O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis Qinghua 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 a Key Laboratory of Pathobiology, Ministry of Education, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China; bDivision of Hematology and Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294; cDepartment of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118; dDepartment of Systems Biology, University of Texas MD Anderson 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)

O-linked GlcNAcylation HPV oncogenicity

| HPV E6 | c-MYC | cervical cancer |

glucose to UDP-N-acetylglucosamine (UDP-GlcNAc), the donor for the O-GlcNAc modification (6). The enzyme O-GlcNAc transferase (OGT) catalyzes the addition of the amino sugar to target proteins, whereas the enzyme O-GlcNAcase (OGA) catalyzes the removal of the sugar (5, 6). Some substrates of O-GlcNAc are alternatively targeted by kinases (7–9). Thus, there is an extensive crosstalk between O-GlcNAc and pathways or mechanisms that are 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, it has been proposed that O-GlcNAc serves primarily to modulate cellular signaling and transcription regulatory pathways in response to metabolic regulation (10, 11). As a nutrient sensor, O-GlcNAc relays the effects of excessive nutritional intake, an important cancer 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. For example, DNA methylation enzymes of the Tet family, which is involved in epigenetic alterations and cancer, interact with and target OGT to multiple chromatin-remodeling complexes (9). Moreover, histones are subject to O-GlcNAc modification, leading to the alteration of their functions (18). O-GlcNAc and OGT levels are elevated in breast, prostate, colon, bladder, and several other Significance

P

ersistent infections of high-risk (HR) human papillomavirus (HPV)-16, HPV-18, and closely related genotypes are etiologically associated with the development of several human cancers, including anogenital and head and neck cancers (1–3). Two HR HPV genes, E6 and E7, are potent oncogenes based on their immortalizing and transforming activities in cell culture systems and their capacities to induce tumors in animal models. The HR HPV E7 oncoprotein binds to more than 20 cellular targets and interferes with multiple cellular processes, leading to deregulated cell cycle, centrosome amplification, DNA damage, anoikis resistance, anchorage-independent cell growth and malignant transformation as well as immune surveillance evasion. E6 is also a multifunctional protein. Constitutive expression of the HR HPV E6 abrogates cell growth arrest and apoptosis, induces genomic instability and somatic mutations, activates telomerase to promote immortalization, disrupts cell polarity, and prevents anoikis. These properties suggest that HPV-associated carcinogenesis involves a coordinated targeting of multiple pathways with each pathway having a distinct but complementing role in malignant transformation (4). O-linked GlcNAcylation (O-GlcNAc) is a reversible posttranslational modification, transferring an amino sugar moiety to serine/threonine residues of cytosolic or nuclear proteins (5). The hexosamine biosynthetic pathway (HBP) converts intracellular www.pnas.org/cgi/doi/10.1073/pnas.1606801113

O-linked GlcNAcylation is a reversible posttranslational protein modification on serine or threonine residues and regulates multiple cellular signaling pathways. We discovered elevated O-GlcNAc and O-GlcNAc transferase (OGT) in HPV-associated cervical neoplasms relative to the normal cervix. We show that HPV E6 upregulates OGT, increases O-GlcNAc, stabilizes c-MYC via O-GlcNAc, and enhances HPV oncogene activities. Conversely, suppression of O-GlcNAc in HPV-transformed cells by knocking down or inhibiting OGT impairs HPV oncogene-induced activities and impedes tumor growth in animal models. Thus, O-GlcNAc plays a critical role in HPV-induced carcinogenesis, and targeting O-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., and Z.-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; and T.-C.W., Johns Hopkins University. The authors declare no conflict of interest. 1

Q.Z. and R.X.Z. contributed equally to this work.

2

To whom correspondence may be addressed. Email: [email protected], [email protected], or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1606801113/-/DCSupplemental.

