Overexpressed Bmi1 Induced By High Glucose Through Histone H4 Acetylation Promotes Aerobic Glycolysis And Immune Suppression In Human Pancreatic Cancer

ObjectivePancreatic Cancer(PC)is intricately associated with diabetes mellitus(DM).Around 80%of pancreatic cancer patients have glucose intolerance or frank DM.More importantly,patients with PC with DM have increased tumor size and lower overall survival than those without DM.However,the role of hyperglycemia in the progression of PC has not been fully understood.In this study,we detected the effects of hyperglycemic microenvironment on glycolysis of pancreatic cancer cells.Through examining intracellular acetyl-Co A levels,ATP-citrate lyase(ACLY)expression,histone acetylation levels,Bmi1 expression,UPF1 expression,Hexokinase 2(HK2)expression of pancreatic cancer cells after cultured in medium with different glucose concentrations,we explored relevant underlying mechanisms by which high glucose promotes the progression of pancreatic cancer.MethodsELISA was adopted to detect the intracellular acetyl-Co A levels of pancreatic cancer cells cultured in medium with different glucose concentrations.Western blotting experiments were conducted to determined ACLY expression,histone acetylation levels,Bmi1 expression,UPF1 expression and HK2 expression in pancreatic cancer cells.Polymerase chain reaction(PCR)experiments were performed to test Bmi1,UPF1,HK2 m RNA levels.Chromatin immunoprecipitation(Ch IP)was performed to detect the histone H4 acetylation levels in the Bmi1 gene promoter region.Lactate production was determined by Lactic Acid assay kit from Nanjing Jiancheng.Glucose uptake was determined by flow cytometry.Glucose uptake potency of pancreatic cancer cells was detected by flow cytometry.RNA immunoprecipitation experiment was performed to detect the relative enrichment levels of HK2 m RNA by anti-UPF1 antibody.The diabetic pancreatic cancer mice model was built to detect the role of Bmi1 played in the progression of pancreatic cancer with diabetes.Flow cytometry was performed to detect the activity of cytotoxic T-cells.ELISA was adopted to examine the levels of IL-2 secreted by Jurkat T cells.ResultsHere we demonstrated that with the increasing of glucose concentration in the medium,intracellular acetyl-Co A levels of pancreatic cancer cells increased in a dose-dependent way.Moreover,ACLY protein expression,histone H3、H4 acetylation levels and Bmi1 protein expression were upregulated by high glucose treatment.Besides,m RNA levels of ALCY and Bmi1 gene were also upregulated by high glucose treatment.ACLY silencing could suppress the effects of high glucose treatment on upregulating histone H3、H4 acetylation levels and Bmi1 expression.Consistently,acetate supplementation,which could effectively upregulate the intracellular acetyl-Co A levels,enhanced histone H3、H4 acetylation levels and Bmi1 expression.Application of Valproic acid(VPA)or C646 could upregulate or downregulate histone H3、H4acetylation levels and Bmi1 expression respectively.Furthermore,KAT2A silencing in pancreatic cancer cells could effectively downregulate histone H3 acetylation levels,but not Bmi1 expression.While KAT5 silencing could downregulate both histone H4acetylation levels and Bmi1 expression.Ch IP experiment showed that the histone H4acetylation levels were significantly higher at the promotor region of Bmi1 gene of high glucose-treated group compared with that of normal glucose-treated group.Animal experiment showed that Bmi1 silencing could inhibit the tumorigenesis of pancreatic cancer cells induced by diabetes.After culturing in medium with different glucose concentrations for 48h,we found that lactate production and glucose uptake of pancreatic cancer cells were increased with the increasing of glucose concentrations in the medium.Western blotting experiments and PCR experiments demonstrated that Hexokinase 2(HK2)expression of pancreatic cancer cells were induced by high glucose treatment.Moreover,Bmi1knock-down could inhibit the upregulation of HK2 expression,lactate production and glucose uptake induced by high glucose.Inhibition of HK2 activity could effectively suppress lactate production of pancreatic cancer cells.Besides,western blotting experiments showed that overexpression of Bmi1 could induce the expression of UPF1and HK2.Knockdown of UPF1 could inhibit the expression of HK2 induced by overexpression of Bmi1.RNA immunoprecipitation experiment demonstrated that the relative enrichment levels of HK2 m RNA was increased in the anti-UPF1 group than that in the anti-normal group.We co-cultured SW1900 cells and CFPAC-1 cells with activated primary human T-cells in medium with different glucose concentrations for 48h,the results showed the population of interferon gamma(IFNγ)-positive cytotoxic T-cells was lower in the high glucose-treated groups compared with that in the normal glucose-treated groups.Moreover,the concentration of IL-2 secreted by Jurkat T cells increased with the increasing of glucose concentrations in the medium.Yet,when co-cultured with pancreatic cancer cells,secreted IL-2 levels of high glucose-treated groups were decreased.Bmi1 silencing could effectively suppress the inhibitive effects of high glucose on CD8~+T cells and Jurkat T cells when co-cultured with pancreatic cancer cells.ConclusionsHere,we demonstrated that in PC cells high glucose effectively promotes aerobic glycolysis via up-regulating Bmi1-UPF1-HK2 axis,thereby increasing lactate production which contributes to inhibition of cytotoxic T cell activity.Further mechanistic studies revealed that high glucose promotes the expression of Bmi1through elevating the cellular acetyl-Co A levels and histone acetylation levels.Gain-and loss-of-function studies demonstrated that Bmi1 up-regulates the expression of UPF1 which enhanced the stability of HK2 m RNA,and thus facilitated the expression of HK2.Taken together,the findings of this study improve understanding of the relationship between PC and DM and the underlying mechanisms would be the key to identify novel and potential therapeutic target for PC.