Cancer Therapy: Preclinical

BAP1 Is a Novel Target in HPV-Negative Headand Neck CancerXiyou Liu1,2, Manish Kumar1, Liangpeng Yang1, David P. Molkentine3,David Valdecanas3, Shiying Yu2, Raymond E. Meyn3, John V. Heymach4, andHeath D. Skinner1

Abstract

Purpose: This study examined the potential role of the nucleardeubiquitinating enzyme BRCA1-associated protein-1 (BAP1) inradioresistance in head and neck squamous cell cancer (HNSCC).

Experimental Design: We overexpressed, knocked down, andrescued BAP1 expression in six HNSCC cell lines, three humanpapillomavirus (HPV)–negative and three HPV-positive, andexamined the effects on radiosensitivity in vitro and in an HNSCCmouse xenograft model. Radiosensitivity was assessed by clono-genic cell survival and tumor growth delay assays; changes inprotein expression were analyzed by immunofluorescence stain-ing and Western blotting. We also analyzed The Cancer GenomeAtlas HNSCC database to test for associations between BAP1expression and outcome in patients.

Results: Overexpression of BAP1 induced radioresistance inboth cell lines and xenograft models; conversely, BAP1 knock-

down led to increased ubiquitination of histone H2A, whichhas been implicated in DNA repair. We further found thatBAP1 depletion suppressed the assembly of constitutiveBRCA1 foci, which are associated with homologous recombi-nation (HR), but had minimal effect on g-H2AX foci and didnot affect proteins associated with nonhomologous end join-ing, suggesting that BAP1 affects radiosensitivity in HNSCC bymodifying HR. Finally, in patients with HNSCC, overexpres-sion of BAP1 was associated with higher failure rates afterradiotherapy.

Conclusions:BAP1 can induce radioresistance inHNSCC cells,possibly via deubiquitination of H2Aub and modulation of HR,and was associated with poor outcomes in patients with HNSCC.BAP1 may be a potential therapeutic target in HNSCC. Clin CancerRes; 24(3); 600–7. �2017 AACR.

IntroductionHead and neck squamous cell carcinoma (HNSCC) is the sixth

most common type of cancer worldwide, with an annual inci-dence of approximately 600,000 new cases and approximately320,000 deaths each year (1). Radiotherapy is the primary treat-ment modality for most patients with locally advanced HNSCC.Unfortunately, about half of such patients experience recurrenceafter treatment and ultimately succumb to the disease, suggestingthe existence or emergence of radioresistant clones that surviveafter radiotherapy (2, 3). Finding the means of overcoming

resistance to radiation is critical to improving survival for patientswith HNSCC.

Recently, our group used proteomic and transcriptomic analysisto screen for potential targets associated with poor outcomefollowing radiation in human papillomavirus (HPV)–negativeHNSCC clinical samples (4). One novel candidate identified onreverse-phase protein array analysis (RPPA) screening was theBRCA1-associated protein-1 (BAP1), a nuclear deubiquitinatingenzymeconsistingof729aminoacids. BAP1 regulates anumberofcellular processes, including cell cycle, cell differentiation, tran-scription, andDNA damage response (DDR). Moreover, germlinemutations in BAP1 can lead to a tumor predisposition syndrome,possibly due to altered DDR (5). BAP1 canmodulate the DDR viaseveral different interactions, including deubiquitinylation ofhistone H2A (6, 7). BAP1 regulates the DDR by interacting withthe BRCA1/BARD1 heterodimer and with RAD51, a BRCA1/BARD1 complex with E3 ubiquitin ligase activity (8, 9).

Because of its ability to modulate DDR, BAP1 may also mod-ulate response to radiation. Indeed, Nishikawa and colleagues (8)first found that depletion of BAP1 in HeLa cells resulted inhypersensitivity to ionizing radiation. This discovery, in combi-nation with our own identification of BAP1 in an RPPA screen forproteins associated with treatment failure following radiation inHPV-negative HNSCC, led us to hypothesize that BAP1 partici-pates in the development of radioresistance in HNSCC, poten-tially via modulation of DDR after irradiation. In testing thishypothesis, we found that BAP1-knockdown (KD) and knockout(KO) cells were more sensitive to radiation regardless of HPV orp53 status, and this sensitivity was reversed by forced reexpression

