Volume 67, Issue 2, Pages 310-320 (August 2017) O-GlcNAc transferase promotes fatty liver-associated liver cancer through inducing palmitic acid and activating endoplasmic reticulum stress  Weiqi Xu, Xiang Zhang, Jian-lin Wu, Li Fu, Ken Liu, Dabin Liu, George Gong Chen, Paul Bo-san Lai, Nathalie Wong, Jun Yu  Journal of Hepatology  Volume 67, Issue 2, Pages 310-320 (August 2017) DOI: 10.1016/j.jhep.2017.03.017 Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Journal of Hepatology 2017 67, 310-320DOI: (10. 1016/j. jhep. 2017. 03 Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Enhanced OGT expression in human NAFLD-HCC. (A) Transcriptome sequencing analysis of 18 pairs of NAFLD-HCC tumor tissues and their adjacent non-tumor tissues identified upregulated OGT in human NAFLD-HCC tumor tissues. (B) OGT mRNA levels in 8 NAFLD-HCC tumor samples and 13 normal controls from the Cancer Genome Atlas (TCGA) data. (C) OGT protein levels in six human NAFLD-HCC tumor tissues and adjacent normal liver tissues by Western blot analysis. (D) OGT protein levels in 6 HCC cell lines, 2 NAFLD-HCC cell lines, two immortalized normal hepatocyte cell lines and four normal human livers by Western blot. Statistical significance was determined by Mann-Whitney U test and unpaired Student’s t test. Journal of Hepatology 2017 67, 310-320DOI: (10.1016/j.jhep.2017.03.017) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 2 OGT promotes cell proliferation. (A) Ectopic expression of OGT in LO2 and MIHA cells was confirmed by Western blot, cell growth was increased by ectopic expression of OGT in LO2 and MIHA cells. (B) Ectopic expression of OGT in LO2 and MIHA cells promoted colony formation. (C) Stable knockdown OGT expression in NAFLD-HCC cell lines HKCI-2 and HKCI-10 cells inhibited cell growth. The knockdown efficiency of OGT was confirmed by Western blot. (D) Stable knockdown OGT expression in HKCI-2 and HKCI-10 cells inhibited colony formation. Statistical significance was determined by unpaired Student’s t test, one-way ANOVA and two-way ANOVA. Journal of Hepatology 2017 67, 310-320DOI: (10.1016/j.jhep.2017.03.017) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 3 OGT aggravates tumor growth in vivo. (A) Ectopic expression of OGT increased the tumor volume and tumor weight in nude mice. Tumor growth curve (left panel) and tumor weight (right panel) of OGT plasmid and control transfected LO2 cells in nude mice are shown. Images of the tumors are shown in the middle. (B) Tumor growth curve of MHCC97L-shOGT cells in nude mice compared with MHCC97L-NTC cells. (C) Tumor growth curve of HKCI-2-shOGT cells in nude mice compared with HKCI-2-NTC cells. Data were expressed as mean±SEM, n=5/group. Statistical significance was determined by two-way ANOVA and unpaired Student’s t test. Journal of Hepatology 2017 67, 310-320DOI: (10.1016/j.jhep.2017.03.017) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 4 OGT promotes cell migration and invasion in vitro and facilitates tumor metastasis in nude mice. (A) Representative results of scratch healing assay on LO2 cells with OGT transfection. (B) Representative results of scratch healing assay on HKCI-2 and HKCI-10 cells with OGT knockdown. (C) OGT promoted HCC cell invasion. Matrigel invasion assay of LO2 and MIHA cells with OGT transfection and HKCI-2 and HKCI-10 cells with OGT knockdown. (D) The effect of OGT expression on protein expression of SIRT1/FOXM1 and key epithelial- mesenchymal transition (EMT) markers by Western blot. EV, empty vector; NTC, non-template control. (E1) Knockdown of OGT in MHCC97L significantly inhibited HCC tumorigenicity in vivo as demonstrated by an orthotopic tumor implantation experiment in nude mice. (E2) Tumor weight (left panel) and tumor volume (right panel) are shown. Data were expressed as mean±SEM, n=5/group. (F) Knockdown of OGT diminished lung metastasis in an orthotopic tumor implantation model in nude mice. Representative images of lung (left panel), hematoxylin and eosin staining of HCC tumor foci in the lungs (middle panel), and incidences of lung metastasis are shown (right panel). Statistical significance was determined by unpaired Student’s t test. Journal of Hepatology 2017 67, 310-320DOI: (10.1016/j.jhep.2017.03.017) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 5 OGT mediates lipid metabolism in NAFLD-HCC cell lines. (A) The change of lipid profile by OGT knockdown in HKCI-2 cells identified by liquid chromatography-mass spectrometry (LC-MS) (multivariate analysis). (B) Orthogonal partial least squares discriminant analysis (OPLS-DA) of the lipid metabolites in HKCI-2. (C) Palmitic acid was identified to be one of the most significantly altered parameter in HKCI-2 after OGT-shRNA knockdown by m/z at 255.2332 (left and middle panels). The decreased palmitic acid was confirmed in HKCI-10 shOGT cells (right panel). (D) OGT inhibitor ST045849 significantly decreased palmitic acid level in HKCI-2 cells. (E1) Palmitic acid (10μM) significantly promoted cell proliferation in HKCI-2 and HKCI-10 cells. (E2) mRNA and protein expression of fatty acid synthase (FASN) in HKCI-2 and HKCI-10 cell lines with OGT knockdown. (F1) Fatty acid synthase inhibitor C75 inhibited cell proliferation in HKCI-2 cells. (F2) FASN inhibitor C75 inhibited cell proliferation in OGT overexpressing LO2 and MIHA cells. Statistical significance was determined by unpaired Student’s t test, one-way ANOVA and and two-way ANOVA. *p<0.05, **p<0.01 vs. control cells. Journal of Hepatology 2017 67, 310-320DOI: (10.1016/j.jhep.2017.03.017) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 6 OGT activates endoplasmic reticulum (ER) stress, JNK and NF-κB pathways. (A) The protein levels of the glucose-regulated protein 78 (GRP78), inositol-requiring enzyme 1α (IRE1α), phospho-JNK, JNK, phospho-c-Jun and c-Jun in OGT overexpressed LO2 and MIHA cells and OGT knockdown HKCI-2 and HKCI-10 cells were determined by Western blot. (B) Luciferase activity was determined by dual luciferase activity assay at 48h post transfection with AP-1 luciferase plasmids in OGT overexpression and knockdown cell lines. (C) Protein expression of phospho-IKKα/β, IKKα, phospho-IκBα, IκBα, phospho-NF-κB p65 and phospho-NF-κB p50 in OGT overexpressed LO2 cell line and HKCI-2 cell line with OGT knockdown. (D) NF-κB DNA binding activity and mRNA expression of TNFα in LO2 cells with OGT overexpression and HKCI-2 cells with OGT knockdown. (E) Palmitic acid treatment activates ER stress, JNK and NF-κB pathway in HKCI-10 cells. (F1) Knockdown efficiency of siGRP78 in LO2 and MIHA cells with or without OGT overexpression. (F2) Knockdown of GRP78 by siRNA diminished the tumor-promoting effect of OGT in OGT overexpressing LO2 and MIHA cells. Statistical significance was determined by unpaired Student’s t test. Journal of Hepatology 2017 67, 310-320DOI: (10.1016/j.jhep.2017.03.017) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions

Fig. 7 (A) OGT inhibitor ST045849 suppressed cell proliferation of HKCI-2 and HKCI-10. (B) Tumor growth curve of HKCI-2 in nude mice treated with OGT inhibitor ST045849. Statistical significance was determined by unpaired Student’s t test and two-way ANOVA. (C) Schematic diagram for the mechanisms of the oncogenic effect of OGT in NAFLD-associated-HCC. Journal of Hepatology 2017 67, 310-320DOI: (10.1016/j.jhep.2017.03.017) Copyright © 2017 European Association for the Study of the Liver Terms and Conditions