Yongxia Qiao,1 Xiao Zhang,2 Yue Zhang,3 Yulan Wang,2 Yanfeng Xu,4 Xiangfan Liu,5

Fenyong Sun,2 and Jiayi Wang2,6

HighGlucose Stimulates Tumorigenesisin Hepatocellular Carcinoma CellsThrough AGER-DependentO-GlcNAcylation of c-JunDiabetes 2016;65:619–632 | DOI: 10.2337/db15-1057

Epidemiologic studies suggest that hepatocellular car-cinoma (HCC) has a strong relationship with diabetes.However, the underlying molecular mechanisms stillremain unclear. Here, we demonstrated that high glucose(HG), one of the main characteristics of diabetes, wascapable of accelerating tumorigenesis in HCC cells.Advanced glycosylation end product–specific receptor(AGER) was identified as a stimulator during this process.Mechanistically, AGER activated a hexosamine biosyn-thetic pathway, leading to enhanced O-GlcNAcylationof target proteins. Notably, AGER was capable of in-creasing activity and stability of proto-oncoprotein c-Junvia O-GlcNAcylation of this protein at Ser73. Interestingly,c-Jun can conversely enhance AGER transcription.Thereby, a positive autoregulatory feedback loop thatstimulates diabetic HCC was established. Finally, wefound that AG490, an inhibitor of Janus kinase, hasthe ability to impair AGER expression and its functionsin HCC cells. In conclusion, AGER and its functions tostimulate O-GlcNAcylation are important during livertumorigenesis, when high blood glucose levels areinadequately controlled.

Diabetes has been established as one of the mostimportant risks in progression of hepatocellular carcinoma(HCC) (1,2). As the prevalence of both diabetes and HCCis substantially increasing worldwide (3), it is urgent to

understand the underlying molecular mechanisms ofhow diabetes promotes HCC. It is well established thatliver is fundamentally important for glucose metabolism,and hyperglycemia is one of the main characteristics ofdiabetes. However, whether and how high glucose (HG)stimulates liver tumorigenesis remain largely unclear.

HG reacts with long-lived proteins to form advancedglycation end products. Advanced glycation end productsaccount for many diabetes complications through theirengagement to advanced glycosylation end product–specific receptor (AGER) (4–6). Inhibition of AGER isregarded as a therapeutic option to prevent diabetescomplications (7). Despite an epidemiologic study thathas demonstrated that AGER may be involved in liverinjury and subsequent carcinogenesis (8), the exact rolesof AGER in liver tumorigenesis are still unknown.

Glucose, glutamine, and acetyl-coA are required for thehexosamine biosynthetic pathway (HBP) to synthesize uri-dine diphosphate (UDP)–N-acetyl-D-glucosamine (UDP-GlcNAc) (9). The HBP couples metabolic flux to controlcell proliferation and participates in O-GlcNAcylation oftarget proteins through O-linked N-acetylglucosamine(GlcNAc) transferase (OGT) (9–11). Despite the fact thatOGT and O-GlcNAcylation have been implicated as veryimportant in the promotion of tumorigenesis in varioustypes of cancer (10,11), very few studies have focused ontheir function in HCC. Given that HBP and subsequent

1School of Public Health, Shanghai Jiao Tong University School of Medicine,Shanghai, China2Department of Clinical Laboratory Medicine, Shanghai Tenth People’s Hospital ofTongji University, Shanghai, China3Department of Central Laboratory, Shanghai Tenth People’s Hospital of TongjiUniversity, Shanghai, China4Department of Pharmacy, Shanghai Municipal Hospital of Traditional ChineseMedicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China5Faculty of Medical Laboratory Science, Shanghai Jiao Tong University School ofMedicine, Shanghai, China6Tongji University Advanced Institute of Translational Medicine, Shanghai, China

Corresponding authors: Fenyong Sun, sunfenyong@126.com, and Jiayi Wang,karajan2@163.com.

Received 30 July 2015 and accepted 24 November 2015.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1057/-/DC1.

Y.Q., X.Z., and Y.Z. are co–first authors.

© 2016 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, andthe work is not altered.

Diabetes Volume 65, March 2016 619

SIG

NALTRANSDUCTIO

N

O-GlcNAcylation are critical for both glucose metabolismand tumorigenesis, we supposed that they might be in-volved in HG-associated HCC.

Here, we demonstrate that HG is capable of pro-moting liver tumorigenesis via AGER. AGER stimulatedO-GlcNAcylation and activation of c-Jun via enhancingHBP and OGT. We also uncovered that c-Jun could con-versely upregulate AGER. Altogether, we establish a posi-tive feedback loop from HG, AGER, and HBP to c-Jun,which may be helpful in the treatment of diabetic HCC.

RESEARCH DESIGN AND METHODS

Mice ExperimentsInsulin-deficient HG and control mice models were con-structed by intraperitoneal injection of streptozocin (STZ)(resolved in solution of 0.1 mol/L citrate acids, pH 4.4) andsaline, respectively, into 5-week Balb/c male mice (Bikai,Shanghai, China). To investigate dose-dependent effects, weinjected STZ at a final concentration of 40 mg/kg i.p. (STZ1)and 200 mg/kg i.p. (STZ2), respectively, into the mice. Themice were treated with STZ1 once a day for four consecutivedays or treated with STZ2 only once. The STZ2-treated micewere also injected with or not with insulin (Sigma-Aldrich,St. Louis, MO) at a final concentration of 2 units/kg i.p. eachday. For xenograft mouse experiments, Bel-7402 cells (5 3106 cells) under different treatments were subcutaneouslyinjected into STZ1-, STZ2-, combined STZ2- and insulin-, orsaline-treated 8-week-old athymic nude mice (Bikai). Thetumor size was measured 8 weeks after injection, and thetumor volume was calculated as 0.5 3 L 3 W2, where L islength and W is width. All mouse experiments were per-formed according to the institution guidelines of ShanghaiTenth People’s Hospital.

Tissue Samples, Cell Culture, and VectorsSlides of tissue microarray analysis (TMA) were purchasedfrom U.S. Biomax (Xi’an, China). Fresh HCC and adjacentnormal liver tissues were acquired at Shanghai RuijinHospital with institutional approval. The HCC cell linesBel-7402, SMMC-7721, Huh7, HepG2, SK-Hep1, andBel-7404 and hepatocyte line HL-7702 cells were culturedin DMEM. Cells were treated with AG490 (Beyotime,Haimen, China) at a final concentration of 50–150 mmol/L,glucose (Sigma-Aldrich) at a final concentration of 5.5–50 mmol/L, PUGNAc (Sigma-Aldrich) at a final concen-tration of 25 mmol/L, GlcNAc (Sigma-Aldrich) at aconcentration of 4 mmol/L, and cycloheximide (CHX)(Sigma-Aldrich) at a final concentration of 0.1 mg/mL,respectively. The cDNA encoding human AGER was pur-chased from OriGene (Beijing, China) and subcloned intothe pLJM-based lentiviral vector with a COOH-terminalMyc tag. The expressing plasmids of COP1, OGT-Myc,c-Jun–Myc, and OGT–short hairpin RNA (sh)4 wereobtained from OriGene. AGER-sh11, AGER-sh12, OGT-sh7,c-Jun–sh2, c-Jun–sh3, and c-Jun–sh4 were obtainedfrom GeneChem (Shanghai, China). Luciferase (LUC) re-porter of c-Jun was purchased from Beyotime. Promoter

regions of human AGER gene were PCR amplified fromgDNA of Bel-7402 cells and cloned into pGL4.21 (Promega,Madison, WI) vectors. The wild-type (WT) and S73A c-Jun–FLAG were subcloned into pcDNA3.1 (+) plasmids. Themutants were constructed by overlapping PCR. The pri-mers used for this study are listed in SupplementaryTable 1.

Immunohistochemistry, Immunofluorescence, andWestern BlottingThe primary antibodies used in immunohistochemistry(IHC) were anti-AGER (no. ab54741; Abcam, Hong Kong,China), anti–cleaved caspase 3 (no. 9664; Cell SignalingTechnology [CST], Boston, MA), anti-Ki67 (no. ab15580;Abcam), anti–c-Jun (no. 9165; CST), and anti–O-GlcNAc(no. ab2739; Abcam).

