The Roles And Mechanisms Of TSP50 In Hepatocyte Glucose Metabolism And Tumorformation By Promoting Acetylation Of PKM2 K433 Site

Hepatocellular carcinoma(HCC)is the fifth most common cancer and the second most frequent cause of cancer-related death worldwide.Many risk factors contribute for HCC initiation,such as infection with either hepatitis B virus or hepatitis C virus,nonalcoholic steatohepatitis,alcoholic cirrhosis,and exposure to environmental toxins.Despite many progresses in the diagnosis and treatment of HCC,the prognosis remains dismal due to high rates of recurrence and intrahepatic metastasis after surgical resection.Therefore,uncovering the mechanisms underlying HCC progression to improve clinical outcomes and develop better therapeutic strategies is of paramount importance.Accumulating evidences have shown that reprogrammed energy metabolism is a hallmark of tumor cells and a critical contributor to tumor development.Differ from most normal cells,cancer cells metabolize glucose to lactate even in the presence of sufficient oxygen,a phenomenon termed aerobic glycolysis,also known as“Warburg effect”.In the past decade,there is a considerable resurgence of interest in the oncogenic role of aerobic glycolysis in cancers.The aerobic glycolysis is characterized by a much higher rate of glucose uptake,consumption and lactate release in cancer cells.Increased glycolysis facilitates cancer cells to rapid utilization of glucose to produce abundant ATP.Through the pentose phosphate pathway,glucose metabolism provides cancer cells with abundant biosynthesis,including nucleotides,lipids,and nonessential amino acids.Moreover,glycolysis-derived lactate can lead to an acidic tumor microenvironment,which is profoundly implicated in tumor progression by modulating tumor metastasis and immune response.Thus,targeting tumor glycolysis holds distinct promise for development of therapeutic strategies for cancer patients.However,the molecular mechanism for the Warburg effect in HCC is far from explored.Testes-specific protease 50(TSP50)is a new member of cancer/testicular antigen(CTA).It is specifically expressed in testicular tissue and is not expressed in other normal tissues or in small amounts.Further studies have found that TSP50 is highly expressed in breast cancer,gastric cancer,colorectal cancer,laryngeal cancer,cervical cancer and lung cancer,and is involved in the proliferation,apoptosis,migration and metastasis of various tumor cells.So far,there is no research on the involvement of TSP50 in metabolic reprogramming of tumor cells,especially hepatic carcinoma cells.Therefore,this study intends to explore whether TSP50 can affect the proliferation of hepatic carcinoma cells by changing the cells metabolic reprogramming,so as to provide a theoretical basis for tumor therapy by targeting TSP50.TSP50 is elevated expressed in HCC and survival-associatedWe evaluated TSP50 transcription levels in HCC tissues via UALCAN database.Analysis results reveal that m RNA expression of TSP50 is significantly higher in TCGA-LIHC tissues than in adjacent normal tissues.Further sub-group analysis of multiple clinic-pathological features of TCGA-LIHC samples based on disease stages,gender,age,and tumor grade show that the expression of TSP50 is significantly higher in HCC patients than normal controls.Then,Kaplan-Meier survival curve was plotted by GEPIA database.The HCC patients were separated into two groups according to TSP50 m RNA expression level.Finally,we find that the high TSP50expression group has a shorter relapse free survival(RFS).Thus,TSP50 expression may serve as a potential diagnostic indicator in HCC.The correlation between the effect of TSP50 on cell glucose metabolism and its tumor-promoting effectIn order to further explore the function of TSP50,the over-expression vector PE-GFP-TSP50/pc DNA3.1-TSP50 was constructed and transfected into L02 cells with low expression of TSP50.The gene silencing vector sh TSP50 was constructed and transferred into Huh7 cells and Bel-7402 cells to knock down the expression level of TSP50.The effects of TSP50 on cell proliferation was examined by MTT and Brd U experiments.The results show that TSP50 can promote cell proliferation.In order to confirm the effect of TSP50 on glucose metabolism,we further detected the changes of related indexes of glucose metabolism.The m RNA detection results