Gluconeogenic Enzyme PCK1 Deficiency Promotes Chk2 O-glcnacylation And Hepatocellular Carcinoma Growth Upon Glucose Deprivation

Objective:Primary liver cancer is currently the sixth most commonly diagnosed cancer and the third leading cause of death globally.Primary liver cancers include hepatocellular carcinoma（HCC）（comprising 75%-85%of cases）and intrahepatic cholangiocarcinoma,as well as other rare types.The main risk factors for HCC are chronic infection with hepatitis B virus（HBV）or hepatitis C virus（HCV）,aflatoxin-contaminated foods,heavy alcohol intake,excess body weight,type 2 diabetes,and smoking.Because early-stage HCC is often asymptomatic,patients with HCC are often diagnosed at an intermediate or advanced stage and often miss opportunities for curative treatment（hepatic resection or liver transplantation）.For patients with advanced HCC,treatment options include local ablation,radiotherapy,chemotherapy,and molecularly targeted therapy.Among them,a multi-target tyrosine kinase inhibitor,sorafenib,is the earliest first-line drug for the treatment of advanced HCC.But due to drug resistance,sorafenib only extended patients’overall survival for an extra 2.8 months.Due to the limited efficacy of current chemotherapeutic drugs,there is an urgent need to deeply understand the mechanism of tumorigenesis and develop new targeted small-molecule chemotherapeutics.It is well known that persistent proliferation is one of the most important hallmarks of cancer.In solid tumors,these limitless replicative tumor cells excessively consume nutrients,and factors such as malformation of blood vessels often lead to nutrient limitations in the micorenvironment（eg,glucose deprivation）.Thus,tumor cells undergo metabolic remodeling to supply energy requirements and biomolecule synthesis for maintaining cell proliferation and survival.In 1920,Otto Warburg firstly showed that tumor cells tend to convert glucose into lactate even in conditions of sufficient oxygen.This phenomenon was termed as aerobic glycolysis or the Warburg effect,and is characterized by enhanced glucose uptake and lactate production,and although ATP production is less efficient,metabolic intermediates produced during this process can be used to synthesize biological macromolecules needed for tumor growth.Being the opposite pathway to glycolysis,gluconeogenesis is the process of synthesizing glucose or glycogen from non-sugar precursors（such as pyruvate,lactate,and glycerol,etc.）,mainly in the liver,and also plays an important regulatory role in tumor growth and metabolic reprogramming.Phosphoenolpyruvate carboxykinase 1（PCK1）is the first rate-limiting enzyme in gluconeogenesis,catalyzing oxaloacetate（OAA）to phosphoenolpyruvate（PEP）.It has been reported that PCK1 expression is low in HCC cells,and glucocorticoids are used to upregulate PCK1expression and inhibit cells proliferation.Our research group also found that under low glucose conditions,overexpressing PCK1 can activate the AMPK/p27 signaling pathway,block the transition from G1 to S phase of the cell cycle and inhibit HCC proliferation.However,PCK1overexpression in melanoma cells promotes the reverse flow of glucose metabolism,increasing the synthesis of more intermediate metabolites to meet their vigorous growth demands.PCK1 is highly expressed in colon cancer and can promote the growth of colon cancer.In conclusion,PCK1is closely related to tumorigenesis,but plays different roles in tumors drived from different tissue origins.PCK1 exhibits anti-tumor effects in gluconeogenic organs（liver and kidney）,but pro-tumor effects in tumors derived from non-gluconeogenic organs.However,it remains to be unclear that the aberrant expression and functional roles of PCK1 have the underlying mechanisms in multiple types of tumors.O-Glc NAcylation is an important post-translational modification of proteins,which regulates protein stability,enzymatic activity,protein-protein interactions and nuclear translocation,etc.,thereby regulating various