HDAC6 Mediated TALDO1 Deacetylation Remodels Glucose Metabolism To Promote Proliferation And Metastasis Of Nasopharyngeal Carcinoma

Background and Purposes: Nasopharyngeal carcinoma(NPC)is a common malignant tumor in southern China,and its distant metastasis is the most challenging problem in clinical treatment.Metabolic reprogramming,a key feature of cancer,is involved in the occurrence and development of tumors,and acetylation regulation of metabolic enzymes plays an important role in tumor metabolic reprogramming.Transaldolase(TALDO1),a non-oxidative enzyme in the pentose phosphate pathway(PPP)of glucose metabolism,is dysregulated in various tumors,but its function and regulatory mechanism in NPC are still unclear.Studies have shown that most cancer suppressors in malignant tumors are often silenced by deacetylation modification.Therefore,exploring the regulatory mechanism of TALDO1 by deacetylation and its role in NPC proliferation and metastasis from this perspective will provide new insights into the molecular mechanism and intervention targets of NPC malignancy progression.Methods:(1)TMT acetylation quantitation proteomics identified proteins with deacetylation modification in NPC cells,KEGG pathway analysis determined the pathways of non-histone proteins enriched in deacetylation,immunoprecipitation(IP)was used to detect the acetylation of TALDO1 exogenous and endogenous,and Western blot was used to detect the protein level of TALDO1 in NPC cells HK1,SUNE1,and HONE1 after treatment with the broad-spectrum deacetylase inhibitor LBH589/PXD101.Western blot was used to detect the expression levels of TALDO1 in nasopharyngeal epithelial cell lines NP69/NP460 and NPC cells,immunohistochemistry(IHC)was used to detect the expression of TALDO1 in NPC tissues,adjacent tissues,and tissues with metastasis,Kaplan-Meier survival curve analysis was used to analyze the correlation between TALDO1 expression and NPC patient prognosis,and ELISA was used to detect the level of TALDO1 in the serum of NPC patients with and without distant metastasis.(2)CCK-8 assay,colony formation assay,scratch assay,Transwell assay,and flow cytometry were used to investigate the biological functions of TALDO1 in NPC cells.Nude mice transplantation and metastasis models were used to investigate the roles of TALDO1 in NPC tumorigenesis and metastasis.(3)IP,Western blot,and immunofluorescence were used to analyze the acetylation and deacetylation enzymes that regulate TALDO1,and IHC was used to detect the correlation between HDAC6 and TALDO1 expression in NPC tissue samples.(4)Using sh RNA or the HDAC6-specific inhibitor CAY10603 to downregulate HDAC6,CCK-8,colony formation,and Transwell assays were used to investigate the biological functions of HDAC6 in NPC cells,and the nude mouse transplantation and metastasis models were used to investigate the roles of HDAC6 in NPC tumorigenesis and metastasis.CCK-8,colony formation,and Transwell assays were also used to investigate the role of TALDO1 in the promotion of NPC cell proliferation,migration,and invasion mediated by HDAC6.(5)IP and Western blot were used to detect the correlation between K7 site deacetylation and TALDO1 protein level.Cycloheximide(CHX)and ubiquitination assays were used to detect the correlation between TALDO1 acetylation and ubiquitination levels.IP was used to detect the interaction between TALDO1 and E3 ubiquitin ligase SMURF1 and the effects of K7 site deacetylation on the binding of TALDO1 and SMURF1,as well as the effect on SMURF1-mediated K48-linked and K63-linked ubiquitination of TALDO1.(6)Targeted metabolomics was used to identify the role of TALDO1 in energy metabolism in NPC cells.Glucose uptake experiment,lactate production experiment,extracellular acidification rate(ECAR)experiment,and Western blot experiment was used to detect the effect of TALDO1 on glycolytic capacity in NPC cells.Use 2-DG combined with CCK-8,colony formation,and Transwell experiment to detect the role of glycolysis in TALDO1-mediated NPC cell proliferation and migration.