Targeting the High-Mobility Group Box 3 Protein ... · showed high ( 100%) clonogenic survival (Fig. 1D), as expected. Interestingly, when HMGB3 was depleted in the cisplatin-resistant

Priority Report

Targeting the High-Mobility Group Box 3 ProteinSensitizesChemoresistantOvarianCancerCells toCisplatinAnirban Mukherjee, Van Huynh, Kailee Gaines,Wade Alan Reh, and Karen M. Vasquez

Abstract

Chemotherapeutic regimens for ovarian cancer ofteninclude the use of DNA interstrand crosslink–inducing agents(e.g., platinum drugs) or DNA double-strand break–inducingagents. Unfortunately, themajority of patients fail tomaintaina durable response to treatment, in part, due to drug resistance,contributing to a poor survival rate. In this study, we reportthat cisplatin sensitivity can be restored in cisplatin-resistantovarian cancer cells by targeting the chromatin-associatedhigh-mobility group box 3 (HMGB3) protein. HMGBproteinshave been implicated in the pathogenesis and prognosis ofovarian cancer, and HMGB3 is often upregulated in cancercells, making it a potential selective target for therapeuticintervention. Depletion of HMGB3 in cisplatin-sensitive andcisplatin-resistant cells resulted in transcriptional downregu-

lation of the kinases ATR and CHK1, which attenuated theATR/CHK1/p-CHK1 DNA damage signaling pathway.HMGB3 was associated with the promoter regions of ATRandCHK1, suggesting anew role forHMGB3 in transcriptionalregulation. Furthermore, HMGB3 depletion significantlyincreased apoptosis in cisplatin-resistant A2780/CP70 cellsafter cisplatin treatment. Taken together, our results indicatethat targeted depletion of HMGB3 attenuates cisplatin resis-tance in human ovarian cancer cells, increasing tumor cellsensitivity to platinum drugs.

Significance: This study shows that targeting HMGB3 is apotential therapeutic strategy to overcome chemoresistance inovarian cancer.

IntroductionOvarian cancer is the fifth most common cause of cancer-

related deaths among women worldwide. In the United Statesalone, it is estimated that approximately 22,000 new cases ofovarian cancer will be diagnosed, leading to approximately14,000 deaths in 2018 (1). Ovarian cancer is difficult to detectand once it has progressed to stage IIIC and IV, the 5-year survivalrate is dismal at only approximately 33%(2). Further contributingto the highmortality rate in the patients with ovarian cancer is thecommon development of resistance to cisplatin- or carboplatin-based chemotherapy (3), after which, patients have an averageprogression-free survival of 3–4 months and a median overallsurvival of 9–12 months (4). Unfortunately, fewer than 15% ofthese patients will respond to further treatment (reviewed inref. 4).

The mechanisms underlying cisplatin resistance in ovariancancer cells include, but are not limited to, increased repair ofcisplatin-DNA interstrand crosslinks (ICL), increased DNA dam-age tolerance, and increased drug efflux (5, 6). The nucleotide

excision repair (NER) mechanism and translesion DNA synthesisare involved in processing ICLs (7), and are thought to be moreefficient in cells resistant to cisplatin chemotherapy (reviewed inref. 8). Cisplatin-induced DNA damage activates the checkpointkinases ATM and ATR. Such activation can lead to the phosphor-ylation of CHK2 at Thr68, CHK1 at Ser345, and both CHK1 andCHK2 can induce cell-cycle arrest and apoptosis by phosphory-lating proapoptotic proteins (9).

We have previously demonstrated that the high-mobility groupbox 1 (HMGB1) protein binds with high affinity to ICLs and actsas anNER cofactor in human cells (10, 11). Othermembers of theHMGB family, HMGB2 and HMGB3, share sequence and struc-tural similarities with HMGB1 and possess two box domains,boxes A andB;whereboxAbindsDNAandboxBbendsDNA, andacidic C-terminal tails. When we depleted HMGB1, HMGB2, orHMGB3 separately in human osteosarcoma cells and then sub-jected them to psoralen and UVA irradiation (to induce ICLformation), depletion of eachwas found to be cytotoxic. HMGB3,unlike HMGB1 and HMGB2, is expressed at low levels in normalcells, but is often overexpressed (up to 20-fold) in cancer cells,making it a potential selective therapeutic target; thus, we focusedthis study onHMGB3 (12). Importantly,HMGB3has been shownto be associated with disease prognosis in a wide variety ofcancers (13).

