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O-GlcNAc transferase promotes fatty liver-associated liver cancer through inducing palmitic acid and activating endoplasmic reticulum stress

  • Weiqi Xu
    Affiliations
    Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China
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  • Xiang Zhang
    Affiliations
    Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China
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  • Jian-lin Wu
    Affiliations
    State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau, China
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  • Li Fu
    Affiliations
    Shenzhen Key Laboratory of Translational Medicine of Tumor and Cancer Research Centre, School of Medicine, Shenzhen University, Shenzhen, China
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  • Ken Liu
    Affiliations
    Faculty of Medicine, The University of Sydney, Sydney, NSW, Australia
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  • Dabin Liu
    Affiliations
    Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China
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  • George Gong Chen
    Affiliations
    Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China
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  • Paul Bo-san Lai
    Affiliations
    Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China
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  • Nathalie Wong
    Affiliations
    Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China
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  • Jun Yu
    Correspondence
    Corresponding author. Address: Institute of Digestive Disease and Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China. Tel.: +852 37636099; fax: +852 21445330.
    Affiliations
    Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China
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Open AccessPublished:March 24, 2017DOI:https://doi.org/10.1016/j.jhep.2017.03.017

      Background & Aims

      O-GlcNAc transferase (OGT) is a unique glycosyltransferase involved in metabolic reprogramming. We investigated the functional role of OGT in non-alcoholic fatty liver disease-associated hepatocellular carcinoma (NAFLD-HCC).

      Methods

      The biological function of OGT in NAFLD-HCC was determined by gain- or loss- of OGT functional assays in vitro and in nude mice. OGT target factors and pathways were identified by liquid chromatography-tandem mass spectrometry (LC-MS), promoter luciferase assay, DNA binding activity assay and Western blot.

      Results

      OGT was upregulated in 12 out of 18 (66.7%) NAFLD-HCC tumor tissues by transcriptome sequencing, which was confirmed in additional NAFLD-HCC tumor tissues and cell lines. Biofunctional investigation demonstrated that OGT significantly increased cell growth (p <0.001), clonogenicity (p <0.01), migration and invasion (p <0.05) ability in vitro, and promoted xenograft tumor growth as well as lung metastasis in nude mice. The oncogenic effect of OGT was investigated, we found that OGT significantly induced palmitic acid production identified by LC-MS, which enhanced the protein expression of endoplasmic reticulum (ER) stress masters of glucose-regulated protein 78 and inositol-requiring enzyme 1α. Consequently, OGT significantly activated JNK/c-jun/AP-1 cascade by increasing protein expression of p-JNK, p-c-Jun and activation of AP-1; and induced NF-κB pathway through enhancing the protein levels of p-IKKα/ p-IKKβ, p-p65, p-p50 and the NF-κB DNA binding activity. Notably, OGT inhibition by its antagonist (ST045849) suppressed cell proliferation in vitro (p <0.001) and in xenograft mice models (p <0.05).

      Conclusions

      OGT plays an oncogenic role in NAFLD-associated HCC through regulating palmitic acid and inducing ER stress, consequently activating oncogenic JNK/c-jun/AP-1 and NF-κB cascades.

      Lay summary

      OGT, a unique glycosyltransferase enzyme, was identified to be upregulated in non-alcoholic fatty liver disease-associated hepatocellular carcinoma tissues by transcriptome sequencing. Here, we found that OGT plays a role in cancer by promoting tumor growth and metastasis in both cell models and animal models. This effect is mediated by the induction of palmitic acid.

      Graphical abstract

      Keywords

      Introduction

      Hepatocellular carcinoma (HCC) is one of the most common human malignancies and the third leading cause of cancer death worldwide.
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      With the control of viral hepatitis and the escalating obesity epidemic, non-alcoholic fatty liver disease (NAFLD) associated HCC is occupying a greater proportion of HCC incidence in recent decades.
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      In view of a projected increase of HCC incidence in NAFLD patients in the coming decade, there is a compelling need for understanding the pathogenic mechanisms of NAFLD-associated HCC (NAFLD-HCC), and therefore provide new insights into mechanism-based therapies.
      The underlying mechanisms, especially the genomic and metabolic perturbations, linking obesity associated-NAFLD to HCC are still unclear. However, inflammation, insulin resistance, oncogenic factors and pathways, have been reported to be involved in the molecular pathogenesis of NAFLD-associated-HCC.
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      Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S.
      Using transcriptome sequencing, we found that OGT (O-GlcNAc transferase), a gene involved in insulin resistance,
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      is upregulated in NAFLD-HCC compared to their adjacent non-tumor tissues. OGT is a glycosyltransferase that catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in cytoplasmic and nuclear proteins, resulting in their modification with a beta-linked N-acetylglucosamine (O-GlcNAc). The intracellular protein level of O-GlcNAc correlates with abnormal glucose and lipid metabolism.
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      O-GlcNAcylation: a bridge between glucose and cell differentiation.
      Aberrant O-GlcNAcylation expression contributes to metabolic disorders such as insulin resistance.
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      NFkappaB activation is associated with its O-GlcNAcylation state under hyperglycemic conditions.
      OGT also participates in transcriptional and epigenetic regulation by regulating transcription factors.
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      O-GlcNAcylation increases ChREBP protein content and transcriptional activity in the liver.
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      The role of O-linked GlcNAc modification on the glucose response of ChREBP.
      OGT was found to be overexpressed in human cancers of breast, lung and colon
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      and participates in the altered metabolism occurring in cancer cells.
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      However, the functional significance of OGT in NAFLD-HCC is largely unclear. In this study, we explored the molecular pathogenic mechanism of OGT in NAFLD-HCC.

