NOS1 Expression Promotes The Warburg Effect In Ovarian Cancer By The S-nitrosylation Of PFKM

Background and ObjectivesNitric oxide(NO)is synthesized by nitric oxide synthase(NOS1,NOS2,NOS3)using arginine and molecular oxygen through a redox reaction.NOS/NO modifies the protein cysteine(Cys)sulfhydryl-SH to form nitroso(-SNO),a NO-specific,reversible redox signal transduction regulatory mechanism that regulates protein positioning,structure,activity and function.Even under aerobic conditions,tumor cells still use the glycolysis pathway as the main energy source,namely the Warburg effect.Phosphofructokinase(PFK)catalyzes the production of fructose 6-phosphate from fructose 1,6-diphosphate,which is the most critical rate-limiting enzyme for glycolysis.The mammalian cells express three kinds of PFK1(PFKL,PFKP and PFKM),and the three subtypes form a homotetramer or a heterologous active tetramer,which participates in the glycolysis process.PFK1 is an allosteric regulatory enzyme that metabolic end products such as ATP,lactic acid and citric acid inhibit the formation of PFK1 tetramer,thereby inhibiting PFK1 activity and controlling glycolysis flux,while ADP,AMP and F2-6BP can stabilize PFK1 tetramer structure and enhance PFK1 activity.The N-terminal PDZ domain of NOS 1 and the C-terminal PDZ domain binding sequence(-EAAV-COOH)of PFKM specifically bind.This topic will explore NOS1 expression promotes Warburg effect in ovarian cancer by the S-nitrosylation of PFKM and provide new ideas for revealing the malignant progress of ovarian cancer.Results1?NOS1 promotes ovarian cancer cell proliferation and tumor growth dependent on PFKM.We matched NOS1 content in 426 human ovarian tumor tissues(T)from TCGA database compared with 88 normal tissues(N)from GTEx data to know that NOS1 is highly expressed in tumor tissues through the GEPIA website.We established a stable overexpression of NOS 1 ovarian cancer cell line SKOV3(OE-NOS1)by lentiviral vector.At the same time,we established a ovarian cancer cell line that stably knocks out NOS1(KO-NOS1)using CRISPR/Cas9 gene editing technology.We examined the Glycolytic rate and lactate production of three SKOV3 cells(KO-NOS1,wild type,OE-NOS1).The results showed that overexpression of NOS1 promoted glucose uptake and lactic acid production in cells,whereas the corresponding glycolysis rate of SKOV3 knocked out of NOS1 decreased.As the expression level of NOS1 increases,the rate of cell proliferation also increases significantly by CCK-8 assay.We performed subcutaneous tumors using SKOV3 cells,KO-NOS1 SKOV3 cells and OE-NOS1 SKOV3 cells in three different parts of nude mice,respectively.After 18 days of monitoring,we found that the size of the tumor was directly proportional to the amount of NOS 1,and there was no tumor formation in the KO-NOS1 group.On the 45th day,there were still no tumors growing in the KO-NOS1 group.Similarly,the weight of the tumor was the largest in the OE-NOS1 group.To test whether PFKM is the key to NOS1 promoting metabolic flux,we established a stable overexpression of NOS1 in KO-PFKM SKOV3 cell lines.The cell proliferation assay showed that the cell proliferation rate increased with the increase of NOS 1 expression,but this phenomenon did not exist when the cell PFKM expression was absent by EdU(5-Ethyny1-2'-deoxyuridine)cell proliferation assay.We examined the Glycolytic rate and lactate production of four kinds of SKOV3 cells(wt SKOV3,OE-NOS1 cells,KO-PFKM cells,and KO-PFKM-OE-NOS1 SKOV3 cells).Lossing of PFKM reduces the promotion of metabolic flux by NOS 1.The activity of PFKM was gradually increasing when the NOS1 content was gradually increased.In overexpressing NOS1 SKOV3 cells,the expression level of PFKM tetramer(active expression form)was increased.We transferred the wt-Flag-PFKM plasmid and showed the same result using the FLAG monoclonal antibody by Western blot.As the content of substrate F-6-P increased,the reaction rate of PFKM increased faster in OE-NOS1 group than in the control group.To assess the role of NOS 1 in regulating the negative feedback regulation of PFK1,we evaluated the PFKM activity respectively in SKOV3 cells,OE-NOS1 cells,and SKOV3 cells with N-PLA(100uM)for 48 h.NOS1 can promote PFKM activity to resist the inhibition of a wide Citrate concentration range in the SKOV3 cells.Similar effects on PFKM activity were observed when the above three cells were treated under different concentrations of ATP and Lactate.After that,we used ultrasonic disruption of cells to extract total protein for detection of PFK1 enzyme activity.In the reaction system for detecting PFK1 activity,different concentrations of Citrate,ATP and Lactate were added to observe the tolerance of PFK1 to negative feedback inhibition in SKOV3 cells,OE-NOS1 cells,and KO-NOS1 SKOV3 cells.Similarly,the higher the content of NOS 1,the stronger the resistance of PFK1 to negative feedback inhibition.Then we added ATP and Citrate(PH=7.5)to the medium to simulate the negative feedback effect under physiological conditions.The results show that NOS1 can promote PFK1 activity to resist the inhibition of a wide Citrate concentration range for 24h in the medium.When NOS1 expression was absent,the concentration of PFK1 inhibited by ATP was reduced.Thus,the results of the above three different levels are consistent,supporting the idea that NOS1 promotes PFK1 activity and enhances PFK1 resistance to negative feedback inhibitors.In order to verify whether NOS1 promotes PFK1 by promoting PFK2,We examined the