In this study, we investigated the effects of HMGB3 depletionon cisplatin sensitivity in cisplatin-sensitive (A2780) or cisplatin-resistant (A2780/CP70) human ovarian cancer cells. We foundthat HMGB3 depletion sensitized cisplatin-resistant ovarian can-cer cells to cisplatin. In addition, apoptosis was increased in thecisplatin-resistant, HMGB3-depleted cells following cisplatintreatment. Furthermore, we found that HMGB3 was associatedwith the ATR and CHK1 promoters contributing to their

Division of Pharmacology and Toxicology, College of Pharmacy, Dell PediatricResearch Institute, The University of Texas at Austin, Austin, Texas.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Karen M. Vasquez, The University of Texas at Austin,1400 Barbara Jordan Blvd., Austin, TX 78723. Phone: 512-495-3040; Fax: 512-495-4945; E-mail: [email protected]

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expression levels. Our novel findings indicate that HMGB3 mayserve as a novel target for combination therapy to attenuatecisplatin resistance in patients with ovarian cancer.

Materials and MethodsCell culture and determination of cisplatin LD50 values

Cells were purchased from ATCC where they perform shorttandem repeat profiling for cell line authentication. Cells werecultured as described previously (11). Cells were grown to 80%–

90% confluency and were passaged at least three times afterthawing before any experiments were performed and were cul-tured for a period of 6months to perform all the experiments andrepetitions. The A2780/CP70 cells were treated with 1 mmol/Lcisplatin every third passage to maintain cisplatin resistance.Testing for Mycoplasma was not performed. LD50 values weredetermined usingMTT assays (Promega). ForMTT assays, approx-imately 50,000 A2780 or A2780/CP70 cells were plated per wellin a 96-well plate and were treated with 0, 5, 10, 15, 20, or 25mmol/L cisplatin and cell survival was measured 72 hours post-incubation, as recommended by the manufacturer.

siRNA transfection, cisplatin treatment, and induction ofpsoralen ICLs

siRNA treatments and induction of psoralen ICLs were per-formed as described previously (10, 11). Cisplatin solutions wereprepared by dissolving 1 mg of cisplatin in 1 mL of 1� PBSsupplemented with 140 mmol/L NaCl to generate a 3.3 mmol/Lstock, stored at 4�C in an amber tube for no longer than 30 days.

To assess DNA damage checkpoint signaling as a function ofHMGB3 depletion, A2780 and A2780/CP70 cells were plated in60-mm dishes and were treated with either HMGB3 siRNA,nontargeted siRNA, or left untreated. The siRNA sequences usedare shown in Supplementary Table S1. Subsequently, A2780 cellswere treated with 2 mmol/L, and A2780/CP70 cells were treatedwith 10 mmol/L cisplatin, corresponding to their LD50 values. Toassess the total protein levels, cellswere collected at 24, 48, 72, and96 hours postcisplatin treatment, and subjected to Western blotanalyses.

Western blot analysisWestern blots were performed as described previously (11)

using primary anti-HMGB3 rabbit polyclonal antibody, ATM andp-ATM (Ser 1981), secondary anti-b actin rabbit polyclonal anti-body (Abcam Biotechnology Company), CHK2, p-CHK2(Thr68), ATR, p-ATR (Ser428), and p-CHK1 (Ser317; Cell Signal-ing Technology), and CHK1 (Santa Cruz Biotechnology).

Clonogenic assayA total of 4�105U2OS cells were platedwith orwithout siRNA

treatment. SmartPool siGENOME HMGB1, HMGB2, HMGB3siRNA, or nontargeting siRNA (Thermo Fisher Scientific) wereused at 20 nmol/L final concentrations for each transfectionas described above. siRNA-transfected A2780/CP70 cells weretreated with 2 mmol/L cisplatin 48 hours following the secondsiRNA treatment. Nontransfected A2780/CP70 and A2780 cellswere seeded at 4 � 105 cells in 60-mm dishes and treatedwith 2 mmol/L cisplatin as controls. All samples were incubatedwith cisplatin for 72 hours, then treated cells were reseeded infour replicates of 1,000 cells each in 60-mm dishes. Platingefficiency was calculated at approximately 60% for both cell lines.

Untreated, nontransfected A2780/CP70 and A2780 cells wereseeded in the same manner to provide untreated controls. Col-onies were allowed to form for 15 days and were subsequentlyvisualized by fixing the cells with 95% ethanol for 10minutes andthen staining with 0.05% crystal violet for 30 minutes.

