| Lung cancer is one of the most lethal malignant tumors,accounting for approximately 25%of all cancer mortality,among all types of tumors worldwide,approximately 75%of lung cancer are non-small cell lung cancer（NSCLC）.Due to the rising incidence and mortality rates and limited therapeutic targets,further understanding of the molecular mechanism of lung cancer development and searching for effective therapeutic targets could be of paramount significance for the diagnosis,treatment,and prognosis of lung cancer patients.Energy metabolic reprogramming is a typical feature of cancer.Tumor cells rely mainly on glycolysis for energy supply even with sufficient oxygen,which is termed aerobic glycolysis or the Warburg effect.Altered energy metabolism not only provides cancer cells with adequate energy but also generates essential metabolic intermediates to support the rapid growth of tumor cells and prevent apoptosis.Accumulating evidence suggests that the cancer-specific modulation of expression or enzymatic activity of glycolytic enzymes is important for tumor cells to maintain a high level of aerobic glycolysis and contribute to carcinogenesis.Therefore,a better understanding of the molecular mechanism by which glycolytic enzymes mediate tumor pathogenesis and progression by regulating aerobic glycolysis could be of paramount significance for the development of new therapeutics for NSCLC.Triosephosphate isomerase（TPI）is a glycolytic enzyme that plays an important catalytic function in glycolysis by catalyzing the reversible conversion between dihydroxyacetone phosphate（DHAP）and D-glyceraldehyde-3-phosphate（GAP）.Both DHAP and GAP are generated from the catabolism of fructose 1,6-bisphosphate,and only GAP can be used by the remaining steps in glycolysis.Therefore,the isomerization conversion of these two metabolites elevates the efficiency of glycolysis.It shows high expression of TPI in the tissues with increased glycolytic capacity,such as esophageal cancer,lung cancer,and gastric cancer.In addition,TPI silencing suppresses proliferation,migration,and invasion as well as induces apoptosis and G2/M arrest in gastric cancer cells.However,the specific regulation of TPI in NSCLC,especially its modification,requires further elucidation.Post-translation modification（PTM）refers to the covalent binding of small molecular groups on the amino acid side chain of proteins,such as phosphorylation,acetylation,methylation,and glycosylation.In addition to these commonly known modifications,more unknown modifications need to be further explored.PTMs can specifically regulate protein function by dynamically modulating its activity,conformation states,localization,interactions,and participate in various physiological and pathological processes,including tumor occurrence,development,and prognosis,and are also closely related to tumor diagnosis and treatment.Recent studies have found that protein modifications can directly regulate glycolysis by changing the structure or activity of metabolic enzymes,thereby playing an important role in the occurrence and development of cancers.Therefore,a systematic and comprehensive analysis of the PTMs of metabolic enzymes and their dysregulation in NSCLC could contribute to further elucidation of the molecular mechanism of cancer-specific regulation of metabolic enzymes.In this study,we analyzed the potential protein modifications throughout the proteome of patients with NSCLC,especially glycolytic enzymes,and further explored the role and mechanism of TPI Ser58 phosphorylation in promoting the development of lung cancer by regulating glycolytic metabolism.Part Ⅰ Diverse protein modifications mapped to the glycolytic enzymes in lung cancer[Objects]We adapted a high-throughput mass spectrometry workflow based on shotgun proteomics to identify delta masses between the actual residues and the coding amino acids,which reflects the occurrence of potential protein modifications in the proteome.Next,we focus on statistical analysis of the changes in total abundance of various delta mass clusters between tumor and adjacent tissues and bioinformatics analysis of the differences in abundance of possible modifications on protein positions and the pathways enriched by differentially modified proteins in NSCLC.Finally,we lay a detailed analysis on all possible modifications mapped to glycolytic enzymes.The modification omics will preliminarily clarify the specific regulation of protein modifications in lung cancer and lay a foundation for further exploring the mechanism of PTMs regulating tumorigenesis and development by mediating the function of metabolic enzymes.