      Materials and methods

      Human samples

      Human NAFLD-HCC tumor tissues and their adjacent normal tissues were collected from patients with biopsy-proven NAFLD-HCC in Prince of Wales Hospital, the Chinese University of Hong Kong. Normal Liver tissues were obtained from liver transplant donors in Queen Mary Hospital, the University of Hong Kong. Written informed consent was obtained from all subjects and the study protocol was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong and the University of Hong Kong.

      Cell lines, cell culture and treatment

      Immortalized hepatocyte cell lines (LO2, MIHA) and liver cancer cell lines (BEL7404, Hep3B, HepG2, Huh7, PLC5 and SK-Hep1) were obtained from American Type Culture Collection (ATCC, Manassas, VA), Cell bank of Chinese Academy of Sciences (Shanghai, China) and Japanese Collection of Research Bioresources Cell Bank (JCRB, Japan), and cultured in Dulbecco's modified Eagle's medium (DMEM). Two NAFLD-HCC cell lines, HKCI-2 and HKCI-10, were established from NAFLD-HCC patients by Dr. Nathalie Wong from Department of Anatomical and Cellular Pathology, the Chinese University of Hong Kong,
      • Chan K.Y.
      • Lai P.B.
      • Squire J.A.
      • Beheshti B.
      • Wong N.L.
      • Sy S.M.
      • et al.
      Positional expression profiling indicates candidate genes in deletion hotspots of hepatocellular carcinoma.
      which were cultured and maintained in RPMI 1640 growth medium (Invitrogen, Carlsbad, CA). HKCI-2 or HKCI-10 cells were treated with different concentrations of OGT inhibitor ST045849 (Tim Tec, Newark, Delaware). LO2, MIHA and HKCI-2 cells were treated with fatty acid synthase (FASN) inhibitor C75 (Sigma-Aldrich) and MTT assay was performed. GRP78 siRNA (ID:s6980) was obtained from Thermo Fisher Scientific (Waltham, MA) and transfected into LO2 and MIHA cells using Lipofectamine® 2000 Transfection Reagent (Thermo Fisher Scientific).

      Analyses of gene expression profiling in HCCs from RNAseq in TCGA dataset

      The full clinical dataset of liver HCC gene expression by RNAseq (IlluminaHiSeq percentile, n = 423) from the Cancer Genome Atlas (TCGA) Research Network data portal was downloaded and assessed for outlier expression candidates. This study meets the publication guidelines provided by TCGA: http://cancergenome.nih.gov/publications/publicationguidelines.

      Lentivirus packaging and transduction

      The lentivirus was produced by co-transfecting 293T cells with the target vector (non-template control [NTC] or shOGT) and two packaging vectors (Inovogen, Beijing, China). Subsequent purification was performed by ultracentrifugation. HKCI-2, HKCI-10 and MHCC97L cells were plated in 10 mm2 dishes and transduced with lentivirus. The stable cell lines were cultured with medium containing 2 μg/ml puromycin (Invitrogen).

      Colony formation assay and cell viability assay

      Immortalized human liver epithelial cell lines LO2 and MIHA were stably transfected with OGT vector or empty vector NEG plasmid (Genecopoeia, Rockville, Maryland) using Lipofectamine® 2000 (Invitrogen) and selected with puromycin (Invitrogen) at 2 mg/ml for 14 days. Cells were collected and seeded (500–1,000 per well) in a fresh 6-well plate for 7 to 9 days. The resulting cells were then fixed with 70% ethanol and stained with 5% crystal violet, and individual colonies with more than 50 cells were counted. Cell viability was examined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using Vybrant MTT Cell Proliferation Assay Kit (Invitrogen).

      Cell invasiveness

      Cell invasiveness was measured by Matrigel Invasion Chamber (BD, Franklin Lakes, New Jersey). Control or treated cells were seeded at 8 × 104. The invaded cells on the lower surfaces of inserts were fixed in 100% methanol and stained with crystal violet before being mounted on glass slides. More than 20 views were analyzed under light microscopy (400× magnification) and the number of invaded cells was counted.

      In vivo tumorigenicity in subcutaneous xenograft mouse model

      Immortalized liver cell line LO2 (1 × 106 cells in 0.1 ml PBS) stably transfected with OGT expression vector or empty vector was injected subcutaneously into the dorsal flank of 4-week-old male Balb/c nude mice (n = 5/group). MHCC97L (5 × 106 cells in 0.1 ml PBS) and HKCI-2 cells (1 × 107 cells in 0.1 ml PBS) transfected with lentivirus-shOGT or lentivirus-NTC were injected subcutaneously into the dorsal flank of 4-week-old male Balb/c nude mice (n = 5/group), respectively. Tumor diameter was measured every 4 days until 28 days. Tumor volume was calculated by the formula: tumor volume [mm3] = (length [mm]) × (width [mm])2 × 0.5.