For immunofluorescence (IF), the primary antibodiesused were anti-AGER (no. ab54741; Abcam), anti-OGT(no. ab184198; Abcam), anti-histone (no. 4499; CST), anti–p-c-Jun (no. 3270; CST), and anti–c-Jun (no. 9165; CST).

For Western blotting (WB), the primary antibodies usedwere anti-GAPDH (no. 5174; CST), anti-OGT (no. ab184198or no. ab177941; Abcam), anti–Myc-tag (no. 2276 or no.2278; CST), anti-AGER (no. ab54741 or no. ab37647;Abcam), anti-GlcNAc (no. ab2739; Abcam), anti-ubiquitin(no. 3936; CST), anti–c-Jun (no. 9165 or no. 2315; CST),anti–p-c-Jun (for p-S73, no. 3270, and for p-S63, no. 9261;CST), anti-COP1 (no. sc-166799; Santa Cruz Biotechnology,Santa Cruz, CA), anti-PDK1 (no. 3062; CST), anti-EDG2(no. ab166903; Abcam), anti-CD44 (no. ab51037; Abcam),anti–c-Abl (no. 2862; CST), anti-HSP27 (no. ab109376;Abcam), anti-FoxA1 (no. ab170933; Abcam), anti-CD166(no. ab109215; Abcam), anti-MCAM (no. ab134065;Abcam), anti-TRIB2 (no. H00028951-m04; Abnova, Taipei,Taiwan), anti-p70S6K (no. ab32359; Abcam), anti-p38(no. 9218; CST), anti-ERK1/2 (no. 4695; CST), anti-eIF4E(no. ab33766; Abcam), anti-MEK1 (no. 2352; CST), anti-SRSF1 (no. ab129108; Abcam), anti–c-Kit (no. 3074; CST),anti–c-Fos (no. 2250; CST), and anti-FLAG (no. 8146 orno. 2368; CST).

All IHC, IF, and WB were performed conventionally,and the protocols are available elsewhere.

LUC Reporter, Cell Proliferation, Caspase 3/7Activity, Soft Agar Colony Formation, ChromatinImmunoprecipitation, Quantitative RT-PCR, GasChromatography/Mass Spectrometry, andMetabolite AnalysisLUC reporter, cell proliferation, caspase 3/7 activity,soft agar colony formation, and quantitative RT-PCR(qPCR) assays were performed as described previously(12,13). Chromatin immunoprecipitation (ChIP) wasperformed using the ChIP-IT express kit from ActiveMotif (Carlsbad, CA). Protein-DNA complexes were incu-bated with 3 mg anti–c-Jun antibodies (no. 9165; CST)or IgG (no. sc-2345; Santa Cruz Biotechnology). The pri-mers used for qPCR are listed in Supplementary Table 1.Gas chromatography/mass spectrometry was performed by

620 High Glucose Promotes Tumorigenesis via AGER Diabetes Volume 65, March 2016

Bojian Biotechnology, Ltd. (Shanghai, China). Metaboliteswere analyzed by corresponding kits from Sigma-Aldrich.Glucose was analyzed by a kit from Applygen (Beijing, China).

CoimmunoprecipitationCoimmunoprecipitation was performed as previously de-scribed (12,13). The reagents used were protein A/G-Sepharose(Novex, Oslo, Norway) and Western/immunoprecipation(IP) lysis buffer (Beyotime) with 0.1% SDS (used for dena-tured IP for ubiquitin and O-GlcNAc). The antibodies usedfor IP were anti-FLAG (no. 2368; CST), anti–c-Jun (no. 9165or no. 2315; CST), and anti-AGER (ab37647; Abcam).

Protein Ligation AssayThe protein ligation assay was performed to identify theinteraction between OGT and c-Jun using the DuolinkIn Situ Red Starter Kit (mouse/rabbit) (Sigma-Aldrich,Uppsala, Sweden) as previously described (14).

In Vitro O-GlcNAcylation of c-JunReaction mixtures containing 1 mmol/L purified humanc-Jun protein (no. H00003725-P01; Abnova), 0.125 mmol/Lhuman OGT (H00008473-P01; Abnova), and 10 mmol/LUDP-GlcNAc (Sigma-Aldrich) in a buffer of 50 mmol/L Tris/HCl (pH 7.5) and 1 mmol/L dithiothreitol were incubated at37°C for 90 min. Then coimmunoprecipitation was per-formed by anti–c-Jun antibodies (no. 9165; CST) anddetected by WB using anti–c-Jun (no. 2315; CST) andanti-GlcNAc (no. ab2739; Abcam) antibodies.

Enzymatic Labeling of O-GlcNAc SitesFirst, immunoprecipitated c-Jun with protein A/G-Sepharose (Novex, Oslo, Norway) was added into reactionbuffer (20 mmol/L HEPES, pH 7.9; 50 mmol/L NaCl;1 mmol/L PUGNAc; and 5 mmol/L MnCl2 with proteaseand phosphatase inhibitors). Next, 2 mL Gal-T1Y289L(Invitrogen, Carlsbad, CA) and 2 mL 0.5 mmol/L UDP-GalNAz (Invitrogen) were added into reaction buffer to avolume of 20 mL. The reaction was performed overnightat 4°C. The beads were washed twice with reaction bufferto remove excess UDP-GalNAz. The samples were thenreacted with biotin alkyne (Invitrogen) or tetramethyl-6-carboxyrhodamine (TAMRA) alkyne (Invitrogen) accordingto the manufacturer’s instructions. Biotin- or TAMRA-labeled samples were finally detected by WB using horse-radish peroxidase (HRP)–labeled streptavidin (no. A0303;Beyotime) or antibodies against TAMRA (no. A6397;Invitrogen).

Statistical AnalysisTests to examine the differences between groups includedthe Student t test and x2 test. P , 0.05 was regarded asstatistically significant.

RESULTS

AGER Was Essential for HG-Stimulated LiverTumorigenesisCompared with the normal glucose level (5.5 mmol/L),increasing HG levels (15–50 mmol/L) resulted in adose-dependent induced cell proliferation (Fig. 1A)

and colony-formation capacity (Fig. 1B and C), whereas areduced caspase 3/7 activity (Fig. 1D) was seen in HCC celllines Bel-7402 and SMMC-7721, which have shown highcarcinogenic properties in our previous studies (13,14).

Then, mice were intraperitoneally injected with twodifferent concentrations of STZ to induce HG. We foundthat STZ could induce a dose-dependent elevation ofserum glucose. By contrast, the HG that resulted fromSTZ-induced insulin deficiency could be reversed byintraperitoneal injection of insulin (Fig. 1E). Comparedwith the saline-treated control, there were more dividingcells with decreased levels of cleaved caspase 3, butincreased levels of Ki67 can be detected in the liver ofSTZ-treated Balb/c mice (Fig. 1F), suggesting that HGpromotes proliferation but inhibits apoptosis. In xenograftmice models, a dose-dependent increasing xenograft tumorvolume was detected in nude mice treated with increasingconcentrations of STZ (Fig. 1G). Similar to the serum glu-cose level (Fig. 1E), insulin could reverse STZ-acceleratedxenograft growth (Fig. 1G). These results demonstratedthat HG is capable of enhancing liver tumorigenesis.

Next, we investigated whether AGER is glucose inducibleand important for tumorigenesis in HCC cells. AGERexpression was tested in saline- and STZ-treated Balb/cmice, and it was found that STZ could induce a dose-dependent elevation of AGER in the liver but could notin the lung and colon. Interestingly, insulin was able toreverse STZ-induced elevation of AGER in the liver (Fig. 1H).These results suggested that AGER has important func-tions in liver when glucose is elevated. Also, AGER couldbe dose-dependently induced by increasing concentrationsof HG in both Bel-7402 and SMMC-7721 cells (Fig. 1I). Inhuman HCC tissues, AGER was highly upregulated com-pared with the paired adjacent normal liver tissues (Fig.1J and K). Moreover, AGER had much higher expressionlevels in established HCC cell lines than that in the trans-formed hepatocyte line (Fig. 1L). To test whether AGER isimportant for HCC malignant function, we used two in-dependent shRNAs against AGER with high knockdownefficiency (Fig. 1M). Contrary to the procarcinogenicroles of HG, depletion of AGER led to a significant re-duction of cell proliferation (Fig. 1N) and colony-formationcapacity (Fig. 1O and P) accompanied by an induction ofcaspase 3/7 activity in both Bel-7402 and SMMC-7721cells (Fig. 1Q). Notably, HG-enhanced transformativephenotype was remarkably inhibited in AGER-depletedcells compared with the control (Supplementary Fig. 1A–C).In addition, knockdown of AGER in Bel-7402 cells impairedxenograft growth and inhibited a significant increase oftumor volume in STZ-treated nude mice compared withthe saline-treated control (Fig. 1R). Together, these dataillustrated an essential role of AGER in the HG-inducedliver tumorigenesis.