[Methods]1.Collect cancer and adjacent tissues from patients with NSCLC,isolate total proteins,digest with trypsin,fractionate by chromatography,and analyze the peptides by LC-MS/MS.2.The acquired MS and MS/MS data were searched against the human UniProt database（version 20171020）using the Byonic spectral alignment algorithm,and the non-zero delta masses between the actual residues and the coding amino acids were calculated.All identified delta masses were divided into subgroups with 1 Da intervals bounded by n-0.5 and n+0.5 Da（n=-200 to 1000）.Delta masses in each mass window were analyzed by multivariate clustering using Gaussian mixture components to calculate the peak value（delta mass cluster）,which reflected the occurrence of potential protein modifications in the proteome.At the same time,we analyzed whether the delta masses matched the molecular weight of the known modifications in the Unimod database.3.Statistical analysis of the total abundance of various delta mass clusters and compared them between lung cancer and adjacent tissues.4.Bioinformatics analysis of the differences in abundance of multiple and diverse delta masses on protein positions and the pathways enriched by differentially modified proteins in NSCLC and clarified the specific regulation of protein modification in lung cancer.5.Statistical analysis of the total abundance or types of delta mass spanned individual protein identified.We laid a detailed analysis on all possible modifications mapped to glycolytic enzymes and compared it between lung cancer and adjacent tissues.[Results]1.Through mass spectrometry identification and analysis,multiple and diverse delta masses were found distributed in the lung cancer proteome.319 unique delta mass clusters were identified with Gaussian non-linear regression and spread over 10,897 protein positions.Some delta mass clusters matched to known protein modifications,and the unmatched delta masses represented potential unknown new modifications.2.Among 319 delta mass clusters,39 were significantly upregulated or downregulated by two-fold in cancer tissues compared to non-tumor tissues（FDR-adj.p value<0.05）,and+79.967 Da was the most statistically significantly elevated,which best matched the mass deviation of the phosphorylation modification,and mainly occurred at the serine residue.3.Bioinformatics analysis demonstrated that the delta masses in 257 protein positions were significantly upregulated and in 489 protein positions downregulated by two-fold in the tumor（FDR-adj.p value<0.05）,and the proteins with differentially modifications were enriched in many functional pathways,especially glycolysis,which indicated the specific regulation of PTMs on glycolytic enzymes in lung cancer.4.Statistical analysis of the total abundance and types of delta mass spanned individual protein indicated that most of the glycolytic enzymes such as GAP dehydrogenase（GAPDH）,ENO1,PKM2,and TPI,were among the top proteins with high frequencies.5.The abundance of delta masses spanned on many glycolytic enzymes globally increased in NSCLCs.[Summary]1.Widespread potential protein modifications spanned over the proteome in lung cancer.2.+79.967 Da was the most statistically significantly elevated in lung cancer,which matched the phosphorylation modification and mainly occurred at the serine residue.3,Multiple potential protein modifications mapped to the glycolytic enzymes,many of which were significantly differential in lung cancer.Part Ⅱ Glycolytic enzymes are commonly upregulated in lung cancer[Objects]In the first part,we found widespread and diverse potential protein modifications spanned over the proteome in lung cancer.Next,the proteomic analysis will be performed to determine the protein expression level.Bioinformatics analysis of differentially expressed proteins and their biological pathways in lung cancer,and a detailed analysis of the expression level of key glycolytic enzymes and their relationship with patient prognosis,which is of great significance to explore the target proteins that mediate the occurrence and development of lung cancer.[Methods]1.The raw MS and MS/MS data were searched against the human UniProt database（version 20171020）containing forward and reverse sequences using MaxQuant（version 1.5.3.30）.The acquired protein intensities were log2-transformed,and missing value inputated and normalized using Perseus.2.Multiple comparisons were performed between lung cancer and corresponding adjacent tissues with a p-value corrected by FDR.Differentially expressed proteins in lung cancer were determined and their biological pathways were further analyzed.3.Bioinformatics analysis of all the metabolism proteins identified in our data.The biological processes involved in differentially expressed metabolism-related proteins in NSCLC were further analyzed using DAVID database.4.The expression level of glycolytic enzymes in lung cancer was analyzed by our proteomics data,and their mRNA levels were analyzed by the human Gene Expression Omnibus（GEO）dataset（GSE74706）.The correlation between the high expression of glycolytic enzymes and the prognosis of patients was analyzed by KM plotter.