      Orthotopic murine liver tumor model of tumor formation and distant metastasis

      An orthotopic HCC mouse model was established using MHCC97L. MHCC97L cells (5 × 106 cells in 0.1 ml PBS) transduced by shOGT-lentivirus (Lenti-shOGT) and control NTC vector-lentivirus (Lenti-NTC) were injected subcutaneously into the left dorsal flank of 4-week-old male Balb/c nude mice, respectively. Subcutaneous tumors were harvested after two weeks and cut into 1.0 mm3 pieces. One piece of the tumor was then implanted into the left liver lobe in a separate group of 6-week old nude mice (n = 5/group). Four weeks after tumor implantation, the mice were sacrificed and examined. The livers and lungs were dissected and paraffin embedded, and the sections were stained with hematoxylin and eosin. Tumors in the liver and metastatic tumors in the lungs were counted in a blinded manner. All animal experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.

      Liquid chromatography-tandem mass spectrometry (LC-MS)

      HKCI-2 and HKCI-10 cells were stably infected with OGT-shRNA or control-shRNA for 48 h. The cell lysis solution (100 μl) was loaded into the cartridges (Waters Co., Milford, MA). The aqueous plug was pulled from the solid phase extraction (SPE) cartridges with high and low vacuum respectively in the cartridges. SPE cartridges were eluted using methanol and ethyl acetate, respectively, thereafter combined, evaporated, redissolved and centrifuged. The supernatant was transferred to auto-sampler vials and stored at −80°C until analysis. An Agilent 1290 Ultra-high Performance Liquid Chromatography (UHPLC, Agilent Technologies, CA) was employed for the separation of components of each sample. The mass spectrometry was conducted on a 6550 iFunnel Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA) with a dual Agilent Jet Stream electrospray ion source (dual AJS ESI). The mass spectra were recorded across the range of 100–1700 m/z for qualitative analyses in both of negative and positive mode.

      Dual luciferase reporter activity assay

      The effects of OGT overexpression and knockdown on the AP-1 activity were measured by reporter assay. Forty-eight hours after the transfection of plasmids, the cells were harvested and assayed by the Dual Luciferase Reporter Assay System (Promega, Madison, Wisconsin) by using GloMax microplate luminometer (Promega).

      Determination of nuclear factor-κB (NF-κB) p65 nuclear DNA binding activity

      The quantification of DNA binding activity of transcription factors NF-κB p65 was determined by the commercial Transcription Factor Assay kits (Cayman Chemical Company, Ann Arbor, MI) according to manufacturer’s instructions.

      RNA extraction and real-time PCR analysis

      Total RNA was extracted using Trizol reagent (Invitrogen). Complementary DNA was synthesized from total RNA using Transcriptor Reverse Transcriptase (Roche Applied Sciences, Indianapolis, IN). Real-time PCR was performed using SYBR Green master mix (Roche) in the Light Cycler 480 Real-Time PCR system (Roche).

      Western blot

      Protein lysates from cell lines were prepared using protease inhibitor cocktail-containing (Roche) lysis buffer. Protein concentration was determined by the Bradford method (Bio-Rad Laboratories, Hercules, CA). Antibody-antigen complexes were detected using the ECL Plus Western Blotting Detection Reagents (GE Healthcare Piscataway, NJ). Primary antibodies used were rabbit anti-OGT (Proteintech Group Inc., Chicago, MA); mouse anti-O-GlycNAc (CTD110.6), phosphorylated c-Jun N-terminal kinases (p-SAPK/JNK), phospho-c-Jun, c-Jun, Vimentin (D21H3), E-cadherin, phospho-IKKα/IKKβ (Ser176/180), IκB kinase α (IKKα), IKKβ, IκBα, phospho-IκBα (Ser32), inositol-requiring enzyme 1 α (IRE1α), glucose-regulated protein 78 (GRP78), Forkhead Box M1 (FOXM1), NAD-Dependent Protein Deacetylase Sirtuin-1 (SIRT1), Cleaved-caspase 3, Caspase 3, Cleaved-caspase 7, Caspase 7, Cleaved-Poly(ADP-Ribose) Polymerase (PARP), PARP (Cell Signaling Technology, Boston, MA); NF-κB p50, NF-κB p65, phospho-IKKα, phospho-NFκB p50, phospho-NFκB p65, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA).

      Flow cytometry for apoptosis assay

      LO2 and MIHA cells with or without OGT transfection and HKCI2 cells with or without OGT knockdown by shRNA were stained with Annexin V-PE and 7-AAD (BD Biosciences, San Jose, CA), and analyzed by fluorescence-activated cell sorting using FACScan (BD Biosciences). Cell populations were classified as early apoptotic (Annexin V positive, 7-AAD negative), late apoptotic (Annexin V positive, 7-AAD positive), necrotic (Annexin V negative, 7-AAD positive) or viable (Annexin V negative, 7-AAD negative).

      Statistical analysis

      The results were expressed as mean ± standard error of the mean (SEM). Mann-Whitney U test or Student’s t test was performed to compare the variables of the two sample groups. Multiple group comparisons were made by the Kruskal-Wallis test or one-way ANOVA. The difference in cell growth curves was determined by two-way ANOVA. The LC-MS data were processed with the MassHunter Workstation Data Acquisition Software and Agilent Metabolite ID Software. All statistical tests were performed using SPSS or GraphPad Software. A difference was considered statistically significant at p <0.05.
      For further details regarding the materials used, please refer to the CTAT table.

