High Glucose Stimulates Tumorigenesis in Hepatocellular Carcinoma Cells Through AGER ...

14 Pages·2016·3.01 MB·English

Diabetes Volume65,March2016 619
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
High Glucose Stimulates Tumorigenesis
in Hepatocellular Carcinoma Cells
Through AGER-Dependent
O-GlcNAcylation of c-Jun
Diabetes2016;65:619–632|DOI:10.2337/db15-1057
Epidemiologicstudiessuggestthathepatocellularcar- understand the underlying molecular mechanisms of
cinoma (HCC) has a strong relationship with diabetes. how diabetes promotes HCC. It is well established that
However, the underlying molecular mechanisms still liverisfundamentallyimportantforglucosemetabolism,
remainunclear.Here,wedemonstratedthathighglucose and hyperglycemia is one of the main characteristics of
(HG), one of the main characteristics of diabetes, was diabetes. However, whether and how high glucose (HG) S
IG
capable of accelerating tumorigenesis in HCC cells. stimulates liver tumorigenesis remain largely unclear. N
Advancedglycosylationend product–specificreceptor A
HG reacts with long-lived proteins to form advanced
L
(AGER)wasidentifiedasastimulatorduringthisprocess.
glycation end products. Advanced glycation end products T
Mechanistically, AGER activated a hexosamine biosyn- R
accountformanydiabetescomplicationsthroughtheir A
thetic pathway, leading to enhanced O-GlcNAcylation engagement to advanced glycosylation end product– NS
of target proteins. Notably, AGER was capable of in- specific receptor (AGER) (4–6). Inhibition of AGER is D
creasingactivityandstabilityofproto-oncoproteinc-Jun U
regarded as a therapeutic option to prevent diabetes C
viaO-GlcNAcylationofthisproteinatSer73.Interestingly, T
c-Jun can conversely enhance AGER transcription. complications(7).Despiteanepidemiologicstudythat IO
has demonstrated that AGER may be involved in liver N
Thereby,apositiveautoregulatoryfeedbackloopthat
injury and subsequent carcinogenesis (8), the exact roles
stimulates diabetic HCC was established. Finally, we
of AGER in liver tumorigenesis are still unknown.
found that AG490, an inhibitor of Janus kinase, has
Glucose,glutamine,andacetyl-coAarerequiredforthe
theabilitytoimpairAGERexpressionanditsfunctions
inHCCcells.Inconclusion,AGERanditsfunctionsto hexosaminebiosyntheticpathway(HBP)tosynthesizeuri-
stimulate O-GlcNAcylation are important during liver dine diphosphate (UDP)–N-acetyl-D-glucosamine (UDP-
tumorigenesis, when high blood glucose levels are GlcNAc) (9). The HBP couples metabolic flux to control
inadequately controlled. cell proliferation and participates in O-GlcNAcylation of
target proteins through O-linked N-acetylglucosamine
(GlcNAc) transferase (OGT) (9–11). Despite the fact that
Diabetes has been established as one of the most OGT and O-GlcNAcylation have been implicated as very
importantrisksinprogressionofhepatocellularcarcinoma important in the promotion of tumorigenesis in various
(HCC)(1,2).AstheprevalenceofbothdiabetesandHCC types of cancer (10,11), very few studies have focused on
is substantially increasing worldwide (3), it is urgent to their function in HCC. Given that HBP and subsequent
1School of Public Health, Shanghai Jiao Tong University School of Medicine, Corresponding authors: Fenyong Sun, [email protected], and Jiayi Wang,
Shanghai,China [email protected].
2DepartmentofClinicalLaboratoryMedicine,ShanghaiTenthPeople’sHospitalof
Received30July2015andaccepted24November2015.
TongjiUniversity,Shanghai,China
ThisarticlecontainsSupplementaryDataonlineathttp://diabetes
3DepartmentofCentralLaboratory,ShanghaiTenthPeople’sHospitalofTongji
.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-1057/-/DC1.
University,Shanghai,China
4Department of Pharmacy, Shanghai Municipal Hospital of Traditional Chinese Y.Q.,X.Z.,andY.Z.areco–firstauthors.
Medicine,ShanghaiUniversityofTraditionalChineseMedicine,Shanghai,China ©2016bytheAmericanDiabetesAssociation.Readersmayusethisarticleas
5FacultyofMedicalLaboratoryScience,ShanghaiJiaoTongUniversitySchoolof longastheworkisproperlycited,theuseiseducationalandnotforprofit,and
Medicine,Shanghai,China theworkisnotaltered.
6TongjiUniversityAdvancedInstituteofTranslationalMedicine,Shanghai,China
620 HighGlucosePromotesTumorigenesisviaAGER Diabetes Volume65,March2016
O-GlcNAcylation are critical for both glucose metabolism regions of human AGER gene were PCR amplified from
and tumorigenesis, we supposed that they might be in- gDNAofBel-7402cellsandclonedintopGL4.21(Promega,
volved in HG-associated HCC. Madison,WI)vectors.Thewild-type(WT)andS73Ac-Jun–
Here, we demonstrate that HG is capable of pro- FLAG were subcloned into pcDNA3.1 (+) plasmids. The
moting liver tumorigenesis via AGER. AGER stimulated mutants were constructed by overlapping PCR. The pri-
O-GlcNAcylation and activation of c-Jun via enhancing mers used for this study are listed in Supplementary
HBP and OGT. We also uncovered that c-Jun could con- Table 1.
versely upregulate AGER. Altogether, we establish a posi-
Immunohistochemistry,Immunofluorescence, and
tive feedback loop from HG, AGER, and HBP to c-Jun,
WesternBlotting
which may be helpful in the treatment of diabetic HCC.
The primary antibodies used in immunohistochemistry
(IHC) wereanti-AGER (no. ab54741; Abcam, Hong Kong,
RESEARCH DESIGN AND METHODS
China), anti–cleaved caspase 3 (no. 9664; Cell Signaling
MiceExperiments
Technology [CST], Boston, MA), anti-Ki67 (no. ab15580;
Insulin-deficient HG and control mice models were con- Abcam), anti–c-Jun (no. 9165; CST), and anti–O-GlcNAc
structed by intraperitoneal injection of streptozocin (STZ)
(no. ab2739; Abcam).
