Inhibition Of Hepatocellular Carcinoma Progression By Phosphoenolpyruvate Carboxykinase 1 Through AMPK Activation

Objective:Hepatocellular carcinoma(HCC)is one of the most malignant solid tumors.For Non-communicable diseases in 2018,an estimated 9.55million deaths were caused by neoplasms.Deaths from HCC ranked the fourth of cancer mortality.With the spread and prevalence of hepatitis B virus(HBV)and hepatitis C virus(HCV),and increasing incidence of obesity and type 2 diabetes,the HCC incidence rate is increasing globally,which seriously threatens human health.Therefore,exploring the pathogenesis of HCC will provide a novel target in HCC diagnosis and treatment.Metabolic reprogramming is a hallmark of cancer.Tumor cells undergo aerobic glycolysis even in the presence of oxygen,also known as the Warburg effect,which is critical for meeting the metabolic requirements of rapid cancer cell growth and proliferation.The liver is the major site of gluconeogenesis during fasting.The conversion of oxaloacetate(OAA)and GTP to phosphoenolpyruvate(PEP)and GDP by phosphoenolpyruvate carboxykinase(PCK,also known as PEPCK)is the first rate-limiting step in hepatic gluconeogenesis.Two isoforms of PCK have been described:a cytoplasmic form(PCK1,PEPCK-C)and a mitochondrial isoform(PCK2,PEPCK-M).In the mammalian liver,PCK1 accounts for over 95%of the activity.PCK1 is upregulated in colon cancer and melanoma cancer,and is associated with increased glucose consumption to support anabolic metabolism and tumor growth.However,in HCC,the expression levels of PCK1 and other key gluconeogenic enzymes are strikingly suppressed,indicating that PCK1has contrasting roles in tumorigenesis in different organs.Nevertheless,the role of PCK1 in HCC progression remains incompletely understood.In this study,we explored the potential role of PCK1 in the suppression of cellular proliferation and tumor growth in HCC,and elucidated the molecular mechanism of PCK1 inhibiting HCC.Methods:1.The expression of PCK1 in HCC and non-tumor liver tissues were analyzed by bioinformatics methods in TCGA(The Cancer Genome Atlas)database.The correlation between PCK1 expression level and the prognosis of patients with HCC were analyzed.Furthermore,PCK1expression levels in human HCC and adjacent non-tumor tissues were detected by Western blot,qRT-PCR and immunohistochemistry(IHC).2.The effect of PCK1 on the proliferation and cell cycle of hepatoma cell lines was detected:Recombinant adenoviruses encoding PCK1 was obtained using AdEasy system,and the PCK1 gene knockout model was established in hepatoma cell lines with the use of Crispr/Cas9system.The effect of PCK1 on cell proliferation was detected by MTS and colony formation assay.The effect of PCK1 on hepatoma cell cycle was determined by flow cytometry.Then,the subcutaneous transplanted tumor model of hepatoma cell line overexpressing PCK1 was established in nude mice.3.The effect of PCK1 on RB/E2F pathway was detected:Through transcriptome sequencing and reviewing literatures,target genes relevant to RB/E2F signal pathway,such as RB1,E2F2 and CCNE1(Cyclin E1)were selected.To verify the expression of target genes at the RNA and protein levels,the levels of these genes were measured by qRT-PCR and Western blot.Then,the effect of AMPK activated by PCK1 on RB/E2F pathway was detected:First,the protein levels of AMPK and phosphor AMPK in PCK1-overexpressing(OE)and PCK1-knockout(KO)hepatoma cells under culture conditions with different time-point or different glucose concentrations were detected by Western blot.Then,upstream molecules of AMPK pathway were measured by Western blot and ATP detection kit.Last,AMPK expression was suppressed by infecting PCK1-OE hepatoma cells with shAMPK,and AMPK was activated by treatment PCK1-KO hepatoma cells with Metformin.Whether AMPK activation is involved in PCK1-induced hepatoma cells growth inhibition was verified using Western blot,flow cytometry and clone formation assay.4 The AMPK activation and downstream genes changes induced by PCK1 were verified in human and mice HCC samples.The correlation between PCK1 and pAMPK expression and the expression of downstream RB/E2F signaling pathway-related molecules were further detected in human and mice HCC tissues using database analysis,Western blot,qRT-PCR and IHC.5 The effects of PCK1 on Nrf2 signaling pathway and oxidative stress in hepatoma were detected.First,the regulation of PCK1 on the cellular energy environment and oxidative stress was determined by NADH/NADP~+kit and ROS kit.Second,the effects of PCK1 on Nrf2signaling pathway and downstream molecular TXNRD1 in hepatoma were measured by Western blot,transcriptome sequencing and qRT-PCR.Last,the regulation of PCK1 on ROS level and TXNRD1 expression was verified in mouse liver cancer tissue samples.To veified the influence of TXNRD1 on apoptosis-inducing effect of sorafenib(the first drug approved for advanced HCC treatment)in PCK1-knockout cells,PCK1-KO cells were treated with auranfin(an inhibitor of thioredoxin reductase)or sorafenib,alone or in combination.Results:1.The data from TCGA database shows that PCK1 level is lower in HCC tissue than in normal liver tissue.Furthermore,patients with