Sumoylation Of PKM2 Is Crucial For Glycolysis And Proliferation Of Lung Adenocarcinoma Cells

Background and ObjectiveCancer cells preferentially rely on aerobic glycolysis to generate energy,this is called“the Warburg effect”.It was well known that aerobic glycolysis was an inefficient way to generate ATP,but it enabled cancer cells to produce more of the important biomass（e.g.,nucleotides,amino acids and lipids）needed to produce a new cell.Pyruvate kinase is an important rate-limiting enzyme in aerobic glycolysis and catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP.There are four mammalian pyruvate kinase isoforms,PKL,PKR,PKM1 and PKM2,which are expressed in different cell types.PKL is mainly expressed in liver cells.PKR is found specifically in red blood cells.PKM1 and PKM2 come from the same mRNA transcript,but undergo different splicing process.PKM1 is found in normal adult cells,while PKM2 is mainly expressed in many different cancer cells and during embryonic development.PKM2 is a master player in the Warburg effect of cancer cells.However,the mechanisms of regulating PKM2 are not fully elucidated.Post-translational modifications（PTMs）are important mechanisms for regulating protein functions.Recently,several reports have focused on the PTMs of PKM2,including phosphorylation,acetylation and methylation.SUMO1 has been reported to promote glycolysis in cancer cells.However,the mechanism of regulating glycolysis is unclear.The study through a series of experiments finds that SUMO1 modified PKM2promotes glycolysis and cell proliferation.MethodSeventy-three lung adenocarcinoma patients were analyzed in our study.The expression levels of PKM2 were analyzed by immunohistochemistry on tissues.The relationship between PKM2 expression and patients’clinicopathologic characteristics was investigated.We used Kaplan–Meier survival analysis to evaluate the prognosis of 73 lung adenocarcinoma patients.In vivo and in vitro SUMOylation and western immunoblotting analysis were conducted to test whether PKM2 could be modified by SUMO1.SUMOsp 2.0 was used to predict potential SUMOylation sites of PKM2 protein.¹⁸F-FDG uptake and lactate production were detected to test the effect of SUMO1 on glycolysis.Further experiments were conducted to test whether the effect was PKM2 dependent.SUMO1was overexpressed or knockdown in A549 cells,then real-time fluorescent quantitative PCR and Western blot were used to detect the mRNA and protein levels of PKM2.¹⁸F-FDG uptake,lactate production and basal oxygen consumption rate（OCR）were detected to test the effect of SUMO1 modified PKM2 on glucose metabolism and oxygen consumption.Pyruvate kinase activity was tested on recombinant protein from eukaryotic cell.Luciferase reporter gene detection and real-time fluorescent quantitative PCR were conducted to test whether SUMO1 modified PKM2 affected the transcriptional activity of HIF-1α.Cell count,colony formation assay and subcutaneous xenograft tumor model were conducted to observe whether SUMO1modified PKM2 affected the proliferation capacity of A549 cells and the tumorigenesis of subcutaneous xenograft tumor.ResultsPKM2 expression was associated with tumor differentiation,lymph node metastasis,and TNM stage（p<0.05）.The overall survival rate（P=0.017）and disease-free survival rate（P?=?0.027）were significantly lower in the positive PKM2 expression group than in the negative PKM2 expression group.By in vivo and in vitro SUMOylation and western immunoblotting analysis,we found that PKM2 could be modified by SUMO 1,and K336 was the main SUMOylation site of PKM2.SUMO1 knockdown by specific siRNA significantly reduced the expression level of endogenous PKM2 in A549 cells.Overexpression of SUMO1 caused an increase in endogenous PKM2 proteins compared with the vector control.In addition,we found that the mRNA levels of PKM2 were not affected by SUMO1 knockdown.Further experiments confirmed that SUMO1 affected the protein stability of PKM2 by regulating its degradation process.By ¹⁸F FDG uptake and lactate production assays,we found that SUMO1 enhanced activity of glycolysis of A549 cells and this effect was PKM2 dependent.We found that SUMO1modification inhibited PK activity and promoted ¹⁸F-FDG uptake,lactate production of A549 cells.By luciferase reporter gene detection and real-time fluorescent quantitative PCR,we confirmed that SUMO1 modification of PKM2 was controlling PKM2 activity on HIF-1αtranscription.By cell count,colony formation assay and subcutaneous xenograft tumor model,we found SUMO1 modified PKM2 promoted the growth of A549 cells both in vitro and in vivo.ConclusionIn conclusion,the study revealed the molecular mechanism of SUMO1 modified PKM2 promoting glycolysis of cancer cells,thus providing important theoretical foundation for tumor targeting therapy in the further.