O‐GlcNAc transferase activates stem‐like cell potential in hepatocarcinoma through O‐GlcNAcylation of eukaryotic initiation factor 4E

Abstract O‐GlcNAcylation catalysed by O‐GlcNAc transferase (OGT) is a reversible post‐translational modification. O‐GlcNAcylation participates in transcription, epigenetic regulation, and intracellular signalling. Dysregulation of O‐GlcNAcylation in response to high glucose or OGT expression has been implicated in metabolic diseases and cancer. However, the underlying mechanisms by which OGT regulates hepatoma development remain largely unknown. Here, we employed the lentiviral shRNA‐based system to knockdown OGT to analyse the contribution of OGT in hepatoma cell proliferation and stem‐like cell potential. The sphere‐forming assay and western blot analysis of stem‐related gene expression were used to evaluate stem‐like cell potential of hepatoma cell. We found that the level of total O‐GlcNAcylation or OGT protein was increased in hepatocellular carcinoma. OGT activated stem‐like cell potential in hepatoma through eukaryotic initiation factor 4E (eIF4E) which bound to stem‐related gene Sox2 5'‐untranslated region. O‐GlcNAcylation of eIF4E at threonine 168 and threonine 177 protected it from degradation through proteasome pathway. Expression of eIF4E in hepatoma was determined by immunostaining in 232 HCC patients, and Kaplan‐Meier survival analysis was used to determine the correlation of eIF4E expression with prognosis. High glucose promoted stem‐like cell potential of hepatoma cell through OGT‐eIF4E axis. Collectively, our findings indicate that OGT promotes the stem‐like cell potential of hepatoma cell through O‐GlcNAcylation of eIF4E. These results provide a mechanism of HCC development and a cue between the pathogenesis of HCC and high glucose condition.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is the most common primary liver malignancy worldwide, especially in developing countries. [1][2][3] Formation of HCC results from multiple risk factors, including HBV or HCV infection, cirrhosis, excessive alcohol consumption, and a variety of genetic factors. 4 Increasing evidences reveal that diabetes is associated with an increased HCC incidence and has been far more than HBV, HCV, or alcoholic liver disease. A study in Taiwan populations has reported that the incidence of HCC was twice higher in the diabetes patients group compared with the non-diabetes patients. 5 Similarly, HBEl-Serag research showed that the proportion of HCC patients with diabetes (43%) was significantly greater than non-cancer controls (19%). 6 However, the potential mechanism for the pathogenesis of HCC with diabetes is incompletely understood.
High glucose increases intracellular concentrations of UDP-GlcNAc, resulting in increased global O-GlcNAcylation. 7 O-GlcNAc is an O-linked-β-N-acetylglucosamine moiety attached to the residue of serine or threonine on nuclear or cytoplasmic proteins. 8 The addition of O-GlcNAc to proteins is catalysed by O-GlcNAc transferase (OGT), and its removal is catalysed by O-GlcNAcase (OGA). 9 O-GlcNAcylation regulates such diverse cellular processes as nutrient sensing, cell cycle progression, transcription, translation, epigenetic regulations, and protein-protein interactions. [10][11][12][13] O-GlcNAcylation is believed to play a role in a variety of signalling cascades that mediate glucose homoeostasis and stress responses. 14 Elevated O-GlcNAcylation has been described in different cancer types including breast, prostate, liver, colon, lung, and chronic lymphocytic leukaemia. [15][16][17][18][19] These studies indicate that high O-GlcNAcylation seems to be a general feature of cancer cells and contributes to tumorigenesis. In hepatoma, Zhu et al reported that O-GlcNAcylation was higher in cancer tissues compared to normal tissues. Moreover, O-GlcNAcylation was higher again in patients diagnosed a recurrence of hepatocarcinoma. 17 Considering that high glucose increases global O-GlcNAcylation, elucidating the role of O-GlcNAcylation in hepatoma progression helps to improving our understanding of the association between diabetes and HCC. However, the contribution of O-GlcNAcylation in hepatoma development remains largely unknown.
In this study, we investigated the roles of OGT in hepatoma development. We found that OGT was highly expressed in hepatocellular carcinoma. OGT activated stem-like cell potential of hepatoma cell through O-GlcNAcylation of eukaryotic initiation factor 4E. Eukaryotic initiation factor 4E, a key translation factor, bound to stem-related gene Sox2 5'untranslated region. High glucose promoted stem-like cell potential of hepatoma cell through OGT-eIF4E axis. Our findings indicate that OGT promotes the stem-like cell potential of hepatoma cell through O-GlcNAcylation of eIF4E, providing a mechanism of HCC development.

