AMPK Inhibits The Occurrence Of Hepatocellular Carcinoma Through Autophagy Induction

Liver cirrhosis is the end stage of various chronic liver diseases,characterized by diffuse liver fibrosis,regenerative nodules and pseudolobule in pathology.The main causes of death in liver cirrhosis include gastrointestinal bleeding,hepatic encephalopathy and liver cancer.With the development of endoscopic ligation,devascularization,portosystemic shunt and antiviral therapy,the life expectancy of patients with cirrhosis is significantly prolonged.But the number of patients who develop liver cancer has increased surprisingly.Primary liver cancer,specifically hepatocellular carcinoma(HCC),is still the third cause of cancer death in China.Although the treatment of liver cancer is developing rapidly and the curative effect has been improved,the prognosis is still poor.It is of vital importance to identify novel effective molecular target to predict and prevent the occurrence of liver cancer.AMP-activated protein kinase(AMPK),a highly conserved heterotrimeric serine/threonine kinase,is a hetero trimer consisting of α subunit,β subunit and γ subunit.In response to certain stressful conditions,such as energy deprivation,AMPK is activated by phosphorylation at Thr172 in α subunit.Once activated,AMPK regulates various molecules and signaling pathways to modulate adaptive changes and maintain metabolic homeostasis.Recently,emerging evidence has indicated a potential role of AMPK signaling in tumor initiation and progression.AMPK activation not only downregulated mTOR but also suppressed the excess aerobic glycolysis(Warburg effect,characteristic of most transformed cells)and genetic ablation of the a1 catalytic subunit of AMPK accelerated Myc-induced lymphomagenesis.TAK 1 is the upstream activation factor of AMPK.Knockout TAK 1 resulted in spontaneous liver fibrosis and liver cancer in mice.The cirrhotic patients taking long-term metformin therapy,an activator of AMPK,showed significantly lower incidence of liver cancer.This line of investigation suggested a tumor suppressor role for AMPK activation.AMPK is also a promising target for treating fibrotic diseases.AMPK activation reduced thioacetamide induced liver fibrosis in mice and inhibited the activation of cultured hepatic stellate cells and macrophages.These data suggested that AMPK activation was an important factor regulating both fibrosis and cancer.Therefore,it is intriguing to understand the phosphorylation status of AMPK in cirrhotic livers and its role in the process from cirrhosis to HCC.Part Ⅰ The predictive effect of activated AMPK on the occurrence of hepatocellular carcinomaObjective: Cirrhosis is a milieu that is conducive to development of hepatocellular carcinoma,this study aims to explore whether activated AMPK,p-AMPK(Thr172),can be used as a biomarker for predicting the occurrence of hepatocellular carcinoma in patients with cirrhosis.Methods: We screened the records patients who underwent Hassab procedure and liver biopsy for treating liver cirrhosis between January 1999 and June 2011 in the Second Affiliated Hospital of Chongqing Medical University.All patients were followed up until HCC detection or the end of the study.The phosphorylation status of AMPK(Thr172)was determined by immunostaining in tissue microarrays.Survival analysis was performed using the Fine and Gray model by accounting for the competing risk of death.An independent external cohort of West China Hospital was adopted to validate the predictive significance of p-AMPK.Results: A total of 426 patients with liver cirrhosis in our hospital were included in the analysis.The median follow-up time was 87 months.HCC occurrence probability at 1/3/5/10 years after Hassab procedure was 3.1/9.6/13.8/30.6% in patients with p-AMPK low expression and 0/0.3/0.3/8% in patients with p-AMPK high expression,respectively.HCC occurrence risk was significantly higher in patients with p-AMPK low expression in univariable analysis(HR,6.25;95% CI: 3.36–11.60;P<0.001)and multivariable analysis(HR,6.0;95% CI: 3.24–11.10;P<0.001).The independent cohort of West China Hospital(Sichuan University)contained 132 patients with cirrhosis.Similarly,patients with low p-AMPK expression were found to have significantly higher risk of hepatocellular carcinoma than patients with high p-AMPK expression(HR,8.49;95% CI: 2.93–24.6;P<0.001).Low expression of p-AMPK was an independent risk factor for hepatocarcinogenesis in patients with cirrhosis(HR,13.4;95% CI:5.14–34.9;P< 0.001).Conclusion: P-AMPK could be used as a biomarker to predict the occurrence of hepatocellular carcinoma in patients with liver cirrhosis.Part Ⅱ Activation of AMPK can inhibit the occurrence of hepatocellular carcinoma induced by DEN in miceObjective: The first part of the study found that low expression of p-AMPK is an independent risk factor for hepatocellular carcinoma in patients with cirrhosis,but whether AMPK activation can be used as a target for