AGER Linked With HBP in HCC CellsFor investigation of the function of AGER in metabolism,gas chromatography/mass spectrometry was performed.

diabetes.diabetesjournals.org Qiao and Associates 621

Among 366 metabolites tested, 22 metabolites were com-monly changed in both Bel-7402 and SMMC-7721 cellswhen AGER was ectopically expressed (Fig. 2A and Sup-plementary Table 2). Among these metabolites, glutamineand glycogenic amino acids including isoleucine, phenylala-nine, tyrosine, and threonine were elevated, while glutamicacid and lactic acid were reduced (Fig. 2A). All these changedmetabolites are important for HBP (see schematic diagramof HBP [Fig. 2B]). To further verify the results gained fromoverexpression of AGER, we knocked down AGER inBel-7402 and SMMC-7721 cells and reexamined metabolitesinvolved in the HBP. As expected, lactic acid and glutamic

acid were induced, while glutamine, acetyl-coA, andfructose-6-phosphate were reduced (Fig. 2C). However, gly-cogen, a metabolite not directly involved in HBP, wasnot changed (Fig. 2C). We also found that knockdownof AGER reduced both glucose consumption from cul-ture media (Fig. 2D) and intracellular glucose levels(Fig. 2E), while overexpression of AGER led to theopposite effects (Fig. 2D and E). These results sug-gested that AGER may enhance metabolic flux to stim-ulate HBP.

To test whether AGER also regulates expression ofgenes involved in HBP, we performed qPCR to test mRNA

Figure 1—HG stimulated tumorigenesis through upregulation of AGER. A–D: HG stimulated tumorigenesis. Cell proliferation, colony-formationcapacity, and caspase 3/7 activities were measured by a methylthiazolyldiphenyltetrazolium bromide (MTT)–based assay (A), soft agar colony-formation assay (B and C), and caspase 3/7 Glo LUC assay (D) after addition of glucose to a final concentration of 5.5–50 mmol/L in Bel-7402and SMMC-7721 cells. Scale bar, 200 mm. E: Serum glucose concentration of Balb/c mice treated with saline or STZ with or without insulin.n = 5/group. Insulin, 2 units/kg/day. F: Liver sections from saline- or STZ-treated Balb/c mice stained by hematoxylin-eosin (H&E) or IHC usinganti–cleaved caspase 3 or anti-Ki67 antibodies. Scale bar, 500 mm. G: Bel-7402 cells (5 3 106) were subcutaneously injected into nude miceunder the same treatments as in E. Tumor size was measured after injection of cells for 8 weeks. n = 5/group. H: AGER protein expression wasmeasured by WB in liver, lung, and colon from saline- or STZ-treated Balb/c mice. Treatments of mice were the same in E. I: AGER proteinexpression in Bel-7402 and SMMC-7721 cells after addition of glucose to a final concentration from 5.5 to 50 mmol/L. J: AGER proteinexpression in paired HCC and adjacent normal liver tissues. K: Representative IHC images of AGER in HCC and its adjacent normal livertissues. Scale bar, 500 mm. L: AGER protein expression in established hepatocyte (HL-7702) and HCC cell lines as indicated.M: AGER proteinexpression in control and Bel-7402 or SMMC-7721 cells with AGER knocked down. N–Q: Knockdown of AGER reduced tumorigenesis.Cell proliferation, colony-formation capacity, and caspase 3/7 activities were measured by an MTT-based assay (N), soft agar colony-formation assay (O and P), and caspase 3/7 Glo LUC assay (Q) in Bel-7402 and SMMC-7721 cells before and after AGER knockdown.Scale bar, 200 mm. R: Tumor volume of xenografts generated by Bel-7402 cells with or without AGER knocked down in nude mice treatedwith either saline or STZ (STZ2). Tumor size was measured after injection of cells for 8 weeks. n = 5/group. The data are shown as themeans 6 SEM from three independent experiments (including WB). *P < 0.05 and **P < 0.01 using Student t test.

622 High Glucose Promotes Tumorigenesis via AGER Diabetes Volume 65, March 2016

levels of GLUT1, HK1 and -2, NUDT9, GUCY1A3, CANT1,GFPT1, GNPNAT1, PGM1, UAP1, OGT, SLC35A3, andPGM2 and -3 and found only OGT was upregulated byoverexpression of AGER in both Bel-7402 and SMMC-7721 cells (Fig. 2F). By contrast, knockdown of AGER ledto a reduction of OGT mRNA levels (Fig. 2G). Comparedwith the control, WB experiments also indicated an induc-tion of OGT protein accompanied with increased levels ofO-GlcNAcylation, an end event of HBP in both Bel-7402and SMMC-7721 cells with AGER overexpressed (Fig. 2H),while a reduction of OGT protein was accompanied bydecreased levels of O-GlcNAcylation in cells with AGERknocked down (Fig. 2I). These results further demon-strated that AGER may function as a regulator of HBPin HCC cells.

O-GlcNAcylation Promoted Tumorigenesis in HCC CellsBecause O-GlcNAcylation is the terminal effect of HBP,we then investigated the roles of O-GlcNAcylationin liver tumorigenesis. We found elevated levels ofO-GlcNAcylation and OGT in the liver from STZ-treatedBalb/c mice compared with the saline-treated control(Fig. 3A). Increased levels of O-GlcNAcylation and OGTwere also detected in human HCC tissues (Fig. 3B) andestablished HCC cell lines (Fig. 3C), respectively, com-pared with the paired adjacent normal liver tissues andhepatocyte line. These data suggested that elevation ofO-GlcNAcylation may be a common event in the liverfrom both HCC patients and patients with diabetes.

To test the importance of O-GlcNAcylation in livertumorigenesis, we stimulated O-GlcNAcylation by using

Figure 2—AGER linked to HBP. A: GC/MC results in control and Bel-7402 or SMMC-7721 cells with AGER overexpressed. Metaboliteswith decreased (left) or increased (right) levels are listed in the rounded boxes. B: Schematic diagram of HBP. OGT stimulates whileO-GlcNAcase suppresses O-GlcNAcylation. C: Metabolites were measured in Bel-7402 and SMMC-7721 cells (5 3 106 cells for eachexperiment) with or without AGER knocked down. D and E: AGER promoted glucose uptake from culture media (D) and raised intracellularglucose level (E ) in Bel-7402 and SMMC-7721 cells. The glucose consumption levels were calculated as the difference between the initialand the remaining glucose levels of cell culture media in cells under different treatment as indicated, while the intracellular glucose levelswere measured directly from cell lysates. F and G: AGER stimulated OGT expression. mRNA levels of components involved in HBP weremeasured by qPCR in control and Bel-7402 or SMMC-7721 cells with AGER overexpressed (F). mRNA levels of OGT were particularlyretested in control and Bel-7402 or SMMC-7721 cells with AGER knocked down (G). H–I: AGER promoted O-GlcNAcylation. Represen-tative WB images of O-GlcNAcylation and OGT in control and Bel-7402 or SMMC-7721 cells with AGER overexpressed (H) or knockeddown (I). The data are shown as the means6 SEM from three independent experiments (including WB). *P < 0.05 and **P < 0.01 using theStudent t test. GC/MS, gas chromatography/mass spectrometry.

diabetes.diabetesjournals.org Qiao and Associates 623

GlcNAc, an agonist of O-GlcNAcylation, and PUGNAc,an antagonist of O-GlcNAcase (an enzyme that has theopposite function against OGT). We found that treat-ment of GlcNAc and PUGNAc could significantly induceO-GlcNAcylation, and such effects were more obviouswhen GlcNAc and PUGNAc were simultaneously used(Fig. 3D). Because the efficacy of PUGNAc in stimulationof O-GlcNAcylation was more significant than that ofGlcNAc (Fig. 3D), we used PUGNAc in combination with orwithout GlcNAc in the following study. We found stimula-tion of O-GlcNAcylation enhanced cell proliferation (Fig. 3E)and colony-formation capacity (Fig. 3F and G) while inhib-iting caspase 3/7 activity (Fig. 3H). Similar to the endoge-nous O-GlcNAcylation levels (Fig. 3D), the transformativephenotypes stimulated by the combination of PUGNAc andGlcNAc were more obvious than that generated by PUGNAc

alone (Fig. 3E–H). To exclude nonspecific effects by PUGNAcand GlcNAc, we knocked endogenous OGT down by specificshRNAs. We found that depletion of OGT could also lead toa significant reduction of O-GlcNAcylation (Fig. 3I) accom-panied by significant reduction of cell proliferation (Fig. 3J)and colony-formation capacity (Fig. 3K and L) but inductionof caspase 3/7 activity (Fig. 3M). By contrast, overexpressionof OGT could stimulate O-GlcNAcylation and transfor-mative phenotypes in both Bel-7402 and SMMC-7721cells (Fig. 3N–Q). All these data suggested that OGT andO-GlcNAcylation are essential for tumorigenesis in HCC cells.

