Nitric Oxide Synthases Promote Ovarian Cancer Glycolysis By Differentially Regulation Of PKM2

Background and ObjectivesOne of the fundamental properties of cancer cells is that they underwent metabolic reprogramming from oxidative phosphorylation to aerobic glycolysis during carcinogenesis.Aerobic glycolysis is essential for tumor growth and survival.Activation of multiple carcinogenic signals contributes to metabolism reprogramming during malignant transformation of cancer.Nitric oxide(NO)is a highly reactive free radical which is from L-arginine and oxygen by nitric oxide synthase(NOS)in cells.There are three NOS isoforms(NOS1,NOS2 and NOS3)in human cells and varied in their cell biological function through distinct regulatory model and protein interactions.One key mechanism by which NO regulates the function of various target proteins is through the process of S-nitrosylation,the coupling of a mitroso moiety to a reactive thiol group in specific cysteine residues leading to the formation of an S-nitrosothiol(SNO).Numerous S-nitrosylated proteins have been reported to be involved in various cancer-related events,such as apoptosis,angiogenesis,cell cycle modification,tissue invasion and metastasis.It was reported that nitric oxide promotes glycolysis but the mechanism remains elusive.Pyruvate kinase(PK)is a rate-limiting enzyme in the last step of glycolysis pathway which catalyzes the pyruvate production from phosphoenolpyruvate.Among four isoforms of PK(PKL,PKR,PKM1 and PKM2),PKM2 is the predominant isoform in tumor cells and exerts an important role in glycolysis and tumor malignancy.Unlike other isoforms,PKM2 converted from high activity tetramers to low activity dimmers.This transform leads to decreasing of pyruvate generation and accumulation of glycolysis intermediates for the macromolecule biosyntheses and NADPH generation.These productions were required for the cancer proliferation and protected from oxidative stress increased during tumor malignant transformation or chemotherapy.PKM2 was submitted to various posttranslational modifications such as phosphorylation(Y105),acetylation(Lys433),and oxidation(Cys358),which modify the PKM2 struction and regulate the convertion of tetramer to dimmer of PKM2.Recently,endogenous NO was found to regulate glycolysis in ovarian cancer.However,the mechanism of NOS regulated glycolysis and the function of S-nitrosylation on PKM2 has not been studied.In this study,we explore the role of NO/NOSs on glycolysis to meet the demands of ovarian cancer proliferation or survival,and whether NOS target PKM2 for glycolysis promotion,and hence provide clues for further understanding of the role of NO/NOS1 and glycolysis in ovarian cancer,contributing to finding promising target for improving the chemotherapeutic treatment of cancer.Results1.NO/NOS1 promotes glycolysis and proliferation in ovarian cancerWe first detected that supplying NO with DETA-NONOate increased the glucose consumption and lactate secretion in ovarian cancer cells,while inhibition of NOSs by L-NAME decreased them.We found that nitric oxide also promoted cell proliferation,survival and colony formation abilities,these effects of NO were abolished by glycolysis inhibition by using glucose analogue 2-deoxyglucose(2-DG).To test whether NOS1 is responsible for endogenous NOS on glycolysis promotion,we modified NOS1 expression genetically in ovarian cancer cells through transfection of GV358-NOS1 lenti-virus and NOS1 siRNA.We found that NOS1 increased the glycolysis rate,ATP and NADPH production,as well as cell proliferation.The NOS1 promoted cell proliferation was attenuated by glycolysis inhibition with 2-DG.In vivo assay showed the same results.2.NOS1 targets PKM2 for glycolysis promotionIn vivo and in vitro assay showed that overexpression of NOS 1 promotes the PKM2 nuclear translocation in ovarian cancer,breast cancer and colon cancer,while whole cell PKM2 expression kept same level.NOS 1-overexpression also increased the transcript of most glycolytic enzymes and transporters.We exploited the global gene expression profiling from Gene Expression Omnibus(GEO,GSE26712).The expression of NOS1 