O-linked GlcNAcylation elevated by HPV E6 mediatesviral oncogenesisQinghua Zenga,b,1, Rui-Xun Zhaob,1, Jianfeng Chenb, Yining Lib, Xiang-Dong Lib, Xiao-Long Liub, Wei-Ming Zhangb,Cheng-Shi Quana, Yi-Shu Wanga, Ying-Xian Zhaia, Jian-Wei Wanga, Mariam Youssefa, Rutao Cuic, Jiyong Liangd,Nicholas Genovesee, Louise T. Chowe,2, Yu-Lin Lia,2, and Zhi-Xiang Xua,b,2

aKey Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China; bDivision ofHematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294; cDepartment of Pharmacologyand Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118; dDepartment of Systems Biology, University of Texas MDAnderson Cancer Center, Houston, TX 77030; and eDepartment of Biochemistry and Molecular Genetics, University of Alabama at Birmingham,Birmingham, AL 35294

Contributed by Louise T. Chow, June 27, 2016 (sent for review January 13, 2016; reviewed by Gerald W. Hart, Renske D. M. Steenbergen, and Tzyy-Choou Wu)

High-risk human papillomaviruses (HPVs) are causative agents ofanogenital cancers and a fraction of head and neck cancers. Themechanisms involved in the progression of HPV neoplasias to cancersremain largely unknown. Here, we report that O-linked GlcNAcyla-tion (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedlyincreased in HPV-caused cervical neoplasms relative to normal cervix,whereas O-GlcNAcase (OGA) levels were not altered. Transduction ofHPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs)up-regulated OGTmRNA and protein, elevated the level of O-GlcNAc,and promoted cell proliferation while reducing cellular senescence.Conversely, in HPV-18–transformed HeLa cervical carcinoma cells, in-hibition of O-GlcNAc with a low concentration of a chemical inhibitorimpaired the transformed phenotypes in vitro. We showed thatE6 elevated c-MYC via increased protein stability attributableto O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viabilityby a high concentration of O-GlcNAc inhibitor was partially rescuedby elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibitionin HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16E6/E7 and activatedK-RAS, reduced c-MYC and suppressed tumorigen-esis and metastasis. Thus, we have uncovered a mechanism for HPVoncoprotein-mediated transformation. These findings may eventuallyaid in the development of effective therapeutics for HPV-associatedmalignancies by targeting aberrant O-GlcNAc.

O-linked GlcNAcylation | HPV E6 | c-MYC | cervical cancer |HPV oncogenicity

Persistent infections of high-risk (HR) human papillomavirus(HPV)-16, HPV-18, and closely related genotypes are etio-

logically associated with the development of several humancancers, including anogenital and head and neck cancers (1–3).Two HR HPV genes, E6 and E7, are potent oncogenes based ontheir immortalizing and transforming activities in cell culturesystems and their capacities to induce tumors in animal models.The HR HPV E7 oncoprotein binds to more than 20 cellulartargets and interferes with multiple cellular processes, leading toderegulated cell cycle, centrosome amplification, DNA damage,anoikis resistance, anchorage-independent cell growth and ma-lignant transformation as well as immune surveillance evasion.E6 is also a multifunctional protein. Constitutive expression ofthe HR HPV E6 abrogates cell growth arrest and apoptosis,induces genomic instability and somatic mutations, activatestelomerase to promote immortalization, disrupts cell polarity,and prevents anoikis. These properties suggest that HPV-associatedcarcinogenesis involves a coordinated targeting of multiple path-ways with each pathway having a distinct but complementing role inmalignant transformation (4).O-linked GlcNAcylation (O-GlcNAc) is a reversible post-

translational modification, transferring an amino sugar moiety toserine/threonine residues of cytosolic or nuclear proteins (5). Thehexosamine biosynthetic pathway (HBP) converts intracellular

glucose to UDP-N-acetylglucosamine (UDP-GlcNAc), the donor forthe O-GlcNAc modification (6). The enzyme O-GlcNAc transferase(OGT) catalyzes the addition of the amino sugar to target pro-teins, whereas the enzyme O-GlcNAcase (OGA) catalyzes theremoval of the sugar (5, 6). Some substrates of O-GlcNAc arealternatively targeted by kinases (7–9). Thus, there is an extensivecrosstalk between O-GlcNAc and pathways or mechanisms thatare regulated by protein phosphorylation-signaling cascades (7–9).Because the biosynthesis of UDP-GlcNAc involves products

from glucose, amino acid, fatty acid, and nucleotide metabolism, ithas been proposed that O-GlcNAc serves primarily to modulatecellular signaling and transcription regulatory pathways in responseto metabolic regulation (10, 11). As a nutrient sensor, O-GlcNAcrelays the effects of excessive nutritional intake, an importantcancer risk factor, onto protein activities and cellular functions (8).Indeed, major tumor suppressors and oncoproteins, such as p53,MYC, NF-κB, and β-catenin are direct targets of O-GlcNAc (12–17). Chromatin dynamics is also modulated by O-GlcNAc. Forexample, DNA methylation enzymes of the Tet family, which isinvolved in epigenetic alterations and cancer, interact with andtarget OGT to multiple chromatin-remodeling complexes (9).Moreover, histones are subject to O-GlcNAc modification, leadingto the alteration of their functions (18). O-GlcNAc and OGT levelsare elevated in breast, prostate, colon, bladder, and several other