      Results

      OGT is upregulated in human NAFLD-HCC patients and HCC cell lines

      OGT was identified to be upregulated in 12 out of 18 (66.7%) human NAFLD-HCC tumor tissues compared with their adjacent non-tumor tissues by transcriptome sequencing (Fig. 1A). In addition, OGT mRNA expression was significantly increased in 8 NAFLD-HCC tumor tissues compared to 13 normal controls (Fig. 1B, p <0.01) from TCGA dataset. Consistently, OGT protein expression was upregulated in NAFLD-HCC tumor tissues compared to their adjacent normal tissues (Fig. 1C). We then compared the protein expression of OGT in six HCC cell lines including BEL7404, Hep3B, HepG2, Huh7, PLC5, SK-Hep1, and two NAFLD-HCC cell lines HKCI-2 and HKCI-10 as well as two immortalized normal hepatocyte cell lines LO2 and MIHA and normal human liver tissues. OGT was strongly expressed in all HCC cell lines including HKCI-2 and HKCI-10, but silenced in normal liver tissues and weakly expressed in LO2 and MIHA cells (Fig. 1D).
      Figure thumbnail gr1
      Fig. 1Enhanced 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.

      OGT had tumor-promoting ability in liver cells

      Frequent upregulation of OGT in NAFLD-HCC cell lines and in clinical NAFLD-HCC tissues suggested the potential tumor-promoting ability of OGT in NAFLD-HCC. To assess the oncogenic role of OGT in liver, we generated stably OGT overexpressed LO2 and MIHA cells. Ectopic expression of OGT in LO2-OGT and MIHA-OGT cells was confirmed by Western blot (Fig. 2A). Overexpression of OGT in LO2 and MIHA significantly promoted cell growth compared with empty vector-transfected cells LO2-EV (p <0.001) and MIHA-EV (p <0.001) (Fig. 2A). The proliferation promotion effect of OGT was further confirmed by the increased colony formation efficiency in overexpressing LO2 (p <0.0001) and MIHA (p <0.01) cells compared to control cells (Fig. 2B).
      Figure thumbnail gr2
      Fig. 2OGT 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.
      To confirm the tumor-promoting role of OGT in NAFLD-HCC, OGT was knocked down by shRNA in NAFLD-HCC cell lines of HKCI-2 and HKCI-10 with endogenous OGT expression. Knockdown of OGT in HKCI-2 and HKCI-10 cells by two stable knockdown vectors (shOGT1 and shOGT2) (Fig. 2C) consistently suppressed cell growth in HKCI-2 (p <0.0001) and in HKCI-10 (p <0.05) (Fig. 2C) and reduced clonogenicity in both cell lines (p <0.001, p <0.05, respectively) (Fig. 2D). These results collectively suggest that OGT plays a tumor-promoting role in NAFLD-HCC. In addition, the effect of OGT on cell apoptosis was evaluated. Ectopic expression of OGT in LO2 and MIHA cells significantly suppressed early and late cell apoptosis; while knockdown of OGT in HKCI-2 induced early and late apoptosis compared to the control cells (Fig. S1A). The effect of OGT on cell apoptosis was confirmed by reduced protein expression of active forms of cleaved-caspase 3, cleaved-caspase 7 and cleaved-PARP in both LO2 an MIHA with OGT expression; and by increased expression of these proteins in HKCI-2 cells with OGT knockdown by Western blot (Fig. S1B). Collectively, these results suggested that the oncogenic effect of OGT is also associated with the suppression of cell apoptosis.

      OGT promotes tumor growth in nude mice

      To explore the in vivo tumorigenic ability of OGT, we set up subcutaneous xenograft tumor model by subcutaneously injecting OGT transfected LO2 and empty vector-transfected cells into nude mice. The tumor growth rate in the nude mice injected with LO2-OGT cells were significantly higher than in those injected with the LO2-vector control cells (p <0.01) (Fig. 3A), concomitant with significantly higher tumor weight at the end of the experiment (p <0.05) (Fig. 3A). Conversely, knockdown of OGT significantly decreased tumor growth in the xenograft subcutaneous models of HKCI-2 and MHCC97L cells in nude mice (Fig. 3B). These results further confirm that OGT has an oncogenic effect in NAFLD-HCC.
      Figure thumbnail gr3
      Fig. 3OGT 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.

      OGT promotes the invasion and migration abilities in NAFLD-HCC cell lines

      The tumor-promoting effect of OGT was further evaluated on cell migration and invasion. Using the monolayer scratch healing assay, we found that ectopic expression of OGT significantly increased the migration ability of LO2 cells (Fig. 4A). Conversely, stable knockdown OGT in HKCI-2 and HKCI-10 cells had the opposite effect on cell migration (Fig. 4B). Quantitative analyses confirmed a significant reduction in wound closure in OGT knockdown cells compared with control cells for 63% in HKCI-2 cells and 47% in HKCI-10 cells, respectively (Fig. 4B). Martrigel invasion assay also showed that overexpression of OGT promoted the invasion of LO2 (p <0.01) and MIHA (p <0.05) cells (Fig. 4C), whereas OGT knockdown impaired the invasiveness in both HKCI-2 and HKCI-10 cells (both p <0.001) (Fig. 4C). We then examined the protein expression of SIRT1 and FOXM1, which are the key in tumor invasion and metastasis, in OGT overexpression LO2 and MIHA cells and OGT knockdown HKCI-2 and HKCI-10 cells. As shown in Fig. 4D, OGT suppressed SIRT1 expression while induced FOXM1 expression in LO2 and MIHA by Western blot; knockdown OGT in HKCI-2 and HKCI-10 cells enhanced SIRT1 but suppressed FOXM1 expression, indicating that SIRT1 modulation of FOXM1 pathway is involved in the oncogenic process of OGT in NAFLD-HCC. Moreover, we examined the protein expression of the key epithelial-mesenchymal transition (EMT) regulators E-cadherin and vimentin in OGT overexpressing and OGT-repressing cells. OGT reduced the protein levels of E-cadherin, while enhancing Vimentin in OGT expressing-LO2 and -MIHA cells (Fig. 4D). While, OGT knockdown increased E-cadherin and decreased vimentin protein expression in HKCI-2 and HKCI-10 cells (Fig. 4D), further confirming the effect of OGT in promoting migration and the invasive abilities of NAFLD-HCC cells through regulating E-cadherin and vimentin expression.
      Figure thumbnail gr4
      Fig. 4OGT 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.

