Metformin Decreases Hepatoma Cell’s Resistance To MTX Or 5-FU Via Inhibiting DHFR And TS

Metformin has long been the first-line therapy for type Ⅱ diabetes.Intriguingly,researchers have gradually discovered the anticancer effect of metformin during the last two decades.Metformin activates AMPK pathway,thus regulating fatty acid metabolism and autophagy via the inhibition of mTOR.Recently,metformin was also reported of the capacity of inhibiting one carbon metabolism and nucleotide metabolism,however,the underlying mechanisms are not yet fully elucidated.For further understanding of metformin’s anticancer mechanisms and metformin’s effects on one carbon metabolism,we conducted RNA-seq and proteomics analysis in hepatoma cells,through which we discovered that metformin inhibits dihydrofolate reductase(DHFR)and thymidylate synthase(TS),two key enzymes in one carbon metabolism.DHFR is indispensable for the production of tetrahydrofolate,the most important coenzyme in one carbon metabolism.While TS catalyzes the conversion of dUMP to dTMP by utilizing 1-C units generated from one carbon metabolism.Besides,DHFR and TS are respectively the direct targets of traditional antifolate chemotherapy drugs methotrexate and 5-Fluorourical.And we discovered that the combination of metformin and methotrexate/5-Fluorourical sensitized hepatoma cells to chemotherapy.With further experiments,we found that the inhibition of metformin on DHFR and TS was not through the classic AMPK/mTOR pathway,but through the upregulation of transcription factor E2F4 or the inhibition of transcription factor E2F1 thus decreasing DHFR and TS’s mRNA levels.After deep investigation,we also found metformin promotes DHFR’s protein degradation through lysosomal degradation pathway.Furthermore,we proved that DNAJB1,one of the Hsp40 protein family,Beclinl,one of the initiators of autophagy,and Cathepsin B,a cysteine peptidase in lysosome participate in metformin-induced lysosomal degradation of DHFR.Next,we explored the potential clinical significance of metformin’s inhibition on DHFR and TS.Drug resistance to antifolate chemotherapy has always been a difficult problem in the clinical cancer treatment.We established resistant hepatoma cell lines to methotrexate or 5-Fluorouracil and verified that the high expression of DHFR and TS was positively correlated with resistance of chemotherapy.However,by combination of metformin and methotrexate or 5-Fluorourical,we found that metformin re-sensitized resistant hepatoma cells to these two chemotherapeutic agents through inhibition of DHFR and TS.Besides,animal study also suggested that metformin sensitized hepatoma cells to methotrexate through decreasing DHFR in vivo.In conclusion,we discovered metformin inhibits DHFR and TS,two key enzymes in one carbon metabolism,through transcriptional and post-transcriptional mechanisms,thus sensitizing hepatoma cells to traditional chemotherapy.These findings will throw new light on metformin’s anticancer mechanisms and provide new thoughts and approaches for the clinical treatment of hepatocellular carcinoma.