Significance

O-linked GlcNAcylation is a reversible posttranslational proteinmodification on serine or threonine residues and regulatesmultiple cellular signaling pathways. We discovered elevatedO-GlcNAc and O-GlcNAc transferase (OGT) in HPV-associatedcervical neoplasms relative to the normal cervix. We show thatHPV E6 upregulates OGT, increases O-GlcNAc, stabilizes c-MYC viaO-GlcNAc, and enhances HPV oncogene activities. Conversely,suppression of O-GlcNAc in HPV-transformed cells by knockingdown or inhibiting OGT impairs HPV oncogene-induced activitiesand impedes tumor growth in animal models. Thus, O-GlcNAcplays a critical role in HPV-induced carcinogenesis, and targetingO-GlcNAc might prove to be a potential therapeutic approach.

Author contributions: L.T.C., Y.-L.L., and Z.-X.X. designed research; Q.Z., R.-X.Z., J.C., Y.L.,X.-D.L., X.-L.L., W.-M.Z., Y.-X.Z., and N.G. performed research; N.G. contributed new reagents/analytic tools; Y.L., X.-D.L., X.-L.L., C.-S.Q., Y.-S.W., J.-W.W., M.Y., R.C., J.L., L.T.C., Y.-L.L., andZ.-X.X. analyzed data; and Q.Z., L.T.C., Y.-L.L., and Z.-X.X. wrote the paper.

Reviewers: G.W.H., Johns Hopkins University; R.D.M.S., VU University Medical Center; andT.-C.W., Johns Hopkins University.

The authors declare no conflict of interest.1Q.Z. and R.X.Z. contributed equally to this work.2To whom correspondence may be addressed. Email: ltchow@uab.edu, ylli@jlu.edu.cn, orzhixiangxu@uabmc.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1606801113/-/DCSupplemental.

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cancers (16, 19, 20), but neither has been examined previously inthe context of HPV oncogene expression or cervical neoplasms. Inthis work, we asked whether O-GlcNAc might play a role in HPV-induced transformation and carcinogenesis.

ResultsO-GlcNAc and OGT Are Elevated in Cervical Neoplasms. To detectO-GlcNAc in the spectrum of cervical lesions attributed to HPVinfections, we used immunohistochemistry (IHC) to probe thepresence of O-GlcNAc in a large collection of normal, cervicalintraepithelial neoplasias (CINs) and carcinomas of the cervix.As shown in Fig. 1 A–C, normal cervical tissues bore a low levelof O-GlcNAc, whereas tissues from CINs and carcinomasexhibited significantly higher signals. High-grade CINs exhibitedstronger signals and higher H scores compared with low-gradelesions (Fig. S1). Elevated OGT, reduced OGA, or both mayhave contributed to the elevated O-GlcNAc. To distinguish thesepossibilities, we probed for OGT and OGA in serial sections ofcervical cancer tissues using IHC. Consistently, both O-GlcNAc

and OGT were strongly positive in malignant foci (Fig. 1D) com-pared with the adjacent normal epithelia. In contrast, there was noappreciable difference in OGA signals between malignant andnormal tissues (Fig. 1D). Characterization of additional tissuespecimens confirmed these initial findings (Fig. 1 E and F).Thus, elevated OGT is primarily responsible for increasedO-GlcNAc in CINs and cervical cancers. Additionally, the stainingpatterns of OGT and O-GlcNAc mostly overlapped with thosefrom p16INK4A (Fig. S2), a surrogate marker for HR HPV-associated high-grade lesions and cancers (21), suggesting thatO-GlcNAc may contribute to HPV oncogenic activity.