PNAS | August 16, 2016 | vol. 113 | no. 33 | 9333–9338

MEDICAL SCIENCES

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA 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, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.

and OGT were strongly positive in malignant foci (Fig. 1D) compared with the adjacent normal epithelia. In contrast, there was no appreciable difference in OGA signals between malignant and normal tissues (Fig. 1D). Characterization of additional tissue specimens confirmed these initial findings (Fig. 1 E and F). Thus, elevated OGT is primarily responsible for increased O-GlcNAc in CINs and cervical cancers. Additionally, the staining patterns of OGT and O-GlcNAc mostly overlapped with those from p16INK4A (Fig. S2), a surrogate marker for HR HPVassociated high-grade lesions and cancers (21), suggesting that O-GlcNAc may contribute to HPV oncogenic activity. O-GlcNAc Is Induced by and Mediates the Oncogenic Activity of E6 and E6/E7. To determine whether increased O-GlcNAc in CINs and

cervical cancers is directly linked to HPV infection, we transduced primary mouse embryonic fibroblasts (MEFs) (at passage 3) with recombinant lentiviruses or retroviruses expressing HPV16 or HPV18 E6, E7, E6/E7, or GFP and examined O-GlcNAc in cell lysates by immunoblot (Fig. 2A and Fig. S3). Consistent with previous reports (21, 22), expression of HPV E6 and E7 reduced p53 and Rb levels in the cells (Fig. 2A), respectively. Importantly, using a pan-GlcNAc antibody, proteins with O-GlcNAc were markedly increased in MEF/HPV E6 or E6/E7 relative to the control, whereas E7 had no effect (Fig. 2A and Fig. S3). In addition, immunoblots

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

cancers (16, 19, 20), but neither has been examined previously in the context of HPV oncogene expression or cervical neoplasms. In this work, we asked whether O-GlcNAc might play a role in HPVinduced transformation and carcinogenesis. Results O-GlcNAc and OGT Are Elevated in Cervical Neoplasms. To detect O-GlcNAc in the spectrum of cervical lesions attributed to HPV infections, we used immunohistochemistry (IHC) to probe the presence of O-GlcNAc in a large collection of normal, cervical intraepithelial neoplasias (CINs) and carcinomas of the cervix. As shown in Fig. 1 A–C, normal cervical tissues bore a low level of O-GlcNAc, whereas tissues from CINs and carcinomas exhibited significantly higher signals. High-grade CINs exhibited stronger signals and higher H scores compared with low-grade lesions (Fig. S1). Elevated OGT, reduced OGA, or both may have contributed to the elevated O-GlcNAc. To distinguish these possibilities, we probed for OGT and OGA in serial sections of cervical cancer tissues using IHC. Consistently, both O-GlcNAc 9334 | www.pnas.org/cgi/doi/10.1073/pnas.1606801113

Fig. 2. O-GlcNAc mediates 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 and OGA as well as with O-GlcNAcylated proteins with respective antibodies. E6, E7, Rb, and p53 were determined for evaluating the HPV16 oncogene expression and function. GAPDH served as a loading control. (B) Percentages of MEFs in senescence at passage 12 were determined after staining with SA–β-gal. Data are average ± SEM of three independent experiments. *P < 0.01. Impact of O-GlcNAcylation on HPV16-induced cell migration (C) and invasion (D) as described in SI Materials and Methods. *P < 0.01 (n = 3). (E) Anchorage-independent cellular growth in soft agar. C33A/GFP and C33A/HPV16-E6/E7 cells were grown on 0.4% agar with 1 μM DON for 2 wk. Colonies were recorded with an Envision light microscope and counted. *P < 0.01 (n = 3). (F–I) O-GlcNAc mediates oncogenic activities of HeLa. HPV18-positive HeLa cells were treated with 50 μM ST045849. Migration (F) and invasion (G) assays and anchorage-independent growth (H) were performed as above described. *P < 0.01 (n = 3). WCEs were used for immunoblot analyses. GAPDH served as a loading control (I).

Zeng et al.