      OGT aggravates HCC metastasis to the lungs in vivo

      We then investigated whether OGT could alter the orthotopic xenograft tumors and the metastatic potential of MHCC97L cells in vivo. Subcutaneously grown xenografts (1 mm3/each) derived from MHCC97L-NTC or MHCC97L-shOGT2 cells were implanted into the livers of nude mice (Fig. 4E1). After four weeks, the mice were harvested for observation of orthotopic tumor formation and lung metastasis. As shown in Fig. 4E2, the orthotopic liver tumor volume and tumor weight were significantly lower in nude mice implanted with OGT knockdown MHCC97L (MHCC97L-shOGT) cells compared with the non-template control MHCC97L-NTC cells. Histological examination of the tumor metastasis in the lung showed that 60% (3/5) of the mice bearing MHCC97L-NTC cells exhibited lung metastases after orthotopic implantation; while, lung metastases were not identified in mice bearing MHCC97L-shOGT cells (Fig. 4F), inferring that OGT promotes lung metastasis of orthotopically implanted HCC cells in vivo.

      OGT induces palmitic acid in NAFLD-HCC cell lines

      As OGT catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to O-GlcNAc, which is associated with metabolic disorders including insulin resistance.
      • Yang X.
      • Ongusaha P.P.
      • Miles P.D.
      • Havstad J.C.
      • Zhang F.
      • So W.V.
      • et al.
      Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance.
      • Sun C.
      • Shang J.
      • Yao Y.
      • Yin X.
      • Liu M.
      • Liu H.
      • et al.
      O-GlcNAcylation: a bridge between glucose and cell differentiation.
      We therefore evaluated the molecular mechanisms of OGT as an oncogenic factor in NAFLD-HCC through mediating the metabolic parameters. The lipid metabolites in HKCI-2 cells with or without OGT knockdown (HKCI-2 shOGT vs. HKCI-2 NTC) were analyzed using LC-MS (Fig. 5A). The orthogonal partial least squares discriminant analysis (OPLS-DA) of LC-MS data showed that OGT knockdown in HKCI-2 shOGT cells led to a significant right shift of the lipid metabolite map compared to HKCI-2 NTC cells (Fig. 5B). In this regard, we examined the metabolites of HKCI-2 cells with or without OGT knockdown through analyzing peaks of compounds by high resolution MS and MS/MS spectra. We identified palmitic acid, a saturated long-chain fatty acid with a 16-carbon backbone, was the most significantly altered parameter in HKCI-2 shOGT cells compared to control HKCI-2 NTC cells (Fig. 5C). This result was confirmed in HKCI-10 shOGT cells compared to control HKCI-10 NTC cells (Fig. 5C). Moreover, OGT inhibitor ST045849 could cause a significantly decreased palmitic acid level in HKCI-2 cells (Fig. 5D).
      Figure thumbnail gr5
      Fig. 5OGT 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 (10 μM) 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.

      Palmitic acid promotes cell proliferation in NAFLD-HCC cell lines

      The effect of palmitic acid on cell proliferation was examined in HKCI-2 and HKCI-10 cells. As shown in Fig. 5E1, palmitic acid treatment (10 μM) significantly promoted cell proliferation in both cell lines. Since FASN is the enzyme responsible for the synthesis of palmitic acid from acetyl-CoA and malonyl-CoA, we examined the effect of OGT on FASN expression. As shown in Fig. 5E2, OGT knockdown significantly reduced FASN expression in HKCI-2 and HKCI-10 cells; conversely, ectopic expression of OGT increased FASN expression in LO2 and MIHA cells. In addition, HKCI-2 cells treated with FASN inhibitor C75 (5 μg/ml) significantly suppressed cell viability compared with the control cells (Fig. 5F1). To further confirm the involvement of FASN in OGT mediated oncogenic effect in liver cells, we treated OGT overexpressing LO2 and MIHA cells with FASN inhibitor C75. The results showed that C75 significantly diminished the effect of OGT on inducing cell viability in LO2 and MIHA cells (Fig. 5F2), suggesting that FASN and its product palmitic acid contribute to the oncogenic effect of OGT in NAFLD-HCC. Thus, the oncogenic effect of OGT in NAFLD-HCC is mediated at least by causing metabolic changes through inducing intracellular palmitic acid level.