O-GlcNAc Is Induced by and Mediates the Oncogenic Activity of E6 andE6/E7. To determine whether increased O-GlcNAc in CINs andcervical cancers is directly linked to HPV infection, we transducedprimary mouse embryonic fibroblasts (MEFs) (at passage 3) withrecombinant lentiviruses or retroviruses expressing HPV16 orHPV18 E6, E7, E6/E7, or GFP and examined O-GlcNAc in celllysates by immunoblot (Fig. 2A and Fig. S3). Consistent with pre-vious reports (21, 22), expression of HPV E6 and E7 reduced p53and Rb levels in the cells (Fig. 2A), respectively. Importantly, using apan-GlcNAc antibody, proteins with O-GlcNAc were markedly in-creased in MEF/HPV E6 or E6/E7 relative to the control, whereasE7 had no effect (Fig. 2A and Fig. S3). In addition, immunoblots

Fig. 1. O-GlcNAc and OGT are increased in cervical lesions. Human cervicaltissues from healthy controls (n = 22), cervical intraepithelial neoplasias (n = 43),and cervical carcinomas (n = 229) were examined. (A) Representative IHCstaining of O-GlcNAc with antibody RL2. (Scale bar, 50 μm.) (B) Quantification ofO-GlcNAc antigen positivity in the above tissue specimens. Masked reading wasperformed by two investigators using the same criteria to evaluate the staining(low: overall negative or weak staining; high: overall moderate or strongstaining). The Pearson’s χ2-test was used to analyze the distribution differenceof O-GlcNAc among human cervical tissues (P < 0.01). (C) H-scores of O-GlcNAcstaining in tissues from normal cervix, CINs, and carcinomas (Pearson’s χ2-test, *P<0.01). (D–F) OGT is elevated in cervical lesions. (D) Representative IHC stainingof O-GlcNAc, OGT, and OGA in serial sections of a cervical cancer tissue. (Scalebar, 50 μm.) Black arrows point to the tumor tissue. Red arrows point to thenormal cervix. (E) Quantification of OGT and OGA antigen positivity in cervicaltissues following the approaches in B (P < 0.01). (F) H-scores of OGT and OGAin normal tissues and cervical diseases were calculated (*P < 0.01).

Fig. 2. O-GlcNAcmediates HPV oncogenic activities. (A and B) MEFs (passage 3)were infected with lentiviruses expressing HPV16 E6, E7, E6/E7, or GFP (control).(A) Whole-cell extracts (WCEs) were analyzed with immunoblots for OGT andOGA as well as with O-GlcNAcylated proteins with respective antibodies. E6, E7,Rb, and p53 were determined for evaluating the HPV16 oncogene expressionand function. GAPDH served as a loading control. (B) Percentages of MEFs insenescence at passage 12 were determined after staining with SA–β-gal. Dataare average ± SEM of three independent experiments. *P < 0.01. Impact ofO-GlcNAcylation on HPV16-induced cell migration (C) and invasion (D) as describedin SI Materials and Methods. *P < 0.01 (n = 3). (E) Anchorage-independentcellular growth in soft agar. C33A/GFP and C33A/HPV16-E6/E7 cells were grownon 0.4% agar with 1 μM DON for 2 wk. Colonies were recorded with an En-vision light microscope and counted. *P < 0.01 (n = 3). (F–I) O-GlcNAc mediatesoncogenic activities of HeLa. HPV18-positive HeLa cells were treatedwith 50 μMST045849. Migration (F) and invasion (G) assays and anchorage-independentgrowth (H) were performed as above described. *P < 0.01 (n = 3). WCEs wereused for immunoblot analyses. GAPDH served as a loading control (I).

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confirmed that OGT, but not OGA, was elevated in MEF/HPV16E6 or E6/E7, but not in MEF/E7 cells (Fig. 2A). Thus, E6 is pri-marily responsible for the elevation of O-GlcNAc and OGT.To determine whether increased O-GlcNAc might affect the

activities of HPV oncogenes, we modulated O-GlcNAc withchemicals in MEF/HPV16 E6/E7 or GFP (Ctrl). Early passage(p3) of MEFs infected with an empty lentivirus vector haltedgrowth and entered senescence at passages 9–11. In contrast,MEF/HPV16 E6/E7 continued to grow, and clusters of spindle-shaped cells were observed as early as passage 6 (Fig. S4).Application of 1.0 μM 6-diazo-5-oxo-L-norleucine (DON), a gluta-mine antagonist that inhibits O-GlcNAc, did not markedly affectcell viability at passage 6 (Fig. S5A), but strikingly blocked themorphological changes of the E6/E7-transduced MEFs (Fig. S4).In addition, MEF/HPV16 E6/E7 had a reduced senescent cellpopulation, but the addition of 1.0 μM DON continuously frompassage 6 increased senescent cells (Fig. 2B). These resultssuggest that O-GlcNAc could affect the oncogenic activities ofHPV16 E6/E7. Similar alterations in the MEFs with or withoutHPV E6/E7 were also observed after exposure to 50 μM ST045849[3-(2-adamantanylethyl)-2-[(4-chlorophenyl)azamethylene]-4-oxo-1,3-thiazaperhydroine-6-carboxylic acid], which inhibits OGTspecifically (23–25) (Figs. S5B and S6).To substantiate this interpretation, we transduced viral onco-