1Department of Radiation Oncology, The University of Texas MD AndersonCancer Center, Houston, Texas. 2Department of Oncology, Tongji MedicalCollege, Tongji Hospital, Huazhong University of Science and Technology,Wuhan, China. 3Department of Experimental RadiationOncology, TheUniversityof Texas MDAnderson Cancer Center, Houston, Texas. 4Department of Thoracicand Head and Neck Medical Oncology, The University of Texas MD AndersonCancer Center, Houston, Texas.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

X. Liu and M. Kumar contributed equally to this article.

Corresponding Author: Heath D. Skinner, Department of Radiation Oncology,The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard,Unit 97, Houston, TX 77030. Phone: 713-563-3508; Fax: 713-563-2331; E-mail:hskinner@mdanderson.org

doi: 10.1158/1078-0432.CCR-17-1573

�2017 American Association for Cancer Research.

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of BAP1-WT in the BAP1-KD cells. We also observed a significantenhancement of radiosensitivity after BAP1-KD in a mouse xeno-graft model of HNSCC. Finally, in the TCGA HNSCC cohort, wefound that BAP1 was associated with disease-free survival. Col-lectively, our findings suggest that BAP1 promotes radioresistancein HNSCC and is a possible target for therapeutic development.

Materials and MethodsCell line and cell culture

The HNSCC cell lines used in this study were generouslysupplied by Dr. Jeffrey Myers via The University of Texas MDAnderson Cancer Center Head and Neck cell line repository.Of the six cell lines tested, HN31, HN30, and TR146 were HPVnegative and UMSCC47, SCC154, and SCC152 were HPVpositive. Cells were maintained in DMEM (Gibco), supplemen-ted with 10% FBS, 1% penicillin/streptomycin, 1% glutamine,1% sodium pyruvate, 1% nonessential amino acids, and 1%vitamins and incubated at 37�C in a humidified atmospherecontaining 5% CO2.

Plasmids and shRNA transfectionCell lines were transfected with shRNAs specific for BAP1 or

control scrambled shRNA via lentiviral vectors containing puro-mycin resistance gene (GE Dharmacon). Lentiviral-transfectedcontrol (pGIPZ; shControl) cells and BAP1 shRNA KD cells weresubjected to puromycin selection. After antibiotic screening,pooled clones of control cells and BAP1-KD cells were assessedfor BAP1 protein expression by immunoblotting. HN31 andUMSCC47 BAP1-KD cell lines were further transduced withlentiviral vectors (pLVX) encoding wild-type (WT) BAP1 vectors(kindly shared by Dr. Boyi Gan from The University of Texas MDAnderson Cancer Center), to rescue BAP1 expression. BAP1expression was confirmed by immunoblotting in these cells aswell. BAP1 CRISPR/Cas9 knockout plasmid and control CRISPR/Cas9 plasmids were constructed as described previously (10). TheBAP1 KO gRNA sequences are shown in the SupplementaryInformation. Knockout of BAP1 expression was confirmed inthese cell lines via immunoblotting.

Ionizing radiationCell cultures were irradiated with an X-Rad 320 Biological

Irradiator (320 KV, 12.5 mA, SSD 50 cm, Precision X-Ray), andin situ tumors were irradiated with a Shepherd Mark I 137Csirradiator (662 keV Model, 68-A), with a custom block. For theShepard Mark I, absolute dosimetry was performed employingion chambers calibrated by the Accredited Dosimetry Calibration

Laboratory, in-air employing AAPM protocol TG61. For mouseirradiation, EBT3 (Gafchromic-Film)was employed for dosimetryin simulated irradiation geometry. The mouse was simulated bytissue-equivalent Gel "Superflab." Ratio of EBT3 response insimulated geometry versus ion chamber calibration in referencegeometry provides the dose rate in the animal.

Colony formation assayRadioresponse (sensitivity or resistance) was assessed via col-

ony formation assay. Known numbers of cells were plated in 6-well plates or 6 dishes for 12 to 14 hours and then irradiated at theindicated doses of radiation. The cells were allowed to formcolonies over a 10- to 14-day incubation period and were thenfixed in a 1.5% crystal violet/25%methanol solution. Numbers ofcolonies containingmore than 50 cells each were then counted todetermine surviving fraction.