      OGT activates ER stress and its associated JNK and NF-κB signaling cascades in HCC cell lines

      To clarify the mechanism(s) underlying the oncogenic effect of OGT, we investigated the involvement of ER stress in NAFLD-HCC mediated by OGT because others have shown that metabolic changes activate ER stress and consequently induces NAFLD and triggers HCC development.
      • Garcia-Ruiz C.
      • Mato J.M.
      • Vance D.
      • Kaplowitz N.
      • Fernandez-Checa J.C.
      Acid sphingomyelinase-ceramide system in steatohepatitis: a novel target regulating multiple pathways.
      • Nakagawa H.
      • Umemura A.
      • Taniguchi K.
      • Font-Burgada J.
      • Dhar D.
      • Ogata H.
      • et al.
      ER stress cooperates with hypernutrition to trigger TNF-dependent spontaneous HCC development.
      We found that the protein expression of ER stress markers GRP78 and IRE1α were significantly induced by OGT in LO2 and MIHA cells; while OGT knockdown reduced the protein levels of GRP78 and IRE1α in HKCI-2 and HKCI-10 cells (Fig. 6A).
      Figure thumbnail gr6
      Fig. 6OGT 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 48 h 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.
      JNK and NF-κB signaling pathways are the major ER stress-related oncogenic signaling pathways, which contributes to both NASH and HCC development, we thus determined the effect of OGT on the activities of JNK/c-Jun/AP-1 and NF-κB cascades. As shown in Fig. 6A, OGT transferase increased the protein levels of phosphorylated-JNK (p-JNK) and p-c-Jun in LO2 and MIHA cells, while knockdown OGT reduced the protein levels of p-JNK and p-c-Jun in HKCI-2 and HKCI-10 cells. In keeping with this, OGT increased the reporter activity of AP-1 signaling pathway, whereas OGT knockdown showed an opposite effect (Fig. 6B). Like JNK, OGT transferase also enhanced levels of NF-κB subunits p-p65 and p-p50, p-IKKα and p-IκBα, indicating the activation of NF-κB cascade by OGT (Fig. 6C). This was further confirmed by increased NF-κB nuclear binding activity and increased expression of NF-κB downstream target genes TNF-α in OGT transfected LO2 cells compared with the control LO2 cells (Fig. 6D). Knockdown OGT in HKCI-2 reduced protein levels of p-p65, p-p50, p-IKKα and p-IκBα and mRNA expression of TNF-α (Fig. 6C and D). Consistent with this role of OGT in ER stress and its related signaling, HKCI-10 cells treated with palmitic acid could also increase protein expression of GRP78, IRE1α and p-JNK, p-NF-kB p65 and decrease IκBα protein expression (Fig. 6E). To confirm the contribution of ER stress in the oncogenic effect of OGT, GRP78 was knocked down by siRNA in OGT overexpressing LO2 and MIHA cells (Fig. 6F1). The result showed that cell viabilities were significantly reduced in LO2 and MIHA cells with GRP78 knockdown (Fig. 6F2). Collectively, our results suggested that the oncogenic effect of OGT is mediated at least in part by inducing ER stress in NAFLD-HCC.

      Specific OGT antagonist suppresses cell proliferation in vitro and in vivo

      The enhanced expression and tumor-promoting effect of OGT in NAFLD-HCC led us hypothesize that OGT could be a potential therapeutic target in this disorder, OGT specific inhibition might dampen or abrogate the growth of NAFLD-HCC. NAFLD-HCC cell lines HKCI-2 and HKCI-10 were treated with a specific OGT antagonist ST045849 at 0, 20, 50 and 100 nM. We found that ST045849 significantly suppressed cell growths of HKCI-2 and HKCI-10 cells in a dose-dependent fashion (p <0.001, p <0.0001, respectively) (Fig. 7A). Moreover, ST045849 suppressed tumorigenicity of HKCI-2 cells in vivo (Fig. 7B).
      Figure thumbnail gr7
      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.