genes into C33A cells, a rare cervical cancer cell line devoid ofoncogenic HPVs. C33A/HPV16 E6/E7 cells exhibited an elevationin O-GlcNAc (Fig. S7 A and B). We then performed assays inTranswell with or without precoated Matrigel. These cellsexhibited increased migration and invasion relative to the parentalcells. The effects were abrogated by the treatment with 1.0 μMDON overnight (Fig. 2 C and D), a condition that did not affectcell proliferation and viability. The ability to grow in the absenceof anchorage to the extracellular matrix is one of the most im-portant oncogenic properties of HPV-positive cancer cells. Wenext examined the growth of C33A/HPV16 E6/E7 cells in soft agarin the presence or absence of DON for 2 wk. C33A cells formedfew colonies (per 5,000 cells) when plated in soft agar (Fig. 2E).Transduction of HPV16 E6/E7 markedly increased the size andnumber of the colonies. Application of 1 μM DON substantiallyreversed the viral effects (Fig. 2E). Similar inhibitory effects onmigration, invasion, and soft agar colony formation were alsoobserved in ST045849-treated HeLa cells, an HPV18-positive cellline established from a cervical adenocarcinoma (Fig. 2 F–H). Theexpression of E6/E7 was not affected (Fig. 2I). Taken together,our data show that O-GlcNAc mediates or promotes the onco-genic activities of the HR HPVs.

HPV E6/E7 Enhances the Transcription of OGT. Three to five percentof the intracellular glucose directly enters into the HBP, where it

is converted into UDP-GlcNAc, the donor for the O-GlcNAcmodification (5). Thus, it is possible that O-GlcNAc is affectedby glucose metabolism. Our recent data showed that MEF/HPV16 E7, but not MEF/HPV16 E6, exhibited appreciably el-evated glucose consumption compared with MEFs, consistentwith the report that HPV16 E7 promotes glycolytic metabolism(26). Thus, our present results would suggest that the increasedO-GlcNAc in E6- or E6/E7-containing cells may not be attrib-uted to an alteration in glucose metabolism.To examine the mechanism by which HR HPV oncogenes up-

regulate OGT, we determined the half-life of OGT. It was notaffected by HPV16 oncogene expression (Fig. S8). Rather, theexpression of HPV16 E6 or E6/E7 in MEFs increased OGTmRNA levels by about threefold, whereas HPV16 E7 had nosignificant impact (Fig. 3A). HPV infection influences the ex-pression or activity of multiple transcription factors, includingAP-1, SP-1, NF-κB, p53, and c-MYC (21, 22). Interestingly, thepromoter region of the OGT gene possesses binding sites for thesetranscriptional factors. Thus, we cloned human OGT promotersequences −1010 to +10 into a pGL3 vector and transfected theconstruct into C33A cells expressing HPV16 oncoproteins (Fig. 3B)or cotransfected the reporter into MEF/HPV16 E6, E7, or E6/E7(Fig. 3C). The results showed that expression of HPV16 E6 or E6/E7 up-regulated the OGT promoter, whereas E7 had little or noeffect (Fig. 3 B and C). We further showed that, in C33A cells,cotransfection with an expression vector of Foxo3a, SP1, NF-κBp65, or c-MYC increased the OGT promoter activity, whereas p53expression was suppressive and CREB and E2F1 had no significantimpact (Fig. S9). Our results are consistent with a report by Neeset al. (27), who examined genes in differentiating cervical kerati-nocytes infected with retroviruses carrying HPV16 E6 or E7; theyfound that HPV16 E6 stimulated expression of multiple genesknown to be regulated by NF-κB and AP-1, whereas E7 was lesseffective.

Knockdown of OGT Reduces O-GlcNAc and Xenograft Growth of HPV-Transformed Cervical Cancer Cells in SCID Mice. To substantiate thein vitro significance of HPV-mediated up-regulation of OGT withregard to viral oncogenic activities, we knocked down (kd) OGT orOGA in HeLa cells with lentiviruses expressing shRNA-OGT orshRNA-OGA. Cells were selected with puromycin (1 μg/mL). Ex-pression of OGT was reduced by ∼75% (Fig. 4A). OGT knockdownmarkedly reduced O-GlcNAcylated proteins in the cells (Fig. 4A).Importantly, cell migration, invasion, and anchorage-independentcellular growth in soft agar were markedly reduced following OGTkd (Fig. 4 B–D). In contrast, each of these activities was enhanced inthe presence of shRNA-OGA, which severely depleted OGA whileelevating O-GlcNAc (Fig. 4 A–D). These observations support theimportance of O-GlcNAc in mediating the viral oncogenic activities.To validate the role of OGT in promoting oncogenic prop-