HG Increased c-Jun Activity in an AGER/OGT/O-GlcNAcylation–Dependent MannerNext, we tried to figure out a target protein that is essentialfor HG and AGER/OGT/O-GlcNAcylation–dependent liver

Figure 3—OGT and O-GlcNAcylation participated in liver tumorigenesis. A–C: Representative WB images of OGT and O-GlcNAcylation in the liverof Balb/c mice under the same treatments as those in Fig. 1E (A), HCC and its corresponding adjacent normal liver tissues (B), and establishedhepatocyte (HL-7702) and HCC cell lines as indicated (C). D: Verification of the stimulation of O-GlcNAcylation by GlcNAc and PUGNAc. Cells asindicated were stimulated by DMSO, GlcNAc (to a final concentration of 4 mmol/L), or PUGNAc (to a final concentration of 25 mmol/L) for 24 h beforeharvest for WB using indicated antibodies. E–H: Stimulation of O-GlcNAcylation by GlcNAc and PUGNAc promoted tumorigenesis, as measured bya methylthiazolyldiphenyltetrazolium bromide (MTT)–based assay (E), soft agar colony-formation assay (F and G), and caspase 3/7 Glo LUC assay(H). I–M: Knockdown of OGT inhibited O-GlcNAcylation and tumorigenesis. Levels of O-GlcNAcylation, cell proliferation, colony-formation capacity,and caspase 3/7 activity weremeasured byWB (I), MTT-based assay (J), soft agar colony-formation assay (K and L), and caspase 3/7 Glo LUC assay(M), respectively. N–Q: Overexpression of OGT stimulated O-GlcNAcylation and tumorigenesis, as measured by WB (N), MTT-based assay (O), softagar colony-formation assay (P), and caspase 3/7 Glo LUC assay (Q), respectively, in cells with or without overexpression of OGT. The data areshown as the means 6 SEM from three independent experiments (including WB). *P < 0.05 and **P < 0.01 using the Student t test.

624 High Glucose Promotes Tumorigenesis via AGER Diabetes Volume 65, March 2016

tumorigenesis. Firstly, we did a screen for proteinsincluding SRSF1, MEK1, eIF4E, c-Jun, c-Fos, ERK1/2,p38/MAPK14, p70S6K, TRIB2, MCAM, CD166, FoxA1,HSP27, c-Abl, CD44, EDG2, PDK1, and c-Kit. These pro-teins are involved in tumorigenesis. We found that onlyproto-oncoprotein c-Jun was a candidate that could beupregulated by AGER overexpression in both Bel-7402and SMMC-7721 cells (Fig. 4A and Supplementary Fig. 2).By contrast, c-Jun could be downregulated by AGERknockdown (Fig. 4B). The phosphorylation levels at Ser63and Ser73, two major phosphorylation sites of c-Jun (15),were tested simultaneously. Opposite the levels of c-Jun,the levels of phosphorylation at Ser73 (hereafter p-c-Jun)were decreased by overexpression of AGER while being in-creased by knockdown of AGER (Fig. 4A and B). However,

the levels of phosphorylation at Ser63 were changed inthe same direction as c-Jun (Supplementary Fig. 3A–C),suggesting the changes of phosphorylation at Ser63 byAGER were due to the changes of c-Jun. These data alsosuggested that only phosphorylation at Ser73 and notat Ser63 is controlled by AGER. LUC reporter assaysalso demonstrated that AGER had a positive influence onthe c-Jun activity (Fig. 4C). By adding increasing concen-trations of glucose, we found c-Jun was dose-dependentlyincreased, while p-c-Jun was dose-dependently decreased(Fig. 4D). Also, c-Jun activity was increased as a result ofHG (Fig. 4E). Because c-Jun exerts its function mainly inthe nucleus, we then detected nuclear localization of c-Junusing a microscope; we found that nuclear c-Jun wasdose-dependently accumulated by glucose (Fig. 4F).

Figure 4—c-Jun was an effector of AGER/OGT/O-GlcNAcylation. A–C: AGER stimulated expression and activity of c-Jun. RepresentativeWestern blots of p-c-Jun and c-Jun in control and Bel-7402 or SMMC-7721 cells with AGER overexpressed (A) or knocked down (B). The activityof c-Jun was tested using a LUC-based reporter assay (C). D–F: Glucose-stimulated expression, activity, and nuclear accumulation of c-Jun asmeasured by WB (D), LUC-based reporter assay (E), and confocal microscopic assay (F), respectively. Cells as indicated were treated withglucose at an indicated final concentration for 24 h before harvest for examinations. Scale bar, 20 mm. G and H: Expression and subcellularlocalization of c-Jun in mice liver. Representative Western blots of p-c-Jun and c-Jun in the liver, lung, and colon from Balb/c mice (G).Subcellular localizations of c-Jun and p-c-Jun were detected in the liver of Balb/c mice by confocal microscopic experiments (H). Mice weretreated under the same conditions as in Fig. 1E. Scale bar, 200 mm. *Cytosolic localization of c-Jun. Arrows indicate nuclear localization of c-Jun.I and J: Stimulation of O-GlcNAcylation increased c-Jun expression and activity, as measured by WB (I) and LUC-based reporter assay (J),respectively, in Bel-7402 and SMMC-7721 under the same treatment as in Fig. 3D. K–M: OGT stimulated c-Jun, as measured by WB in controland Bel-7402 or SMMC-7721 cells with OGT overexpressed (K) or knocked down (L). c-Jun activity was measured by a LUC-based assay (M).The data are shown as the means 6 SEM from three independent experiments (including WB). **P < 0.01 using the Student t test.

diabetes.diabetesjournals.org Qiao and Associates 625

Similar to the levels of serum glucose and AGER expres-sion (Fig. 1E and H), a dose-dependent elevation of c-Junand OGT accompanied by a dose-dependent downregu-lation of p-c-Jun was observed only in the liver fromSTZ-treated Balb/c mice compared with the saline-treatedcontrol (Fig. 4G), further demonstrating that the HG/AGER/OGT/c-Jun signaling axis is liver specific. More-over, treatment of insulin was able to partially reversesuch effects (Fig. 4G). Similar effects of STZ and insulinon the expressions of c-Jun and p-c-Jun were also de-tected in the mouse liver using IF experiments (Fig. 4H).These results suggested that c-Jun may be a downstreameffector of AGER controlled by glucose metabolism.

Then we tested whether c-Jun is also regulated by OGTand O-GlcNAcylation. We found that treating Bel-7402and SMMC-7721 cells with combined PUGNAc and GlcNAchad more obvious effects than treating PUGNAc alone onthe induction of c-Jun but reduction of p-c-Jun (Fig. 4I).Moreover, c-Jun activity was increased after stimulation ofO-GlcNAcylation (Fig. 4J). By gain and loss of function ofOGT, we found that c-Jun was upregulated, while p-c-Junwas downregulated by OGT overexpression in bothBel-7402 and SMMC-7721 cells (Fig. 4K). By contrast,OGT knockdown had the opposite effects (Fig. 4L).Furthermore, c-Jun activity was also positively correlatedwith the OGT expression levels (Fig. 4M). These data sug-gested that OGT and O-GlcNAcylation are also critical inthe regulation of c-Jun.

c-Jun Was Stabilized by AGER and OGTNext, we further investigated how AGER and OGT regulatec-Jun. We first excluded the possibility that AGER andOGT regulate mRNA levels of c-Jun because qPCRdata indicated no changes of c-Jun mRNA before andafter overexpression/knockdown of AGER or OGT(data not shown). However, we found that overexpressionof OGT in Bel-7402 cells resulted in a prolonged half-lifetime of c-Jun protein as indicated by a CHX chase analysis(Fig. 5A and B). By contrast, knockdown of OGT accelerateddegradation of c-Jun (Fig. 5C and D). Similarly, over-expression of AGER also extended the half-life time ofc-Jun compared with the control (Fig. 5E and F), support-ing a critical role of AGER and its downstream effectorOGT in the regulation of protein stability of c-Jun.