prevention of hepatocellular carcinoma is not yet clear,this section intends to investigate whether activation of AMPK can inhibit hepatocarcinogenesis induced by DEN in mice.Methods: Hepatocarcinogenesis mice model was induced by DEN combined with CCl4.Liver biopsy was performed in mice at 8 weeks in the model process.The expression of p-AMPK was detected by immunohistochemical staining.According to the expression of p-AMPK,17 mice were p-AMPK high expression and 20 mice were p-AMPK low expression.In mice with p-AMPK high expression,Dor(n=8)or PBS(n = 9)were used to inhibit AMPK activation or as control.In p-AMPK low-expression group,Met(n=7)or AICAR(n=6)was used to activate AMPK,PBS(n=7)was used as control.All mice were killed at 32 weeks.Liver index,visible tumor number and total tumor diameter was compared.The levels of alanine aminotransferase(ALT),total bilirubin(TBIL),albumin(ALB)and triglycerides were detected by an automatic biochemical analyzer.Liver collagen deposition was detected by Sirius red staining.The expression level of α-SMA,F4-80 and P62 was detected by immunohistochemical staining.The expression level of ACC 、 RAGE,DLK-1,CD44,LC3 B and P62 was tested by WB.Autophagic corpuscles were counted by transmission electron microscopy.Results: When treated with PBS,mice with low p-AMPK expression showed higher liver index,visible tumor number and total tumor diameter.Futher more,in p-AMPK high-expression mice,AMPK inhibition by Dor lead to a significant increase in liver index,visible tumor number and total tumor diameter.By contrast,in p-AMPK low-expression mice,a significant decrease in tumor number and total tumor diameter was observed in the Met or AICAR treated mice compared to PBS.HSC activation,fibrosis and myeloid cell were drastically increased when AMPK inhibited by Dor in p-AMPK high expression mice.Also,AMPK activation by either Met or AICAR resulted in reduced HSC activation,fibrosis and myeloid cells.In addition,hepatic serologic injury indicators were negatively related to p-AMPK activation.It was reported that diminished hepatic lipogenesis or hepatic progenitor cell activation was involved in the protective process of Met on hepatocarcinogenesis.The protein levels of ACC,the rate limiting enzyme involved in fatty acid synthesis,or serum triglycerides levels were not different significantly in neither different p-AMPK expression level mice treated with PBS nor different p-AMPK modulators.We also did not observe any significant differences in the expression of RAGE,DLK-1 and CD44(marker of hepatic progenitor cell activation)between the different groups.Taken together,our results suggested that mechanisms other than diminished hepatic lipogenesis or hepatic progenitor cell activation were also responsible for the decreased incidence of HCC.AMPK activation significantly increased LC3 B II/I ratio and decreased p62 expression when analyzed by Western blotting.Correspondingly,analysis of autophagy using transmission electron microscope showed increased numbers of autophagosomes in the liver of high AMPK activation group.Conclusion: AMPK activation can inhibit the occurrence of hepatocellular carcinoma in mice,and its mechanism may be related to the enhancement of autophagy.Part Ⅲ Activated AMPK can inhibit the occurrence of hepatocellular carcinoma by regulating autophagyObjective: To explore the underlying mechanism of AMPK to inhibit the occurrence of hepatocellular carcinoma.Methods: Hepatocarcinogenesis mice model was induced by DEN combined with CCl4.Liver biopsy was performed in mice at 8 weeks in the model process.The expression of p-AMPK was detected by immunohistochemical staining.Mice with low expression of p-AMPK were selected as the research object.The mice were divided into 3 groups: blank control(N=8),Met group(N=8)or Met+CQ group(N=8).All mice were killed at 32 weeks.Liver index,visible tumor number and total tumor diameter was compared.The levels of ALT,TBIL and ALB were detected by an automatic biochemical analyzer.Liver collagen deposition was detected by sirius red staining.The expression level of α-SMA,F4-80 and P62 was detected by immunohistochemical staining.The expression level of LC3 B and P62 was tested by WB.Autophagic corpuscles were counted by transmission electron microscopy.Results: CQ decreased numbers of autophagosomes in the liver compared with Met alone and promoted accumulation of p62 without suppressed LC3B-II production.A significant increase in tumor number and tumor diameter was observed in Met+CQ group than Met group.However,there was no significant difference between the Met+CQ group and the control group.The HSC activation,liver fibrosis and myeloid cell reduction by metformin were also blocked by CQ.Conclusion: Notably,CQ removed the Met mediated protective action against hepatic carcinogenesis.These data suggested that AMPK activation inhibits hepatic carcinogenesis in vivo,and the mechanism may be related to the induction of autophagy.