erties in an in vivo system, we performed s.c. inoculation ofHeLa/shRNA-Ctrl, shRNA-OGT, and shRNA-OGA cells intoSCID mice. Knockdown of OGT substantially reduced the tumorvolume (Fig. 4E) and the proportion of cells expressing theproliferating cell nuclear antigen (PCNA) (Fig. S10). Apoptotictumor cells increased, as detected with an antibody to cleavedcaspase-3 (Fig. 4F). In stark contrast, depletion of OGA stimu-lated s.c. tumor growth (Fig. 4E) and increased PCNA-positivecell number (Fig. S10), but had no effect on the cell populationpositive for cleaved caspase-3 (Fig. 4F). Taken together, our datasupport the notion that HPV oncogene-induced elevation ofOGT and O-GlcNAc have a significant impact on tumor cellsurvival and growth.

Suppression of O-GlcNAcylation Impedes Tumor Growth and Metastasisof HPV-Transformed TC-1 Cells in Syngeneic Mice. To verify furtherthe significance of O-GlcNAc in HPV-induced tumor growth,we examined the effects of DON in the TC-1 cell-induced mice

Fig. 3. HPV16 E6 or E6/E7 enhancesOGT transcription. (A) MEFs were infectedwith lentiviruses expressing HPV16 E6, E7, E6/E7, or GFP (control). mRNA levelsof OGT in the transduced cells were determined by qPCR. The relative mRNAlevels were calculated. *P < 0.01 (n = 3). HPV activated the promoter of OGT inC33A cells (B) and in MEFs (C). Human OGT promoter sequences −1010 to +10were cloned to the pGL3 vector and transfected to the cells expressing HPV16oncoproteins. The pRL vector expressing wild-type Renilla luciferase was usedas a control reporter. Relative luciferase unit (RLU) was the ratio of the OGTpromoter-driven luciferase activity to Renilla activity. *P < 0.01 (n = 3).

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tumor model. TC-1 cells were derived from primary lung epi-thelial cells of C57BL/6 mice transformed by HPV16 E6/E7and activated K-RAS (28). These cells induce tumors in syn-geneic mice and preferentially metastasize to the lungs (28).TC-1 cells were inoculated into the flank of syngeneic mice andtreated with DON as described in Materials and Methods. Themouse body weight was stable (Fig. S11), but the s.c. tumorvolumes were significantly smaller than those in PBS-treated controlmice (Fig. 5 A–C). Immunoblots of tumor tissues showed that DONreduced O-GlcNAc relative to controls (Fig. 5D). IHC of tumortissues confirmed this result and also revealed that the population ofPCNA-positive cells was significantly reduced, whereas cells positivefor cleaved caspase-3 were greatly increased in DON-treated mice(Fig. 5E).Because inhibition of O-GlcNAc suppresses migration, invasion,

and anchorage-independent cell growth of HPV-positive cells (Fig.2 C–H), O-GlcNAc might also contribute to HPV-promoted tumormetastasis. To test this hypothesis, we used the TC-1 cells in themouse lung metastasis model (28). TC-1 cells were injected intothe tail vein of C57BL/6 mice. Application of DON strikingly re-duced the number of TC-1 tumor foci in the lungs as well as thesizes of the foci (Fig. 5 F and G; P < 0.01). No notable toxic sideeffect (such as leucopenia, lethargy, diarrhea, etc.) was observed.Thus, our results show that suppression of O-GlcNAc reducesHPV-positive tumor growth and metastasis in mouse models.

c-MYC Is O-GlcNAcylated and Stabilized in HPV Infections. The basisfor substrate specificity of O-GlcNAc is unclear, making the iden-tification of key substrates challenging. However, c-MYC, an on-cogenic transcriptional factor, is known to be O-GlcNAcylated (12,13, 25). To examine whether c-MYC and other cellular factorsknown to be activated by HPVs are O-GlcNAcylated due to theinduction of OGT by HPV E6 or E6/E7, we used wheat germ ag-glutinin affinity purification to pull down O-GlcNAc proteins from

MEF/GFP (control) and MEF/HPV16 oncogenes. Immunoblotsshowed that, relative to the control, both total and O-GlcNAcylatedc-MYC were markedly increased in MEF/HPV16 E6 or E6/E7, butnot in MEF/HPV16 E7 (Fig. 6A). To confirm the O-GlcNAcylationof c-MYC, we performed a reciprocal pull-down using HeLa cellextracts. Immunoprecipitated c-MYC was detected with a pan-O-GlcNAc antibody, in particular in cells with elevated O-GlcNAcdue to OGA kd (Fig. 6B). In our experimental settings, there wasno detectible O-GlcNAcylation of GAPDH and S6 (Fig. 6A).However, we cannot rule out the possibility that other host proteinsin HPV-infected cells are O-Glycosylated and play a role in medi-ating HPV oncogenic activities.To validate that O-GlcNAc is responsible for elevated c-MYC

levels in HPV-infected cells, we exposed MEF/HPV16 E6E7 orMEF/GFP to 10 μM DON for 16 h. As before, the level of c-MYCwas elevated upon E6/E7 expression relative to the control (Fig. 6C).However, it was dramatically reduced in the presence of DONregardless of the presence or absence of E6/E7 (Fig. 6C). This