and NOS3 were correlated positively with PKM2,Kaplan-Meier survival analysis showed that the high NOS1 predicts a poor survival of the 185 ovarian cancer patients.The expression of PKM2 was increased in 185 primary ovarian tumors as compare to the normal ovarian tissues.The PKM2 expression positively correlated with c-MYC and HIF-la as well as the glycolytic genes,supporting the PKM2 regulated network in glycolysis of ovarian cancer.3.NOS1 and NOS3 differentially S-nitrosylates PKM2 at distinct sitesBiotin-switch assay showed that PKM2 S-nitrosylation level increased in NOS1 over-expressed SKOV3 cells and reduced in NOS1 knockdown cells.Applying wild type(Flag-PKM2-WT)and mutated PKM2(Flag-PKM2-C326S or Flag-PKM2-C423-424S),we found that NOS1 only S-nitrosylated PKM2 at Cys326 but not Cys423-424.Cys423-424S mutated PKM2 increased significantly the level of PKM2-SNO induced by NOS 1-overexpression.Interestingly we found that NOS3 was the source of NO to S-nitrosylated Cys423-424 of PKM2 due to knockdown of NOS3 mimicked the effect of Cys423-424 mutation on the increased level of SNO-PKM2.NOS3 repressed the SNO-PKM2 at C326 and the increased SNO-PKM2 by si-NOS3 was abolished by Cys-326 mutation.Co-immunoprecipitation assay showed that NOS 1-overexpression inhibited combination of the Flag-PKM2 subunit,and this effect was abolished by Cys326 mutation.Moreover,NOS1 overexpression converted all tetramers to dimmers and the effect was abolished by C326-mutation.NOS 1-induced PKM2 nuclear translocation was also interfered by C326S-mutation.Thus,NOS 1-induced SNO-C326 inhibited the formation of PKM2 tetramer and promoted the nuclear translocation.4.NOS2 activated glycolysis through EGFR/ERK1/2 signalingWe also found the dual role of nitric oxide in glycolysis:low/physiological nitric oxide(≤100 nM)promotes glycolysis for ATP production,oxidative defense and cell proliferation of ovary cancer cells,whereas excess nitric oxide(≥ 500 nM)inhibits it.Nitric oxide has a positive effect on glycolysis by inducing PKM2 nuclear translocation in an EGFR/ERK2 signalingdependent manner.Moreover,NOS2 induced by mild inflammatory stimulation increased glycolysis and cell proliferation by producing low doses of nitric oxide,while hyper inflammation induced NOS2 inhibited it by producing excess nitric oxide.Our study indicated that NOS2/NO play a dual role of in tumor glycolysis and progression,and established a bridge between NOS2/NO signaling pathway and EGFR/ERK2/PKM2 signaling pathway,suggesting that interfering glycolysis by targeting the NOS2/NO/PKM2 axis may be a valuable new therapeutic approach of treating ovarian cancer.5.Clinical associations of NOS1 and NOS2 in ovarian cancer specimensWe analyzed the expression of NOS1,NOS2 and PKM2 in tissue microarrays containing 150 ovarian carcinomas and 10 normal ovarian epithelial tissues.Immunohistochemistry analysis showed that NOS1,NOS2 and PKM2 were significantly increased in ovarian malignant specimens than that in non-cancerous ovarian epithelial biopsies.Correlation of NOS 1 and NOS2 expression with various clinicopathologic characteristics of ovarian cancer patients showed that,overexpressed-NOS 1 and NOS2 were peositively correlated with aggressive phenotypes ovarian tumors.Both NOS1 and NOS2 were significantly correlated with increased PKM2 expression.ConclusionWe demonstrated that increased expression of NOS isoforms in cancer distinctly modified PKM2 to promote glycolysis and differentially regulated the energy production.Constitutive expression of NOS 1 addicts cancer cells to glycolysis for the intensive proliferation though PKM2 S-nitrosylation at Cys326.In addition,we found that NOS3 and activated NOS2 drive glycolysis for ATP production.NOS3 is activated by energy senor AMPK and AKT signal pathway,and NOS2 was induced by inflammation factors.The activation of NOS3 and NOS2 provide energy for cancer biological functions such as survival,metastasis,autophagy and chemothereputic-resistance.Thus,NOS isoforms differentially promotes glycolysis and fine tune the sub-flux of glycolysis in response to various cellular stresses for the progression of cancer.