biological effects.Unlike the phosphorylation,the O-Glc NAcylation consists of a unique OGT（O-Glc NAc transferase）or OGA（O-Glc NAcase）transferring（or removing）N-acetylglucosamine（Glc NAc）to（or from）the serine/threonine of the target protein.Uridine diphosphate-N-acetylglucosamine diphosphate（UDP-Glc NAc）is a donor substrate for protein O-Glc NAcylation,derived from the Hexosamine biosynthetic pathway（HBP）,and composed of glucose,glutamine,Acetyl-Co A and nucleotide.The O-Glc NAcylation levels mainly depends on the concentration of cellular UDP-Glc NAc,which is proposed to be a nutrient sensor.Glutamine-Fructose-6-Phosphate Amidotransferase 1（GFAT1）is the first rate-limiting enzyme and key regulatory node of the HBP pathway,which can convert fructose-6-phosphate（F6P）and Glutamine catalyzes glucosamine 6-phosphate and glutamate.It has been reported that activation of AMPK can phosphorylate serine 243 of GFAT1 protein,thereby inhibiting GFAT1 enzymatic activity,reducing HBP flux and protein O-Glc NAcylation levels.O-Glc NAcylation is significantly increased in various types of tumors and is closely related to the occurrence and development of tumors.It is regarded as one of the important new hallmarks of tumor cells.More cancer-related proteins,such as c-Myc,β-Catenin,and snail1,have been shown to be modified by O-Glc NAc and regulate tumorigenic signaling pathways through various mechanisms,including:cell proliferation,energy synthesis,angiogenesis,invasion and metastasis,cell death,oxidative and metabolic stress,etc.The study of O-Glc NAcylation will help us to understand the role of protein modification in tumorigenesis,explain how tumor cells sense nutritional status and coordinate gene expression,further deepen our understanding of tumorigenesis mechanism,and help us find the prognostic markers of tumor at early stage,and help develop new therapeutic and preventive strategies.Therefore,we proposed a hypothesis whether PCK1,a key gluconeogenesis enzyme,affects the proliferation of hepatoma cells by regulating the cellular HBP and protein O-Glc NAcyaltion under glucose-deficient conditions.Methods:1.To observe the effect of PCK1 knockout on the O-Glc NAcylation levels in HCC cells under low-glucose conditions.Firstly,PLC/PRF/5,SK-Hep1,Huh7 and MHCC-97H（HCC cell lines）accompanied knockouting or overexpressing PCK1 were treated with different concentrations of glucose medium for 12 hours to observe the effects on protein O-Glc NAcylation levels and cell proliferation.At the same time,we used a DEN/CCl₄-induced tumor model of Pck1-knockout mice and an orthotopic liver tumor model of nude mice to verify our results in vitro.q PCR and Western Blot analysis were performed to obsever the effects of PCK1 on the m RNA and protein expression levels of key enzymes OGT,OGA and GFAT1 which regulating HBP and O-Glc NAcylation.In addition,G309R（an enzymatically deficient mutant of PCK1）was overexpressed to observe the O-Glc NAcylation levels in HCC cells,confirming whether the enzymatic activity of PCK1 plays a key role in regulating the O-Glc NAcylation levels.2.To observe the levels of OAA and UDP-Glc NAc in PCK1-knockout cells.Untargeted metabolome and targeted metabolite profiling techniques were used to detect the changes of PCK1 on intermediate metabolites of TCA cycle,HBP（eg,UDP-Glc NAc）and UTP de novo synthesis under low glucose conditions.HCC cells were treated with OAA and aspartate（Asp）to observe the effects of UDP-Glc NAc synthesis,O-Glc NAcylation levels and cells proliferation.GOT2 is an important metabolic enzyme that catalyzes the generation of Asp from OAA.PCK1-Knockout cells were treated with AOA（the GOT2 inhibitor）to observe whether this process could be blocked,and confirme that knockouting PCK1 promoted the accumulation of OAA and Asp and the biosynthesis of UDP-Glc NAc.Labeling metabolites,such as m+4 OAA and m+3 UDP-Glc NAc,were further detected by isotope labeling of ₁₃C⁵-glutamine assay.3.To observe that overexpression of PCK1 