[Results]1.Proteomic analysis revealed a total of 4426 proteins（FDR<1%）that demonstrated a significant spatial separation based on proteome intensity between lung cancer and adjacent tissues.2.634 differentially expressed proteins（DEPs）were found in the NSCLC cohort through less stringent supervised comparisons（FDR-adj.p<0.05）.Of these proteins,418 were upregulated and 117 were downregulated by two-fold in lung cancer.Kyoto Encyclopedia Genes and Genomes pathway analysis indicated that the differentially expressed proteins were distributed in many metabolism pathways,including glycolysis,carbon metabolism,and pyruvate metabolism.3.Further bioinformatics analysis demonstrated that 101 metabolism proteins were dysregulated in NSCLC（FDR-adj.p<0.05）.Detailed Gene ontology analysis by the DAVID database indicated that these differentially expressed metabolic proteins were also enriched in glycolysis.4.The protein expression and mRNA levels of almost all glycolytic enzymes were consistently upregulated in NSCLC,and the expression of these glycolytic enzymes was negatively correlated with the prognosis of patients with lung cancer.[Summary]1.There was a significant spatial separation based on proteome intensity between lung cancer and adjacent tissues.2.The differentially expressed proteins in lung cancer were significantly enriched in the glycolysis pathway.3.The mRNA and protein expression levels of key glycolytic enzymes were commonly upregulated in NSCLC,and their high levels were negatively correlated with the prognosis of patients with lung cancer.Part Ⅲ TPI Ser58 phosphorylation promotes glycolysis and tumor development in NSCLC[Objects]In the first two parts,the omics analysis both emphasized the dysregulation of glycolysis in lung cancer,which further illustrates the important role of metabolic reprogramming in tumorigenesis and development.In addition,widespread and diverse potential protein modifications mapped to the glycolytic enzymes,which indicates the specific regulation of PTMs on lung cancer by mediating the function of metabolic enzymes.Next,the delta masses of glycolytic enzymes were further analyzed,leading to the observation that the changes of TPI@58S@+79.967,GAPDH@313W@+15.995,GAPDH@65G@+1.968 are the most obvious in NSCLC.Accumulating studies have revealed the mechanism relationship between protein kinase signal transduction and metabolic pathways,which indicates that phosphorylation modification is of great significance in regulating cancer cell metabolism and tumor development.Then,we focused on the TPI@58S+79.967,which matched the serine 58 phosphorylation of TPI.This part will validate that TPI@58S+79.967 is the phosphorylation modification and its elevation in tumors,and further explore the regulation of TPI Ser58 phosphorylation on glycolysis and tumor development.[Methods]1.A549 cells were transfected with exogenous TPI,the transiently expressed or endogenous TPIs were immunoprecipitated,resolved using sodium dodecyl sulfatepolyacrylamide gel electrophoresis（SDS-PAGE）,and visualized by Coomassie staining,cut the target TPI band,and analyzed by LC-MS/MS,and confirmed whether Ser58 was the key site for+79.967 Da mass shift of TPI in lung cancer.2.Prepare the specific antibody to recognize the phosphorylated Ser58 in TPI（TPI pS58）.The efficiency and specificity of the three batches of anti-TPI-pS58 antibodies were analyzed by Dot blot.WT TPI and its mutant expression plasmids were constructed and transfected into HEK293T cells,and the Ser58 phosphorylation was examined to evaluate the efficiency and specificity of the generated TPI pS58 antibody and further confirmed the existence of TPI Ser58 phosphorylation.3.The levels of TPI Ser58 phosphorylation in lung,breast,gastric,and pancreatic cancer and their corresponding normal epithelial cells were examined using the specific TPI pS58 antibody by Western blot.TPI Ser58 phosphorylation was examined in lung,gastric,pancreatic,and breast cancer tissue arrays by IHC.The correlation between TPI Ser58 phosphorylation level and the prognosis of patients with lung cancer was analyzed according to clinicopathological data.4.The TPI enzyme activity in various lung cancer and their corresponding normal epithelial cell lines was assessed using the TPI Activity Colorimetric Assay Kit,and the changes of TPI enzyme activity in lung cancer cells were analyzed.5.Lentivirus vector pLKO.1 was used to construct TPI knockdown plasmids（pLKO.1TPI）,and lentivirus was packaged and infected H157 and A549 cells,separately.The knockdown efficiency of TPI was examined by Western blot.The ATP level and lactate production were examined by commercial detection kits,and the rate of extracellular acidification（ECAR）was monitored with a