FACS analysisA total of 4 � 105 A2780 and A2780/CP70 cells were plated

with or without HMGB3 and nontargeted siRNA (20 nmol/L)and then were treated with 2 mmol/L cisplatin. Forty-eighthours after cisplatin treatment, cells were collected usingTrypsin-Ethylenediaminetetraacetic acid, washed twice withchilled 1�PBS, and fixed with 70% ethanol for 2 hours at 4�C.Subsequently, cells were stained with 20 mg/mL propidiumiodide (final concentration) in PBS with 0.5% Triton-X and20 mg/mL RNase A (final concentration) for 1 hour at 37�C.Cells were sorted using a BD FACS ARIA II Cell Sorter (BDBiosciences) and DNA content was measured.

qRT-PCRTotal RNA was isolated using the TRIzol Reagent (Thermo

Fisher Scientific) as per themanufacturer's recommendation. Twomicrograms of purified RNA for each experimental sample wasused for reverse transcription assays using the High-CapacitycDNA Reverse Transcription Kit (Thermo Fisher Scientific) in a20-mL reaction volume following the manufacturer's recommen-dation. cDNA (100 ng) was used for qPCR using iTaq UniversalSYBR Green Supermix (Bio-Rad Laboratories) in 10-mL reactionvolumes and samples were amplified using a ViiA 7 Real-TimePCR system (Thermo Fisher Scientific) using the machine defaultsetup for amplification, and data were visualized and analyzedusing the ViiA 7 software. The primer sequences used to amplifyDNA are shown in Supplementary Table S2.

Chromatin immunoprecipitation assayChromatin immunoprecipitation assays were performed as

described previously (11). In brief, 106 A2780 and A2780/CP70cells were plated. Twenty-four hours later, cells were fixed andchromatin preps were immunoprecipitated with ATR (CellSignaling Technology) and CHK1 (Santa Cruz Biotechnology)antibodies using the SimpleChip Enzymatic Chromatin IP Kit(Cell Signaling Technology). Samples were amplified using a ViiA7 Real-Time PCR system (Thermo Fisher Scientific) using primersshown in the Supplementary Table S3. A 321-bp region wasamplifiedwith theATR1 andCHK1 primers and a 285-bp productwas amplified with the ATR2 primers (�147 to þ158 from thetranscription start site).

Statistical analysisStatistical analyses were carried out using GraphPad Prism

software. Tests performed to determine P values are indicated inthe figure legends.

Analysis of The Cancer Genome Atlas for HMGB proteinexpression and gene alterations

Alterations in copy numbers, mutations, and expression levelsof different HMGB genes were analyzed on the basis of thesequence data from The Cancer Genome Atlas (TCGA; ref. 14;PanCancer Atlas; TCGA provisional) using the cBioPortal (http://www.cbioportal.org; ref. 12). HMGB protein levels were analyzedusing The Human Protein Atlas (15).

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ResultsIn U2OS cells, we found an increased sensitivity to psoralen

ICLs in the absence of the HMGB proteins (Fig. 1A; Supplemen-tary Fig. S1) with HMGB3 depletion showing a similar effect oncell sensitivity compared with HMGB1 depletion. Importantly,analysis of the alterations in copy numbers, mutations, andexpression levels fromTCGA indicated up to20-fold upregulationofHMGB3expression in cancer cells (Supplementary Fig. S2A andS2B). HMGB1 is ubiquitously expressed at high levels comparedwith HMGB3 in normal cells, but the levels of HMGB1and HMGB3 are very similar in cancer cells (SupplementaryFig. S2C and S2D). This overexpression of HMGB3 in cancer cellsmakes it a potential selective target for therapeutic intervention.Furthermore, we observed an increased gene alteration frequencyof HMGB3 (more than 6%, predominantly in gene amplificationevents) in human serous ovarian cancer compared with HMGB1(less than 2% gene alteration) and HMGB2 (slightly over4%; Fig. 1B).

We confirmed that the cisplatin-resistant A2780/CP70 cellswere approximately 10-fold more resistant to cisplatin(Supplementary Fig. S3), as published previously (16).Targeting HMGB3 using an siRNA-based approach consis-tently achieved approximately 90% reduction in proteinlevels (Fig. 1C). Subsequently, we treated the cells withcisplatin and measured colony formation. The A2780 cellstreated with 2 mmol/L cisplatin showed nearly undetectablelevels of colony formation relative to the untreated controlcells, while A2780/CP70 cells treated with 2 mmol/L cisplatinshowed high (�100%) clonogenic survival (Fig. 1D), asexpected. Interestingly, when HMGB3 was depleted in thecisplatin-resistant A2780/CP70 cells, cisplatin treatment at aconcentration (at 2 mmol/L) approximately 5-fold lower thanthat of the LD50 values, significantly reduced clonogenicsurvival by approximately 50% (Fig. 1D; SupplementaryFig. S3). These results indicated that depletion of HMGB3substantially sensitized cisplatin-resistant A2780/CP70 cellsto cisplatin treatment.

