression in glioblastoma caused by LHPP overexpression.Conclusion:LHPP is low expressed in glioblastoma tissue,and this is associated with survival prognosis in glioma patients.Overexpression of LHPP could inhibits the malignant progression of gliblastoma by impeding the energy supply by suppressing glycolysis and respiration via inducing unbiquitin-mediated protein degradation of PKM2.