ImmunoblottingProtein expression levels were assessed by immunoblotting

from whole-cell lysates. Total protein was extracted by usingnuclear and cytoplasmic extraction reagents with protease andphosphatase inhibitors according to the manufacturer's instruc-tions (Thermo Fisher Scientific). The protein concentrations oflysates were measured by the ABC assay (Bio-Rad). The fol-lowing primary antibodies were used: BAP1 (C-4), p53 (DO-1),and BRCA1 from Santa Cruz Biotechnology; gH2AX, H2Aub(K119), 53-BP1, Chk1, Chk2, p-Chk1, p-Chk2, and b-actinfrom Cell Signaling Technology. Goat anti-mouse and anti-rabbit secondary antibody conjugated to horseradish peroxi-dase (Santa Cruz Biotechnology) were used, and signal wasgenerated with the SuperSignal West Chemiluminescence Sys-tem (Pierce Biotechnology).

Immunofluorescence stainingImmunofluorescence was measured as described previously

(11). In brief, cells were cultivated on cover slips placed in 35-mmcell culture dishes. At specified time points after exposure toradiation (2 Gy), cells were fixed in 4% paraformaldehyde for15 minutes at room temperature, briefly washed in PBS (Media-tech), and placed in 70% ethanol overnight at 4�C. Then, fixedcells were permeabilized with 0.1% IGEPAL (octylphenoxypo-lyethoxyethanol) for 20minutes at room temperature, blocked in2% BSA (Sigma) for 60 minutes, and then incubated in anti-gH2AX (1:400) or anti-BRCA1 primary antibody (1:400) over-night at 4�C. To assessH2Aub level, cells were permeabilizedwith0.4% Triton X-100 for 5minutes at room temperature, blocked in5%normal goat serum for 60minutes, and then incubated in anti-H2Aub (1:1,000) overnight at 4�C. Cells were then washed fourtimes with PBS and incubated for 1 hour in secondary anti-rabbitantibody conjugated with Cy3 (Jackson ImmunoResearch) tovisualize gH2AX foci and H2Aub or in secondary anti-mouseantibody conjugated to Cy3 to visualize BRCA1. DNAwas stainedwith DAPI (40, 6-diamidino-2-phenylindole; Sigma) followed bymounting on labeled slides with mounting media (DAKO).Immunoreaction results were visualized with a Leica Microsys-tems microscope, and foci were counted with ImageJ software(https://imagej.nih.gov/ij/).

Homologous recombination repair assayIn HEK293T cells, the pDR-GFP plasmid (3 mg) was transfected

using PEI transfection reagent in Opti-MEM Reduced SerumMedia (5 ug/mL). After an incubation of 4 to 6 hours at 37�C,

Translational Relevance

HPV-negative head and neck cancer is the most aggressiveform of the disease and is often resistant to curative therapywith radiation. We have examined a novel biomarker of pooroutcome in this disease, BRCA1-associated protein-1 (BAP1).We have validated the clinical significance of this protein inpatient samples as well as showed that modulating BAP1expression directly affects response to radiation both in vitroand in vivo. BAP1 is a clinically significant and potentiallytargetable marker of radioresistance in head and neck cancer.

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media containing 10% FBS were added to the cells. Stablyexpressing cells harboring the pDR-GFP plasmid were selectedwith (2 mg/mL) puromycin. BAP1 expression in these cells wastransiently knocked down by transfecting 10 pmol ON-TARGETplus siRNA Human BAP1 (Dharmacon) by electroporation(Nucleofactor II, Amaxa). After 24 hours, cells were transfectedwith SceI-expressing plasmid (as described above) to inducedouble-stranded breaks. Two days after transfection with SceIplasmid, the dishes were washed with PBS and cells wereharvested and transferred to filter-top FACS tubes. Flow cyto-metric analysis for GFP was performed immediately using a BDAccuri C6 Flow Cytometer (BD Biosciences) and the resultsanalyzed using the BD Accuri C6 Software.

Mouse xenograft modelForty male athymic nude mice (6–8 weeks old, ENVIGO/

HARLAN) were randomly assigned to one of 4 groups of 10 miceeach: untreated HN31 shControl, untreated HN31 BAP1-KD,irradiated HN31-shControl, and irradiated HN31 BAP1-KD.