show that TSP50 over-expression promotes GLUT1 expression in L02 cells,and TSP50knockdown inhibits GLUT1,HK2,PKM2,LDHA,GPI,PFKL,PGK1,ENO1,ALDOA,PGAM1,GAPDH,PFKM and PFKP expression in Huh7 and Bel7402tumor cells,but there is no significant difference in GPI,PGK1,ENO1,PGAM1,GAPDH,PFKM and PFKP expression in Huh7 cells.The western blot detection results show that the protein levels of GLUT1,HK2,PKM2,LDHA,GPI,PGK1,ENO1,ALDOA and PGAM1 are increased after overexpression of TSP50,while the levels of GLUT1,HK2,PKM2,LDHA,GPI,PGK1,ENO1,ALDOA and PGAM1 are decreased after TSP50 is knocked down in hepatic carcinoma cells,and TSP50 has no effect on the expression levels of PFKL,PFKM,PFKP and GAPDH proteins.Compared with control cells,L02 cells overexpressing TSP50 have lower OCR levels,higher ECAR levels,significantly increased glucose uptake,lactate production,ATP content and metabolites of 2-PG and G6P and enhanced LDH activity with statistically significant differences.However,the knockdown of TSP50 expression in Huh7 and Bel-7402 cells significantly reduces the levels of the above glycolysis-related indicators.These results indicate that TSP50 is involved in the regulation of aerobic glycolysis.In order to study the correlation between the effect of TSP50 on glucose metabolism and its tumor-promoting effect,a small molecular glycolysis inhibitor,2-deoxy-d-glucose(2-DG),6mmol/L,was selected and applied to L02 cells overexpressing TSP50 for 48h,and the changes in cell proliferation level and glycolysis related indicators were detected.The results show that,after blocking glycolysis,the proliferation ability of L02 cells and the level of glycolysis related indicators decrease significantly,but do not fall back to the level of the cells treated only with 2-DG.These results suggest that the tumor-promoting effect of TSP50 is partially dependent on cellular glycolysis.Screening and verification of TSP50 interaction proteinsWe further investigated the molecular mechanism of TSP50 regulating the aerobic glycolysis pathway.LC-MS/MS results show that TSP50 can interact with a variety of proteins,including PKM2.To verify the result,we performed immunofluorescence,Co-IP and GST pull-down assays to confirm the interaction.GFP-TSP50 and flag-PKM2 plasmids were co-transfected into Huh7 and Bel-7402cells.As confirmed by immunofluorescence,TSP50 and PKM2 are co-localized in the cytoplasm.The results of endogenous Co-IP show that TSP50 protein can interact with PKM2 protein in cells.Furthermore,it has been revealed that GST-tagged TSP50 protein can pull-down Flag-tagged PKM2 protein by western blotting upon GST pull-down assays,indicating that there is a close direct interaction between TSP50 and PKM2 protein.Meanwhile,our results show that TSP50 can not bind to other PK subtypes such as PKM1 or PKLR.Therefore,we speculate that TSP50 may partially participate in the rapid proliferation of hepatic carcinoma cells by regulating PKM2 through the aerobic glycolysis pathway.We then knocked down the expression of PKM2 in L02,Huh7 and BEL7402 cells that overexpressed TSP50,and the results show that the pro-proliferation and aerobic glycolysis effects of TSP50 are significantly reduced after knockdown of PKM2,suggesting that the pro-proliferation and aerobic glycolysis effects of TSP50 are dependent on PKM2.TSP50-mediated acetylation is essential for maintaining PKM2 activity inhibitionIn cancer cells,a constitutively low PKM2 activity is essential for aerobic glycolysis.The oligomers of PKM2 exist in high activity tetramer,low activity dimer and inactive monomer forms.Multiple covalent modifications in tumor cells can reduce PKM2 activity by destroying the oligonucleotide state of PKM2,leading to increased anabolism.Therefore,we investigated a possible relationship between PKM2 and TSP50.We first compared the activity of PKM2 in different cells,and the results show that,compared with L02 cells,the highly active tetramer form in Huh7cells and Bel7402 cells are reduced,and correspondingly,the PKM2 activity is significantly reduced.Furthermore,TSP50-overexpressed L02 cells show a low PKM2 activity,while,TSP50 knockdown in Huh7 and Bel7402 cells significantly increase PKM2 activity.After Tepp-46 was added to Huh7 and Bel 7402 cells,the tetramer form of PKM2 is increased,and the cell proliferation and aerobic glycolysis levels are significantly