(resolvedinsolutionof0.1mol/Lcitrateacids,pH4.4)and For immunofluorescence (IF), the primary antibodies
saline, respectively, into 5-week Balb/c male mice (Bikai,
used were anti-AGER (no. ab54741; Abcam), anti-OGT
Shanghai,China).Toinvestigatedose-dependenteffects,we (no.ab184198;Abcam),anti-histone(no.4499;CST),anti–
injectedSTZatafinalconcentrationof40mg/kgi.p.(STZ1) p-c-Jun (no. 3270; CST), and anti–c-Jun (no. 9165; CST).
and 200 mg/kg i.p. (STZ2), respectively, into the mice. The
For Western blotting (WB), the primary antibodies used
miceweretreatedwithSTZ1onceadayforfourconsecutive
wereanti-GAPDH(no.5174;CST),anti-OGT(no.ab184198
daysortreatedwithSTZ2onlyonce.TheSTZ2-treatedmice or no. ab177941; Abcam), anti–Myc-tag (no. 2276 or no.
were also injected with or not with insulin (Sigma-Aldrich,
2278; CST), anti-AGER (no. ab54741 or no. ab37647;
St.Louis,MO)atafinalconcentrationof2units/kgi.p.each
Abcam), anti-GlcNAc (no. ab2739; Abcam), anti-ubiquitin
day. For xenograft mouse experiments, Bel-7402 cells (5 3 (no. 3936; CST), anti–c-Jun (no. 9165 or no. 2315; CST),
106 cells) under different treatments were subcutaneously anti–p-c-Jun(forp-S73,no.3270,andforp-S63,no.9261;
injectedintoSTZ1-,STZ2-,combinedSTZ2-andinsulin-,or
CST),anti-COP1(no.sc-166799;SantaCruzBiotechnology,
saline-treated 8-week-old athymic nude mice (Bikai). The
Santa Cruz, CA), anti-PDK1 (no. 3062; CST), anti-EDG2
tumor size was measured 8 weeks after injection, and the
(no. ab166903; Abcam), anti-CD44 (no. ab51037; Abcam),
tumor volume was calculated as 0.5 3 L 3 W2, where L is anti–c-Abl (no. 2862; CST), anti-HSP27 (no. ab109376;
length and W is width. All mouse experiments were per-
Abcam), anti-FoxA1 (no. ab170933; Abcam), anti-CD166
formed according to the institution guidelines of Shanghai
(no. ab109215; Abcam), anti-MCAM (no. ab134065;
Tenth People’s Hospital.
Abcam),anti-TRIB2(no.H00028951-m04;Abnova,Taipei,
Taiwan), anti-p70S6K (no. ab32359; Abcam), anti-p38
TissueSamples,CellCulture,andVectors
(no. 9218; CST), anti-ERK1/2 (no. 4695; CST), anti-eIF4E
Slides of tissue microarray analysis (TMA) were purchased
from U.S. Biomax (Xi’an, China). Fresh HCC and adjacent (no. ab33766; Abcam), anti-MEK1 (no. 2352; CST), anti-
SRSF1(no.ab129108;Abcam),anti–c-Kit(no.3074;CST),
normal liver tissues were acquired at Shanghai Ruijin
anti–c-Fos (no. 2250; CST), and anti-FLAG (no. 8146 or
Hospital with institutional approval. The HCC cell lines
no. 2368; CST).
Bel-7402, SMMC-7721, Huh7, HepG2, SK-Hep1, and
All IHC, IF, and WB were performed conventionally,
Bel-7404andhepatocytelineHL-7702cellswerecultured
and the protocols are available elsewhere.
in DMEM. Cells were treated with AG490 (Beyotime,
Haimen,China)atafinalconcentrationof50–150mmol/L,
LUC Reporter, Cell Proliferation, Caspase 3/7
glucose (Sigma-Aldrich) at a final concentration of 5.5–
Activity, Soft Agar Colony Formation, Chromatin
50 mmol/L, PUGNAc (Sigma-Aldrich) at a final concen- Immunoprecipitation, Quantitative RT-PCR, Gas
tration of 25 mmol/L, GlcNAc (Sigma-Aldrich) at a Chromatography/Mass Spectrometry, and
concentration of 4 mmol/L, and cycloheximide (CHX) Metabolite Analysis
(Sigma-Aldrich) at a final concentration of 0.1 mg/mL, LUC reporter, cell proliferation, caspase 3/7 activity,
respectively. The cDNA encoding human AGER was pur- soft agar colony formation, and quantitative RT-PCR
chased from OriGene (Beijing, China) and subcloned into (qPCR) assays were performed as described previously
the pLJM-based lentiviral vector with a COOH-terminal (12,13). Chromatin immunoprecipitation (ChIP) was
Myc tag. The expressing plasmids of COP1, OGT-Myc, performed using the ChIP-IT express kit from Active
c-Jun–Myc, and OGT–short hairpin RNA (sh)4 were Motif (Carlsbad, CA). Protein-DNA complexes were incu-
obtained from OriGene. AGER-sh11, AGER-sh12, OGT-sh7, bated with 3 mg anti–c-Jun antibodies (no. 9165; CST)
c-Jun–sh2, c-Jun–sh3, and c-Jun–sh4 were obtained or IgG (no. sc-2345; Santa Cruz Biotechnology). The pri-
from GeneChem (Shanghai, China). Luciferase (LUC) re- mers used for qPCR are listed in Supplementary Table 1.
porter of c-Jun was purchased from Beyotime. Promoter Gaschromatography/massspectrometrywasperformedby
diabetes.diabetesjournals.org QiaoandAssociates 621
Bojian Biotechnology, Ltd. (Shanghai, China). Metabolites and colony-formation capacity (Fig. 1B and C), whereas a
were analyzed by corresponding kits from Sigma-Aldrich. reducedcaspase3/7activity(Fig.1D)wasseeninHCCcell
GlucosewasanalyzedbyakitfromApplygen(Beijing,China). lines Bel-7402 and SMMC-7721, which have shown high
carcinogenic properties in our previous studies (13,14).