HN31 tumor cells (4 � 106 in 0.1 mL of serum-free medium)were injected subcutaneously in the right dorsal flank of eachmouse. After palpable tumors had developed, tumor diameterswere measured with digital calipers, and tumor volume wascalculated asA� B2� 0.5, whereA represents the largest diameterand B the smallest diameter of tumor. When the tumor volumesreached approximately 150 mm3, tumors were irradiated with10Gy (2Gy/day for 5 consecutivedays) and tracked for3weeks. Atthat time, the experiment was terminated and tumors harvested.

Clinical data analysisClinical data and BAP1 protein expression were abstracted

from the Head and Neck Cancer cohort of The Cancer GenomeAtlas (TCGA). Of the 530 patients in the cohort, informationon BAP1 protein expression and disease-free survival data wereavailable for 248, and these patients formed the basis for thisanalysis. Survival curves were generated with the Kaplan–Meiermethod, with log-rank statistics used to identify significantdifferences between groups. Patients were grouped by upper,

Figure 1.

BAP1 promotes proliferation in HNSCCcell lines. A and B, Proliferation wastested with MTT assay in HN31 cells(A; HN31-shControl, HN31-BAP1-KD,and HN31-BAP1-WT) and UMSCC47cells (B; UMSCC47-shControl,UMSCC47-BAP1-KD, and UMSCC47-BAP1-WT). Data, means � SEM.� , P < 0.05. C–F, Baseline clonogenicsurvival was tested in colony formationassays in HN31 cells (C; HN31-shControl,HN31-BAP1-KD, and HN31-BAP1-WT)and UMSCC47 cells (E; UMSCC47-shControl, UMSCC47-BAP1-KD, andUMSCC47-BAP1-WT), withcorresponding data normalized toeach control and expressed as means� SEM (D and F). � , P < 0.05.

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middle, and lower tertiles of BAP1 protein expression for thisanalysis.

Statistical analysisData were reported as mean � SEM, and Student t tests

(unpaired, unequal variance) were used to compare two groupsof independent samples for in vitro radiosensitivity, gH2A foci,and BRCA1 foci. GraphPad Prism (v6.0) and SPSS Statistics (v22)were used to analyze the data. A P value <0.05 was considered toindicate statistical significance in all analyses.

ResultsBAP1 promotes proliferation inHNSCC cells regardless of HPVstatus

Studies of the effects of BAP1 on cell proliferation have shownconflicting results, with some reporting that BAP1 suppresses cellproliferation (12, 13), and others reporting that BAP1 enhancesproliferation (14, 15). To investigate the role of BAP1 in HNSCCcell proliferation and clonogenic survival, we tested HN31 (HPV-negative) and UMSCC47 (HPV-positive) HNSCC cell lines trans-fected with BAP1 shRNA to knock down BAP1 (BAP1-KD). Weperformed standardMTT assay to access the effect of BAP1-KD oncell proliferation and colony formation assaywas used to examinebaseline clonogenic survival (Fig. 1). In both cell lines, knock-down of BAP1 significantly inhibited cell proliferation (P <0.05; Fig. 1A and B) and clonogenic survival (Fig. 1C–F). Todetermine whether this phenomenon could be reversed, werescued BAP1 expression via transfection of stably expressingBAP1-WT in HN31-BAP1-KD and UMSCC47-BAP1-KD cell lines.

In both sets of experiments, forced reexpression of BAP1-WTreversed the effect of BAP1-KD phenotype.

BAP1 modulates radioresponse regardless of molecularbackground in HNSCC cells

We previously observed that clinical failure after radiation isassociated with high BAP1 expression in tumors. We next exam-ined the role of BAP1 on radioresponsiveness in HNSCC cell lines.To do so, we utilized stable knockdown-BAP1 versions of severalcell lines. Because the two most important biomarkers of radio-resistance in HNSCC are HPV and p53 mutation, we tested celllines of different HPV and p53 status, namely HN31 (HPV�/p53mutant), TR-146 (HPV�/p53 mutant), HN30 (HPV�/p53 WT),and UMSCC47 (HPVþ/p53 WT). We also generated cells rescuedfrom BAP1-KD by forced expression of BAP1 WT. Colony forma-tion assays after irradiation were used to examine the relationshipbetween BAP1 expression and response to radiation. Interestingly,we found that inhibition of BAP1 universally increased sensitivityto radiation in all cell line variants tested and that forced expres-sion of BAP1 WT reversed this phenomenon (Fig. 2). To rule outshRNA off-target effects, BAP1 gene was knocked out usingCRISPR (KO) in UMSCC47 cells and confirmed the KO of BAP1expression by immunoblotting. Colony formation assay revealed asignificant reduction in the survival fraction in BAP1 KO cellsafter radiation treatment as compared with control cells (P <0.05; Fig. 2E).