Increased stabilization was usually accompanied bydecreased ubiquitination (16,17); thereby, we hypothe-sized that OGT decreases ubiquitination of c-Jun. Toaddress this, we overexpressed COP1, a known ubiquitinE3 ligase of c-Jun (18), in the absence or presence ofincreasing concentration of OGT in Bel-7402 cells andfound that OGT was able to reverse ubiquitination andfollowed degradation of c-Jun by COP1 (Fig. 5G). Bycontrast, knocking OGT down resulted in an accumu-lated ubiquitination of c-Jun in both Bel-7402 andSMMC-7721 cells (Fig. 5H). These results further sup-ported a positive role of OGT in the maintenance ofc-Jun stability.

Then we performed function tests to further investigatethe relationship between OGT and c-Jun. Despite the factthat overexpression of c-Jun alone could induce a signif-icant elevation of c-Jun (Fig. 5I), transformative pheno-types were not enhanced as significantly as expected(Fig. 5J–L), suggesting that c-Jun had already overloadedin HCC cells. However, simultaneous overexpression ofc-Jun was able to reverse impaired c-Jun expression (Fig. 5I),cell proliferation (Fig. 5J), colony-formation capacity(Fig. 5K), and increased caspase 3/7 activity (Fig. 5L) thatwas induced by knockdown of OGT, suggesting thatOGT-stimulated tumorigenesis in HCC cells may bec-Jun dependent.

O-GlcNAcylation of c-Jun in HCC CellsBecause O-GlcNAcylation interplays with ubiquitinationand followed degradation of target proteins (19), weinvestigated whether and how c-Jun was O-GlcNAcylatedin HCC cells. We found that O-GlcNAcylation of c-Jun wasdetectable in both Bel-7402 and SMMC-7721 cells whenoverexpression of OGT was induced (Fig. 6A), while knock-down of OGT reduced O-GlcNAcylation of c-Jun (Fig. 6B).Stimulation of O-GlcNAcylation by PUGNAc and GlcNAcalso resulted in an increased level of O-GlcNAcylation ofc-Jun protein (Fig. 6C). In addition, by adding increasingconcentrations of glucose, O-GlcNAcylation of c-Junwas dose-dependently induced (Fig. 6D). Furthermore,overexpression of AGER was capable of inducingO-GlcNAcylation of c-Jun (Fig. 6E). These results sug-gested that c-Jun can be O-GlcNAcylated in HCC cellsin a glucose/AGER/OGT-dependent manner.

Confocal microscopic analysis indicated that c-Junand OGT were colocalized (Fig. 6F). Moreover, the directinteractions between c-Jun and OGT were further con-firmed by performing PLA analysis using a Duolink kit(Fig. 6G), suggesting that O-GlcNAcylation of c-Jun mayoccur through its interaction with OGT. Enzymatic label-ing of O-GlcNAc site by using anti-TAMRA antibodies andHRP-labeled streptavidin, respectively, in Bel-7402 andSMMC-7721 cells provided evidence that c-Jun can beO-GlcNAcylated (Fig. 6H and I). In vitro O-GlcNAcylationexperiments by mixing purified c-Jun and OGT proteinswith UDP-GlcNAc also resulted in an O-GlcNAcylation ofc-Jun (Fig. 6J), further supporting that c-Jun can beO-GlcNAcylated by OGT.

We have described a negative association between c-Junand p-c-Jun, and stimulation of O-GlcNAcylation elevatesc-Jun expression (Fig. 4); therefore, we hypothesized thatO-GlcNAc and phosphate may competitively occupy Ser73.To address this, we replaced this serine by an alanine in thec-Jun protein and generated the S73A mutant. We foundthat O-GlcNAcylation of S73A mutant was much reducedcompared with the WT one as indicated by enzymaticlabeling of O-GlcNAc sites using anti-TAMRA antibodiesand HRP-labeled streptavidin, respectively (Fig. 6K). Wealso found that stimulation of O-GlcNAcylation by PUGNAcwith or without GlcNAc could result in a dose-dependent

626 High Glucose Promotes Tumorigenesis via AGER Diabetes Volume 65, March 2016

increase in O-GlcNAcylation of WT c-Jun; however, sucheffects were much reduced for the S73A mutant (Fig. 6L).These data revealed that O-GlcNAcylation of c-Jun islargely due to the O-GlcNAcylation at Ser73. Then weinvestigated whether O-GlcNAcylation of c-Jun is accom-panied by dephosphorylation at Ser73. It was observedthat O-GlcNAcylation levels of c-Jun were dose-dependentlyinduced, while phosphorylation levels at Ser73 were dose-dependently reduced in the IPs pulled down by anti–c-Junantibodies under the treatment of PUGNAc with orwithout GlcNAc (Fig. 6M). Therefore, we concluded thatO-GlcNAcylation at Ser73 might play a large part in sup-pressing phosphorylation of c-Jun at the same site.

By performing TMA using IHC, we also revealed a sig-nificant positive correlation between O-GlcNAcylation andc-Jun in HCCs (Fig. 6N and O), further supporting the

importance of the relationship between O-GlcNAcylationand c-Jun in clinical samples.

Then we tested whether AGER has direct impacts on thephosphorylation of c-Jun. We found that overexpression ofAGER led to a significant decreased level of phosphoryla-tion at Ser73 in WT c-Jun. However, due to the fact thatO-GlcNAc and phosphate can occupy at the same site, therewere no signals of phosphorylation at Ser73 in the S73Amutant c-Jun before or after overexpression of AGER(Supplementary Fig. 4). These data suggested that AGERmay directly inhibit phosphorylation of c-Jun at Ser73.

AGER Was Conversely Regulated by c-JunWe investigated whether c-Jun has a feedback on AGER.We found knockdown of c-Jun to be reduced (Fig. 7A),while overexpression of c-Jun dose-dependently induced

Figure 5—AGER/OGT enhanced protein stability of c-Jun. A and B: OGT overexpression extended half-life time of c-Jun, as measured by aCHX chase assay (A), and the relative protein levels of c-Jun were normalized to the levels of GAPDH (B). C and D: OGT knockdownstimulated protein degradation of c-Jun. Representative Western blots of c-Jun in Bel-7402 and SMMC-7721 cells treated with CHX for0 or 4 h are shown in C, and the relative protein levels of c-Jun were normalized to the levels of GAPDH, with the data shown in D. E and F:AGER protected c-Jun from degradation, as measured by a CHX chase assay (E), and the relative protein levels of c-Jun were normalizedto the levels of GAPDH (F ). G: OGT reversed COP1-induced ubiquitination (Ub) of c-Jun, as measured by IP using an anti–c-Jun antibody inBel-7402 cells under different treatments as indicated. H: OGT knockdown increased ubiquitination of c-Jun, as measured in control andBel-7402 or SMMC-7721 cells with OGT knocked down. I–L: Knockdown of OGT-reduced transformative phenotypes could be reversed byc-Jun. Expression of c-Jun under indicated treatment was measured by WB (I). Transformative phenotypes were measured by a methylthia-zolyldiphenyltetrazolium bromide (MTT)–based assay (J), soft agar colony-formation assay (K), and caspase 3/7 Glo LUC-based assay (L). Thedata are shown as the means 6 SEM from three independent experiments (including WB). **P < 0.01 using the Student t test.

diabetes.diabetesjournals.org Qiao and Associates 627

protein levels of AGER (Fig. 7B). qPCR experiments indi-cated that AGER mRNA could also be reduced by knock-down of c-Jun (Fig. 7C) while induced by overexpression ofc-Jun (Fig. 7D), suggesting that c-Jun may regulate AGERlargely through a transcription-dependent mechanism.

