CIP2A Reprograms Cancer Metabolism Via Stabilizing PKM2 Oligomerization In Non Small Cell Lung Cancer Cells/Transcriptional Regulation Of ACE2 In Lung Epithelial Cells

Background:Pyruvate kinase M2(PKM2),the final rate-limiting enzyme in glycolysis,catalyzes the conversion of phosphoenolpyruvate to pyruvate with concomitant formation of ATP.PKM2 exists in a dynamic population of dimer and tetramer that determine its pyruvate kinase activity and several small molecules have been shown to modulate PKM2 conformational change.However,the roles of oncoproteins,such as the cancerous inhibitor of protein phosphatase PP2A(CIP2A)that is overexpressed in most human malignancies,including non small cell lung cancer(NSCLC),in modulation of PKM2 dimer-tetramer switching and tumour metabolic reprogramming,remain largely unknown.Methods:The interaction between CIP2A and PKM2 in NSCLC cell lines H1299 and A549 was analyzed by Co-Immunoprecipitation(Co-IP)and Immunofluorescence(IF)staining assays.The effects of CIP2A on PKM2 dimer-tetramer switching was evaluated by size-exclusion chromatography and protein crosslinking assays.XFe Seahorse energy metabolic stress assay was applied to determine the impact of CIP2A on glucose metabolism.The expression of CIP2A and phosphorylated PKM2 S287 in NSCLC was detected by immunohistochemistry assay,and the therapeutic efficacy of agents targeting CIP2A and PKM2 was tested in NSCLC murine model.Results:We reported that CIP2A could interact with PKM2 in NSCLC cells.CIP2A induced formation of PKM2 tetramer by phosphorylation of PKM2 at Ser287,a novel phosphorylation site that was essential for PKM2 dimer-tetramer switching and for maintaining the activity of PKM2.In contrast,depletion of CIP2A promoted PKM2 dimer accumulation and translocation into the nucleus,resulting in the enhancement of glycolysis.Clinically,the expression of CIP2A was positively associated with the level of phosphorylated PKM2 Ser287 in human lung adenocarcinoma tissues.Pharmacological inhibition of glycolysis or activation of PKM2 synergistically inhibited NSCLC cell growth when combined with CIP2A-targeting compounds both in vitro and in vivo.Conclusions:In conclusion,we identified CIP2A as a novel binding partner and modulator of PKM2.Further more,the phosphorylation of PKM2 at the Ser287 site is critical for CIP2A to regulate PKM2 functions and suppress the Warburg effect in NSCLC cells.The combination of a glycolysis inhibitor or PKM2 activator with the CIP2A-targeted compounds leads to a synergistic antitumour effect and might serve as a novel and promising strategy for treating patients with NSCLC.Background:Angiotensin converting enzyme 2(ACE2),is an important hydrolase of renin-angiotensin-aldosterone system,which catalyzes angiotensin II to angiotensin 1-7.ACE2 is expressed in multiple organs,including lung.In addition,ACE2 was found to be the receptor of Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)to bind to its spike glycoprotein and enter the host cells.The expression of ACE2 can be modified and regulated by histone methylation,acetylation and ubiquitination.However,the potential transcription factors(TFs)that control the expression of ACE2,have not been systematically dissected.Methods and results:Here,we employed the online datasets to identify potential TFs that regulate ACE2 expression,and found that 24 TFs may be ACE2 regulators,with Signal transducer and activator of transcription 3(Stat3)as the most significant one.We next tested the association between the expression of ACE2 and the active form of Stat3,the phosphorylated Stat3(p-Stat3),in lung biopsy samples of 12 patients with benign disease and 49 normal lung tissues of patients with lung cancer and found that ACE2 expression level was positively associated with p-Stat3 expression.By analyzing the promoter sequence of ACE2,we found a Stat3-binding site,locates at 874 bp upstream of its transcription start site.By electrophoretic mobility shift assay(EMSA),we showed that at the presence of interleukin-6(IL-6),the biotin-labeled Stat3-binding(BD)DNA of ACE2 promoter formed complex with Stat3 protein.In 16HBE cells,silencing of Stat3 by siRNAs significantly downregulated ACE2 expression at mRNA and protein,whereas overexpression of Stat3 by transfection of Flag-Stat3 plasmids into the cells resulted in increased expression of Stat3,p-Stat3,and ACE2.In addition,IL-6 stimulation could upregulate ACE2 expression in 16HBE cells by activating Stat3.Conclusions:These results indicate that Stat3 represents an important TF that controls ACE2 expression.This study provides a theoretical basis for targeting ACE2 to develop therapeutics to treat SARS-CoV-2 infection.