Functional Characterization of Glycine N-Methyltransferase and Its Interactive Protein DEPDC6/DEPTOR in Hepatocellular Carcinoma
Glycine N-methyltransferase (GNMT) is a tumor suppressor for hepatocellular carcinoma (HCC). High rates of Gnmt knockout mice developed HCC. Epigenetic alteration and dysregulation of several pathways including wingless-type MMTV integration site (Wnt), mitogen-activated protein kinase (MAPK) and Janus kinase and signal transducer and activator of transcription (JAK-STAT) are associated with HCC development in Gnmt knockout mice. We hypothesized that GNMT may regulate signal transduction through interacting with other proteins directly. In this report, we identified a mammalian target of rapamycin (mTOR) inhibitor (DEP domain containing MTOR-interacting protein [DEPDC6/DEPTOR]) as a GNMT-binding protein by using yeast two-hybrid screening. Fluorescence resonance energy transfer assay demonstrated that the C-terminal half of GNMT interact with the PSD-95/Dlg1/ZO-1 (PDZ) domain of DEPDC6/DEPTOR. Immunohistochemical staining showed that 27.5% (14/51) of HCC patients had higher expression levels of DEPDC6/DEPTOR in the tumorous tissues than in tumor-adjacent tissues, especially among HCC patients with hepatitis B viral infection (odds ratio 10.3, 95% confidence interval [CI] 1.05–11.3) or patients with poor prognosis (death hazard ratio 4.51, 95% CI 1.60–12.7). In terms of molecular mechanism, knockdown of DEPDC6/DEPTOR expression in HuH-7 cells caused S6K and 4E-BP activation, but suppressed Akt. Overexpression of DEPDC6/DEPTOR activated Akt and increased survival of HCC cells. Overexpression of GNMT caused activation of mTOR/raptor downstream signaling and delayed G2/M cell cycle progression, which altogether resulted in cellular senescence. Furthermore, GNMT reduced proliferation of HuH-7 cells and sensitized them to rapamycin treatment both in vitro and in vivo. In conclusion, GNMT regulates HCC growth in part through interacting with DEPDC6/DEPTOR and modulating mTOR/raptor signaling pathway. Both GNMT and DEPDC6/DEPTOR are potential targets for developing therapeutics for HCC.
We thank TLCN for providing the HCC tissue samples and related clinical data. This network currently includes five major medical centers (National Taiwan University Hospital, Chang-Gung Memorial Hospital-Linko, Veteran General Hospital-Taichung, Chang-Gung Memorial Hospital-Kaohsiung and Veteran General Hospital-Kaohsiung). TLCN was supported by grants from the National Science Council since 2005 (NSC 100-2325-B-182-006) and the National Health Research Institutes, Taiwan. In addition, we thank the Sequencing Core Facility of the National Yang-Ming University Genome Research Center and the Confocal Microscope Lab of National Taiwan University for technical support and for assistance in the FRET experiments. This work was supported in part by grants from the National Science Council of the Republic of China (National Research Program for Biopharmaceuticals [NRPB], grant NSC100-2325-B-010-008) and the Ministry of Education of the Republic of China (Aim for the Top University Plan).
- 42.Lizcano JM, et al. (2002) Molecular basis for the substrate specificity of NIMA-related kinase-6 (NEK6): evidence that NEK6 does not phosphorylate the hydrophobic motif of ribosomal S6 protein kinase and serum- and glucocorticoid-induced protein kinase in vivo. J. Biol. Chem. 277:27839–49.CrossRefGoogle Scholar