confirmed that OGT, but not OGA, was elevated in MEF/HPV16 E6 or E6/E7, but not in MEF/E7 cells (Fig. 2A). Thus, E6 is primarily 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 with chemicals in MEF/HPV16 E6/E7 or GFP (Ctrl). Early passage (p3) of MEFs infected with an empty lentivirus vector halted growth and entered senescence at passages 9–11. In contrast, MEF/HPV16 E6/E7 continued to grow, and clusters of spindleshaped cells were observed as early as passage 6 (Fig. S4). Application of 1.0 μM 6-diazo-5-oxo-L-norleucine (DON), a glutamine antagonist that inhibits O-GlcNAc, did not markedly affect cell viability at passage 6 (Fig. S5A), but strikingly blocked the morphological changes of the E6/E7-transduced MEFs (Fig. S4). In addition, MEF/HPV16 E6/E7 had a reduced senescent cell population, but the addition of 1.0 μM DON continuously from passage 6 increased senescent cells (Fig. 2B). These results suggest that O-GlcNAc could affect the oncogenic activities of HPV16 E6/E7. Similar alterations in the MEFs with or without HPV E6/E7 were also observed after exposure to 50 μM ST045849 [3-(2-adamantanylethyl)-2-[(4-chlorophenyl)azamethylene]-4-oxo1,3-thiazaperhydroine-6-carboxylic acid], which inhibits OGT specifically (23–25) (Figs. S5B and S6). To substantiate this interpretation, we transduced viral oncogenes into C33A cells, a rare cervical cancer cell line devoid of oncogenic HPVs. C33A/HPV16 E6/E7 cells exhibited an elevation in O-GlcNAc (Fig. S7 A and B). We then performed assays in Transwell with or without precoated Matrigel. These cells exhibited increased migration and invasion relative to the parental cells. The effects were abrogated by the treatment with 1.0 μM DON overnight (Fig. 2 C and D), a condition that did not affect cell proliferation and viability. The ability to grow in the absence of anchorage to the extracellular matrix is one of the most important oncogenic properties of HPV-positive cancer cells. We next examined the growth of C33A/HPV16 E6/E7 cells in soft agar in the presence or absence of DON for 2 wk. C33A cells formed few colonies (per 5,000 cells) when plated in soft agar (Fig. 2E). Transduction of HPV16 E6/E7 markedly increased the size and number of the colonies. Application of 1 μM DON substantially reversed the viral effects (Fig. 2E). Similar inhibitory effects on migration, invasion, and soft agar colony formation were also observed in ST045849-treated HeLa cells, an HPV18-positive cell line established from a cervical adenocarcinoma (Fig. 2 F–H). The expression of E6/E7 was not affected (Fig. 2I). Taken together, our data show that O-GlcNAc mediates or promotes the oncogenic activities of the HR HPVs. HPV E6/E7 Enhances the Transcription of OGT. Three to five percent of the intracellular glucose directly enters into the HBP, where it Zeng et al.

Knockdown of OGT Reduces O-GlcNAc and Xenograft Growth of HPVTransformed Cervical Cancer Cells in SCID Mice. To substantiate the

in vitro significance of HPV-mediated up-regulation of OGT with regard to viral oncogenic activities, we knocked down (kd) OGT or OGA in HeLa cells with lentiviruses expressing shRNA-OGT or shRNA-OGA. Cells were selected with puromycin (1 μg/mL). Expression of OGT was reduced by ∼75% (Fig. 4A). OGT knockdown markedly reduced O-GlcNAcylated proteins in the cells (Fig. 4A). Importantly, cell migration, invasion, and anchorage-independent cellular growth in soft agar were markedly reduced following OGT kd (Fig. 4 B–D). In contrast, each of these activities was enhanced in the presence of shRNA-OGA, which severely depleted OGA while elevating O-GlcNAc (Fig. 4 A–D). These observations support the importance of O-GlcNAc in mediating the viral oncogenic activities. To validate the role of OGT in promoting oncogenic properties in an in vivo system, we performed s.c. inoculation of HeLa/shRNA-Ctrl, shRNA-OGT, and shRNA-OGA cells into SCID mice. Knockdown of OGT substantially reduced the tumor volume (Fig. 4E) and the proportion of cells expressing the proliferating cell nuclear antigen (PCNA) (Fig. S10). Apoptotic tumor cells increased, as detected with an antibody to cleaved caspase-3 (Fig. 4F). In stark contrast, depletion of OGA stimulated s.c. tumor growth (Fig. 4E) and increased PCNA-positive cell number (Fig. S10), but had no effect on the cell population positive for cleaved caspase-3 (Fig. 4F). Taken together, our data support the notion that HPV oncogene-induced elevation of OGT and O-GlcNAc have a significant impact on tumor cell survival and growth. Suppression of O-GlcNAcylation Impedes Tumor Growth and Metastasis of HPV-Transformed TC-1 Cells in Syngeneic Mice. To verify further

the significance of O-GlcNAc in HPV-induced tumor growth, we examined the effects of DON in the TC-1 cell-induced mice PNAS | August 16, 2016 | vol. 113 | no. 33 | 9335

