IDDF2018-ABS-0140 The crosstalk of MTORC1 and DNA methylation in hepatocellular carcinoma

Background Metabolic reprogramming in cancer cells is likely to interact with the epigenetic landmarks, which may modulate tumorigenesis. However, the exact mechanism is still largely unknown. In our previous study, inhibition of activity of mechanistic target of rapamycin (mTORC1), a nutrient sensor that affects cellular metabolism, was able to attenuate the DNA methylation level. In this study, we aim to clarify the correlation between the metabolism and DNA methylation and evaluate their interactive roles in the pathogenesis of hepatocellular carcinoma (HCC). Methods We treated HCC cell lines with rapamycin to inhibit mTORC1 activity and performed immunofluorescence (IF) and Enzyme-linked immune sorbent assay (ELISA) to detect the levels of 5-methylcytosine (5-mC). Western-blot and IF assays were used to detect the protein levels of DNA methyltransferases (DNMTs) and RT-PCR was performed to test transcription of DNMTs. Cycloheximide was used to suppress the protein translation. Moreover, MG132 and BafA1 were used to inhibit the functions of ubiquitin-proteasome and autophagy-lysosome respectively to evaluate the effect of posttranslational modification for DNMTs degradation. Finally, we selected twenty liver cancer cell lines and combined western-blot and CCK8 assays to observe the association between mTORC1 inhibition-induced DNMTs degradation and cell viabilities. Results Rapamycin treatment reduced 5 mC levels in HCC cell lines. In parallel, mTORC1 inhibition attenuated the expressions of DNMT1. The subsequent experiments revealed that transcriptional regulation and posttranslational modification is not likely to participate in the process of DNMT1 degradation induced by mTORC1 inhibition. However, cycloheximide half-life assay indicated that mTORC1 might adjust the expression DNMT1 through regulating initial translational efficiency. We found that mTORC1-mediated DNMT1 degradation is closely linked to cell proliferation. Conclusions In conclusion, we demonstrated that mTORC1 could affect DNA methylation levels in HCC probably through regulating DNMTs expression. Besides, this interactive role seems to be closely linked to HCC cell viability. Investigation of the relationship between mTORC1 signalling and DNA methylation will deepen our insight on the pathogenesis of HCC and develop a novel therapeutic strategy for HCC patients.