Coimmunoprecipitation
Then, mice were intraperitoneally injected with two
Coimmunoprecipitation was performed as previously de-
different concentrations of STZ to induce HG. We found
scribed(12,13).ThereagentsusedwereproteinA/G-Sepharose
that STZ could induce a dose-dependent elevation of
(Novex, Oslo, Norway) and Western/immunoprecipation
serum glucose. By contrast, the HG that resulted from
(IP) lysis buffer (Beyotime) with 0.1% SDS (used for dena-
STZ-induced insulin deficiency could be reversed by
tured IP for ubiquitin and O-GlcNAc). The antibodies used
forIPwereanti-FLAG(no.2368;CST),anti–c-Jun(no.9165 intraperitoneal injection of insulin (Fig. 1E). Compared
with the saline-treated control, there were more dividing
or no. 2315; CST), and anti-AGER (ab37647; Abcam).
cells with decreased levels of cleaved caspase 3, but
ProteinLigationAssay increased levels of Ki67 can be detected in the liver of
The protein ligation assay was performed to identify the STZ-treated Balb/c mice (Fig. 1F), suggesting that HG
interaction between OGT and c-Jun using the Duolink promotesproliferationbutinhibitsapoptosis.Inxenograft
In Situ Red Starter Kit (mouse/rabbit) (Sigma-Aldrich, micemodels,adose-dependentincreasingxenografttumor
Uppsala, Sweden) as previously described (14). volume was detectedin nude micetreatedwith increasing
concentrations of STZ (Fig. 1G). Similar to the serum glu-
InVitroO-GlcNAcylationofc-Jun
Reaction mixtures containing 1 mmol/L purified human cose level (Fig. 1E), insulin could reverse STZ-accelerated
c-Junprotein(no.H00003725-P01;Abnova),0.125mmol/L xenograft growth (Fig. 1G). These results demonstrated
human OGT (H00008473-P01; Abnova), and 10 mmol/L that HG is capable of enhancing liver tumorigenesis.
Next,weinvestigatedwhetherAGERisglucoseinducible
UDP-GlcNAc (Sigma-Aldrich) in a buffer of 50 mmol/L Tris/
HCl(pH7.5)and1mmol/Ldithiothreitolwereincubatedat and important for tumorigenesis in HCC cells. AGER
37°Cfor90min.Thencoimmunoprecipitationwasper- expression wastestedinsaline- andSTZ-treatedBalb/c
formed by anti–c-Jun antibodies (no. 9165; CST) and mice, and it was found that STZ could induce a dose-
detected by WB using anti–c-Jun (no. 2315; CST) and dependent elevation of AGER in the liver but could not
in the lung and colon. Interestingly, insulin was able to
anti-GlcNAc (no. ab2739; Abcam) antibodies.
reverseSTZ-inducedelevationofAGERintheliver(Fig.1H).
Enzymatic LabelingofO-GlcNAcSites These results suggested that AGER has important func-
First, immunoprecipitated c-Jun with protein A/G-
tions in liver when glucose is elevated. Also, AGER could
Sepharose (Novex, Oslo, Norway) was added into reaction
be dose-dependently induced by increasing concentrations
buffer (20 mmol/L HEPES, pH 7.9; 50 mmol/L NaCl; ofHGinbothBel-7402andSMMC-7721cells(Fig.1I).In
1 mmol/L PUGNAc; and 5 mmol/L MnCl with protease
2 human HCC tissues, AGER was highly upregulated com-
and phosphatase inhibitors). Next, 2 mL Gal-T1Y289L pared with the paired adjacent normal liver tissues (Fig.
(Invitrogen, Carlsbad, CA) and 2 mL 0.5 mmol/L UDP- 1J and K). Moreover, AGER had much higher expression
GalNAz(Invitrogen)wereaddedintoreactionbuffertoa
levels in established HCC cell lines than that in the trans-
volumeof20mL. Thereaction wasperformedovernight formedhepatocyte line (Fig. 1L).TotestwhetherAGERis
at4°C.Thebeadswerewashedtwicewithreactionbuffer
important for HCC malignant function, we used two in-
to remove excess UDP-GalNAz. The samples were then
dependent shRNAs against AGER with high knockdown
reacted with biotin alkyne (Invitrogen) or tetramethyl- efficiency (Fig. 1M). Contrary to the procarcinogenic
6-carboxyrhodamine(TAMRA)alkyne(Invitrogen)according roles of HG, depletion of AGER led to a significant re-
to the manufacturer’s instructions. Biotin- or TAMRA- ductionofcellproliferation(Fig.1N)andcolony-formation
labeledsampleswerefinallydetectedbyWBusinghorse- capacity(Fig.1OandP)accompaniedbyaninductionof
radish peroxidase (HRP)–labeled streptavidin (no. A0303;
caspase 3/7 activity in both Bel-7402 and SMMC-7721
Beyotime) or antibodies against TAMRA (no. A6397; cells (Fig. 1Q). Notably, HG-enhanced transformative
Invitrogen).
phenotype was remarkably inhibited in AGER-depleted
cellscomparedwiththecontrol(SupplementaryFig.1A–C).
Statistical Analysis
Teststoexaminethedifferencesbetweengroupsincluded Inaddition,knockdownofAGERinBel-7402cellsimpaired
the Student t test and x2 test. P , 0.05 was regarded as xenograft growth and inhibited a significant increase of
statistically significant. tumor volume in STZ-treated nude mice compared with
the saline-treated control (Fig. 1R). Together, these data
RESULTS illustrated an essential role of AGER in the HG-induced
AGERWasEssentialforHG-StimulatedLiver liver tumorigenesis.
Tumorigenesis
Compared with the normal glucose level (5.5 mmol/L), AGERLinkedWithHBPinHCCCells
increasing HG levels (15–50 mmol/L) resulted in a ForinvestigationofthefunctionofAGERinmetabolism,
dose-dependent induced cell proliferation (Fig. 1A) gas chromatography/mass spectrometry was performed.