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Fig. 3. HPV16 E6 or E6/E7 enhances OGT transcription. (A) MEFs were infected with lentiviruses expressing HPV16 E6, E7, E6/E7, or GFP (control). mRNA levels of OGT in the transduced cells were determined by qPCR. The relative mRNA levels were calculated. *P < 0.01 (n = 3). HPV activated the promoter of OGT in C33A cells (B) and in MEFs (C). Human OGT promoter sequences −1010 to +10 were cloned to the pGL3 vector and transfected to the cells expressing HPV16 oncoproteins. The pRL vector expressing wild-type Renilla luciferase was used as a control reporter. Relative luciferase unit (RLU) was the ratio of the OGT promoter-driven luciferase activity to Renilla activity. *P < 0.01 (n = 3).

is converted into UDP-GlcNAc, the donor for the O-GlcNAc modification (5). Thus, it is possible that O-GlcNAc is affected by glucose metabolism. Our recent data showed that MEF/ HPV16 E7, but not MEF/HPV16 E6, exhibited appreciably elevated glucose consumption compared with MEFs, consistent with the report that HPV16 E7 promotes glycolytic metabolism (26). Thus, our present results would suggest that the increased O-GlcNAc in E6- or E6/E7-containing cells may not be attributed to an alteration in glucose metabolism. To examine the mechanism by which HR HPV oncogenes upregulate OGT, we determined the half-life of OGT. It was not affected by HPV16 oncogene expression (Fig. S8). Rather, the expression of HPV16 E6 or E6/E7 in MEFs increased OGT mRNA levels by about threefold, whereas HPV16 E7 had no significant impact (Fig. 3A). HPV infection influences the expression or activity of multiple transcription factors, including AP-1, SP-1, NF-κB, p53, and c-MYC (21, 22). Interestingly, the promoter region of the OGT gene possesses binding sites for these transcriptional factors. Thus, we cloned human OGT promoter sequences −1010 to +10 into a pGL3 vector and transfected the construct 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 no effect (Fig. 3 B and C). We further showed that, in C33A cells, cotransfection with an expression vector of Foxo3a, SP1, NF-κB p65, or c-MYC increased the OGT promoter activity, whereas p53 expression was suppressive and CREB and E2F1 had no significant impact (Fig. S9). Our results are consistent with a report by Nees et al. (27), who examined genes in differentiating cervical keratinocytes infected with retroviruses carrying HPV16 E6 or E7; they found that HPV16 E6 stimulated expression of multiple genes known to be regulated by NF-κB and AP-1, whereas E7 was less effective.

MEF/GFP (control) and MEF/HPV16 oncogenes. Immunoblots showed that, relative to the control, both total and O-GlcNAcylated c-MYC were markedly increased in MEF/HPV16 E6 or E6/E7, but not in MEF/HPV16 E7 (Fig. 6A). To confirm the O-GlcNAcylation of c-MYC, we performed a reciprocal pull-down using HeLa cell extracts. Immunoprecipitated c-MYC was detected with a pan-OGlcNAc antibody, in particular in cells with elevated O-GlcNAc due to OGA kd (Fig. 6B). In our experimental settings, there was no detectible O-GlcNAcylation of GAPDH and S6 (Fig. 6A). However, we cannot rule out the possibility that other host proteins in HPV-infected cells are O-Glycosylated and play a role in mediating HPV oncogenic activities. To validate that O-GlcNAc is responsible for elevated c-MYC levels in HPV-infected cells, we exposed MEF/HPV16 E6E7 or MEF/GFP to 10 μM DON for 16 h. As before, the level of c-MYC was elevated upon E6/E7 expression relative to the control (Fig. 6C). However, it was dramatically reduced in the presence of DON regardless of the presence or absence of E6/E7 (Fig. 6C). This Fig. 4. Knockdown of OGT reduces O-GlcNAc and tumor growth. HeLa cells were stably transduced with lentiviruses expressing shRNA-Ctrl, shRNA-OGT, or shRNA-OGA. (A) Immunoblots were used to detect O-GlcNAc, OGT, and OGA in cell lysates. GAPDH served as a loading control. (B and C) Migration and invasion assays in HeLa cells in which OGT or OGA expression was stably knocked down. *P < 0.01 (n = 3). (D) Anchorage-independent cellular growth in soft agar. *P < 0.01 (n = 3). (E and F) Depletion of OGT reduced tumor growth in SCID mice, whereas OGA knockdown promoted tumor growth. Two million HeLa/shRNA-Ctrl, /shRNA-OGT, or /shRNA-OGA cells were inoculated s.c. under the flank. (E) Tumor volumes were measured every 3 d. *P < 0.01 and **P < 0.05 compared with shRNA-Ctrl group (n = 10). (F) The tumors were removed from euthanized mice. IHC was used to detect cells positive for active cleaved caspase-3 in the tumor tissues. At least 200 cells were counted in each group for determining the percentage of positive cells. *P < 0.01.