model XF96 Flux Analyzer.6.The TPI-WT or its S58A mutant was expressed in the endogenous TPI-depleted A549 cells,the TPI and its Ser58 phosphorylation were examined by Western blot and immunofluorescence,and the TPI activity,the lactate production,the ATP level,and the rate of ECAR were further examined to evaluate the effects of TPI Ser58 phosphorylation on glycolysis.7.shTPI/TPIWT and shTPI/TPIS58A cells were cultured supplemented with 13C6-labeled glucose and metabolites were extracted for LC-MS/MS analysis to evaluate the effects of TPI Ser58 phosphorylation on glycolysis flux in lung cancer.8.The pLVX,TPI-WT or its S58A,S58D mutant was stably expressed in H1299 and A549 cells,and the TPI and its Ser58 phosphorylation were examined by Western blot.The effects of Ser58 phosphorylation on lactate production,the ATP level,and the rate of ECAR were further examined.9.MTT and colony formation analyses of the effect of TPI knockdown on H157 and A549 cell proliferation,and the migration ability as determined by the Transwell and wound healing assays.Nude mice were hypodermically injected with TPI silent cells or control A549 cells,and the tumor volumes were measured.Approximately 4-5 weeks later,tumors were dissected,photographed,and their weights were measured.The indicated cell lines were injected into the tail vein and liver tissues were dissected after 40-50 days.The total number of distant metastasis were quantified.10.The effects of TPI Ser58 phosphorylation on the proliferation and migration of lung cancer cells were detected by MTT,clone formation,and Transwell assays using the established shTPl/TPIWT and shTPI/TPIS5BA cell lines.The effects of TPI Ser58 phosphorylation on the growth and metastasis of lung cancer cells in vivo were evaluated by subcutaneous injection and tail vein injection of indicated cell lines into nude mice.[Results]1.Omics data analysis showed that the abundance of TPI@58S@+79.967 among glycolytic metabolic enzymes was elevated most significantly,and the+79.967 Da mass shift was confirmed to be phosphorylation using the generated TPI pS58 antibody,and further found that Ser58 was the main phosphorylation site of TPI by LC-MS/MS analysis.2.TPI Ser58 phosphorylation was increased in lung cancer cells and tissues,and the TPI Ser58 phosphorylation level was negatively correlated with the survival of the patients with NSCLC.TPI pS58 level was also increased in different types of cancers,including breast,gastric,and pancreatic cancer cells.3.The enzymatic activity of TPI was much higher in lung cancer cells than in normal cells.The ATP level and lactate production,and the rate of ECAR decreased in TPI silencing cell lines.4.The enzymatic activity of TPI was reduced in Ser58 phosphorylation depleted cells（shTPI/TPIS58A）compared to shTPI/TPIWT cells,and the ATP level and lactate production,and the rate of ECAR also decreased.13C-glucose labeled shTPI/TPIS58A cells exhibited an accumulation of TPI upstream glycolytic intermediates 13C-G6P,13C-FBP,and 13C-DHAP,and a reduction of TPI downstream intermediates 13C-BPG,13C-PEP,and 13C-Pvruvate.5.A lower rate of glycolysis and ATP and lactate production were observed in cells stably overexpressing phosphorylation-silencing TPI S58A compared with the cells stably overexpressing WT TPI,which were elevated in cells stably overexpressing phosphorylationmimicking TPI S58D.6.TPI silencing inhibited the proliferation and migration abilities in H157 and A549 cells compared to control cells.TPI knockdown inhibited the growth and metastasis of lung cancer by the in vivo experiments.7.TPI Ser58 phosphorylation deletion inhibited the proliferation and migration in lung cancer.Ser58 phosphorylation deletion inhibited the growth and metastasis of lung cancer cells by the in vivo experiments.[Summary]1.Ser58 was the main phosphorylation site of TPI,and TPI Ser58 phosphorylation level was significantly elevated in multiple tumors.2.TPI enzyme was critical for aerobic glycolysis and lung cancer development.3.TPI Ser58 phosphorylation can mediate TPI enzyme activity,thereby promoting glycolysis metabolism in lung cancer.4.TPI Ser58 phosphorylation promoted the growth and metastasis of lung cancer cells.Part Ⅳ PRKACA kinase directly phosphorylates TPI at Ser58[Objects]In the third part,our study found that TPI Ser58 phosphorylation was significantly increased in lung cancer and regulated glycolysis and tumor development.Therefore,in this part,we further explore the upstream kinase responsible for the phosphorylation of TPI Ser58 to clarify the molecular regulation mechanism of TPI Ser58 phosphorylation in lung cancer cells.