Then, we constructed LUC reporters containing trun-cated versions of AGER promoter from –923 to 120 nt

relative to the transcription start site (TSS, +1) of humanAGER gene (gene identification no. 117). We found over-expression of c-Jun was unable to induce AGER promoteractivity when the –334 to –257 region was lost because2256 LUC reporter had no response to c-Jun comparedwith the 2334 LUC reporter in Bel-7402 cells (Fig. 7E).Hereafter, we named the –334 to –257 region as a c-Jun

Figure 6—O-GlcNAcylation of c-Jun in HCC cells. A and B: OGT promoted O-GlcNAcylation of c-Jun. O-GlcNAcylation of c-Jun wasmeasured in control and Bel-7402 or SMMC-7721 cells with OGT overexpressed (A) or knocked down (B). C: O-GlcNAcylation of c-Jun inBel-7402 and SMMC-7721 cells treated with DMSO or PUGNAc (final concentration of 25 mmol/L) with or without GlcNAc (final concen-tration of 4 mmol/L) for 24 h. D: O-GlcNAcylation of c-Jun was stimulated by increasing concentrations of glucose as indicated in Bel-7402and SMMC-7721 cells for 24 h. E: AGER stimulated O-GlcNAcylation of c-Jun, as measured in control and Bel-7402 or SMMC-7721 cellswith AGER overexpressed. F: Colocalization of c-Jun and OGT in Bel-7402 and SMMC-7721 cells, as measured by a confocal microscopicanalysis. Scale bar, 20 mm. G: PLA analysis of the direct interaction between c-Jun and OGT. The areas with rectangle boxes were enlargedat the right side. Arrows indicate the signals of interaction in the nucleus. Scale bar, 50 mm. Rb, rabbit origin; Ms, mouse origin. H and I:Enzymatic labeling of O-GlcNAc in c-Jun as analyzed by anti-TAMRA antibodies (H) and HRP-labeled streptavidin (I), respectively, in Bel-7402 and SMMC-7721 cells. J: In vitro O-GlcNAcylation of c-Jun was analyzed by incubation of purified c-Jun and OGT proteins with UDP-GlcNAc at 37°C for 90 min before IP using anti–c-Jun antibodies followed by WB using antibodies against O-GlcNAc. K: Enzymatic labelingof O-GlcNAc in WT and S73A c-Jun–FLAG proteins as analyzed by anti-TAMRA antibodies and HRP-labeled streptavidin, respectively, in Bel-7402 cells. L: Mutation of S73 reduced O-GlcNAcylation of c-Jun. Bel-7402 cells expressing WT and S73A c-Jun–FLAG, respectively, weretreated with DMSO or PUGNAc (final concentration of 25 mmol/L) with or without GlcNAc (final concentration of 4 mmol/L) for 24 h beforeharvest for IP using anti-FLAG antibodies. O-GlcNAcylation of exogenous c-Jun–FLAG was then measured by WB using anti–O-GlcNAcantibodies.M: Stimulation of O-GlcNAcylation reduced phosphorylation of c-Jun. Bel-7402 and SMMC-7721 cells were treated with DMSOor PUGNAc (final concentration of 25 mmol/L) with or without GlcNAc (final concentration of 4 mmol/L) for 24 h before harvest for IP usinganti–c-Jun antibodies. O-GlcNAcylation and phosphorylation of endogenous c-Jun were then measured by WB using anti–O-GlcNAc andanti–p-c-Jun antibodies, respectively. N and O: TMA of O-GlcNAc and c-Jun in HCC. Represent IHC images are shown in N. Data wereanalyzed by x2 and are shown in O. Scale bar, 500 mm. Sections were scored using a semiquantitative scale for each individual tumortissue on the array slide as follows: -, negative staining (i.e., none of cells showing intensity staining); -/+, weak staining (i.e., $10% ofcells showing very intense staining or $30% of cells showing weak-to-moderate intense staining in an appropriate subcellular distribution);+, strong staining (i.e., $30% of cells showing very intense staining). Scoring results were simplified into -, -/+, and + categories. The datashown here are representative images from three independent experiments except TMA. Ab, antibody.

628 High Glucose Promotes Tumorigenesis via AGER Diabetes Volume 65, March 2016

response element (JRE) in the following studies. We alsofound that overexpression of OGT had a similar stimula-tion of the AGER promoter activity as seen with overex-pression of c-Jun (Fig. 7E), supporting the idea that OGThas a positive impact on c-Jun, as described above. Datafrom SMMC-7721 cells were also compromised (Fig. 7F).We then knocked down c-Jun and OGT, respectively, andfound that promoter activities from the2334 LUC reporterwere remarkably reduced, whereas no changes of pro-moter activities were detected from the 2256 LUC reporter(Fig. 7G). Potential transcription factor binding sites, as in-dicated in Fig. 7H, were then predicated by PROMO onlinesoftware (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) within the JRE. Despite thefact that no c-Jun binding site was predicated, deletion

of JRE in the 2923 LUC reporter resulted in no responseto either overexpression or knockdown of c-Jun/OGTcompared with the WT one (Fig. 7I), suggesting that c-Junmay indirectly bind with JRE within the AGER promoter.Furthermore, physical binding of c-Jun around JRE wasconfirmed by ChIP; however, parallel ChIP analysis on the22k and 2k regions, either upstream or downstream of thehuman AGER gene, showed no occupancy of c-Jun (Fig. 7J).These results demonstrated that c-Jun is capable of inducingAGER expression through activation of its promoter.

AG490 Served as a Potential Inhibitor to AGERAGER may be regarded as a promising therapeutic targetin HCC cells because AGER was detected mainly at the cellmembrane or cytoplasm (Fig. 8A). Therefore, we tried to

Figure 7—AGER was conversely regulated by c-Jun. A–D: c-Jun promoted AGER expression. AGER proteins were analyzed by WB incontrol and Bel-7402 or SMMC-7721 cells with c-Jun knocked down (A) or overexpressed (B). mRNA levels of AGER were analyzed byqPCR in control and Bel-7402 or SMMC-7721 cells with c-Jun knocked down (C). mRNA levels of AGER were also analyzed by qPCR incontrol and Bel-7402 cells transfected with increasing concentration of c-Jun–expressing plasmids (D). E: Promoter/LUC activity analysisof the region from 2923 to 120 nt relative to the TSS of the human AGER gene in control and Bel-7402 cells with either c-Jun or OGToverexpressed. Rectangle boxes indicate a region containing JRE at 2334 to 2257 nt relative to the TSS. **P < 0.01 vs. empty control.F: LUC activities of the2334 LUC,2256 LUC, and empty (pGL4.21) reporters were reanalyzed in control and SMMC-7721 cells with eitherc-Jun or OGT overexpressed. G: Promoter activities from the2334 LUC to the 2256 LUC reporters were analyzed in control and Bel-7402or SMMC-7721 cells with either c-Jun or OGT knocked down. H: Predication of potential transcription factor binding sites within JRE byPROMO online software. I: Promoter activities from 2923 LUC reporters with or without JRE in control and Bel-7402 or SMMC-7721 cellswith either c-Jun/OGT overexpressed or knocked down. J: Occupancy of c-Jun around JRE within the AGER promoter as analyzed by ChIPusing anti–c-Jun or IgG control antibodies in Bel-7402 and SMMC-7721 cells. One 2k upstream of TSS region (22k) and one 2k down-stream of AGER gene region (2k) were parallel tested. The data are shown as the means 6 SEM from three independent experiments(including WB). Del, deleted. In all panels but E, **P < 0.01 using the Student t test.

diabetes.diabetesjournals.org Qiao and Associates 629

figure out a possible inhibitor by testing a series of com-monly used chemical inhibitors. As shown in Fig. 8B, onlytreatment of AG490, a Janus kinase inhibitor, was foundto significantly reduce AGER expression in Bel-7402 cells.In SMMC-7721 cells, AGER could also be dose-dependentlyreduced when increasing concentrations of AG490 wereused (Fig. 8C). qPCR and CHX chase analysis demonstratedthat AG490 was only able to reduce protein half-life timeand not mRNA levels of AGER (data not shown and Fig. 8Dand E). We further found that using AG490 could induce asignificant ubiquitination of AGER (Fig. 8F). These resultssuggested that AG490 affects AGER expression maybelargely through inducing ubiquitination and reducing pro-tein stability of AGER.

