Metformin Promotes 2-Deoxy-2-[¹⁸F]Fluoro-D-Glucose Uptake In Hepatocellular Carcinoma Cells Through FoxO1-Mediated Downregulation Of Glucose-6-Phosphatase

Background:Hepatocellular carcinoma（HCC）is the second highest cause of death from cancer worldwide,ranking as the third biggest cause of cancer mortality in China.The 5-year survival rates for early-stage HCC patients average around 70 %;however,the median survival period for advanced HCC patients is normally less than a year.Hence,early diagnosis is crucial to improve the treatment of patients with HCC and increase their survival chances.Currently,the National Comprehensive Cancer Network（NCCN） guidelines recommend the use of four-phase X-ray computed tomography（CT）and dynamic magnetic resonance imaging（MRI）for the diagnosis of HCC.However,the low sensitivity of these techniques（62.0–78.4 %）,especially for the diagnosis of small tumors（≤ 2 cm3）,means that these approaches are not suitable as early diagnostic techniques for HCC.Positron emission tomography（PET）using 2-deoxy-2-[¹⁸F]fluoro-D-glucose(¹⁸F-FDG)as a tracer in combination with CT imaging is recognized as being a superior approach to traditional anatomical imaging for the early diagnosis and location of malignant tumors.However,the accumulation levels of ¹⁸F-FDG in HCC cells are often low,especially in differentiated HCC cells,which means that ¹⁸F-FDG PET/CT imaging is not currently recommended as a diagnostic technique for HCC tumors.¹⁸F-FDG is transported into cells by glucose transporter 1（GLUT1）,where it is then phosphorylated by hexokinase 2（HK2）to afford charged ¹⁸F-FDG-6-phosphate that accumulates within the cell.HCC tumors are known to overexpress the enzyme glucose 6-phosphatase（G6Pase）,which can hydrolyze ¹⁸F-FDG-6-phosphate to ¹⁸F-FDG that may then be transported back out of cancer cells by glucose transporters and P-glycoproteins.Therefore,selective inhibition of G6Pase activity could potentially result in higher concentrations of ¹⁸F-FDG accumulating in HCC cells,which would then allow PET/CT imaging to be used for the diagnosis of HCC tumors.Metformin（Met）is used for the treatment of type 2 diabetes,where it is known to exert a range of pharmacologic effects on cellular glucose metabolism in both healthy and cancer cells.Met is thought to effectively downregulate G6Pase by reducing its m RNA expression levels.Forkhead box protein O1（FoxO1）is a transcription factor that plays an important role in regulating glucose metabolism and tumor progression.In its non-phosphorylated state,FoxO1 increases the rate of production of hepatic glucose by triggering an increase in the transcription levels of G6Pase.Studies have shown that inhibition of FoxO1 activity can reduce the expression levels of G6Pase in the liver,with Met also having been reported to have a strong inhibitory effect on FoxO1 expression in macrophages.Consequently,it was decided to investigate whether the treatment of HCC cells with Met would result in an increase in their overall ¹⁸F-FDG uptake.Objective:Improve the status of ¹⁸F-FDG uptake in hepatocellular carcinoma cells and enhance the value of PET in the early diagnosis of hepatocellular carcinoma,and provide a new alternative to fully reveal the role of metformin and FoxO1 in the metabolism of liver cancer.Methods:1.The effects of metformin and FoxO1-siRNA on FoxO1 and G6Pase and the glucose transporter proteins GLUT1 and HK2 in hepatocellular carcinoma SMMC-7721cells were detected by quantitative RT-PCR and Western blotting.2.After verifying the validity of FoxO1-siRNA,Inducible lentivirus expressing FoxO1 siRNA（FoxO1-KD）and lentivirus overexpressing FoxO1（FoxO1-OE）under the control of a doxycycline-dependent Tet On/Tet Off switch were constructed by Genechem（Shanghai,China）.3.The effect of metformin and FoxO1-siRNA on the uptake of hepatoma cells SMMC-7721 and HepG2 ¹⁸F-FDG in vitro was examined.4.Establishment of SMMC-7721 xenograft model of FoxO1 knockout and FoxO1overexpression.Intraperitoneal injection of metformin（250 mg/kg/day）and induction of FoxO1 knockdown and overexpression of FoxO1 by gavage of doxycycline（0.2 mL（2mg/mL））demonstrated that metformin increased ¹⁸F-FDG uptake by hepatoma cells via FoxO1.Tumor uptake was measured by ROI method and quantified by%ID/g.5.The effect of metformin on the expression of FoxO1,G6Pase,GLUT1 and HK2in hepatocellular carcinoma was confirmed by immunohistochemistry and Western blotting.Results:1.Studies on the treatment of FoxO1 with Met were carried out in vitro,with treatment of FoxO1 with FoxO1-knockdown siRNA being used as a control.The relative expression levels of FoxO1 were significantly reduced in both the 10 mM Met group（1.000±0.058 vs.0.561±0.052,P=0.0048）and the FoxO1-siRNA group（1.000±0.058vs.0.495±0.0232,P=0.0031）.However,the relative transcription levels of FoxO1 were not significantly altered in every group that was treated with Met（F=0.5684,P=0.6512）,suggesting that Met reduces FoxO1 expression levels via a post-transcriptional