decreased,suggesting that TSP50 can affect cell proliferation and aerobic glycolysis levels by regulating the oligomeric form of PKM2.Furthermore,we used immunoprecipitation assay to verify the effect of TSP50on PKM2 protein modification.The results show that the TSP50 promote the acetylation level of PKM2,with negligible effects on phosphorylation and glycosylation.However,TSP50 only has the activity of threonine hydrolase,so we speculate that TSP50 may affect the binding ability of PKM2 to acetylase or deacetylase.Further detection results show that TSP50 can reduce deacetylase SIRT2level and increase acetylase KAT9 level,which both interact with PKM2.These results indicate that TSP50 inhibits PKM2 activity by mediating acetylation of PKM2.PKM2 is acetylated at K433We next sought to identify the PKM2 acetylation site(s)affected by TSP50.The acetylation sites of PKM2 are usually K62 lysine,K305 lysine and K433 lysine.Therefore,the mutant plasmids of PKM2-K62R,K305R and K433R were constructed.Subsequently,after transfecting TSP50 in PKM2-WT,K62R,K305R and K433R cells,different forms of PKM2 protein were purified by immunoprecipitation,and the acetylation level was detected.The results show that the transfection of TSP50 do not affect PKM2 K433R activity and acetylation level in PKM2 K433R cells.After overexpression of TSP50 in L02 cells and knockdown of TSP50 in Huh7 and Bel-7402 cells,the acetylation level of PKM2 K433 was detected using the specific PKM2 K433 acetylation antibody.The results show that TSP50 significantly promotes the acetylation level of PKM2 K433.Together,these results demonstrate that TSP50 regulate the acetylates of PKM2 at K433.PKM2 K433 acetylation partially mediates the cell proliferation induced by TSP50 through aerobic glycolysisTo confirm that PKM2 K433 acetylation affects TSP50-induced cell proliferation,we constructed a K433Q mutant plasmid to simulate the continuous acetylation of PKM2.The PKM2 K433R activity remains unchanged,while the PKM2 K433Q activity is significantly reduced after the PKM2 K433Q and K433R mutant plasmids were transfected into HEK-293T cells.Transfection of PKM2 K433Q into TSP50knockdown Huh7 and Bel-7402 cells significantly increases cell proliferation and glycolysis levels.Furthermore,we co-transfected TSP50 with PKM2 K433R into L02and hepatic carcinoma cells,the TSP50+PKM2-WT and TSP50+PKM2 K62R were used as controls.After transfection for 48h,MTT and Brd U results show that the proliferation level of cells in the K433R group is significantly lower than that in the PKM2-WT and PKM2 K62R groups.The level of aerobic glycolysis was further detected,and the results show that glucose uptake,lactate production,ATP levels as well as ECAR levels are decreased significantly in K433R group.Taken together,these results suggest that the regulation of TSP50 on cell proliferation through the glycolysis pathway depends on the acetylation of PKM2 K433 site.PKM2 K433 acetylation is required for TSP50-induced tumor growth in nude miceTo extend our studies into an animal model,we examined the role of PKM2K433 acetylation in regulating the growth of TSP50-induced tumor in immune-deficient mice in vivo and assayed for tumor formation(n=6/group).The TSP50,PKM2 WT,TSP50+PKM2 WT and TSP50+PKM2 K433R-stable-expressed L02 cells were prepared by lentivirus infection and 5×10~7 L02 cells were used for female athymic nude mice dorsal subcutaneous injection.Except for the empty group,all animals in the other injection groups demonstrate tumor formation by 4 weeks post-injection.TSP50+PKM2 WT significantly increases tumor size and weight,as well as the aerobic glycolysis level in tumor tissues compared with PKM2 WT group.In NC,TSP50,TSP50+PKM2 WT and TSP50+PKM2 K433R groups,the mean tumor size and tumor weight are dramatically increase in the animals injected with TSP50 overexpressing L02 cells compared with empty vector group.On the other hand,in TSP50-overexpressing cells,PKM2 K433R transfection group demonstrate a smaller tumor size,accompanied by a dramatically reduce tumor weight(compared with TSP50+PKM2 WT group).Furthermore,L02 cells with TSP50 and wild-PKM2transfected shows a higher aerobic glycolysis level.Overall,these findings indicate that TSP50 play a key role in cell proliferation and tumor growth,at least in part,by regulating PKM2 K433 acetylation through aerobic glycolysis pathway.