Then, we tested whether AG490 affects the functionof AGER in HCC cells. O-GlcNAcylation could be dose-dependently reduced by increasing concentrations of

AG490 in both Bel-7402 and SMMC-7721 cells (Fig.8G). In addition, the levels of p-c-Jun were induced, whilethe levels of c-Jun were reduced (Fig. 8H), accompaniedby reduced c-Jun activity (Fig. 8I) after addition of AG490into the cultured Bel-7402 and SMMC-7721 cells. Fur-thermore, de-O-GlcNAcylation of c-Jun was also observedafter treatment of cells with AG490 (Fig. 8J). By test-ing malignant phenotypes in Bel-7402 and SMMC-7721 cells, we found that cell proliferation (Fig. 8K)and colony-formation capacity (Fig. 8L) were reduced,while caspase 3/7 activities (Fig. 8M) were inducedafter AG490 was used. Interestingly, the inhibitoryeffects of AG490 on the malignant phenotypes weremuch reduced in cells with AGER ectopically expressedcompared with the control (Fig. 8K–M), further dem-onstrating that AG490 maybe served as a potentialinhibitor to AGER.

Figure 8—AG490 could be treated as an inhibitor to AGER. A: Subcellular localization of AGER in Bel-7402 and SMMC-7721 cells. Nucleuswas indicated by staining with anti-histone antibodies and DAPI. Scale bar, 20 mm. B: Screening of potential inhibitors to AGER in Bel-7402cells. Cells were treated with DMSO or agents as indicated for 24 h before harvest for WB. Particularly, the final concentration of AG490 is50 mmol/L. C: Verification of the inhibition of AGER by AG490 at a final concentration of 50–150 mmol/L in SMMC-7721 cells. D andE: AG490 (50 mmol/L) shortened half-life time of AGER in Bel-7402 cells, as measured by a CHX chase assay (D). The relative protein levelsof AGER were normalized to those of GAPDH (E). F: AG490 stimulated ubiquitination (Ub) of AGER, as measured in Bel-7402 and SMMC-7721 cells treated with DMSO or AG490 at a final concentration of 50 mmol/L for 24 h. G: AG490 reduced O-GlcNAcylation, as measured inBel-7402 and SMMC-7721 cells treated with DMSO or AG490 at a final concentration of 50–150 mmol/L for 24 h. H–J: AG490 reducedexpression, activity, and O-GlcNAcylation of c-Jun, as measured by WB (H), LUC-based reporter assay (I), and IP assay (J) at a final con-centration of 50–150 mmol/L for 24 h. K–M: AG490 inhibited transformative phenotypes, as measured by a methylthiazolyldiphenyltetrazoliumbromide (MTT)–based assay (K), soft agar colony-formation assay (L), and caspase 3/7 Glo LUC assay (M) at a final concentration of50 mmol/L in Bel-7402 and SMMC-7721 cells with or without AGER overexpressed. N: Schematic diagram of a possible mechanismunderlying how HG promotes liver tumorigenesis through a positive autoregulatory feedback between AGER and c-Jun. The data areshown as the means 6 SEM from three independent experiments (including WB). **P < 0.01 using the Student t test.

630 High Glucose Promotes Tumorigenesis via AGER Diabetes Volume 65, March 2016

DISCUSSION

HCC has a strong relationship with diabetes (3). Despitethe fact that the influences of some risk factors of diabe-tes (such as insulin, IGF-I, and chronic inflammation) oncancer initiation and progression have been extensivelystudied, the mechanism of how hyperglycemia promotestumorigenesis in cancer, especially in HCC, has receivedless attention. Here, we only focused on the potentialroles of HG in mice with STZ-induced diabetes and HCCcell lines. The risk factor of insulin was excluded becauseSTZ is used to destroy pancreatic b-cells in mice and in-sulin was not added into the cell culture media. We foundthat HG supports cell proliferation, colony-formation ca-pacity, and in vivo tumor growth while inhibiting apopto-sis in HCC cells, which is in agreement with several largecohort and case-control studies that indicate that hyper-glycemia stimulates carcinogenesis in other cancer types(20–22). HG not only can provide a high-energy source (23)but also can enhance expression of genes that are critical inmaintaining transformative phenotypes of cancer cells (24).We identified that AGER, which has been demonstrated toaccount for diabetes complications (4), is also glucose induc-ible and critical for liver tumorigenesis. Therefore, we sug-gest that AGER and its downstream signaling may beresponsible for diabetes-induced HCC. Interestingly, a recentstudy has shown that the expression of AGER is significantlyhigher in HCC tissues than in adjacent normal liver tissuesand is closely associated with pathological staging andlymph-vascular space invasion (25), which further supportsthe importance of AGER in liver tumorigenesis.

It is known that metabolic disorders lead to HCC(26,27); however, it is still uncertain how AGER regu-lates metabolism in HCC cells. Evidence from our studysuggests that AGER enhances glucose uptake, a well-established malignant phenotype of cancer cells (28),and acts as a stimulator of HBP and O-GlcNAcylation oftarget proteins in HCC cells. Recent work indicates thatincreased O-GlcNAcylation is a general feature of cancerand contributes to transformed phenotypes (29). In thecurrent study, we concluded that there is a similar impor-tance of O-GlcNAcylation in the maintenance of transfor-mative phenotype in HCC cells. We further revealed thatO-GlcNAcylation of c-Jun is a critical downstream eventof AGER signaling and found that OGT-dependent induc-tion of liver tumorigenesis is indispensible for c-Jun.Analogous to phosphorylation, O-GlcNAcylation playscrucial regulatory roles in cellular signaling. Emerging ev-idence has demonstrated at least up to four types of in-terplay between O-GlcNAcylation and phosphorylation ontarget proteins. These modifications can competitivelyand alternatively occupy either the same site or differentsites within a protein. They can also simultaneously oc-cupy different sites. At some occasions, competitive andsimultaneous occupancies of O-GlcNAc and phosphatemay occur within the same protein (30). Experimentalanalysis of c-Jun indicated that O-GlcNAc and phosphate

competitively occupy Ser73. However, selective enrich-ment of low-abundance O-GlcNAcylated species, followedby high-technology mass spectrometry methods, were notinvolved in the current study, and mapping and quantifi-cation of multiple O-GlcNAc sites in c-Jun protein areneeded in our future studies.

We also have revealed that HG can stimulateO-GlcNAcylation of c-Jun. Furthermore, the c-Jun protein canbe stabilized by both AGER and OGT. As the procarcinogenicrole of c-Jun has been implicated in liver tumorigenesis(31,32), we suppose that hyperglycemia stimulates liver tu-morigenesis maybe via O-GlcNAcylation and stabilizationof c-Jun in an AGER/OGT-dependent manner. IncreasedAGER signaling has been demonstrated to promote signal-ing nodes including p38, ERK1/2, and nuclear factor-kB(33–35). However, numerous studies also have suggestedthat AGER signaling is ligand- and cell type–dependent (4).This can explain why only c-Jun was found obviouslyupregulated under the stimulation of AGER in HCC cells.Interestingly, c-Jun can conversely increase AGER ex-pression; therefore, a novel positive autoregulatory feed-back was established in HCC cells. It is also not difficultto conclude that sustained activation of AGER, HBP, andc-Jun may lead to hyperglycemia-induced liver tumori-genesis. Notably, a follow-up study has demonstratedthat inadequate maintenance of blood glucose in patientswith diabetes is a significant risk factor for recurrence ofHCC (36), supporting our finding that HG stimulates HCC.

In the current study, we also found that AG490 is apotential inhibitor of AGER because it can block AGERexpression and its function. AG490 is a Janus kinaseinhibitor that has been shown to inhibit cell growth inHCC cells (37). However, no studies have linked AG490 toglucose metabolic disorders in HCC cells. To the best ofour knowledge, we are the first to report that AG490 cansuppress the malignant property of HCC cells throughblocking AGER-dependent O-GlcNAcylation of c-Jun.Therefore, AG490 may serve as a potential therapeuticagent for treatment of HCC in patients with diabetes.

Taken together, we report a potential mechanismunderlying how diabetes boosts HCC. A signaling cascadefrom AGER to HBP to proto-oncoprotein c-Jun has beenrevealed that plays an important protumorigenic role inHG-induced liver tumorigenesis (Fig. 8N). Blocking thiscascade may be helpful in the treatment of diabetic HCC.