activates the p-AMPK/p-GFAT signaling axis.Western Blot was use to dectect the p-AMPK/p-GFAT1signal axis and O-Glc NAcylation in HCC cells with knockouting or overexpressing PCK1.Intervention of p-AMPK/p-GFAT1 signaling axis by treating AMPK activator or knockdowning gene to determine whether PCK1 inhibits the activity of GFAT1,the levels of O-Glc NAcylation and the proliferation of HCC cells,which is dependent on p-AMPK.Targeted mass spectrometry was used to detect the level of UDP-Glc NAc in PCK1-knockout cells treated with inhibitor DON to intervene p-GFAT1.4.To observe the effect of PCK1 knockout on the levels of CHK2 O-Glc NAcylation.In order to further find out which important proteins undergo O-Glc NAcylation in PCK1-knockout cells to promote the proliferation of HCC cells,we had constructed an OGT fusion Flag-tagged recombinant plasmid.Target proteins were immunoprecipitated using anti-Flag in PCK1-knockout hepatoma cells overexpressing Flag-tagged OGT proteins treated with low glucose,and then detected by mass spectrometry（MS）.MS data were used to enrich signaling pathways and screen out the important regulatory of cell cycle,CHK2.Co-IP and IP experiments were used to verify whether CHK2 interacts with OGT and occurs O-Glc NAcylation.The important sites of CHK2 O-Glc NAcylation were analyzed by MS and IP experiments.Mutant plasmids of important site of CHK2 O-Glc NAcylation were constructed to verify the effect of important sites.5.To observe the functions of important sites of CHK2 O-Glc NAcylation.IP ubiquitination assay was used to observe the effect of PCK1 on CHK2ubiquitination.The stability of CHK2 modified by O-Glc NAc was observed by treatment with protein synthesis inhibitor（CHX）.Co-IP,oligomerization and molecular dynamics simulation assay were performed to dectect the dimers formation of O-Glc NAc-modified CHK2.HCC cells were treated with knockouting or overexpressing PCK1,or inhibitors ST or PUGNAc,or PEP or OAA to observe the activation of CHK2 and downstream p-CDK2/p-Rb pathway,and the proliferation of HCC.Restoration of wild-type CHK2 or site-mutated CHK2 in PCK1/CHK2double-knockout HCC cells was preformed to detect the downstream signaling axes and cell proliferation.At the same time,the orthotopic implantation model was constructed to verify the results in vitro.6.Analysis of the correlation between PCK1 and tumor size,O-Glc NAcylation and p-Rb expression in clinical human HCC samples.The expression levels of PCK1,protein O-Glc NAcylation and CHK2 O-Glc NAcylation were analyzed by clinical data and Western blot in 40 pairs of tumor tissues and adjacent tissue samples.These were also analyzed about the correlation of PCK1 expression with tumor size,O-Glc NAcylation,and p-Rb expression.7.To observe tumor growth after HBP intervention in DEN/CCl₄-induced HCC model of Pck1 liver-knockout（LKO）mouse.LKO mice were bred and screened,and then DEN combined with CCl₄ was used to induce hepatoma formation.After 8 months of induction,the number of tumor nodules,UDP-Glc NAc,protein O-Glc NAcylaton and CHK2 O-Glc NAcylation were detected in liver cancers of LKO mice,comparing with the wild type.To observe the effects of HBP-related inhibitors AOA and DON on the growth of HCC in mice.Results:1.PCK1 knockout strengthen the protein O-Glc NAcylation level in hepatoma cells under low glucose condition.Knockout or overexpression of PCK1 in PLC/PRF/5,SK-Hep1,Huh7 and MHCC-97H HCC cell lines were treated with different concentrations of glucose medium for 12 hours.It was found that under the condition of 5m M glucose,PCK1 knockout significantly promoted O-Glc NAcylation and cells proliferation,whereas PCK1 overexpression reduces these.DEN/CCl₄-induced HCC model of Pck1-knockout mice of and orthotopic tumor model of nude mice had verified the result in vitro.It was further found that PCK1 did not affect the m RNA level and protein expression of the key enzymes OGT,OGA and GFAT1 to regulate the HBP and protein O-Glc NAcylation.Interestingly,overexpression