(7)Nucleuscytoplasm separation experiment was performed to detect the effect of K7 site deacetylation on TALDO1 nuclear translocation.IP experiment was used to detect the effect of K7 site deacetylation on the interaction between TALDO1 and BRCA1.q RT-PCR and Western blot experiments was used to detect the role of BRCA1 in TALDO1-mediated metabolic enzyme expression inhibition.Use glucose uptake experiment,lactate production experiment to detect the role of BRCA1 in TALDO1-mediated glycolysis inhibition,and use CCK-8 and Transwell experiments to analyze the role of BRCA1 in TALDO1-mediated NPC cell proliferation and migration.Results: 1.TMT acetylation quantitative proteomic data analysis showed that the deacetylated non-histone protein clusters in NPC cells were clustered in the cell metabolic pathway,and TALDO1 was the most significantly differentially expressed metabolic enzyme among them;Western blot results showed that TALDO1 expression was significantly higher in the nasopharyngeal epithelial cell lines NP69 and NP460 than in NPC cells;tissue microarray data analysis showed that TALDO1 expression was significantly lower in NPC tissues than in paired cancer adjacent tissues,and low TALDO1 expression was positively associated with poor prognosis in NPC patients;moreover,TALDO1 expression was lower in NPC tissues with distant metastases than in those without metastases;the content of TALDO1 in the serum of NPC patients with distant metastases was significantly lower than that in patients without metastases.2.Cellular and animal experiments showed that upregulation of TALDO1 expression inhibited NPC proliferation and metastasis.3.HDAC6 regulates TALDO1 deacetylation.Knockdown of HDAC6 upregulated TALDO1 protein levels;the acetyltransferase p300 bound to and regulated the acetylation of TALDO1,and overexpression of p300 upregulated TALDO1 protein levels.4.HDAC6 was highly expressed in NPC,and cellular and animal experiments showed that downregulation of HDAC6 inhibited NPC proliferation and metastasis;knockdown of TALDO1 on the basis of downregulating HDAC6 restored the inhibition of NPC cell proliferation and migration caused by downregulation of HDAC6.5.TMT acetylation quantitative proteomic data and acetylation IP experiments showed that TALDO1 was acetylated only at the K7 site;after simulating deacetylation(K7R),the protein level of TALDO1 was significantly lower than that of the wild type(WT);CHX experiment results showed that downregulation of HDAC6 increased the stability of TALDO1 protein;ubiquitination experiment results showed that downregulation of HDAC6 or overexpression of p300 significantly increased the ubiquitination level of TALDO1,and the ubiquitination level of the K7 R mutant was significantly lower than that of the WT group;IP experiments showed that compared with WT,the K7 R mutation had a stronger interaction with SMURF1,and the K7 R mutation promoted SMURF1-mediated K48-linked ubiquitination of TALDO1 and inhibited K63-linked ubiquitination of TALDO1.6.Metabolomic data showed that overexpression of TALDO1 inhibited NPC cell glycolysis and the TCA cycle;TALDO1 inhibited glucose uptake and lactate production in NPC cells,and downregulation of TALDO1 promoted NPC cell proliferation and migration.Adding the glycolysis inhibitor 2-DG significantly inhibited this phenomenon on the basis of downregulating TALDO1 expression;Western blot results showed that downregulation of TALDO1 increased the expression of key enzymes in the glycolysis process,and adding 2-DG partially restored the expression of metabolic enzymes.7.The K7 R mutation inhibited the nuclear translocation of TALDO1 and its interaction with BRCA1.ECAR experiment results showed that the glucose uptake and lactate production capacity of K7 R mutant cells were significantly higher than those of the WT group;q RT-PCR and Western blot results showed that overexpression of TALDO1 inhibited the expression of key enzymes in the glycolysis process,and after knocking down BRCA1 on this basis,this phenomenon was reversed;glucose uptake and lactate production experiments showed that deacetylation at the K7 site inhibited the TALDO1/BRCA1 axis and promoted glycolysis;CCK-8 and Transwell results showed that TALDO1 inhibited NPC cell proliferation and migration through BRCA1.Conclusion: In this study,we clarified that the deacetylase HDAC6 mediated TALDO1 K7 site deacetylation,promoted the interaction of E3 ligase SMURF1 with TALDO1,which enhanced the degradation of K48-linked ubiquitination and inhibited K63-linked ubiquitination,thereby inhibiting TALDO1 stability,and K7 site deacetylation inhibited TALDO1 nuclear translocation,inhibited its binding to BRCA1 The K7 site deacetylation inhibited TALDO1 nuclear translocation,inhibited its binding to BRCA1,promoted c-Myc transcriptional activation of glycolytic metabolic enzymes HK2,LDHA and PDK1 expression,enhanced cellular glycolysis,and thus promoted NPC proliferation and metastasis.This study will promote a new perspective on the molecular mechanism of NPC development and provide new markers and intervention strategies for the clinical treatment of NPC.