622 HighGlucosePromotesTumorigenesisviaAGER Diabetes Volume65,March2016
Figure1—HGstimulatedtumorigenesisthroughupregulationofAGER.A–D:HGstimulatedtumorigenesis.Cellproliferation,colony-formation
capacity,andcaspase3/7activitiesweremeasuredbyamethylthiazolyldiphenyltetrazoliumbromide(MTT)–basedassay(A),softagarcolony-
formationassay(BandC),andcaspase3/7GloLUCassay(D)afteradditionofglucosetoafinalconcentrationof5.5–50mmol/LinBel-7402
andSMMC-7721cells.Scalebar,200mm.E:SerumglucoseconcentrationofBalb/cmicetreatedwithsalineorSTZwithorwithoutinsulin.
n=5/group.Insulin,2units/kg/day.F:Liversectionsfromsaline-orSTZ-treatedBalb/cmicestainedbyhematoxylin-eosin(H&E)orIHCusing
anti–cleavedcaspase3oranti-Ki67antibodies.Scalebar,500mm.G:Bel-7402cells(53106)weresubcutaneouslyinjectedintonudemice
underthesametreatmentsasinE.Tumorsizewasmeasuredafterinjectionofcellsfor8weeks.n=5/group.H:AGERproteinexpressionwas
measuredbyWBinliver,lung,andcolonfromsaline-orSTZ-treatedBalb/cmice.TreatmentsofmicewerethesameinE.I:AGERprotein
expressioninBel-7402and SMMC-7721 cellsafter addition ofglucose toa final concentration from5.5 to50mmol/L. J: AGERprotein
expressioninpairedHCCandadjacentnormallivertissues.K:RepresentativeIHCimagesofAGERinHCCanditsadjacentnormalliver
tissues.Scalebar,500mm.L:AGERproteinexpressioninestablishedhepatocyte(HL-7702)andHCCcelllinesasindicated.M:AGERprotein
expressionincontrolandBel-7402orSMMC-7721cellswithAGERknockeddown.N–Q:KnockdownofAGERreducedtumorigenesis.
Cell proliferation, colony-formation capacity, andcaspase3/7 activities were measuredby an MTT-based assay(N), soft agar colony-
formationassay(OandP),andcaspase3/7GloLUCassay(Q)inBel-7402andSMMC-7721cellsbeforeandafterAGERknockdown.
Scalebar,200mm.R:TumorvolumeofxenograftsgeneratedbyBel-7402cellswithorwithoutAGERknockeddowninnudemicetreated
witheithersalineorSTZ(STZ2).Tumorsizewasmeasuredafterinjectionofcellsfor8weeks.n=5/group.Thedataareshownasthe
means6SEMfromthreeindependentexperiments(includingWB).*P<0.05and**P<0.01usingStudentttest.
Among366metabolitestested,22metaboliteswerecom- acid were induced, while glutamine, acetyl-coA, and
monly changed in both Bel-7402 and SMMC-7721 cells fructose-6-phosphatewerereduced(Fig.2C).However,gly-
when AGER was ectopically expressed (Fig. 2A and Sup- cogen, a metabolite not directly involved in HBP, was
plementaryTable2).Amongthesemetabolites,glutamine not changed (Fig. 2C). We also found that knockdown
and glycogenicamino acids including isoleucine,phenylala- of AGER reduced both glucose consumption from cul-
nine,tyrosine,andthreoninewereelevated,whileglutamic ture media (Fig. 2D) and intracellular glucose levels
acidandlacticacidwerereduced(Fig.2A).Allthesechanged (Fig. 2E), while overexpression of AGER led to the
metabolitesare important for HBP (see schematicdiagram opposite effects (Fig. 2D and E). These results sug-
ofHBP[Fig.2B]).Tofurtherverifytheresultsgainedfrom gestedthatAGERmayenhance metabolicfluxtostim-
overexpression of AGER, we knocked down AGER in ulate HBP.
Bel-7402andSMMC-7721cellsandreexaminedmetabolites To test whether AGER also regulates expression of
involved in the HBP. As expected, lactic acid and glutamic genesinvolvedinHBP,weperformedqPCRtotestmRNA
diabetes.diabetesjournals.org QiaoandAssociates 623
Figure2—AGERlinkedtoHBP.A:GC/MCresultsincontrolandBel-7402orSMMC-7721cellswithAGERoverexpressed.Metabolites
with decreased (left) or increased (right) levels are listed in the rounded boxes. B: Schematic diagram of HBP. OGT stimulates while
O-GlcNAcase suppresses O-GlcNAcylation. C: Metabolites were measured in Bel-7402 and SMMC-7721 cells (5 3 106 cells for each
experiment)withorwithoutAGERknockeddown.DandE:AGERpromotedglucoseuptakefromculturemedia(D)andraisedintracellular
glucoselevel(E)inBel-7402andSMMC-7721cells.Theglucoseconsumptionlevelswerecalculatedasthedifferencebetweentheinitial
andtheremainingglucoselevelsofcellculturemediaincellsunderdifferenttreatmentasindicated,whiletheintracellularglucoselevels
weremeasureddirectlyfromcelllysates.FandG:AGERstimulatedOGTexpression.mRNAlevelsofcomponentsinvolvedinHBPwere
measuredbyqPCRincontrolandBel-7402orSMMC-7721cellswithAGERoverexpressed(F).mRNAlevelsofOGTwereparticularly
retestedincontrolandBel-7402orSMMC-7721cellswithAGERknockeddown(G).H–I:AGERpromotedO-GlcNAcylation.Represen-
tativeWBimagesofO-GlcNAcylationandOGTincontrolandBel-7402orSMMC-7721cellswithAGERoverexpressed(H)orknocked
down(I).Thedataareshownasthemeans6SEMfromthreeindependentexperiments(includingWB).*P<0.05and**P<0.01usingthe
Studentttest.GC/MS,gaschromatography/massspectrometry.