tumor model. TC-1 cells were derived from primary lung epithelial cells of C57BL/6 mice transformed by HPV16 E6/E7 and activated K-RAS (28). These cells induce tumors in syngeneic mice and preferentially metastasize to the lungs (28). TC-1 cells were inoculated into the flank of syngeneic mice and treated with DON as described in Materials and Methods. The mouse body weight was stable (Fig. S11), but the s.c. tumor volumes were significantly smaller than those in PBS-treated control mice (Fig. 5 A–C). Immunoblots of tumor tissues showed that DON reduced O-GlcNAc relative to controls (Fig. 5D). IHC of tumor tissues confirmed this result and also revealed that the population of PCNA-positive cells was significantly reduced, whereas cells positive for 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 tumor metastasis. To test this hypothesis, we used the TC-1 cells in the mouse lung metastasis model (28). TC-1 cells were injected into the tail vein of C57BL/6 mice. Application of DON strikingly reduced the number of TC-1 tumor foci in the lungs as well as the sizes of the foci (Fig. 5 F and G; P < 0.01). No notable toxic side effect (such as leucopenia, lethargy, diarrhea, etc.) was observed. Thus, our results show that suppression of O-GlcNAc reduces HPV-positive tumor growth and metastasis in mouse models. c-MYC Is O-GlcNAcylated and Stabilized in HPV Infections. The basis for substrate specificity of O-GlcNAc is unclear, making the identification of key substrates challenging. However, c-MYC, an oncogenic transcriptional factor, is known to be O-GlcNAcylated (12, 13, 25). To examine whether c-MYC and other cellular factors known to be activated by HPVs are O-GlcNAcylated due to the induction of OGT by HPV E6 or E6/E7, we used wheat germ agglutinin affinity purification to pull down O-GlcNAc proteins from 9336 | www.pnas.org/cgi/doi/10.1073/pnas.1606801113

Fig. 5. Suppression of O-GlcNAcylation impedes HPV-positive tumor growth and metastasis in TC-1–induced cancer models in syngeneic mouse. (A–D) The 2 × 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 for PBS- and DON-treated mice. *P < 0.01, comparison of the tumor volumes between DON and PBS treatment from day 9. (B) Tumors in mice. Arrows indicate the locations of the tumors. (C) Tumors recovered from euthanized mice. (D) O-GlcNAc and c-MYC were reduced in tumors from mice treated with DON. Fresh tumor tissues from the mice were frozen in liquid nitrogen and were kept at −80°. WCEs from the tumor tissues were isolated for immunoblot analyses. GAPDH served as a loading control. (E) IHC detection of O-GlcNAc, PCNA, active cleaved caspase-3, and c-MYC in the tumor tissues. Representative 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 collected from euthanized mice, fixed, and stained with H&E. (F) Representative photographs of lung tumor foci. (Scale bar, 100 μm.) Lung metastatic foci in the maximum horizontal layer for each mouse were counted (G). *P < 0.01.