Fig. 4. Knockdown of OGT reduces O-GlcNAc and tumor growth. HeLa cellswere stably transduced with lentiviruses expressing shRNA-Ctrl, shRNA-OGT, orshRNA-OGA. (A) Immunoblots were used to detect O-GlcNAc, OGT, and OGAin cell lysates. GAPDH served as a loading control. (B and C) Migration andinvasion assays in HeLa cells in which OGT or OGA expression was stablyknocked down. *P < 0.01 (n = 3). (D) Anchorage-independent cellular growthin soft agar. *P < 0.01 (n = 3). (E and F) Depletion of OGT reduced tumorgrowth in SCID mice, whereas OGA knockdown promoted tumor growth. Twomillion HeLa/shRNA-Ctrl, /shRNA-OGT, or /shRNA-OGA cells were inoculateds.c. under the flank. (E) Tumor volumes were measured every 3 d. *P < 0.01and **P < 0.05 compared with shRNA-Ctrl group (n = 10). (F) The tumors wereremoved from euthanized mice. IHC was used to detect cells positive for activecleaved caspase-3 in the tumor tissues. At least 200 cells were counted in eachgroup for determining the percentage of positive cells. *P < 0.01.

Fig. 5. Suppression of O-GlcNAcylation impedes HPV-positive tumor growthand metastasis in TC-1–induced cancer models in syngeneic mouse. (A–D) The2 × 106 TC-1 cells were inoculated s.c. under the flank of C57BL6 mouse.Mice were treated with PBS or DON (five mice/group). (A) Tumor volumes forPBS- and DON-treated mice. *P < 0.01, comparison of the tumor volumesbetween DON and PBS treatment from day 9. (B) Tumors in mice. Arrows in-dicate the locations of the tumors. (C) Tumors recovered from euthanizedmice. (D) O-GlcNAc and c-MYC were reduced in tumors frommice treated withDON. Fresh tumor tissues from the mice were frozen in liquid nitrogen andwere kept at −80°. WCEs from the tumor tissues were isolated for immunoblotanalyses. GAPDH served as a loading control. (E) IHC detection of O-GlcNAc,PCNA, active cleaved caspase-3, and c-MYC in the tumor tissues. Representa-tive stained tissue sections were presented for each group. (Scale bar, 50 μm.)(F and G) C57BL6 mice were injected with 2 × 105 TC-1 cells via the tail vein.Mice were administered with PBS or DON as in A–D. Lung tissues were col-lected from euthanized mice, fixed, and stained with H&E. (F) Representativephotographs of lung tumor foci. (Scale bar, 100 μm.) Lung metastatic foci inthe maximum horizontal layer for each mouse were counted (G). *P < 0.01.

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O-GlcNAc–mediated regulation of c-MYC levels was further ver-ified in mouse TC-1 cells (Fig. 6D). Suppression of O-GlcNAc with10 μM DON for 16 h in TC-1 cells markedly reduced the level ofc-MYC (Fig. 6D). In contrast, elevating O-GlcNAc with thiamet-G(TG), an inhibitor of OGA, or 2-deoxyglucose (2-DG), a glucose-mimic, correlated with increased levels of c-MYC (Fig. 6D). Wenoted that depletion of glucose (−G), a means previously reportedto promote O-GlcNAc, did not do so in our experimental setting(Fig. 6D). Interestingly, the levels of OGT and OGA changedin opposite directions and correlated with levels of intracellularO-GlcNAc (Fig. 6 C and D), possibly due to a feedback regulation(5). The reduction of c-MYC by DON treatment was also con-firmed in TC-1 tumors by IHC (Fig. 5E). Collectively, our datasuggest that HPV-up–regulated O-GlcNAcylation is responsiblefor the elevation of c-MYC protein.To examine the mechanism by which O-GlcNAc elevated

c-MYC protein levels, we determined the protein stability in MEFstransduced with HPV16 E6, E7, E6/E7, or GFP. Cells were

exposed to 20 μg/mL cycloheximide (CHX) for up to 8 h, and thelevels of c-MYC were determined by immunoblots. The half-life ofc-MYC in the control or E7-expressing cells was shorter than30 min. In contrast, its half-lives in E6- or E6/E7-expressing cellswere substantially longer, reaching 60 min (Fig. 6 E and F). c-MYCwas reported to be O-GlcNAcylated on Thr58 (8, 12, 13). Weobserved that O-GlcNAc on his-tagged ectopic c-MYC Thr58A(Thr-to-ala mutation) was dramatically reduced in HeLa cells rel-ative to wild-type his-tagged c-MYC (Fig. S12A). Moreover, unlikeendogenous wild-type c-MYC (Figs. 5E and 6 C and D), the his-c-MYC Thr58A level was not affected by O-GlcNAc modulatorsST045849 or TG (Fig. S12B). Taken together, our results suggestthat c-MYC was stabilized in E6- and E6/E7-expressing cells uponO-GlcNAcylation on Thr58.