levelsofGLUT1,HK1and-2,NUDT9,GUCY1A3,CANT1, O-GlcNAcylationPromotedTumorigenesisinHCCCells
GFPT1, GNPNAT1, PGM1, UAP1, OGT, SLC35A3, and Because O-GlcNAcylation is the terminal effect of HBP,
PGM2 and -3 and found only OGT was upregulated by we then investigated the roles of O-GlcNAcylation
overexpression of AGER in both Bel-7402 and SMMC- in liver tumorigenesis. We found elevated levels of
7721 cells (Fig.2F).By contrast, knockdown ofAGERled O-GlcNAcylation and OGT in the liver from STZ-treated
to a reduction of OGT mRNA levels (Fig. 2G). Compared Balb/c mice compared with the saline-treated control
with the control,WBexperimentsalso indicatedan induc- (Fig. 3A). Increased levels of O-GlcNAcylation and OGT
tion of OGT protein accompanied with increased levels of were also detected in human HCC tissues (Fig. 3B) and
O-GlcNAcylation, an end event of HBP in both Bel-7402 established HCC cell lines (Fig. 3C), respectively, com-
andSMMC-7721cellswithAGERoverexpressed(Fig.2H), pared with the paired adjacent normal liver tissues and
while a reduction of OGT protein was accompanied by hepatocyte line. These data suggested that elevation of
decreased levels of O-GlcNAcylation in cells with AGER O-GlcNAcylation may be a common event in the liver
knocked down (Fig. 2I). These results further demon- from both HCC patients and patients with diabetes.
strated that AGER may function as a regulator of HBP To test the importance of O-GlcNAcylation in liver
in HCC cells. tumorigenesis, we stimulated O-GlcNAcylation by using
624 HighGlucosePromotesTumorigenesisviaAGER Diabetes Volume65,March2016
Figure3—OGTandO-GlcNAcylationparticipatedinlivertumorigenesis.A–C:RepresentativeWBimagesofOGTandO-GlcNAcylationintheliver
ofBalb/cmiceunderthesametreatmentsasthoseinFig.1E(A),HCCanditscorrespondingadjacentnormallivertissues(B),andestablished
hepatocyte(HL-7702)andHCCcelllinesasindicated(C).D:VerificationofthestimulationofO-GlcNAcylationbyGlcNAcandPUGNAc.Cellsas
indicatedwerestimulatedbyDMSO,GlcNAc(toafinalconcentrationof4mmol/L),orPUGNAc(toafinalconcentrationof25mmol/L)for24hbefore
harvestforWBusingindicatedantibodies.E–H:StimulationofO-GlcNAcylationbyGlcNAcandPUGNAcpromotedtumorigenesis,asmeasuredby
amethylthiazolyldiphenyltetrazoliumbromide(MTT)–basedassay(E),softagarcolony-formationassay(FandG),andcaspase3/7GloLUCassay
(H).I–M:KnockdownofOGTinhibitedO-GlcNAcylationandtumorigenesis.LevelsofO-GlcNAcylation,cellproliferation,colony-formationcapacity,
andcaspase3/7activityweremeasuredbyWB(I),MTT-basedassay(J),softagarcolony-formationassay(KandL),andcaspase3/7GloLUCassay
(M),respectively.N–Q:OverexpressionofOGTstimulatedO-GlcNAcylationandtumorigenesis,asmeasuredbyWB(N),MTT-basedassay(O),soft
agarcolony-formationassay(P),andcaspase3/7GloLUCassay(Q),respectively,incellswithorwithoutoverexpressionofOGT.Thedataare
shownasthemeans6SEMfromthreeindependentexperiments(includingWB).*P<0.05and**P<0.01usingtheStudentttest.
GlcNAc, an agonist of O-GlcNAcylation, and PUGNAc, alone(Fig.3E–H).ToexcludenonspecificeffectsbyPUGNAc
an antagonist of O-GlcNAcase (an enzyme that has the andGlcNAc,weknockedendogenousOGTdownbyspecific
opposite function against OGT). We found that treat- shRNAs.WefoundthatdepletionofOGTcouldalsoleadto
mentofGlcNAcandPUGNAccouldsignificantlyinduce a significant reduction of O-GlcNAcylation (Fig. 3I) accom-
O-GlcNAcylation, and such effects were more obvious paniedbysignificantreductionofcellproliferation(Fig.3J)
when GlcNAc and PUGNAc were simultaneously used andcolony-formationcapacity(Fig.3KandL)butinduction
(Fig. 3D). Because the efficacy of PUGNAc in stimulation ofcaspase3/7activity(Fig.3M).Bycontrast,overexpression
of O-GlcNAcylation was more significant than that of of OGT could stimulate O-GlcNAcylation and transfor-
GlcNAc(Fig.3D),weusedPUGNAcincombinationwithor mative phenotypes in both Bel-7402 and SMMC-7721
without GlcNAc in the following study. We found stimula- cells (Fig. 3N–Q). All these data suggested that OGT and
tionofO-GlcNAcylationenhancedcellproliferation(Fig.3E) O-GlcNAcylationareessentialfortumorigenesisinHCCcells.