      Discussion

      In this study, we demonstrated that OGT was silenced in normal human liver tissues, but frequently expressed in NAFLD-HCC patients and NAFLD-HCC cell lines. Similar upregulation of OGT expression in tumor tissue compared with adjacent non-tumorous tissues has also been observed in human breast, lung, colon, bladder and prostate cancer.
      • Gu Y.
      • Mi W.
      • Ge Y.
      • Liu H.
      • Fan Q.
      • Han C.
      • et al.
      GlcNAcylation plays an essential role in breast cancer metastasis.
      • Slawson C.
      • Pidala J.
      • Potter R.
      Increased N-acetyl-beta-glucosaminidase activity in primary breast carcinomas corresponds to a decrease in N-acetylglucosamine containing proteins.
      • Mi W.
      • Gu Y.
      • Han C.
      • Liu H.
      • Fan Q.
      • Zhang X.
      • et al.
      O-GlcNAcylation is a novel regulator of lung and colon cancer malignancy.
      • Rozanski W.
      • Krzeslak A.
      • Forma E.
      • Brys M.
      • Blewniewski M.
      • Wozniak P.
      • et al.
      Prediction of bladder cancer based on urinary content of MGEA5 and OGT mRNA level.
      OGT is a unique glycosyltransferase that catalyzes the O-GlcNAcylation. As a nutrient sensor, O-GlcNAcylation can relay the effects of excessive nutritional intake and play a significant role in cancer development through different mechanisms.
      • Hanover J.A.
      • Krause M.W.
      • Love D.C.
      Bittersweet memories: linking metabolism to epigenetics through O-GlcNAcylation.
      Induction of OGT in NAFLD-HCC patients and NAFLD-HCC cell lines suggested that OGT may behave as an oncogenic factor and its upregulation may contribute to the development and progression of NAFLD-associated-HCC.
      To investigate the functional significance of OGT in NAFLD-HCC, a series of in vitro and in vivo experiments were performed. The ectopic expression of OGT could significantly promote the cell proliferation in two normal hepatocyte cell lines LO2 and MIHA, while the knockdown of OGT suppressed the cell proliferation in two NAFLD-HCC cell lines HKCI-2 and HKCI-10 (Fig. 2). Furthermore, both subcutaneous xenograft and orthotropic liver implantation mouse models confirmed that OGT significantly promoted tumorigenicity of liver cancer (Fig. 3). The results collectively suggest that OGT acts as a tumor-promoting factor in NAFLD-HCC. The effects of OGT on cell migration and invasion were further evaluated. Ectopic expression of OGT in LO2 and MIHA cells promoted cell migration and invasion, while knockdown of OGT in HKCI-2 and HKCI-10 inhibited their migration and invasion abilities (Fig. 4). In keeping with these findings, OGT significantly promoted lung metastases in an orthotopic HCC xenograft model (Fig. 4E). The molecular mechanisms by which OGT exerts its pro-migration and pro-invasive abilities was shown to be mediated via inhibiting the expression of E-cadherin, an invasion suppressor
      • Vleminckx K.
      • Vakaet Jr., L.
      • Mareel M.
      • Fiers W.
      • van Roy F.
      Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role.
      and enhancing the expression of vimentin, a marker of cell invasion and metastasis.
      • Vuoriluoto K.
      • Haugen H.
      • Kiviluoto S.
      • Mpindi J.P.
      • Nevo J.
      • Gjerdrum C.
      • et al.
      Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer.
      We also found that OGT suppressed SIRT1 expression while induced FOXM1 expression (Fig. 4D), which is in keeping with previous study in breast cancer.
      • Ferrer C.M.
      • Lu T.Y.
      • Bacigalupa Z.A.
      • Katsetos C.D.
      • Sinclair D.A.
      • Reginato M.J.
      O-GlcNAcylation regulates breast cancer metastasis via SIRT1 modulation of FOXM1 pathway.
      As FOXM1 transcription factor is one of the key genes inducing tumor invasion and metastasis, our results confirmed the involvement of tumor invasion and metastasis in the oncogenic process of OGT in NAFLD-HCC. Taken together, these findings indicate that OGT promotes the metastatic and invasive potential of HCC through modulating the key elements of EMT E-cadherin and vimentin that contribute to its tumor-promotive effect in NAFLD-HCC, adding further weight for the oncogenic role of OGT in NAFLD-HCC.
      Altered cellular metabolism is one of the most important hallmarks of cancer. Cancer cells exhibit aberrant glucose metabolism characterized by aerobic glycolysis. Accelerated glucose uptake and glycolysis are main characteristics of cancer cells that allow them for intensive growth and proliferation. Recent evidences suggest that OGT may link hexosamine biosynthesis pathway to oncogenic signaling through regulating factors involved in glucose and lipid metabolism.
      • Itkonen H.M.
      • Minner S.
      • Guldvik I.J.
      • Sandmann M.J.
      • Tsourlakis M.C.
      • Berge V.
      • et al.
      O-GlcNAc transferase integrates metabolic pathways to regulate the stability of c-MYC in human prostate cancer cells.
      • Ferrer C.M.
      • Lynch T.P.
      • Sodi V.L.
      • Falcone J.N.
      • Schwab L.P.
      • Peacock D.L.
      • et al.
      O-GlcNAcylation regulates cancer metabolism and survival stress signaling via regulation of the HIF-1 pathway.
      Therefore, it is reasonable to speculate that upregulation of OGT in metabolic abnormalities may link the cancer cell metabolic reprogramming to the malignant progression of NAFLD-HCC. To investigate whether OGT confers an oncogenic role via abnormal lipid metabolism, we performed LC-MS in NAFLD-HCC cell lines. Palmitic acid, a saturated free fatty acid, was identified as the most significantly altered parameter mediated by OGT in HKCI-2 and HKCI-10 cells. In keeping with this, treatment with OGT inhibitor ST045849 decreased palmitic acid level (Fig. 5). Palmitic acid was recently reported to induce steatosis and cytotoxicity on rat hepatocytes,
      • Moravcova A.
      • Cervinkova Z.
      • Kucera O.