BAP1 deubiquitinates H2AubBAP1 has been reported to deubquitinate H2Aub (7), which in

turn inhibits transcription in chromatin regions flanking DNA

Figure 2.

BAP1-KD enhanced sensitivity to radiation in HNSCC cells. A–D, Radiation sensitivity was tested with colony formation assays in HN31 (A), UMSCC47 (B),HN30 (C), and TR146 (D) cell lines. BAP1-KD led to increased sensitivity to radiation, which was reversed by reexpression of WT BAP1. E, BAP1 KO also led tosignificant radiosensitization in UMSCC47 cells. Survival curves, means � SEM. �, P < 0.05.

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double-strand breaks, facilitating DNA repair (16, 17). Thus,BAP1 is thought to modulate DDR through H2Aub. To verifythis assumption in HNSCC, we examined H2Aub expression inBAP1-KD HNSCC lines. Specifically, we transfected HN31,HN30, TR146, UMSCC47, SCC154, and SCC152 cell lines withBAP1 shRNA to generate BAP1-KD cell lines and notedincreased H2Aub in these cell lines (Fig. 3A). To determinewhether this phenomenon could be reversed, we transfectedHN31-BAP1-KD cells and UMSCC47-BAP1-KD cells with stablyexpressed BAP1-WT and found that reexpression of BAP1-WTsuccessfully reversed the effect of BAP1-KD on H2Aub expression(Fig. 3B). Moreover, in a coculture experiment in which controlHN31 or UMSCC47 cells were cultured with their BAP1-KDcounterparts, BAP1 was inversely associated with H2Aub expres-sion (Fig. 3C). In addition, BAP1 KO in UMSCC47 cells led toincreased levels ofH2Aubas comparedwith control cells (Fig. 3D).Thus, BAP1 seems to regulateH2Aub inHNSCCcells andmaybe a

mechanism by which BAP1 induces radioresistance via facilitatingDNA repair.

BAP1 regulates response to radiation via modulatinghomologous recombination

Tobetter understand themechanismunderlying BAP1-inducedradioresistance in HNSCC cells, we counted numbers of g-H2AXfoci (indicative of double-strand breaks) at different times afterirradiation. We found that the number of foci in all variants hadincreased to their peak levels in all cells at 1 hour (Fig. 4A);however, the number of g-H2AX foci was no different amongvariants in either UMSCC47 cells or in HN31 cells (Fig. 4B),findings confirmed by Western blotting (Supplementary Fig. S1).Because BRCA1 has an important role in DDR, specifically inhomologous recombination (HR)–mediated repair, we alsoexamined BRCA1 foci after irradiation at different time intervals(Fig. 4B). We found significantly fewer numbers of BRCA1 foci in

Figure 3.

BAP1 deubiquitinates H2Aub. A, H2Aub expression in HN31, HN30, TR146, UMSCC47, SCC154, and SCC152 cell lines after BAP1-KD. B, Rescue of BAP1 viatransfecting BAP1-WT vector into HN31-BAP1-KD and UMSCC47-BAP1-KD reversed the effect of BAP1-KD on H2Aub levels, as determined by immunoblotting.C, Controls and BAP1-KD cells (HN31 or UMSCC47) were cultured in a 1:1 ratio, fixed, and stained for BAP1 (green), H2Aub (red), and the nuclear stain40 ,6-diamidino-2-phenylindole (blue). Individual cells with inhibited BAP1 expression had higher levels of H2Aub in both cell lines. D, Increased H2Aub expressionin UMSCC47 BAP1 KO cells (gBAP1 #1 and gBAP1 #2) after radiation treatment compared with control cells.