regulatory mechanism.To further understand this mechanism,the phosphorylation levels of both FoxO1 and the upstream AMPK-Akt-4EBP1 pathway that controls FoxO1posttranscriptional expression levels were determined.The expression ratio of phosphorylated FoxO1 to total FoxO1 was unchanged in the treatment with 10 mM Met（F=0.1116,P=0.9756）.However,the use of 10 mM Met did result in increased levels of phosphorylated AMPK（1.000±0.0833 vs.1.424±0.3789,P=0.0097）,while the levels of phosphorylated Akt and phosphorylated 4EBP1 were decreased（1.000±0.1217 vs.0.3758±0.0372,P=0.0083;1.000±0.0752 vs.0.364±0.0347,P=0.0016）.This indicates that Met has an overall inhibitory effect on the AMPK-Akt-4EBP1 pathway.2.The effect of Met and FoxO1 on the expression of G6Pase and FoxO1-knockdown（FoxO1-KD）wasinvestigatedusingsiRNAand FoxO1-overexpressed（FoxO1-OE）using a Dox inducible Tet on/Tet off switch system.The relative transcription levels of G6Pase were significantly reduced on treatment with Met（0 mM 1.000±0.0893 vs.1 mM Met 0.5271±0.048,P=0.0096;5 mM Met 0.053±0.0055,P=0.0005;10 mM Met 0.0448±0.0068,P=0.0004）and FoxO1-KD（0 mM 1.000±0.0893 vs.0.0287±0.0037,P=0.0004）.The relative expression level of G6Pase was also downregulated by treatment with Met（0 mM 1.000±0.0921 vs.1 mM Met1.008±0.1101,P=0.9602;5 mM Met 0.6655±0.0652,P=0.0413;10 mM Met0.5621±0.0974,P=0.0309）and FoxO1-KD（0 mM 1.000±0.0921 vs.0.4247±0.0841,P=0.0099）.FoxO1-OE manipulation using Dox increased the expression of FoxO1,resulting in increased transcription and expression of G6Pase.Additionally,the combined intervention of Met and Dox partially rescued the positive effect of FoxO1-OE manipulation on FoxO1（2.656±0.0959 vs.3.548±0.1700,P=0.0038）and G6Pase expression（1.323±0.0492 vs.2.177±0.1348,P=0.0011）.This demonstrates that both Met and FoxO1 have a regulatory effect on the transcriptional and expression levels of G6Pase.Details of the transcriptional verification of the FoxO1-OE manipulation system.Meanwhile,metformin had no significant effect on the expression of GLUT1 and HK2（F=0.8935 P=0.5027,N=3;F=0.3387 P=0.8459,N=3）.3.In vitro cell uptake assay and in vivo microPET/CT imaging studies were carried out to determine whether Met-induced alteration of G6Pase levels resulted in an increase in the uptake of ¹⁸F-FDG in SMMC-7721 cells.Treatment with Met resulted in a dose-dependent increase in the uptake of ¹⁸F-FDG into HCC cells in vitro.The ¹⁸F-FDG uptake ratio in SMMC-7721 cells was found to be 3.5-fold higher in the 10 mM Met group than in the untreated control group（1.474±0.0946 vs.0.421±0.0487,P=0.0006）.Furthermore,the ¹⁸F-FDG uptake ratio in the FoxO1-siRNA group was also found to be significantly higher than in the control group（1.244±0.0630 vs.0.421±0.0487,P=0.0005）.4.Met treatment of mice,bearing inducible FoxO1-KD or FoxO1-OE HCC tumors,also resulted in elevated ¹⁸F-FDG uptake levels.Our result reveals that the SUV_Max value and T/NT ratios observed during NC imaging of Met-treated mice were significantly higher in both FoxO1-KD mice(SUV_Max value 1.907±0.139 vs.0.443±0.022,P=0.0005;T/NT ratio 3.678±0.131 vs.1.037±0.0185,P<0.0001)and FoxO1-OE mice(SUV_Maxax value 1.764±0.057 vs.0.407±0.018,P<0.0001;T/NT ratio 3.769±0.098 vs.1.019±0.018,P<0.0001).These findings confirm the in vitro results,demonstrating that the treatment with Met has a positive effect on the incorporation of ¹⁸F-FDG into HCC cells in vivo.Dox-induced knockout of FoxO1 in FoxO1-KD mice was also shown to result in a significant increase in the tumor uptake of ¹⁸F-FDG(SUV_Maxax value 1.013±0.055 vs.0.443±0.022,P=0.0006;T/NT ratio:3.065±0.049 vs.1.037±0.0185,P<0.0001).However,simultaneous overexpression of FoxO1 in FoxO1-OE mice did not result in Met having any positive effects on the uptake of ¹⁸F-FDG into tumor cells(SUV_Max value0.533±0.064 vs.1.764±0.057,P=0.0001;T/NT ratio 1.287±0.099 vs.3.769±0.098,P<0.0001).Taken together,these results confirm that FoxO1 plays an important role in regulating the rate of ¹⁸F-FDG accumulation in HCC cells.5.In order to probe the mechanism of action of Met and FoxO1-KD in increasing intracellular levels of ¹⁸F-FDG in vivo,it was decided to examine the expression levels of GLUT1,HK2,and G6Pase in tumor cells.Western blot analysis of tumor tissues revealed a significant decrease in G6Pase expression in both Mettreated（NC1.000±0.1574 vs.Met 0.3156±0.06117 p=0.0154）and FoxO1-KD mice（NC1.000±0.1574 vs.FoxO1-KD 0.2750±0.03244 P=0.0107）.This decrease corresponded with the expression levels of GLUT1 and HK2 remaining unchanged for all groups studied,with immunohistochemical analysis also confirming these results.Conclusion:Metformin can significantly increase the uptake of ¹⁸F-FDG in SMMC-7721 HCC cells in vitro and in vivo,by decreasing G6Pase expression.FoxO1 is a key factor that mediates the regulation of G6Pase and ¹⁸F-FDG uptake by metformin.Metformin treatment and FoxO1 inhibition may be potential strategies to improve the performance of ¹⁸F-FDG PET/CT in early detection of HCC.