andcolony-formationcapacity(Fig.3FandG)whileinhib-
iting caspase 3/7 activity (Fig. 3H). Similar to the endoge- HGIncreased c-JunActivityinanAGER/OGT/
nous O-GlcNAcylation levels (Fig. 3D), the transformative O-GlcNAcylation–Dependent Manner
phenotypesstimulatedbythecombinationofPUGNAcand Next,wetriedtofigureoutatargetproteinthatisessential
GlcNAcweremoreobviousthanthatgeneratedbyPUGNAc for HG and AGER/OGT/O-GlcNAcylation–dependent liver
diabetes.diabetesjournals.org QiaoandAssociates 625
tumorigenesis. Firstly, we did a screen for proteins the levels of phosphorylation at Ser63 were changed in
including SRSF1, MEK1, eIF4E, c-Jun, c-Fos, ERK1/2, the same direction as c-Jun (Supplementary Fig. 3A–C),
p38/MAPK14, p70S6K, TRIB2, MCAM, CD166, FoxA1, suggesting the changes of phosphorylation at Ser63 by
HSP27, c-Abl, CD44, EDG2, PDK1, and c-Kit. These pro- AGER were due to the changesof c-Jun. These data also
teins are involved in tumorigenesis. We found that only suggested that only phosphorylation at Ser73 and not
proto-oncoprotein c-Jun was a candidate that could be at Ser63 is controlled by AGER. LUC reporter assays
upregulated by AGER overexpression in both Bel-7402 also demonstrated that AGER had a positive influence on
and SMMC-7721 cells (Fig. 4A and Supplementary Fig. 2). the c-Jun activity (Fig. 4C). By adding increasing concen-
By contrast, c-Jun could be downregulated by AGER trations of glucose, we found c-Jun was dose-dependently
knockdown (Fig. 4B). The phosphorylation levels at Ser63 increased, while p-c-Jun was dose-dependently decreased
and Ser73, two major phosphorylation sites ofc-Jun (15), (Fig. 4D). Also, c-Jun activity was increased as a result of
were tested simultaneously. Opposite the levels of c-Jun, HG (Fig. 4E). Because c-Jun exerts its function mainly in
the levels of phosphorylation at Ser73 (hereafter p-c-Jun) thenucleus,wethendetectednuclearlocalizationofc-Jun
weredecreasedbyoverexpressionofAGERwhilebeingin- using a microscope; we found that nuclear c-Jun was
creased byknockdown ofAGER (Fig. 4A andB).However, dose-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. Representative
Westernblotsofp-c-Junandc-JunincontrolandBel-7402orSMMC-7721cellswithAGERoverexpressed(A)orknockeddown(B).Theactivity
ofc-JunwastestedusingaLUC-basedreporterassay(C).D–F:Glucose-stimulatedexpression,activity,andnuclearaccumulationofc-Junas
measuredbyWB(D),LUC-basedreporterassay(E),andconfocalmicroscopicassay(F),respectively.Cellsasindicatedweretreatedwith
glucoseatanindicatedfinalconcentrationfor24hbeforeharvestforexaminations.Scalebar,20mm.GandH:Expressionandsubcellular
localization 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).
Subcellularlocalizationsofc-Junandp-c-JunweredetectedintheliverofBalb/cmicebyconfocalmicroscopicexperiments(H).Micewere
treatedunderthesameconditionsasinFig.1E.Scalebar,200mm.*Cytosoliclocalizationofc-Jun.Arrowsindicatenuclearlocalizationofc-Jun.
IandJ:StimulationofO-GlcNAcylationincreasedc-Junexpressionandactivity,asmeasuredbyWB(I)andLUC-basedreporterassay(J),
respectively,inBel-7402andSMMC-7721underthesametreatmentasinFig.3D.K–M:OGTstimulatedc-Jun,asmeasuredbyWBincontrol
andBel-7402orSMMC-7721cellswithOGToverexpressed(K)orknockeddown(L).c-JunactivitywasmeasuredbyaLUC-basedassay(M).
Thedataareshownasthemeans6SEMfromthreeindependentexperiments(includingWB).**P<0.01usingtheStudentttest.
626 HighGlucosePromotesTumorigenesisviaAGER Diabetes Volume65,March2016
Similar to the levels of serum glucose and AGER expres- Thenweperformedfunctionteststofurtherinvestigate
sion(Fig.1EandH),adose-dependentelevationofc-Jun the relationship between OGT and c-Jun. Despite the fact
and OGT accompanied by a dose-dependent downregu- that overexpression of c-Jun alone could induce a signif-
lation of p-c-Jun was observed only in the liver from icant elevation of c-Jun (Fig. 5I), transformative pheno-
STZ-treated Balb/c mice compared with the saline-treated types were not enhanced as significantly as expected
control (Fig. 4G), further demonstrating that the HG/ (Fig. 5J–L), suggesting that c-Jun had already overloaded
AGER/OGT/c-Jun signaling axis is liver specific. More- in HCC cells. However, simultaneous overexpression of
over, treatment of insulin was able to partially reverse c-Junwasabletoreverseimpairedc-Junexpression(Fig.5I),
such effects (Fig. 4G). Similar effects of STZ and insulin cell proliferation (Fig. 5J), colony-formation capacity
on the expressions of c-Jun and p-c-Jun were also de- (Fig. 5K), and increased caspase 3/7 activity (Fig. 5L) that
tected in the mouse liver using IF experiments (Fig. 4H). was induced by knockdown of OGT, suggesting that
These results suggested that c-Jun may be a downstream OGT-stimulated tumorigenesis in HCC cells may be
effector of AGER controlled by glucose metabolism. c-Jun dependent.
Thenwetestedwhetherc-JunisalsoregulatedbyOGT
and O-GlcNAcylation. We found that treating Bel-7402 O-GlcNAcylationofc-JuninHCCCells
andSMMC-7721cellswithcombinedPUGNAcandGlcNAc Because O-GlcNAcylation interplays with ubiquitination
had more obvious effects than treating PUGNAc alone on and followed degradation of target proteins (19), we
the induction of c-Jun but reduction of p-c-Jun (Fig. 4I). investigated whether and how c-Jun was O-GlcNAcylated
Moreover,c-Junactivitywasincreasedafterstimulationof inHCCcells.WefoundthatO-GlcNAcylationofc-Junwas
O-GlcNAcylation (Fig. 4J). By gain and loss of function of detectable in both Bel-7402 and SMMC-7721 cells when
OGT, we found that c-Jun was upregulated, while p-c-Jun overexpressionofOGTwasinduced(Fig.6A),whileknock-
was downregulated by OGT overexpression in both downofOGT reduced O-GlcNAcylation ofc-Jun(Fig. 6B).