Overexpression of c-MYC Partially Rescues HPV-Mediated Cell ViabilityBlocked by a High Concentration of O-GlcNAc Inhibitor. We then setout to determine the effect of O-GlcNAc on c-MYC function. Wegenetically manipulated the expression of c-MYC in HeLa cellsand determined cell viability under the influence of the O-GlcNAcinhibitor DON. As shown in Fig. 6G, stable expression of c-MYCincreased HeLa cell number and partially reversed cellular via-bility suppressed by 10 μM of DON for 72 h. These results suggestthat O-GlcNAc enhances HPV-mediated cell survival and thatthis effect is at least in part mediated through elevated c-MYCupon O-GlcNAcylation.

DiscussionAlthough HPV E6 and E7 proteins possess multiple biochemicalactivities, including inactivating the two major tumor suppressorsp53 and pRb, these interactions cannot completely account forHPV carcinogenesis. Therefore, it is of interest to identify cellularfactors that contribute to lesion progression to cancer. In thisstudy, we demonstrated that O-GlcNAc and OGT were markedlyincreased in cervical neoplasms associated with HR HPV infec-tions, whereas OGA was not significantly altered (Fig. 1). Wedemonstrated in vitro that this increase was due to HR HPV E6-stimulated transcriptional up-regulation of OGT (Figs. 2A and 3and Fig. S3), and a number of transcription factors were impli-cated (Fig. S9). Some of these factors, such as NF-κB, AP-1, andp53, are known targets of the viral E6 protein for activation orinactivation (21, 22, 27). We further demonstrated that O-GlcNAcenhanced HPV oncogenic activities, including cell proliferation,migration, invasion, and anchorage-independent cell growthin vitro (Figs. 2 and 4), as well as tumorigenesis and metastasis ofHPV-transformed cells in mouse models (Figs. 4 and 5). Consis-tent with these observations, suppression of O-GlcNAc by thechemical reagents DON and ST045849 or by OGT knockdownsubstantially inhibited these transformed phenotypes in vitro andin vivo. In contrast, knockdown of OGA elevated O-GlcNAc andenhanced the transformed phenotypes in vitro and in vivo (Figs. 2,4, and 5). Collectively, our results support the conclusion thatO-GlcNAc mediates the oncogenic activity of HPV oncogenes.Yew et al. previously identified an isoform of mixed lineage

leukemia 5 (MLL5β), which regulates HPV E6/E7 oncogenetranscription through its interaction with AP-1 at the 5′ segment ofthe HPV16/18 long control region (LCR) (29). Interestingly, Ninet al. (30) reported that MLL5β was O-GlcNAcylated at T440residue and this modification is necessary for its interaction withAP-1 and its recruitment to the HPV16/18-LCR. Thus, MLL5βO-GlcNAcylation is an important initiation step in E6/E7 transcrip-tion. Inhibition of O-GlcNAcylation by azaserine decreased E6/E7levels and selectively suppressed the survival of HPV-positive cervicalcancer cells (30). Our current data suggest that O-GlcNAcylationalso mediates the HPV downstream oncogenic activities. It wouldseem that there is a feed-forward action for O-GlcNAcylation inHPV infection.

