Atg7 Regulated The Warburg Effect By Inhibiting PKM2 Tyrosine Phosphorylation Involving In EMT

Objective: Autophagy is a self-digestion process,highly conserved during biological evolution.Cells can form autophagosome to degrade damaged organelles,aged or misfolded proteins,thereby providing enough energy and materials for metabolic demand.Thereby Autophagy is induced in a variety of stress condition and also known as type II programmed cell deaths.Functional autophagy is not just a simple biochemical phenomenon.Under physiological conditions,the basal level of autophagy is to maintain the ecological balance of the cells.In the case of metabolic stress,cells obtain energy through "self-eating",exhibiting stress-induced autophagy.Under pathophysiological state,autophagy can be induced via "self-sacrifice".Autophagy can protect the cells away from damage,thereby it is regarded as a suppressive mechanism for tumorigenesis.However,during the process of tumorigenesis,stress-induced autophagy will provide favorable conditions for the growth of tumor cells to help them overcome nutritional deficiencies or adverse hypoxic environment,thus contributing to tumor growth.Therefore,the exact role of autophagy in tumorigenesis remains controversy.The process of autophagy involves many autophagy-related proteins,among which,Atg7 is essential for the formation of autophagy.As an E1-like enzyme,Atg7 can activate Atg12 and promote the integration of Atg12 and Atg5.The deficiency of Atg7 will affect the formation of autophagosome and,result in the occurrence of cancer and some other diseases.In 1920,Otto Warburg found that cancer cells are metabolically changed.In the presence of oxygen,cancer cells produce energy via the conversion of glucose into lactate,a process known as aerobic glycolysis or the Warburg effect.A large number of glycolytic intermediates may be used by tumor cells,to synthesize proteins,nucleic acids,lipids and other biological macromolecules to promote the growth and proliferation of tumor.Pyruvate kinase is a rate-limiting enzyme of glycolysis and plays an important role in the glycolytic pathway.It catalyzes the dephosphorylation of phosphoenolpyruvate(PET)to pyruvate and yields ATP.There are four types pf pyruvate kinase isoenzymes in mammals,M1,M2,L and R type.Pyruvate kinase isoenzyme type M2(PKM2)is mainly expressed in tissues active in nucleic acid synthesis such as embryos and stem cells.A large number of studies have found that PKM2 is highly expressed in tumor cells.There are multiple post-translational modifications sites in PKM2.It was shown that fibroblast growth factor receptor 1(FGFR1)is a direct kinase of PKM2.Thephosphorylation of PKM2(Tyr105 sites)hinder the formation of tetramer and the resultant less activedimeric forms promote Warburg effect.Recent studies have found that the deficiency of ATG7 or ATG5 leads to the reduction of oxygen consumption and the increase of lactate production.Upon the treatment of Rapamycin,the production of lactate is suppressed.Collectively,a close link between autophagy and Warburg effect is suggested.Another study showed that the Tyr105 phosphorylation site of PKM2 promoted Warburg effect.Our data indicated that knockdown of Atg7 promote the phosphorylation of PKM2 Tyr105 site.However,the detailed mechanism remains unclear.This study aimed to to explore the relationship between the relationship between autophagy-related protein Atg7 and the key regulatory molecules PKM2 in Warburg effect.The role of autophagy and the Warburg effect in the development of tumors will be clarified,thus providing a theoretical basis for the prevention and treatment of cancer.Methods:1.HCT116,He La and MEF cells were cultured.Experiment-related plasmids and Atg7 knockdown cell line were constructed.2.Glycolysis related proteins were detected by Western Blot assay in Atg7 knockdown cell lines.3.Detecting the rate of glucose consumption and lactate production in the control group and Atg7 knockdown cell lines by optical absorption method rate.Examine the change of ECAR by Seahorse machine in Atg7 knockdown cells.4.Compare the level of phosphorylated PKM2(p-Tyr105)in different tissues in Atg7-deficient and WT control mice.Compare the level of phosphorylated PKM2(p-Tyr105)in control,Atg7 knockdown cells,Atg7 overexpressing cells and Atg7 knockdown cells rescued with Atg7 overexpression.5.Examine the the direct binding between Atg7 and PKM2 in vitro by using GST pull-down assay.Examine the binding between Atg7 and PKM2 in vivo by co-immunoprecipitation assay.Furthermore,the binding in vivo was accessed in the situation of b FGF stimulation and starvation.6.Accessing the binding between PKM2 with its direct kinase FGFR1 by co-immunoprecipitation assay in Atg7 knockdown cells,Atg7 overexpressing cells and Atg7 knockdown cells rescued with Atg7 over-expression.Examining whether Atg7 bind with FGFR1 upon b FGF stimulation.7.Under starvation or upon b FGF stimulating,examining the level of total and phosphorylated PKM2(Tyr-105 site)in control and Atg7 knockdown cells by Western Blot.8.Detecting the proliferation of control cells,cells stimulated with b FGF and Atg7 knockdown cells by cell counting.9.Detecting the maekers of EMT by Western Blot.Transwell invasion assay to measure cellular capacity for migration with Atg7.Results:1.Eukaryotic and prokaryotic expression vector of Atg7 were successfully constructed.Atg7 stable knockdown cell lines,He La and HCT116 cell lines were successfully constructed and knockdown efficiency was more than 70%.2.Glycolysis-related proteins are up-regulated upon Atg7 knockdown by Western Blot assay.3.Glucose consumption,lactate production and ECAR are down-regulated in Atg7 overexpression cells with no obvious change of OCR;Glucose consumption,lactate production and ECAR are up-regulated in Atg7 knockdown cells with no obvious change of OCR.4.Atg7 inhibitit PKM2 Tyr105 phosphorylation,but does not affect the level of the total PKM2.5.GST-pull down assay showed that Atg7 and PKM2 were directly interacted.The interaction between Atg7 and PKM2 and the interaction between Atg7 and FGFR1 were confirmed by immunoprecipitation assay in vivo.The interactions were enhanced by starvation and b FGF stimulation.6.Atg7 prevents the interaction between PKM2 and FGFR1.Upon b FGF stimulation,the interaction between PKM2 and FGFR1 were enhanced in control cells and the interaction between PKM2 and FGFR1 were suppressed in Atg7 knockdown cells.7.Upon starvation,The phosphorylation level of PKM2 was enhanced,In Atg7 knockdown cells,the enhancement was even more significant.Upon b FGF stimulation,the phosphorylation level of PKM2 was elevated,but the enhancement was not obvious in Atg7 knockdown cells.8.Cell proliferation was faster in Atg7 knockdown cells.b FGF promotes cell proliferation in both control and Atg7 knockdown cells.9.Inhibition of Atg7 promotes epithelial-mesenchymal transition(EMT)through PKM2 mediated Warburg effect.Conclusion: Experiments showed that Atg7 can modulate the Warburg effect and Atg7 inhibits the Warburg effect.Atg7 binds the key regulator of Warburg effect: PKM2 and revents the Tyr105 phosphorylation of PKM2.Atg7 inhibits PKM2 Tyr105 phosphorylation by preventing the interaction between PKM2 and its upstream kinase FGFR1.Inhibition of Atg7 promotes epithelial-mesenchymal transition(EMT)through PKM2 mediated Warburg effect...