Bel-7402 and SMMC-7721 cells (Fig. 4K). By contrast, Stimulation of O-GlcNAcylation by PUGNAc and GlcNAc
OGT knockdown had the opposite effects (Fig. 4L). alsoresultedin an increasedlevelofO-GlcNAcylationof
Furthermore, c-Jun activity was also positively correlated c-Junprotein(Fig.6C).Inaddition,byaddingincreasing
with the OGT expression levels (Fig. 4M). These data sug- concentrations of glucose, O-GlcNAcylation of c-Jun
gested that OGT and O-GlcNAcylation are also critical in was dose-dependently induced (Fig. 6D). Furthermore,
the regulation of c-Jun. overexpression of AGER was capable of inducing
O-GlcNAcylation of c-Jun (Fig. 6E). These results sug-
c-JunWasStabilizedbyAGERandOGT gested that c-Jun can be O-GlcNAcylated in HCC cells
Next,wefurtherinvestigatedhowAGERandOGTregulate in a glucose/AGER/OGT-dependent manner.
c-Jun. We first excluded the possibility that AGER and Confocal microscopic analysis indicated that c-Jun
OGT regulate mRNA levels of c-Jun because qPCR and OGT were colocalized (Fig. 6F). Moreover, the direct
data indicated no changes of c-Jun mRNA before and interactions between c-Jun and OGT were further con-
after overexpression/knockdown of AGER or OGT firmed by performing PLA analysis using a Duolink kit
(datanotshown).However,wefoundthatoverexpression (Fig. 6G), suggesting that O-GlcNAcylation of c-Jun may
of OGT in Bel-7402 cells resulted in a prolonged half-life occur through its interaction with OGT. Enzymatic label-
timeofc-JunproteinasindicatedbyaCHXchaseanalysis ingofO-GlcNAcsitebyusinganti-TAMRAantibodiesand
(Fig.5AandB).Bycontrast,knockdownofOGTaccelerated HRP-labeled streptavidin, respectively, in Bel-7402 and
degradation of c-Jun (Fig. 5C and D). Similarly, over- SMMC-7721 cells provided evidence that c-Jun can be
expression of AGER also extended the half-life time of O-GlcNAcylated (Fig.6HandI).Invitro O-GlcNAcylation
c-Juncomparedwiththecontrol(Fig.5EandF),support- experiments by mixing purified c-Jun and OGT proteins
ing a critical role of AGER and its downstream effector with UDP-GlcNAc also resulted in an O-GlcNAcylation of
OGT in the regulation of protein stability of c-Jun. c-Jun (Fig. 6J), further supporting that c-Jun can be
Increased stabilization was usually accompanied by O-GlcNAcylated by OGT.
decreased ubiquitination (16,17); thereby, we hypothe- Wehavedescribedanegativeassociationbetweenc-Jun
sized that OGT decreases ubiquitination of c-Jun. To and p-c-Jun, and stimulation of O-GlcNAcylation elevates
address this, we overexpressed COP1, a known ubiquitin c-Jun expression (Fig. 4); therefore, we hypothesized that
E3 ligase of c-Jun (18), in the absence or presence of O-GlcNAcandphosphatemaycompetitivelyoccupySer73.
increasing concentration of OGT in Bel-7402 cells and Toaddressthis,wereplacedthisserinebyanalanineinthe
found that OGT was able to reverse ubiquitination and c-Jun protein and generated the S73A mutant. We found
followed degradation of c-Jun by COP1 (Fig. 5G). By that O-GlcNAcylation of S73A mutant was much reduced
contrast, knocking OGT down resulted in an accumu- compared with the WT one as indicated by enzymatic
lated ubiquitination of c-Jun in both Bel-7402 and labeling ofO-GlcNAcsites usinganti-TAMRAantibodies
SMMC-7721 cells (Fig. 5H). These results further sup- and HRP-labeled streptavidin, respectively (Fig. 6K). We
ported a positive role of OGT in the maintenance of alsofoundthatstimulationofO-GlcNAcylationbyPUGNAc
c-Jun stability. with or without GlcNAc could result in a dose-dependent
diabetes.diabetesjournals.org QiaoandAssociates 627
Figure5—AGER/OGTenhancedproteinstabilityofc-Jun.AandB:OGToverexpressionextendedhalf-lifetimeofc-Jun,asmeasuredbya
CHX chase assay (A), andthe relative protein levelsof c-Jun were normalized to the levels of GAPDH (B). C and D: OGT knockdown
stimulatedproteindegradationofc-Jun.RepresentativeWesternblotsofc-JuninBel-7402andSMMC-7721cellstreatedwithCHXfor
0or4hareshowninC,andtherelativeproteinlevelsofc-JunwerenormalizedtothelevelsofGAPDH,withthedatashowninD.EandF:
AGERprotectedc-Junfromdegradation,asmeasuredbyaCHXchaseassay(E),andtherelativeproteinlevelsofc-Junwerenormalized
tothelevelsofGAPDH(F).G:OGTreversedCOP1-inducedubiquitination(Ub)ofc-Jun,asmeasuredbyIPusingananti–c-Junantibodyin
Bel-7402cellsunderdifferenttreatmentsasindicated.H:OGTknockdownincreasedubiquitinationofc-Jun,asmeasuredincontroland
Bel-7402orSMMC-7721cellswithOGTknockeddown.I–L:KnockdownofOGT-reducedtransformativephenotypescouldbereversedby
c-Jun.Expressionofc-JununderindicatedtreatmentwasmeasuredbyWB(I).Transformativephenotypesweremeasuredbyamethylthia-
zolyldiphenyltetrazoliumbromide(MTT)–basedassay(J),softagarcolony-formationassay(K),andcaspase3/7GloLUC-basedassay(L).The
dataareshownasthemeans6SEMfromthreeindependentexperiments(includingWB).**P<0.01usingtheStudentttest.
increase in O-GlcNAcylation of WT c-Jun; however, such importance of the relationship between O-GlcNAcylation
effects were much reduced for the S73A mutant (Fig. 6L). and c-Jun in clinical samples.