of G309R（catalytically inactive mutant of PCK1）did not change O-Glc NAcylation,indicating that the enzymatic activity of PCK1may play a key role in regulating the level of O-Glc NAcylation.2.PCK1 knockout promotes UDP-Glc NAc synthesis by increasing OAA accumulation.Using MS analysis of non-target metabolomics and target metabolism,it was found that PCK1 significantly down-regulated the TCA cycle,UDP-Glc NAc of the key metabolite of HBP,and UTP anabolism under low glucose conditions.OAA and Asp increased the synthesis of UDP-Glc NAc and the levels of O-Glc NAcylation,and promoted the proliferation of HCC cells.AOA can block this process,GOT2 catalyzes OAA to generate Asp,indicating that PCK1 knockout strengthen Asp and UDP-Glc NAc synthesis via OAA accumulation.These results were further verified by isotope tracer labeling of ₁₃C⁵-glutamine assay.3.PCK1 overexpression reduced UDP-Glc NAc synthesis and the levels of O-Glc NAcylation by activating the p-AMPK/p-GFAT1 signaling axis.Metformin（p-AMPK activator）activated p-GFAT1 and inhibit the levels of O-Glc NAcylation and the proliferation of hepatoma cell in PCK1-knockout cells.DON（GFAT1 inhibitor）significantly reduced the levels of UDP-Glc NAc and O-Glc NAcylation,and inhibited the cells proliferation in PCK1-knockout cells.4.PCK1 knockout enhanced CHK2 O-Glc NAcylation.Through IP-MS assay and pathway enrichment analysis,we found that CHK2 may be a potential target protein bound to OGT in PCK1-knockout cells.Co-IP and IP assays verified that CHK2 interacts with OGT,and occurs O-Glc NAcylation.Further assays found that PCK1 overexpression or ST（OGT inhibitor）decreased the levels of CHK2 O-Glc NAcylation,whereas PCK1-knockout,PUGNAc（OGA inhibitor）or metabolite OAA enhanced its modification.MS analysis and IP assay showed that Thr378 was an important site of CHK2 O-Glc NAcylation.5.O-Glc NAcylation increased the protein stability and dimerization of CHK2,activates the p-CDK2/p-Rb signaling axis and promotes the proliferation of hepatoma cell.IP and protein stability assays showed that O-Glc NAc-modified CHK2 at Thr378 reduced its ubiquitination degradation,and increased protein stability.Further assays found that O-Glc NAcylation promoted CHK2 dimer formation.Overexpression of PCK1 or ST reduced CHK2 expression,and inhibited the downstream p-CDK2/p-Rb pathway and HCC proliferation,whereas PCK1-knockout,PUGNAc,or metabolite OAA activated its downstream pathway.Restoring T378A in PCK1/CHK2 double knockout hepatoma cells could not activate the downstream p-CDK2/p-Rb pathway signaling axis and cell proliferation,indicating that the T378 site of CHK2 O-Glc NAcylation plays an important role.The orthotopic tumor model of nude mice was constructed to verify the results in vitro.6.We found that the expression of PCK1 is decreased,while the expression of protein O-Glc NAcylation,CHK2 O-Glc NAcylation and p-Rb is increased in 40 pairs of HCC tissues samples,comparing with those adjacent tissues.Correlation analysis found that PCK1 expression was negatively correlated with tumor size,O-Glc NAcylation modification,and p-Rb expression.7.In the DEN/CCl₄-induced tumor model,the number of tumor nodules in Pck1-knockout mice was significantly higher than that in wild type,and the levels of UDP-Glc NAc,all O-Glc NAcylation and CHK2 O-Glc NAcylation were significantly increased in LKO mice.Inhibitors AOA and DON can significantly inhibit HCC growth.Conclusion:These results reveal that the gluconeogenic enzyme PCK1deficiency enhances HBP and O-Glc NAcylation,and promotes HCC proliferation under low-glucose conditions.We demonstrate that PCK1deletion promotes UDP-Glc NAc synthesis and O-Glc NAcylation through OAA accumulation and activation GFAT1,and describe the importance of CHK2 O-Glc NAcylaiton in HCC development.Importantly,our study revealed a novel link between the gluconeogenic enzyme PCK1 and HBP-mediated O-Glc NAcylation,which provides a new therapeutic strategy for the treatment of HCC.