| Human tumour tissues
Hepatocarcinoma tissues and matching tumour adjacent normal tissues were obtained from the ZhongShan Hospital, Shanghai. The procedures related to human subjects were approved by the Ethics Committee of the Institutes of Biomedical Sciences, Fudan University. Experiments were undertaken with the understanding and written consent of each subject.

| Plasmids and transfection
PCRs were performed to amplify eIF4E cDNA and inserted eIF4E cDNA into Lv-Flag vector with Flag tag at N-terminal region, HA tag at C-terminal region. eIF4E single mutant (T168A or T177A) and double mutant (T168, 177A) were constructed. The pCMV-OGT-myc plasmid was provided as described previously. 20

| Mapping of O-GlcNAc site using mass spectrometry
To map O-GlcNAc sites, Nano-LC-ESI-MS/MS was performed as previously described. 21 Overexpressed wild-type Flag-eIF4E proteins in HEK293T cells were purified using ANTI-FLAG M2 affinity gel (Sigma-Aldrich, A2220) and subjected to SDS-PAGE.

| Tissue microarray and immunohistochemistry
Tissue microarray was constructed and immunohistochemistry was carried out as described previously. 22,23 The eIF4E and O-GlcNAcylation immunostaining intensities were semi-quantitatively scored as: 0, negative; 1, weak; 2, moderate; and 3, strong. All samples were anonymized and independently scored by two investigators. In case of disagreement, the slides were re-examined and a consensus was reached by the observers.

| Immunoprecipitation assays
Cells were washed with cold phosphate-buffered saline (PBS), then lysed on ice with RIPA lysis buffer (10 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100 (v/v), 0.5% sodium deoxycholate (w/ v), 0.1% sodium dodecyl sulfate (w/v), and protease inhibitors) for immunoprecipitation. The cell extracts were then centrifuged at 12 000 g for 10 minutes at 4°C. The supernatants were pre-cleared with sepharose-labelled protein G (Roche) for 2 hours. The beads were discarded after a 1 minute centrifugation at 2500 g, and the supernatants was incubated with interest primary antibodies and rocked at 4°C overnight. Controls for immunoprecipitation specificities were performed with normal mouse or rabbit IgG (Santa Cruz Biotechnology).

| Cell proliferation assays
Cell proliferation assays was analysed using the commercial Cell Counting Kit (CCK8) in accordance with the manufacturer's instructions. In brief, cells were seeded onto 96-well plates (Corning) at a density of 5 × 10 3 cells/well, and incubated for 2 hours to allow cell adherence to the plate. CCK8 reagents (Dojindo, CK04) were added to each well and incubated for 2 hours at 37°C. Absorbance at 450 nm was measured using Microplate Reader (Bio-Tech Instruments, USA). The results were plotted as means ± SD of three separate experiments.