low PCK1 expression have poor overall survival compared with patients with high PCK1 expression.In the 40 pairs of human liver tumor and non-tumor tissues,the level of PCK1 mRNA in the tumor tissues is lower than in the non-tumor tissues.The result of Western blot shows that in77.5%of the 40 pairs,PCK1 protein expression was found to decrease in HCC tissue,compared to in para-cancerous tissue,which is in accordance with the result of immunohistochemistry.2.PCK1 overexpression and knockout hepatoma cell lines were identified by Western blot.The effects of PCK1 on cell proliferation and cell cycle were detected by MTS,colony formation assay,flow cytometry analysis and fluorescent staining.The proliferation of hepatoma cell lines was inhibited,the G1/S transition was delayed by overexpression of PCK1.PCK1-knockout promoted the proliferation of PLC/PRF/5 cells,induced the G1/S transition.In vivo,the growth rate and weight of subcutaneous transplanted tumor of nude mice in PCK1-overespression group were lower than control group.3.The genes related to G1 cell cycle-arrest induction by PCK1 were analyzed by genome-wide RNA-Seq.Then,the RNA-Seq results were validated by qRT-PCR and Western blot.The data demonstrated that enhanced expression of PCK1 induced upregulation of p27 protein hepatoma cells,whereas pRB,pCDK2,and CyclinE1 were markedly downregulated in PCK1-overexpressing cells.In contrast,knockout of PCK1 resulted in increased levels of pRB,pCDK2,E2F2,and CyclinE1,whereas the expression of p27 was significantly decreased.Together,these findings suggested that PCK1 may dampen CDK/Rb/E2F signaling to inhibit G1/S transition in hepatoma cells.4.The protein levels of AMPK and phosphor AMPK in PCK1-OE and PCK1-KO hepatoma cells under different culture conditions were evaluated.The data indicated that after 5-day culture,pAMPK level was significantly upregulated in PCK1-OE cells.Knockout of PCK1 in cells inhibited pAMPK expression compared with parental cells.Furthermore,PCK1 overexpression markedly upregulated pAMPK expression,whereas knockout of PCK1 downregulated pAMPK expression in cells cultured in medium with 1 mM glucose.The results of Western blot assays suggested that the protein levels of LKB1 and CaMKK2 were not affected upon modulation of PCK1 expression.However,the ATP levels in PCK1-OE hepatoma cells were significantly reduced,whereas they were strongly increased in PCK1-KO PLC/PRF/5 cells.These results indicated that a decrease in ATP generation plays an essential role in PCK1-induced AMPK activation.The AMPK protein level was suppressed by infecting hepatoma cells with lentiviruses expressing short hairpin RNAs(shRNAs)targeting AMPK.Then,knockdown of AMPK prevented PCK1-induced upregulation of p27,downregulation of pRB,delay of G1/S transition,and inhibition of cell proliferation.The AMPK activator metformin partially offset PCK1 deficiency-mediated downregulation of p27,upregulation of pRB,and G1/S transition,and promoted hepatoma cell proliferation.5.In vivo,the growth rate and weight of subcutaneous transplanted tumor of nude mice in PCK1-overespression group were lower than control group,and the expression of pAMPK and p27 were increased in tumor tissues in PCK1-overexpression group.In murine orthotopic HCC model,PCK1-KO significantly promoted tumor formation,whereas metformin reverses tumor-promoting effects brought about by PCK1deficiency.In an independent cohort of 371 HCC samples from The Cancer Genome Atlas(TCGA)database,PCK1 expression was positively correlated with p27 expression,while it was negatively correlated with cyclinE1 expression.PCK1,pAMPK,and p27 protein levels were markedly lower in HCC than in non-cancerous tissues collected from 20patients.Furthermore,downregulation of PCK1 and pAMPK expression in HCC tissues was confirmed by IHC staining.6.PCK1 overexpression increased NADPH/NADP~+ratio while decreased cellular ROS level,which results in activation of Nrf2 and expression of TXNRD1.Furthermore,auranofin enhanced the sensitivity to sorafenib via inhibition of TXNRD1 in PCK1-knockout cells in vitro and in vivo.Conclusion:In the current study,we confirmed that the expression of PCK1 is lower in the HCC tissue than in the non-tumor tissue.Furthermore,both gain-and loss-of-function experiments demonstrated that PCK1 deficiency promotes hepatoma cell proliferation through inactivation of AMPK,suppression of p27 expression,and stimulation of the CDK/Rb/E2F pathway,thereby accelerating cell cycle transition from the G1 to S phase under glucose-starved conditions.Overexpression of PCK1 reduced the cellular ATP level and enhanced AMPK phosphorylation and p27 expression,but decreased Rb phosphorylation,leading to cell cycle arrest at G1.Knockdown of AMPK significantly reversed this inhibitory effect of PCK1 on hepatoma cell proliferation.In addition,the AMPK activator metformin remarkably suppressed the growth of PCK1-knockout PLC/PRF/5 cells and inhibited tumor growth in an orthotropic HCC mouse model.This study revealed that PCK1negativelyregulatescell-cycleprogression andhepatomacell proliferation via the AMPK/p27 axis and suggests a potential therapeutic and protective effect of metformin on HCC.