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BAP1-KD cells relative to BAP1 vector control and BAP1-WT celllines at 1, 4, and 24 hours after irradiation in both UMSCC47 andHN31 cell lines, suggesting that BAP1 may affect the recruitmentof BRCA1 to DDR. Because others have found that CHK1, CHK2,53BP1, and ATM proteins have important roles in nonhomolo-gous end joining (NHEJ) inDNAdouble-strandbreak repairDRR,we next examined the effect of BAP1-KD on proteins known to berelated to NHEJ after irradiation with 4 Gy and found that BAP1-KD did not affect levels of Chk1, pCHk1, CHk2, pCHk2, 53-BP1,or ATM(Supplementary Fig. S1), indicating thatNHEJ is not likelythe pathway bywhichBAP1 regulates theDDR.Wenext examinedHR in BAP1-KD and control HEK293T expressing the pDR-GFPplasmid. Inhibition of BAP1 led to a significant reduction in I-SceI–inducedHR (P<0.05; Fig. 4E). Thus, our results indicate thatknockdown of BAP1may lead to radiosensitization via inhibitionof HR.

BAP1-mediated radioresistance in an HNSCC xenograftmodel

To further confirm the association between BAP1 and radio-resistance in vivo, we implanted athymic mice with relativelyradioresistant HN31 shControl and HN31 BAP1-KD tumor cells,allowed tumors to develop, and irradiated the tumors with 2 Gyper day for 5 consecutive days (total dose 10 Gy). We observedthat 2 Gy � 5 days of radiation treatment had minimal effect oncontrol tumors (Fig. 5) but led to a significant decrease in growthof BAP1-KD tumors (Fig. 5; P < 0.05), suggesting that BAP1mediated radioresistance in this xenograft model.

BAP1 expression is associated with disease-free survival inpatients with HNSCC

We previously identified BAP1 as one of several targets asso-ciated with treatment failure following radiation in a screen of

Figure 4.

BAP1 influences DNA damage repair. A and B, Cells were fixed at different times after irradiation with 2 Gy (at 0, 1, 4, and 24 hours) and immunostained forg-H2A foci or BRCA1 foci. A, An example of gH2AX and BRCA1 foci staining in UMSCC47 cells. B, The number of gH2AX foci was not different in control,BAP1-KD, or BAP1-WT variants in either UMSCC47 or HN31 cells. However, knockdown of BAP1 significantly reduced the numbers of BRCA1 foci at all time pointsmeasured in both UMSCC47 and HN31 cells. � , P < 0.05. Data shown are means � SEM from three independent experiments. C, BAP1 siRNA was used totransiently knock down BAP1 expression, with a resultant decrease in GFP expression and, consequently HR, following iScel-mediated DNA damage.� , P < 0.05. Data shown are means � SEM from three independent experiments.

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HNSCC clinical samples (4), leading us to examine potentialassociations between BAP1 protein expression and outcomes inthe HNSCC cohort of The Cancer Genome Atlas database. Weconfirmed that BAP1 was associated with poorer disease-freesurvival in this group (Fig. 6), highlighting the importance ofBAP1 expression in disease control in HNSCC.

DiscussionPatients with radioresistant HNSCC have poor survival out-

comes (2). Increasing the radiotherapy dose may overcomeradioresistance; however, this is not feasible in HNSCC due tonormal tissue toxicity. So, identifying genes that mediate radio-resistance, and finding ways of manipulating these genes so asto enhance radiosensitivity, is critical as a therapeutic strategyfor patients with HNSCC. Previously, we have identified BAP1as a potential biomarker of failure following radiation inHNSCC (4). In the current study, we investigated the abilityof BAP1 to modulate radioresistance in HNSCC as well aspredict outcome in this disease. We found that inhibition ofBAP1 expression led to radiosensitization irrespective of HPVor p53 status, likely due to modulation of HR, and that thisphenomenon can be rescued by forced expression of WT BAP1.Inhibition of BAP1 also led to in vivo radiosensitization, andBAP1 protein expression was associated with disease-free sur-vival in the TCGA HNSCC cohort.

The two predominant DDR repair pathways are classical NHEJand HR. Typically, radiation-induced double-strand breaks inDNA are repaired by NHEJ, but HR can have a significant roleas well. In our study, we found that BAP1 depletion decreased theassembly of constitutive BRCA1 foci, which are associated with

HR, but had minimal effect on g-H2AX foci. Moreover, proteinsassociated with NHEJ were not altered by BAP1 modulation.Thus, in HNSCC, BAP1 seems to be important for radioresponsenot by NHEJ, but rather by modifying HR.