| Statistical analyses
Statistical analysis of the data was calculated by using two-tailed Student's t tests (*P < 0.05, **P < 0.01) on GraphPad Prism. All values included in figures represent mean ± SD Error bars represent SD. Data are representative of at least three independent experiments.
The difference of O-GlcNAcylation and OGT protein level between hepatoma tissues and adjacent normal liver tissues. A, The level of total O-GlcNAcylation was analysed in eight paired HCC specimens with their corresponding non-cancerous specimens by western blotting. β-actin expression was served as a loading control. B, OGT protein expression levels were analysed in 14 paired HCC specimens by western blotting. β-actin expression was served as a loading control. C, OGT values were calculated from images in B. Data represent mean ± SD of at least three independent experiments. **P < 0.01. D, Representative immunohistochemical staining of O-GlcNAcylation in liver cancer tissues. E, Number of different staining of O-GlcNAcylation patients was analysed. F, Representative immunohistochemical staining of O-GlcNAcylation in liver cancer tissues and peri-tumour liver tissues. G, Quantitative analysis of the level of O-GlcNAcylation in liver cancer tissue microarrays showed that the expression of O-GlcNAcylation was increased in liver cancer (P = 0.005). Immunohistochemical staining was estimated, as follows: negative: staining intensity ≤10%; weak: staining intensity in 10%-20%; moderate: staining intensity in 20%-50%; strong: staining intensity > 50%. T: tumour sample; N: non-cancerous sample  Figure 2B). Considering that cancer stem cell is responsible for the initiation of HCC. 24 We next examined the contribution of OGT in stem-like cell potential in hepatocarcinoma. The sphere-forming assays have been widely used to evaluate the self-renewal ability of cancer stem cell. 25 Knockdown of OGT obviously decreased the diameter and number of tumorsphere in Huh7 and PLC/PRF/5 cells in conditional culture ( Figure 2C-H). We further demonstrate that down-regulation of OGT expression decreased stem-like cell potential in Huh7 cells. The population of cancer stem cells was examined by flow cytometry using stem cell marker, like CD133. Flow cytometry analysis showed that the percentage of CD133 + cells decreased from 47.8% to 32.1% after OGT knockdown in Huh7 cells ( Figure 2I).
Meanwhile, some reports have showed that stem-like potential proteins, such as Sox2, OCT4, and KLF4, which can augment stem-cell function. And these proteins can be used as stemness-related markers. [26][27][28][29] The expression of stem-like cell potential proteins (Sox2, OCT4 and KLF4) were also reduced in Huh7 cells with OGT knockdown ( Figure 6J). Together, these data suggest that OGT promotes cell proliferation and activates stem-like cell potential in hepatocarcinoma.

| eIF4E is O-GlcNAcylated in hepatoma
We next aimed to explore the mechanisms underlying OGT promotes stem-like cell potential in hepatocarcinoma. Previous study reported that O-GlcNAc modification of ribosomal subunits contributed to the translational machinery. 12 Of numerous eukaryotic initiation factors in translational machinery, eIF4E is a key player in the regulation of translation initiation and is required for the recruitment of specific mRNAs to the ribosome. 30 eIF4E has been reported to regulate the self-renewal of glioma-initiating cell. 31 These findings promoted us to investigate whether eIF4E was O-

| Mutation of eIF4E O-GlcNAc sites at threonine 168 and threonine 177 reduces its protein stability
To determine the location of the O-GlcNAc site(s) on eIF4E by mass spectrometry analysis, Flag-eIF4E was purified from HEK293T cells F I G U R E 5 OGT knockdown reduces eIF4E protein expression and higher expression of eIF4E indicates a poor prognosis in HCC patients. A, The protein level of eIF4E was analysed by western blotting in Huh7 infected with control shRNA, OGT shRNA1 and OGT shRNA2 lentivirus. β-actin expression was served as a loading control. B, OGT values were calculated from images in A. Data represent mean ± SD of at least three independent experiments. **P < 0.01. C, eIF4E values were calculated from images in A. Data represent mean ± SD of at least three independent experiments. *P < 0.05; **P < 0.01. D, The protein level of eIF4E was analysed by western blotting in PLC/PRF/5 cells infected with control shRNA, OGT shRNA1, and OGT shRNA2 lentivirus. β-actin expression was served as a loading control. E, OGT values were calculated from images in D. Data represent mean ± SD of at least three independent experiments. **P < 0.01. F, eIF4E values were calculated from images in D. Data represent mean ± SD of at least three independent experiments. **P < 0.01; n.s, no significance. G, The protein level of eIF4A and eIF4G were analysed by western blotting in Huh7 and PLC/PRF/5 cells infected with control shRNA or OGT shRNA2 lentivirus. β-actin expression was served as a loading control. H, eIF4G, and eIF4A values were calculated from images in G. Data represent mean ± SD of at least three independent experiments. n.s, no significance. I, Representative immunohistochemical staining of eIF4E in liver cancer tissues and peri-tumour liver tissues. J, Quantitative analysis of liver cancer tissue microarrays showed that the expression of eIF4E was higher in liver cancer tissues than in normal liver tissues (P < 0.