Funding. This work was supported by the National Natural ScienceFoundation of China (81301689, 81201913, and 81202958), the YangfanProject of Shanghai Municipal Science and Technology Commission(14YF1412300), the Shanghai Municipal Health and Family PlanningCommission (20114y009), the Shanghai Jiao Tong University School ofMedicine Outstanding Young Teacher Grant (to Y.Q.), the China centralcolleges and universities basic research–specific cross discipline grant(1501219096 [to J.W.]), the Outstanding Youth Training Program of TongjiUniversity (1501219080), the Shanghai Tenth People’s Hospital ClimbingTraining Program (04.01.13024), and the Young College Teachers’ TrainingScheme of Shanghai (ZZjdyx13007).

diabetes.diabetesjournals.org Qiao and Associates 631

Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. Y.Q. designed the study and researched andanalyzed data. X.Z., Y.Z., and Y.W. researched data. Y.X. contributed to discussion.X.L. researched and analyzed data. F.S. designed the study. J.W. designed thestudy, researched data, and wrote the manuscript. J.W. is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

References1. Mukherjee B, Bhattacharya S, Chakraborty S, Satapathy BS, Dey NS, ShawTK. Is type 2 diabetes mellitus a predisposal cause for developing hepatocellularcarcinoma? Curr Diabetes Rev 2015;11:64–702. El-Serag HB, Tran T, Everhart JE. Diabetes increases the risk of chronic liverdisease and hepatocellular carcinoma. Gastroenterology 2004;126:460–4683. Ryu TY, Park J, Scherer PE. Hyperglycemia as a risk factor for cancerprogression. Diabetes Metab. J 2014;38:330–3364. Zhao J, Randive R, Stewart JA. Molecular mechanisms of AGE/RAGE-mediated fibrosis in the diabetic heart. World J Diabetes 2014;5:860–8675. Piperi C, Goumenos A, Adamopoulos C, Papavassiliou AG. AGE/RAGEsignalling regulation by miRNAs: associations with diabetic complications andtherapeutic potential. Int J Biochem Cell Biol 2015;60:197–2016. Hagiwara S, McClelland A, Kantharidis P. MicroRNA in diabetic nephropathy:renin angiotensin, AGE/RAGE, and oxidative stress pathway. J Diabetes Res2013;2013:1737837. McVicar CM, Ward M, Colhoun LM, et al. Role of the receptor for advancedglycation endproducts (RAGE) in retinal vasodegenerative pathology during diabetesin mice. Diabetologia 2015;58:1129–11378. Moy KA, Jiao L, Freedman ND, et al. Soluble receptor for advanced glycationend products and risk of liver cancer. Hepatology 2013;57:2338–23459. Wellen KE, Thompson CB. A two-way street: reciprocal regulation ofmetabolism and signalling. Nat Rev Mol Cell Biol 2012;13:270–27610. Itkonen HM, Minner S, Guldvik IJ, et al. O-GlcNAc transferase integratesmetabolic pathways to regulate the stability of c-MYC in human prostate cancercells. Cancer Res 2013;73:5277–528711. Slawson C, Hart GW. O-GlcNAc signalling: implications for cancer cell biology.Nat Rev Cancer 2011;11:678–68412. Wang J, Park JS, Wei Y, et al. TRIB2 acts downstream of Wnt/TCF in livercancer cells to regulate YAP and C/EBPa function. Mol Cell 2013;51:211–22513. Wang J, Ma L, Weng W, et al. Mutual interaction between YAP and CREBpromotes tumorigenesis in liver cancer. Hepatology 2013;58:1011–102014. Wang J, Ma L, Tang X, et al. Doxorubicin induces apoptosis by targetingMadcam1 and AKT and inhibiting protein translation initiation in hepatocellularcarcinoma cells. Oncotarget 2015;6:24075–2409115. Cho YY, Tang F, Yao K, et al. Cyclin-dependent kinase-3-mediated c-Junphosphorylation at Ser63 and Ser73 enhances cell transformation. Cancer Res2009;69:272–28116. Tang X, Chen X, Xu Y, et al. CD166 positively regulates MCAM via inhibitionto ubiquitin E3 ligases Smurf1 and bTrCP through PI3K/AKT and c-Raf/MEK/ERKsignaling in Bel-7402 hepatocellular carcinoma cells. Cell Signal 2015;27:1694–170217. Wang J, Zhang Y, Weng W, et al. Impaired phosphorylation and ubiquitinationby p70 S6 kinase (p70S6K) and Smad ubiquitination regulatory factor 1 (Smurf1)promote tribbles homolog 2 (TRIB2) stability and carcinogenic property in livercancer. J Biol Chem 2013;288:33667–33681

18. Shao J, Teng Y, Padia R, et al. COP1 and GSK3b cooperate to promotec-Jun degradation and inhibit breast cancer cell tumorigenesis. Neoplasia 2013;15:1075–1085

19. Zhu Y, Liu TW, Cecioni S, Eskandari R, Zandberg WF, Vocadlo DJ. O-GlcNAcoccurs cotranslationally to stabilize nascent polypeptide chains. Nat Chem Biol2015;11:319–325

20. Muti P, Quattrin T, Grant BJ, et al. Fasting glucose is a risk factor forbreast cancer: a prospective study. Cancer Epidemiol Biomarkers Prev 2002;11:1361–1368

21. Saydah SH, Platz EA, Rifai N, Pollak MN, Brancati FL, Helzlsouer KJ.Association of markers of insulin and glucose control with subsequent colorectalcancer risk. Cancer Epidemiol Biomarkers Prev 2003;12:412–418

22. Stattin P, Björ O, Ferrari P, et al. Prospective study of hyperglycemia andcancer risk. Diabetes Care 2007;30:561–567

23. Warburg O. On the origin of cancer cells. Science 1956;123:309–314

24. Masur K, Vetter C, Hinz A, et al. Diabetogenic glucose and insulin con-centrations modulate transcriptome and protein levels involved in tumour cellmigration, adhesion and proliferation. Br J Cancer 2011;104:345–352

25. Yang Y, Zhao LH, Huang B, et al. Pioglitazone, a PPARg agonist, inhibitsgrowth and invasion of human hepatocellular carcinoma via blockade of the ragesignaling. Mol Carcinog 2015;54:1584–1595

26. Scalera A, Tarantino G. Could metabolic syndrome lead to hepatocarcinomavia non-alcoholic fatty liver disease? World J Gastroenterol 2014;20:9217–922827. Jinjuvadia R, Patel S, Liangpunsakul S. The association between metabolicsyndrome and hepatocellular carcinoma: systemic review and meta-analysis.J Clin Gastroenterol 2014;48:172–17728. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell2011;144:646–67429. Ma Z, Vosseller K. Cancer metabolism and elevated O-GlcNAc in oncogenicsignaling. J Biol Chem 2014;289:34457–3446530. Zeidan Q, Hart GW. The intersections between O-GlcNAcylation andphosphorylation: implications for multiple signaling pathways. J Cell Sci 2010;123:13–2231. Eferl R, Ricci R, Kenner L, et al. Liver tumor development. c-Jun antago-nizes the proapoptotic activity of p53. Cell 2003;112:181–19232. Watanabe T, Hiasa Y, Tokumoto Y, et al. Protein kinase R modulates c-Fosand c-Jun signaling to promote proliferation of hepatocellular carcinoma withhepatitis C virus infection. PLoS One 2013;8:e6775033. Fukami K, Ueda S, Yamagishi S, et al. AGEs activate mesangial TGF-beta-Smad signaling via an angiotensin II type I receptor interaction. Kidney Int 2004;66:2137–214734. Ramasamy R, Yan SF, Schmidt AM. Receptor for AGE (RAGE): signalingmechanisms in the pathogenesis of diabetes and its complications. Ann N Y AcadSci 2011;1243:88–10235. Sun C, Liang C, Ren Y, et al. Advanced glycation end products depressfunction of endothelial progenitor cells via p38 and ERK 1/2 mitogen-activatedprotein kinase pathways. Basic Res Cardiol 2009;104:42–4936. Hosokawa T, Kurosaki M, Tsuchiya K, et al. Hyperglycemia is a significantprognostic factor of hepatocellular carcinoma after curative therapy. World JGastroenterol 2013;19:249–25737. Niwa Y, Kanda H, Shikauchi Y, et al. Methylation silencing of SOCS-3promotes cell growth and migration by enhancing JAK/STAT and FAK signalingsin human hepatocellular carcinoma. Oncogene 2005;24:6406–6417

632 High Glucose Promotes Tumorigenesis via AGER Diabetes Volume 65, March 2016