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exposed to 20 μg/mL cycloheximide (CHX) for up to 8 h, and the levels of c-MYC were determined by immunoblots. The half-life of c-MYC in the control or E7-expressing cells was shorter than 30 min. In contrast, its half-lives in E6- or E6/E7-expressing cells were substantially longer, reaching 60 min (Fig. 6 E and F). c-MYC was reported to be O-GlcNAcylated on Thr58 (8, 12, 13). We observed that O-GlcNAc on his-tagged ectopic c-MYC Thr58A (Thr-to-ala mutation) was dramatically reduced in HeLa cells relative to wild-type his-tagged c-MYC (Fig. S12A). Moreover, unlike endogenous wild-type c-MYC (Figs. 5E and 6 C and D), the his-cMYC Thr58A level was not affected by O-GlcNAc modulators ST045849 or TG (Fig. S12B). Taken together, our results suggest that c-MYC was stabilized in E6- and E6/E7-expressing cells upon O-GlcNAcylation on Thr58. Overexpression of c-MYC Partially Rescues HPV-Mediated Cell Viability Blocked by a High Concentration of O-GlcNAc Inhibitor. We then set

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 immunoblotting. (B) c-MYC was O-GlcNAcylated. c-MYC in HeLa/shRNA-Ctrl, shRNA-OGT, and shRNA-OGA cells was immunoprecipiated with a polyclonal antibody to c-MYC (Upper). The O-GlcNAcylated c-MYC was detected with an O-GlcNAc monoclonal antibody, RL2. (C) HPV16 E6/E7 elevated the O-GlcNAcylated proteins and the c-MYC protein. Both enhancements were abolished by the inhibition of O-GlcNAc with DON. MEFs transduced with GFP or E6/E7 were treated with 10 μM DON overnight. The WCEs were harvested for immunoblot analyses. (D) The amounts of c-MYC correlated with the levels of O-GlcNAc. TC-1 cells were treated with 10 μM DON, 10 mM 2-DG, 10 μM TG, or starved (no glucose, −G) for overnight. The WCEs were collected and subjected to immunoblot analyses. In C and D, GAPDH served as a loading control. (E and F) c-MYC was stabilized in HPV16 E6- and E6/E7-expressing cells. MEF/GFP, E6, E7, and E6/E7 cells were treated with 20 μg/mL CHX for the indicated duration. The WCEs 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-induced repression of cell viability. HeLa cells were stably transfected with empty vector or c-MYC expression construct. The cells were then selected with 500 μg/mL G418 for 2 wk. The survived clones were pooled and subjected to cell viability assay in the presence or absence of DON (10 μM) for 72 h. *P < 0.01 (n = 3).

O-GlcNAc–mediated regulation of c-MYC levels was further verified in mouse TC-1 cells (Fig. 6D). Suppression of O-GlcNAc with 10 μM DON for 16 h in TC-1 cells markedly reduced the level of c-MYC (Fig. 6D). In contrast, elevating O-GlcNAc with thiamet-G (TG), an inhibitor of OGA, or 2-deoxyglucose (2-DG), a glucosemimic, correlated with increased levels of c-MYC (Fig. 6D). We noted that depletion of glucose (−G), a means previously reported to promote O-GlcNAc, did not do so in our experimental setting (Fig. 6D). Interestingly, the levels of OGT and OGA changed in opposite directions and correlated with levels of intracellular O-GlcNAc (Fig. 6 C and D), possibly due to a feedback regulation (5). The reduction of c-MYC by DON treatment was also confirmed in TC-1 tumors by IHC (Fig. 5E). Collectively, our data suggest that HPV-up–regulated O-GlcNAcylation is responsible for 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 MEFs transduced with HPV16 E6, E7, E6/E7, or GFP. Cells were Zeng et al.