      • Mezera V.
      • Rychtrmoc D.
      • Lotkova H.
      The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture.
      and to alter lipid metabolism by multiple mechanisms.
      • Garcia-Ruiz C.
      • Mato J.M.
      • Vance D.
      • Kaplowitz N.
      • Fernandez-Checa J.C.
      Acid sphingomyelinase-ceramide system in steatohepatitis: a novel target regulating multiple pathways.
      We found that palmitic acid induced cell proliferation in NAFLD-HCC cells. On the other hand, inhibiting FASN, an enzyme responsible for the synthesis of palmitic acid, by FASN inhibitor C75 significantly suppressed HKCI-2 cell viability compared with the control cells (Fig. 5F). These findings suggest that the oncogenic effect of OGT in NAFLD-HCC is mediated at least by inducing the production of its metabolic regulator palmitic acid.
      ER stress becomes activated during metabolic alterations and their persistent activation contributes to lipotoxicity and cancer development.
      • Garcia-Ruiz C.
      • Mato J.M.
      • Vance D.
      • Kaplowitz N.
      • Fernandez-Checa J.C.
      Acid sphingomyelinase-ceramide system in steatohepatitis: a novel target regulating multiple pathways.
      To understand the mechanisms of the oncogenic role of OGT in NAFLD-HCC, we investigated the effect of OGT on ER stress and revealed that OGT significantly enhanced ER stress evidenced by increase in expression of ER stress master regulator GRP78 and ER stress responsive protein IRE1α in LO2 and MIHA cells transfected with OGT; which were confirmed by a reduced expression of GRP78 and IRE1α in HKCL-2 and HKCL-10 by OGT knockdown (Fig. 6). Moreover, palmitic acid could also activate ER stress in HKCI-10 cells (Fig. 6), further inferring the effect of OGT in contributing to ER stress in NAFLD-HCC. As such, we further measured the activation of critical ER stress-initiated oncogenic pathways such as JNK/c-jun/AP1 and NF-κB in NAFLD-HCC mediated by OGT. We revealed that OGT induced JNK/c-jun/AP1 signaling cascade by activating JNK, c-Jun and AP-1. Strong activation of JNK/c-jun/AP1 signaling has been observed in NAFLD
      • Farrell G.C.
      • van Rooyen D.
      • Gan L.
      • Chitturi S.
      NASH is an inflammatory disorder: pathogenic, prognostic and therapeutic implications.
      and in HCC
      • Seki E.
      • Brenner D.A.
      • Karin M.
      A liver full of JNK: signaling in regulation of cell function and disease pathogenesis, and clinical approaches.
      from others and in NAFLD-HCC in animal model as reported recently by us.
      • Shen J.
      • Tsoi H.
      • Liang Q.
      • Chu E.S.
      • Liu D.
      • Yu A.C.
      • et al.
      Oncogenic mutations and dysregulated pathways in obesity-associated hepatocellular carcinoma.
      In addition, NF-κB has long been considered a proinflammatory signaling pathway in NASH
      • Zhang X.
      • Shen J.
      • Man K.
      • Chu E.S.
      • Yau T.O.
      • Sung J.C.
      • et al.
      CXCL10 plays a key role as an inflammatory mediator and a non-invasive biomarker of non-alcoholic steatohepatitis.
      and a pro-oncogenic factor in HCC.
      • Han S.W.
      • Roman J.
      Fibronectin induces cell proliferation and inhibits apoptosis in human bronchial epithelial cells: pro-oncogenic effects mediated by PI3-kinase and NF-kappa B.
      In this study, we found that OGT activated NF-κB signaling cascade in NAFLD-HCC cells (Fig. 6). Moreover, palmitic acid induced JNK/c-Jun/AP1 and NF-κB signaling pathways in NAFLD-HCC cells. These results collectively indicated that the tumor-promoting role of OGT is mediated by regulating lipid metabolism at least through increasing palmitic acid, thereby activating ER stress, JNK/c-Jun/AP1 and NF-κB signaling cascades in NAFLD-HCC development (Fig. 7C).
      The observation that OGT plays an oncogenic role in NAFLD-HCC encouraged us to investigate if OGT inhibition could have any effect on suppressing NAFLD-HCC cell growth. Undeniably, OGT inhibitor ST04589 significantly suppressed cell proliferation in NAFLD-HCC both in vitro and inhibited tumor growth in xenograft tumors in nude mice (Fig. 7), inferring that OGT is a potential therapeutic target in NAFLD-HCC.
      In conclusion, this study demonstrated that OGT is commonly upregulated in NAFLD-associated HCC with a functional oncogenic role by promoting tumorigenesis and lung metastasis. We provide mechanistic insights into its oncogenic role in NAFLD-HCC and reveal that OGT regulates palmitic acid through generating palmitic acid and reactive oxygen species (ROS), which consequently activating ER stress, and ER stress-related JNK/c-Jun/AP1 and NF-κB pathways. OGT may serve as a potential NAFLD-HCC therapeutic target of NAFLD-HCC.

      Financial support

      This project was supported by research funds from RGC-GRF Hong Kong (766613, 14106415, 14111216), National Basic Research Program of China (973 Program, 2013CB531401), Theme-based Research Scheme of the Hong Kong Research Grants Council (T12-403-11), Collaborative Research Fund (HKU3/CRF11R, CUHK3/CRF/12R) of the Research Grant Council Hong Kong, Shenzhen Municipal Science and Technology R & D fund (JCYJ20130401151108652), Shenzhen Virtual University Park Support Scheme to CUHK Shenzhen Research Institute, Direct grant for Research 2013/2014, CUHK (4054100), The Macao Science and Technology Development Fund (087/2013/A3).

      Conflict of interest

      The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details.

      Authors’ contributions

      WX was involved in study design, conducted the experiments and drafted the paper; XZ, JW, KL and DL performed the experiments; GGC and PBSL provided NAFLD-HCC human samples; NW performed transcriptome sequencing and provided human NAFLD-HCC cell lines; XZ and LF designed and revised the paper; JY designed, supervised the study and critically revised the paper.

      Supplementary data

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