[Methods]1.The Netphosk program was used to identify the possible protein kinase responsible for TPI Ser58 phosphorylation.Submit the TPI amino acid sequence to the Netphosk database,and select the possible kinase responsible for TPI Ser58 phosphorylation according to the prediction results and literature reports:PKA,RSK,AKT,PKC,and CDK.2.Treat the lung cancer cells with a panel of inhibitors for the predicted protein kinases,and the TPI protein and its Ser58 phosphorylation levels were examined.The results showed only the PKA inhibitor suppressed the phosphorylation of TPI at Ser58.The TPI protein and its Ser58 phosphorylation levels were detected in gastric,breast,and pancreatic cancer cells treated with PKA kinase inhibitors by Western blot.3.Treat H1299 and A549 cells with the PKA inhibitor H89,and the TPI enzymatic activity was evaluated by kit.4.Treat lung,gastric,breast,and pancreatic cancer cells with the PKA kinase activators 8-Br-cAMP and dc-AMP,and the TPI protein and its Ser58 phosphorylation levels were examined by Western blot.5.HEK293T cells were transfected with TPI expressing plasmid 48 h and treated with the PKA inhibitor or activators.Endogenous TPIs were immunoprecipitated,and the endogenous TPI protein and its Ser58 phosphorylation levels were examined by Western blot.6.qRT-PCR analysis was used to determine the mRNA levels of PRKACA,PRKACB.and PRKACG isoforms,and PRKACG was almost undetectable.7.RNA interference was used to knock down PRKACA or PRKACB expression in A549 and H1299 cells,and TPI protein and its Ser58 phosphorylation were examined to determine the PKA isoform responsible for TPI Ser58 phosphorylation.8.An in vitro kinase assay was performed by incubating the selected PKA catalytic subunit with the purified recombinant TPI or TPI S58A mutant proteins in a buffer supplemented with ATP at 30℃ for 1 h,and TPI Ser58 phosphorylation was examined by Western blot.9.Knockdown of the selected PKA kinase subunit with RNAi in H1299 and A549 cells,and the TPI activity and the rate of ECAR were examined.10.Construct the PKA kinase subunit overexpression and knockdown cell lines to evaluate its effect on cell proliferation and migration ability in lung cancer by colony formation and the Transwell assays.[Results]1.PKA,RSK,PKC,or CDK was the possible kinase responsible for TPI Ser58 phosphorylation according to the prediction of the Netphosk database.In addition,the AKT kinase has been reported to regulate glycolysis metabolism.Therefore,the above five kinases were selected for further screening objects.2.Only the PKA inhibitor significantly suppressed the TPI Ser58 phosphorylation,except for RSK,AKT,PKC,and CDK inhibitors.The PKA inhibitor-induced inhibitory effect on TPI Ser58 phosphorylation was also observed in gastric,pancreatic,and breast cancer cells.3.The TPI enzymatic activity was reduced in H1299 and A549 cells treated with the PKA inhibitor.4.The TPI Ser58 phosphorylation was largely increased in H1299 and A549 cells treated with the PKA activators 8-Br-cAMP or deoxy-cAMP.5.The phosphorylation of exogenous TPI at Ser58 was consistently inhibited or activated,respectively after treating with the PKA inhibitor or activator.6.qRT-PCR analysis showed that PRKACG was almost undetectable in lung cancer.Knockdown of PRKACA significantly decreased the level of TPI Ser58 phosphorylation,whereas the knockdown of PRKACB did not.The in vitro kinase assay demonstrated that PRKACA kinase directly phosphorylated TPI at Ser58.7.Knockdown of PRKACA using RNAi in H1299 and A549 cells significantly reduced TPI activity,accompanied by a reduced rate of ECAR.8.PRKACA overexpression had increases in cell proliferation and migration.In addition,silencing PRKACA in lung cancer cells inhibited growth and migration rates.[Summary]1.PKA was the main kinase responsible for TPI Ser58 phosphorylation in cancer cells.2.PRKACA kinase directly phosphorylated TPI at Ser58.3.PRKACA regulated glycolysis metabolism and lung cancer development.Above all,in this study,we systematically identify all delta masses between the actual residues and the coding amino acids by LC-MS/MS,which indicate the occurrence of potential PTMs,at the proteomic scale in cancer and adjacent tissues from patients with NSCLC.Bioinformatics analysis revealed the dysregulated modifications of glycolytic enzymes in human non-small cell lung cancer,particularly the mass shift of 79.967 Da at Ser58 of TPI（TPI@58S@+79.967）,which was confirmed to be phosphorylation and generally elevated in human tumor specimens and correlated with poor survival.Blocking TPI Ser58 phosphorylation dramatically inhibited glycolysis,cancer growth,and metastasis.In addition,the protein kinase PRKACA was demonstrated to directly phosphorylate TPI Ser58,thereby enhancing glycolysis.Therefore,our study identified many cancer-specific protein modifications spanned on glycolytic enzymes and unraveled the significance of TPI Ser58 phosphorylation in glycolysis and lung cancer development. |
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