Discussion Although HPV E6 and E7 proteins possess multiple biochemical activities, including inactivating the two major tumor suppressors p53 and pRb, these interactions cannot completely account for HPV carcinogenesis. Therefore, it is of interest to identify cellular factors that contribute to lesion progression to cancer. In this study, we demonstrated that O-GlcNAc and OGT were markedly increased in cervical neoplasms associated with HR HPV infections, whereas OGA was not significantly altered (Fig. 1). We demonstrated in vitro that this increase was due to HR HPV E6stimulated transcriptional up-regulation of OGT (Figs. 2A and 3 and Fig. S3), and a number of transcription factors were implicated (Fig. S9). Some of these factors, such as NF-κB, AP-1, and p53, are known targets of the viral E6 protein for activation or inactivation (21, 22, 27). We further demonstrated that O-GlcNAc enhanced HPV oncogenic activities, including cell proliferation, migration, invasion, and anchorage-independent cell growth in vitro (Figs. 2 and 4), as well as tumorigenesis and metastasis of HPV-transformed cells in mouse models (Figs. 4 and 5). Consistent with these observations, suppression of O-GlcNAc by the chemical reagents DON and ST045849 or by OGT knockdown substantially inhibited these transformed phenotypes in vitro and in vivo. In contrast, knockdown of OGA elevated O-GlcNAc and enhanced the transformed phenotypes in vitro and in vivo (Figs. 2, 4, and 5). Collectively, our results support the conclusion that O-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 oncogene transcription through its interaction with AP-1 at the 5′ segment of the HPV16/18 long control region (LCR) (29). Interestingly, Nin et al. (30) reported that MLL5β was O-GlcNAcylated at T440 residue and this modification is necessary for its interaction with AP-1 and its recruitment to the HPV16/18-LCR. Thus, MLL5β O-GlcNAcylation is an important initiation step in E6/E7 transcription. Inhibition of O-GlcNAcylation by azaserine decreased E6/E7 levels and selectively suppressed the survival of HPV-positive cervical cancer cells (30). Our current data suggest that O-GlcNAcylation also mediates the HPV downstream oncogenic activities. It would seem that there is a feed-forward action for O-GlcNAcylation in HPV infection. PNAS | August 16, 2016 | vol. 113 | no. 33 | 9337

MEDICAL SCIENCES

out to determine the effect of O-GlcNAc on c-MYC function. We genetically manipulated the expression of c-MYC in HeLa cells and determined cell viability under the influence of the O-GlcNAc inhibitor DON. As shown in Fig. 6G, stable expression of c-MYC increased HeLa cell number and partially reversed cellular viability suppressed by 10 μM of DON for 72 h. These results suggest that O-GlcNAc enhances HPV-mediated cell survival and that this effect is at least in part mediated through elevated c-MYC upon O-GlcNAcylation.

The MYC family proteins are key regulators of cell growth, proliferation, differentiation, and apoptosis by modulating the expression of a significant number of genes (31). MYC also governs events associated with tumor progression, including genetic instability, cell migration, and angiogenesis (31, 32). In particular, c-MYC is a critical mediator for HPV-induced immortalization and transformation. HPV-E6 interacts with c-MYC to reactivate the transcription of human telomerase reverse transcriptase (33–36). However, the effect of HPV oncoproteins on c-MYC protein levels is inconclusive (33– 36). Due to the pleiotropic activities of c-MYC, its expression and function must be fine-tuned. In particular, the protein is subject to posttranslational modifications, including phosphorylation, ubiquitinylation, 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 via O-GlcNAcylation (Fig. 6). c-MYC has been reported to be O-Glycosylated at Thr58, a known phosphorylation site and a mutational hot spot in lymphomas (12, 13). Our current data indicate that c-MYC Thr58 is targeted by HPV oncogene-induced O-Glycosylation (Fig. S12). We showed that stable expression of c-MYC in HeLa cells increased cell viability and partially restored viable cell populations that were reduced by inhibiting O-GlcNAc with a high concentration of DON (Fig. 6G). Importantly, c-MYC and O-GlcNAc levels were coregulated in the mouse tumor model with regard to cell proliferation and survival (Fig. 5 D and E). Our findings are also consistent with previous reports demonstrating that c-MYC is positively correlated to cell proliferation in cervical tissue specimens (39). In summary, we have uncovered a mechanism for HPV carcinogenesis in the form of E6-stimulated OGT expression, leading to 1. Walboomers JM, et al. (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189(1):12–19. 2. zur Hausen H (2009) Papillomaviruses in the causation of human cancers: A brief historical account. Virology 384(2):260–265. 3. Lajer CB, von Buchwald C (2010) The role of human papillomavirus in head and neck cancer. APMIS 118(6-7):510–519. 4. White EA, Howley PM (2013) Proteomic approaches to the study of papillomavirushost interactions. Virology 435(1):57–69. 5. Hart GW, Slawson C, Ramirez-Correa G, Lagerlof O (2011) Cross talk between O-GlcNAcylation and phosphorylation: Roles in signaling, transcription, and chronic disease. Annu Rev Biochem 80:825–858. 6. Hanover JA, Krause MW, Love DC (2012) Bittersweet memories: Linking metabolism to epigenetics through O-GlcNAcylation. Nat Rev Mol Cell Biol 13(5):312–321. 7. Stine ZE, Dang CV (2013) Stress eating and tuning out: Cancer cells re-wire metabolism to counter stress. Crit Rev Biochem Mol Biol 48(6):609–619. 8. Slawson C, Hart GW (2011) O-GlcNAc signalling: Implications for cancer cell biology. Nat Rev Cancer 11(9):678–684. 9. Chen Q, Chen Y, Bian C, Fujiki R, Yu X (2013) TET2 promotes histone O-GlcNAcylation during gene transcription. Nature 493(7433):561–564. 10. Erickson JR, et al. (2013) Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation. Nature 502(7471):372–376. 11. Yang X, et al. (2008) Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance. Nature 451(7181):964–969. 12. Chou TY, Dang CV, Hart GW (1995) Glycosylation of the c-Myc transactivation domain. Proc Natl Acad Sci USA 92(10):4417–4421. 13. Chou TY, Hart GW, Dang CV (1995) c-Myc is glycosylated at threonine 58, a known phosphorylation site and a mutational hot spot in lymphomas. J Biol Chem 270(32):18961–18965. 14. Jang H, et al. (2012) O-GlcNAc regulates pluripotency and reprogramming by directly acting on core components of the pluripotency network. Cell Stem Cell 11(1):62–74. 15. Ha JR, et al. (2014) β-catenin is O-GlcNAc glycosylated at Serine 23: Implications for β-catenin’s subcellular localization and transactivator function. Exp Cell Res 321(2):153–166. 16. Ma Z, Vocadlo DJ, Vosseller K (2013) Hyper-O-GlcNAcylation is anti-apoptotic and maintains constitutive NF-κB activity in pancreatic cancer cells. J Biol Chem 288(21):15121–15130. 17. Yang WH, et al. (2006) Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability. Nat Cell Biol 8(10):1074–1083. 18. Fujiki R, et al. (2011) GlcNAcylation of histone H2B facilitates its monoubiquitination. Nature 480(7378):557–560. 19. Fardini Y, Dehennaut V, Lefebvre T, Issad T (2013) O-GlcNAcylation: A New Cancer Hallmark? Front Endocrinol (Lausanne) 4:99. 20. Ferrer CM, et al. (2014) O-GlcNAcylation regulates cancer metabolism and survival stress signaling via regulation of the HIF-1 pathway. Mol Cell 54(5):820–831. 21. McLaughlin-Drubin ME, Münger K (2009) Oncogenic activities of human papillomaviruses. Virus Res 143(2):195–208.