Fig. 6. c-MYC is O-GlcNAcylated and stabilized by HPV oncogene expression.(A) O-GlcNAcylated proteins in MEFs transduced with GFP or with HPV16 E7, E6,or E6/E7 were pulled down with succinylated wheat germ agglutinin beads.c-MYC and O-GlcNAc in the pull-down complexes were detected by immuno-blotting. (B) c-MYC was O-GlcNAcylated. c-MYC in HeLa/shRNA-Ctrl, shRNA-OGT,and shRNA-OGA cells was immunoprecipiated with a polyclonal antibody toc-MYC (Upper). The O-GlcNAcylated c-MYC was detected with an O-GlcNAcmonoclonal antibody, RL2. (C) HPV16 E6/E7 elevated the O-GlcNAcylated pro-teins and the c-MYC protein. Both enhancements were abolished by the in-hibition of O-GlcNAc with DON. MEFs transduced with GFP or E6/E7 weretreated with 10 μM DON overnight. The WCEs were harvested for immunoblotanalyses. (D) The amounts of c-MYC correlated with the levels of O-GlcNAc.TC-1 cells were treated with 10 μMDON, 10 mM 2-DG, 10 μM TG, or starved (noglucose, −G) for overnight. The WCEs were collected and subjected to immu-noblot analyses. In C and D, GAPDH served as a loading control. (E and F) c-MYCwas stabilized in HPV16 E6- and E6/E7-expressing cells. MEF/GFP, E6, E7, andE6/E7 cells were treated with 20 μg/mL CHX for the indicated duration. TheWCEs were then collected for immunoblots to detect c-MYC and S6 in the cells(E). c-MYC levels at each time point were quantified with NIH ImageJ software(F). (G) Overexpression of c-MYC partially rescued O-GlcNAc inhibition-inducedrepression of cell viability. HeLa cells were stably transfected with empty vectoror c-MYC expression construct. The cells were then selected with 500 μg/mLG418 for 2 wk. The survived clones were pooled and subjected to cell viabilityassay in the presence or absence of DON (10 μM) for 72 h. *P < 0.01 (n = 3).

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The MYC family proteins are key regulators of cell growth, pro-liferation, differentiation, and apoptosis by modulating the expressionof a significant number of genes (31). MYC also governs events as-sociated with tumor progression, including genetic instability, cellmigration, and angiogenesis (31, 32). In particular, c-MYC is a criticalmediator for HPV-induced immortalization and transformation.HPV-E6 interacts with c-MYC to reactivate the transcription ofhuman telomerase reverse transcriptase (33–36). However, the effectof HPV oncoproteins on c-MYC protein levels is inconclusive (33–36). Due to the pleiotropic activities of c-MYC, its expression andfunction must be fine-tuned. In particular, the protein is subjectto posttranslational modifications, including phosphorylation, ubiq-uitinylation, acetylation, and glycosylation (12, 13, 37, 38).We demonstrated that, in HPV E6- and E6/E7-expressing

cells, c-MYC was elevated through protein stabilization viaO-GlcNAcylation (Fig. 6). c-MYC has been reported to beO-Glycosylated at Thr58, a known phosphorylation site and amutational hot spot in lymphomas (12, 13). Our current dataindicate that c-MYC Thr58 is targeted by HPV oncogene-inducedO-Glycosylation (Fig. S12). We showed that stable expressionof c-MYC in HeLa cells increased cell viability and partiallyrestored viable cell populations that were reduced by inhibitingO-GlcNAc with a high concentration of DON (Fig. 6G). Im-portantly, c-MYC and O-GlcNAc levels were coregulated inthe mouse tumor model with regard to cell proliferation andsurvival (Fig. 5 D and E). Our findings are also consistent withprevious reports demonstrating that c-MYC is positively cor-related to cell proliferation in cervical tissue specimens (39).In summary, we have uncovered a mechanism for HPV carci-

nogenesis in the form of E6-stimulated OGT expression, leading to

elevated O-GlcNAcylation. We have identified c-MYC to be oneof the OGT substrates, and c-MYC level is highly elevatedthrough this posttranslational protein stabilization. Because ofthe extensively collaborative activities between O-GlcNAc andHPV oncogenes, we believe that reduction of O-GlcNAc inHPV-infected cells with inhibitors, knockdown of OGT, or over-expression of OGA could be instrumental in reducing HPV on-cogenic actions. The O-GlcNAc targeting in mice tumor modelsdescribed in this study supports this hypothesis and could ulti-mately prove to be of therapeutic value.

Materials and MethodsThe animal protocol was approved by the Institutional Animal Care and UseCommittee at Jilin University and the University of Alabama at Birmingham(UAB). Human cervical tissues were obtained from the Department ofPathology at Jilin University and the University of Alabama at Birminghamtissue bank. Institutional review board approvals for the usage of thesetissues were obtained from Jilin University and UAB. C33A and MEFs wereinfected with lentiviruses expressing HPV16 E6, E7, E6/E7, or GFP. After aselection with puromycin (1.0 μg/mL) for 2 wk, resistant stable clones werepooled and passaged. To knock down OGT or OGA stably in HeLa cells, cellswere infected with lentiviruses containing the OGT, OGA, or scramble shRNAconstructs and selected with puromycin (1 μg/mL) for 2 wk. There were nodeleterious effects on the viability of selected cells, as determined by trypanblue exclusion and ATP levels.

Additional information on materials and methods is presented in SIMaterials and Methods.

ACKNOWLEDGMENTS. We thank Dr. T.-C. Wu (Johns Hopkins University) forproviding TC-1 cells. The study was supported by NIH Grants R01CA133053,R01CA83679, P50CA098252, and U19AI113212 and National Natural ScienceFoundation of China Grants 81271853, 81272243, and 81573087.

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