| Knockdown of OGT reduces eIF4E expression and high expression of eIF4E predicts poor prognosis of HCC
The finding that O-GlcNAcylation participated in eIF4E stability motivated us to investigate whether OGT regulated eIF4E protein F I G U R E 6 Knockdown OGT inhibits proliferation and tumorsphere formation of hepatoma cell through reducing eIF4E expression. A, Huh7 and PLC/PRF/5 cells were infected with control shRNA, OGT shRNA2 alone, or with wild-type eIF4E lentivirus. The cell lysates were harvested for western blotting analysis using indicated antibodies. β-actin expression was served as a loading control. B, Cell proliferation of Huh7 and PLC/PRF/5 cells infected with lentiviruses as in panel (A) were measured with CCK8 assay. (C-H) Huh7 and PLC/PRF/5 cells infected with lentiviruses as in panel (A) were seeded into 96-well plates. After 12 d, tumorsphere were counted and quantified. Representative images of sphere (scale bars, 100 μm) were shown (C, F). The diameter of sphere (D, G) and number of sphere (E, H) were count. Data represent mean ± SD of at least three independent experiments. The two-tailed Student's t tests were used. **P < 0.01. I, Huh7 cells expressing either OGT shRNA2 alone or with wild-type eIF4E lentivirus were incubated with PE-labelled anti-AC133 antibody. The percentages of CD133 + cells in graphs were analysed by flow cytometry. Black line, control IgG staining; red line, CD133 staining. J, Cell lysates were examined by western blotting with indicated antibodies. The right panel showcases relative protein amounts of different groups. Error bars represent ±SD of triplicate experiments. **P < 0.01; n.s, no significance. K, Huh7 cells were collected and subjected to immunoprecipitation with antibody against eIF4E or normal mouse IgG. Total RNAs were purified from immunocomplexes and subjected to RT-PCR to measure Sox2, OCT4, and KLF4 mRNAs associated with eIF4E  OGT activated stem-like cell potential in hepatoma cell partly through up-regulation of eIF4E expression. The eukaryotic translation initiation factor 4E is a key regulator of protein synthesis, which is generally the rate-limiting factor recruits mRNAs to eIF4F. 30 Uncontrolled of eIF4E activity or expression in various cancers stimulates cellular proliferation and malignant transformation. 39,40 Thus, eIF4E has been considered as a therapeutic target in cancer. Previous studies indicate that eIF4E regulates function of common tumour cells. 40 Here, we found that ectopic expression of eIF4E increased the diameter and number of tumorsphere and increased the expression of stem-like cell potential proteins (Sox2, OCT4). Furthermore, 5ʹ-UTR of Sox2 mRNA but not OCT4 mRNA, was tightly bound to eIF4E by RNA-ChIP assay.
The literature suggest that cellular mRNAs most sensitive to alterations in eIF4E availability and eIF4F complex formation. In tumours, elevated eIF4E function selectively and disproportionately increases translation of weak mRNAs. These mRNAs with G/C-rich 5ʹ-UTR had encoded potent growth, and survival factors notoriously involved in malignancy. 40 Accordingly, we found that 5ʹ-UTR of Sox2 had rich G/C bases compared to 5ʹ-UTR of OCT4. Our data indicate that eIF4E regulates the stem-like cell potential of hepatoma cell, providing a new mechanism that eIF4E promotes cancer development.
Our data also provide evidence that O-GlcNAcylation increases the stability of eIF4E protein. The activity or expression of eIF4E is controlled by its binding proteins and by upstream signalling pathways. For example, phosphorylation of eIF4E on S209 by MNK1/2, released eIF4E from eIF4E-binding proteins (4EBP1), resulting in the activation of eIF4E. 41 Phosphorylation of eIF4E on S209 is elevated in human cancer and is associated with tumour aggressiveness and poor patient outcome. 42 In addition, eIF4E is ubiquitinated at Lys159, suggesting the proteasome-dependent proteolysis of eIF4E. 43 Increasing researches suggest OGT participates in protein stability. 32,33 In this study, O-GlcNAcylation of eIF4E at Thr168 or