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elevated O-GlcNAcylation. We have identified c-MYC to be one of the OGT substrates, and c-MYC level is highly elevated through this posttranslational protein stabilization. Because of the extensively collaborative activities between O-GlcNAc and HPV oncogenes, we believe that reduction of O-GlcNAc in HPV-infected cells with inhibitors, knockdown of OGT, or overexpression of OGA could be instrumental in reducing HPV oncogenic actions. The O-GlcNAc targeting in mice tumor models described in this study supports this hypothesis and could ultimately prove to be of therapeutic value. Materials and Methods The animal protocol was approved by the Institutional Animal Care and Use Committee at Jilin University and the University of Alabama at Birmingham (UAB). Human cervical tissues were obtained from the Department of Pathology at Jilin University and the University of Alabama at Birmingham tissue bank. Institutional review board approvals for the usage of these tissues were obtained from Jilin University and UAB. C33A and MEFs were infected with lentiviruses expressing HPV16 E6, E7, E6/E7, or GFP. After a selection with puromycin (1.0 μg/mL) for 2 wk, resistant stable clones were pooled and passaged. To knock down OGT or OGA stably in HeLa cells, cells were infected with lentiviruses containing the OGT, OGA, or scramble shRNA constructs and selected with puromycin (1 μg/mL) for 2 wk. There were no deleterious effects on the viability of selected cells, as determined by trypan blue exclusion and ATP levels. Additional information on materials and methods is presented in SI Materials and Methods. ACKNOWLEDGMENTS. We thank Dr. T.-C. Wu (Johns Hopkins University) for providing TC-1 cells. The study was supported by NIH Grants R01CA133053, R01CA83679, P50CA098252, and U19AI113212 and National Natural Science Foundation of China Grants 81271853, 81272243, and 81573087.

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Zeng et al.

O-linked GlcNAcylation elevated by HPV E6 mediates viral oncogenesis.

High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in...
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