Previously, Calypso and others have demonstrated that, a BAP1homolog in Drosophila, was shown to deubiquitinate H2Aub,following DNA damage and was potentially required for silencingof transcriptionatDSBs (12, 18). Inour study,we found thatBAP1-KD increased expression of H2Aub protein in 6 different HNSCCcell lines of various HPV status and that this increase was reversedby the forced expressionof BAP1-WT. Thus,H2Aubmaybe a targetof BAP1 in HNSCC. Zhou and colleagues reported that histoneH2A ubiquitination/deubiquitination was a critical chromatinmodification involved in regulating gene expression,maintenanceof heterochromatin, and DDR responses (19). Indeed, RNF8 canphysically interact with H2A and catalyze its ubiquitination inresponse to DSBs, leading to BRCA1 and 53BP1 accumulation atflanking regionsofDSBs (19, 20). Yu and colleagues (7) found thatBAP1was recruited toDNAbreak sites after irradiation andH2Aublevels correlated inversely with BAP1 recruitment. In contrast, norecruitment of BAP1 was detected distal to the break sites, wherehigh levels ofH2Aubwere observed.Ourownfindings suggest thatBAP1 inhibition leads to increased level ofH2Aub,which interfereswith chromatin and histone modifications at double-strandbreaks. Moreover, increased H2Aub is inversely proportional tocell proliferation, and indirect stabilization of H2Aub leads toreduced cell proliferation (7, 18, 21).

In conclusion, we found that BAP1 can deubiquitinate H2Auband induces radioresistance in HNSCC cells via modulation ofHR. This effect seems to be independent ofHPV status and to haveclinical significance, as BAP1 protein expression was found to beassociated with poor outcome in HNSCC. BAP1 expression mayserve as a biomarker of radioresponse, and further investigation ofthe interaction between BAP1, HR, and radiation in this disease iswarranted.

Figure 5.

BAP1 mediates radioresistance in an in vivo xenograft model. HN31 cells stablytransfected with control shRNA or shRNA to BAP1 were implanted innudemice and the tumor volumesmeasured every alternate day for 3weeks. Nosignificant difference in tumor growth rate was found in shControl orBAP1-KD tumors; however, exposing BAP1-KD tumors to radiation (2 Gy for5 consecutive days) significantly suppressed tumor growth (P < 0.05).Data are shown asmeans� SEM. Inset: immunoblot showing expression of BAP1expression, H2Aub, and b-actin (loading control) from pooled tumor tissue(�2 tumors).

Figure 6.

BAP1 expression is associated with disease-free survival in TCGA head andneck cancer cohort. Patients are grouped by tertiles of BAP1 expression,with the highest tertile (high BAP1 expression) having the poorest disease-freesurvival.

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Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: L. Yang, H.D. SkinnerDevelopment of methodology: X. Liu, L. Yang, H.D. SkinnerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): X. Liu, M. Kumar, L. Yang, D.P. Molkentine,D. Valdecanas, H.D. SkinnerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Liu, M. Kumar, L. Yang, D. Valdecanas, S. Yu,H.D. SkinnerWriting, review, and/or revision of the manuscript: X. Liu, M. Kumar, L. Yang,S. Yu, R.E. Meyn, J.V. Heymach, H.D. SkinnerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Liu, M. Kumar, L. Yang, J.V. Heymach,H.D. SkinnerStudy supervision: L. Yang, S. Yu, H.D. Skinner

AcknowledgmentsThis work was supported by the following grants from the NCI (NIH):

R01 CA 168484-022 (to J.V. Heymach), HNSCC SPORE 5 P50CA070907-16, CCSG 5 P30 CA01667239 (to J.V. Heymach), R01 CA 168485 (toR.E. Meyn and H.D. Skinner), and R21 CA 182964 (to H.D. Skinner). Thiswork was also supported by the Cancer Prevention Institute of Texas(RP150293; to H.D. Skinner) and The University of Texas MD AndersonCancer Center Multidisciplinary Research Program (to H.D. Skinner andR.E. Meyn).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received June 1, 2017; revised October 3, 2017; accepted November 3, 2017;published OnlineFirst November 7, 2017.

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BAP1 and Head and Neck Cancer

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