These data revealed that O-GlcNAcylation of c-Jun is ThenwetestedwhetherAGERhasdirectimpactsonthe
largely due to the O-GlcNAcylation at Ser73. Then we phosphorylationofc-Jun.Wefoundthatoverexpressionof
investigatedwhetherO-GlcNAcylationofc-Junisaccom- AGER led to a significant decreased level of phosphoryla-
panied by dephosphorylation at Ser73. It was observed tion at Ser73 in WT c-Jun. However, due to the fact that
thatO-GlcNAcylationlevelsofc-Junweredose-dependently O-GlcNAcandphosphatecanoccupyatthesamesite,there
induced, while phosphorylation levels at Ser73 were dose- were no signals of phosphorylation at Ser73 in the S73A
dependently reducedinthe IPs pulled down by anti–c-Jun mutant c-Jun before or after overexpression of AGER
antibodies under the treatment of PUGNAc with or (Supplementary Fig. 4). These data suggested that AGER
without GlcNAc (Fig. 6M). Therefore, we concluded that may directly inhibit phosphorylation of c-Jun at Ser73.
O-GlcNAcylation at Ser73 might play a large part in sup-
pressing phosphorylation of c-Jun at the same site. AGERWasConverselyRegulatedbyc-Jun
By performing TMA using IHC, we also revealed a sig- We investigated whether c-Jun has a feedback on AGER.
nificant positive correlation between O-GlcNAcylation and We found knockdown of c-Jun to be reduced (Fig. 7A),
c-Jun in HCCs (Fig. 6N and O), further supporting the while overexpression of c-Jun dose-dependently induced
628 HighGlucosePromotesTumorigenesisviaAGER Diabetes Volume65,March2016
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 was
measuredincontrolandBel-7402orSMMC-7721cellswithOGToverexpressed(A)orknockeddown(B).C:O-GlcNAcylationofc-Junin
Bel-7402andSMMC-7721cellstreatedwithDMSOorPUGNAc(finalconcentrationof25mmol/L)withorwithoutGlcNAc(finalconcen-
trationof4mmol/L)for24h.D:O-GlcNAcylationofc-JunwasstimulatedbyincreasingconcentrationsofglucoseasindicatedinBel-7402
andSMMC-7721cellsfor24h.E:AGERstimulatedO-GlcNAcylationofc-Jun,asmeasuredincontrolandBel-7402orSMMC-7721cells
withAGERoverexpressed.F:Colocalizationofc-JunandOGTinBel-7402andSMMC-7721cells,asmeasuredbyaconfocalmicroscopic
analysis.Scalebar,20mm.G:PLAanalysisofthedirectinteractionbetweenc-JunandOGT.Theareaswithrectangleboxeswereenlarged
attherightside.Arrowsindicatethesignalsofinteractioninthenucleus.Scalebar,50mm.Rb,rabbitorigin;Ms,mouseorigin.HandI:
EnzymaticlabelingofO-GlcNAcinc-Junasanalyzedbyanti-TAMRAantibodies(H)andHRP-labeledstreptavidin(I),respectively,inBel-
7402andSMMC-7721cells.J:InvitroO-GlcNAcylationofc-Junwasanalyzedbyincubationofpurifiedc-JunandOGTproteinswithUDP-
GlcNAcat37°Cfor90minbeforeIPusinganti–c-JunantibodiesfollowedbyWBusingantibodiesagainstO-GlcNAc.K:Enzymaticlabeling
ofO-GlcNAcinWTandS73Ac-Jun–FLAGproteinsasanalyzedbyanti-TAMRAantibodiesandHRP-labeledstreptavidin,respectively,inBel-
7402cells.L:MutationofS73reducedO-GlcNAcylationofc-Jun.Bel-7402cellsexpressingWTandS73Ac-Jun–FLAG,respectively,were
treatedwithDMSOorPUGNAc(finalconcentrationof25mmol/L)withorwithoutGlcNAc(finalconcentrationof4mmol/L)for24hbefore
harvestforIPusinganti-FLAGantibodies.O-GlcNAcylationofexogenousc-Jun–FLAGwasthenmeasuredbyWBusing anti–O-GlcNAc
antibodies.M:StimulationofO-GlcNAcylationreducedphosphorylationofc-Jun.Bel-7402andSMMC-7721cellsweretreatedwithDMSO
orPUGNAc(finalconcentrationof25mmol/L)withorwithoutGlcNAc(finalconcentrationof4mmol/L)for24hbeforeharvestforIPusing
anti–c-Junantibodies.O-GlcNAcylationandphosphorylationofendogenousc-JunwerethenmeasuredbyWBusinganti–O-GlcNAcand
anti–p-c-Junantibodies,respectively.NandO:TMAofO-GlcNAcandc-JuninHCC.RepresentIHCimagesareshowninN.Datawere
analyzedbyx2andareshowninO.Scalebar,500mm.Sectionswerescoredusingasemiquantitativescaleforeachindividualtumor
tissueonthearrayslideasfollows:-,negativestaining(i.e.,noneofcellsshowingintensitystaining);-/+,weakstaining(i.e.,$10%of
cellsshowingveryintensestainingor$30%ofcellsshowingweak-to-moderateintensestaininginanappropriatesubcellulardistribution);
+,strongstaining(i.e.,$30%ofcellsshowingveryintensestaining).Scoringresultsweresimplifiedinto-,-/+,and+categories.Thedata
shownherearerepresentativeimagesfromthreeindependentexperimentsexceptTMA.Ab,antibody.
protein levels of AGER (Fig. 7B). qPCR experiments indi- relative to the transcription start site (TSS, +1) of human
cated that AGER mRNA could also be reduced by knock- AGER gene (gene identification no. 117). We found over-
downofc-Jun(Fig.7C)whileinducedbyoverexpressionof expression of c-Jun was unable to induce AGER promoter
c-Jun (Fig. 7D), suggesting that c-Jun may regulate AGER activity when the –334 to –257 region was lost because
largely through a transcription-dependent mechanism. 2256 LUC reporter had no response to c-Jun compared
Then, we constructed LUC reporters containing trun- with the 2334 LUC reporter in Bel-7402 cells (Fig. 7E).
cated versions of AGER promoter from –923 to 120 nt Hereafter, we named the –334 to –257 region as a c-Jun