changes in the activity of PFK1 in SKOV3 and OE-NOS1 cells which were treated with PFK15 0 or 5uM.The results showed that the activity of PFK1 was not affected by PFK15 in OE-NOS1 cells.2?PFKM s-nitrosation at Cys351 by NOS1 regulates the enzyme activity.Our results of Co-immunoprecipitation(Co-IP)showed that NOS1 can bind to PFKM in SKOV3 and OVCAR3 cells.We have established the PDZ domain of GST-tagged NOS1 in vitro by recombinant protein.Western blot results showed that PDZ domain of GST-tagged can bind to PFKM by GST pull-down assay.Phosphofructokinase-M,one proposed nNOS binding partner,display a class ? PDZ domain binding motif at its C termini.To verify that PFK1 is a s-nitrosated protein,We converted the s-nitroso to a biotin label using "biotin switch",then purified all s-nitrosylated proteins in SKOV3 and OVCAR3 cells using Streptavidin Agarose Resins.We found that the s-nitrosation-modified PFKM protein increased when the cells overexpressed NOS1.Identification of the S-nitrosylation site on PFKM using mass spectrometry.According to the data obtained by the mass spectrometer,the search database was searched by Thermo Proteome Discoverer 2.2.We identified a single site of S-nitrosylation at Cys 351.The mutation of cysteine 351 to serine(C351S)reduced the S-nitrosylation of PFKM in SKOV3 cells.The mutation of cysteine 351 to serine(C351S)reduced the activity of PFKM and the tetramer content in SKOV3-KO-PFKM cells.13C-glucose tracer combined with gas chromatography-mass spectrometry(GC-MS)technique was used to analyze the effect of S-nitrosylation of PFKM on glucose metabolism of ovarian cancer SKOV3 cells.The result show that the glycolysis and TCA cycle were decreased in SKOV3-KO-PFKM-C351S cells.Next,we studied the effect of s-nitrosylation-modified PFKM on cell energy metabolism using the seahorse XF96 cell energy technology.The results showed that SKOV3-KOPFKM-C351S cells group reduced basal level OCR,ATP-related OCR and maximal OCR,suggesting that S-nitrosylated PFKM enhances mitochondrial oxidative phosphorylation.3?Subcutaneously transplanted tumors in BALB/c mice and lung metastasis models in C57bl/6 mice further demonstrated that the promotion of NOS1 after Cys351 mutation was weakened.We respectively established stably expressing PFKM/WT and PFKM/C351S proteins in SKOV3-KO-PFKM and SKOV3-KO-PFKM-OENOS1 cells.We examined the Glycolytic rate and lactate production of WT group,C351S group,OE-NOS1-WT group and OE-NOS1-C351S group.The results showed that overexpression of NOS 1 promoted glucose uptake and lactic acid production in cells,but this increase disappeared when Cys351 mutation to serine(C351S)was stably expressed in SKOV3-KO-PFKM-OENOS1 cells.We used cell counting experiments to verify that NOS 1 could not promote cell proliferation after mutating the s-nitrosated modification site.In order to verify whether the in vivo experiments are consistent,we performed a subcutaneous tumor formation experiment in nude mice.After 35 days of monitoring,C351S reduces cancer cell growth and NOS1 promotes tumor growth reduction in OENOS1-C351S.We measured cell survival rate in ovarian cancer cells based on the IC50 value of DDP.It was found that the sensitivity of the C351S group increased regardless of whether DDP was applied for 24 h or 48 h.The NOS1-S-nitrosation-modified PFKM signaling axis coordinates glycolysis and TCA cycle to modulate cancer cell growth.PFKM-C351S reduces melanoma lung metastasis survival in C57b1/6 mice.4?NOS1 regulates S-nitrosation modification of glycolysis-related proteins.To quantify the S-nitrosylation level in overexpression of cells and controls,we used the biotin switch method with the Western blot for the amount of the S-nitrosoproteome.The level of S-nitrosylation in the OE-NOS1 group was significantly higher than that in the control group.To explore which S-nitrosylated proteins regulated by NOS1,we propose a simple strategy that combines our biotin switch method with the labelfree strategy for the site-specific identification and quantitation of the S-nitrosoproteome.Ovarian cancer cells strain stably overexpressing NOS1 was established,and proteins of OE-NOS1 and CON cell line were respectively extracted.A biotin replacement method replaces the nitroso with biotin.The protein is digested into peptides by trypsin,the biotin peptide is purified by streptavidin resin,then desalted by C18 desalting column,LC.MS/MS analysis.According to the data obtained by the mass spectrometer,the search database was searched by Thermo Proteome Discoverer 2.2.The results show that the S-nitrosylated protein up-regulated by NOS1 are most strongly associated with metabolism.We use the DAVID website to detect which pathways are associated with these proteins.The results show that the up-and down-regulated proteins are related to metabolic pathways and immune-related pathways.We limited the ratio to more than 2 times,using the DAVID website to analyze that the protein regulated by NOS1 is most closely related to glycolysis.ConclusionNOS1 depends on PFKM to promote cellular glycolysis.NOS1 S-nitrosation modified the Cys351 site of PFKM.Mutation at the Cys351 site reduced PFKM activity,while NOS1 promoted glycolysis was also reduced.Blocking S-nitrosation of PFKMCys351 can reduce cancer cell proliferation in vitro and damage tumor formation in vivo.It is hoped to elucidate the role and mechanism of NOS 1-induced S-nitrosation in regulating tumor glycolysis,and to provide new markers for tumor diagnosis and treatment.