Figure 1.

HMGB3 depletion increases cisplatin sensitivity in cisplatin-resistant human ovarian cancer cells. A, Clonogenic survival of U2OS cells, treated with psoralen and1.8 J/cm2 UVA (365 nm) to induce ICLs, as a function of HMGB protein depletion. NTC, nontreated control; NSsi, nonspecific siRNA; HMGB1–3, specific siRNAsagainst each protein as listed. B, Alteration of the HMGB genes. Red, gene amplification; blue, deletions; green, point mutations. C, Schematic outline of thesiRNA treatment and clonogenic survival assay along with siRNA-mediated depletion of HMGB3 in A2780/CP70 cells, evaluated byWestern blot analysis. Onaverage, approximately 90% HMGB3 depletion was detected from three independent experiments. KD, knockdown; NT, cells treated with nontargeting siRNA(Mock KD). D, Colony formation was evaluated using a clonogenic assay and visualized by fixing the cells with 95% alcohol and staining with 0.05% crystal violet.Various treatments are listed on the right side of the panel. The bar graph represents quantification of colony numbers from three independent experiments.Error bars,� SD. The P values were determined via t test and P values of 0.05 or lower were considered significant. ���� , P < 0.00005. CP, cisplatin.

HMGB3 Modulates Cisplatin Sensitivity

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Consistent with the clonogenic survival assays (Fig. 1D),HMGB3-depleted, cisplatin-treated A2780 cells showed anapproximately 6% increase in the sub-G1 population (Fig. 2A)while the A2780/CP70 cells showed an approximately 24%increase in the sub-G1 cell population compared with the controlcells within 24hours of treatment (Fig. 2B), suggesting an increasein the apoptotic cell population. Average sub-G1 values for non-targeted siRNAandHMGB3-siRNA–treatedA2780 cells followingcisplatin treatment were 39.6% and 33.4%, respectively (Fig. 2A).The nontargeted siRNA-treated cells showed an increase in thesub-G1 population (average sub-G1 20.2% compared with6.7%; Fig. 2B) but it was less than that in the HMGB3-siRNA–treated cells and could be due to the toxic nature of the siRNAtransfection method itself.

To examine a potential role of HMGB3 depletion in increasedchemosensitivity of the resistant A2780/CP70 cells, we evaluatedDNA damage responses following cisplatin treatment by mea-

suring the levels of the ATM, phospho-ATM, ATR, phospho-ATR,CHK2, phospho-CHK2, CHK1, and phospho-CHK1 checkpointkinases at different time points in both the A2780 and A2780/CP70 cells.Western blot analysis ofwhole-cell lysates (Fig. 3A andB) and subsequent densitometric quantification of the proteinlevels revealed that the ATR, CHK1, and p-CHK1 kinase levelswere significantly reduced (by�50% at 24 hours and >50% at 48,72, and 96 hours) up to 96 hours after cisplatin treatment as afunction of HMGB3 depletion in both cell lines (Fig. 3C and D).Our results indicated thatHMGB3depletion significantly loweredthe distribution of the averages of ATR/p-ATR (P < 0.0001) andCHK1/pCHK1 (P < 0.0001) in both the cisplatin-sensitive andcisplatin-resistant cells when compared with the nontargetingsiRNA-treated groups, suggesting a disruption in DNA damagesignaling as a probable cause of increased cell death (as shownin Fig. 1D). Interestingly, we observed a significantly lowerdistribution of the averages of the p-ATM levels in both cell lines

Figure 2.

HMGB3 depletion increases apoptosis in A2780/CP70 cells following treatment with cisplatin. A, A2780 cells were subjected to cell-cycle analysis without orwith 2 mmol/L cisplatin, and sub-G1 cell populations were measured. Similarly, A2780 cells were treated with nontargeted siRNA (NT siRNA) or HMGB3 siRNA and2 mmol/L cisplatin. Cisplatin treatment increased the apoptotic population of the A2780 cells, but no significant difference was observed as a function of HMGB3depletion. B, A2780/CP70 cells were subjected to cell-cycle analysis treated without or with 2 mmol/L cisplatin and the sub-G1 cell populations were measured.As described above, A2780/CP70 cells were treated with NT siRNA or HMGB3 siRNA and then treated with 2 mmol/L cisplatin. HMGB3 depletion increased theapoptotic population of the A2780/CP70 cells after 2 mmol/L cisplatin treatment. The sub-G1 and the G1 values presented as insets are an average of threemeasurements from three independent experiments. CP, cisplatin.

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(P¼ 0.005 for A2780 cells and 0.0048 for the A2780/CP70 cells)but not of the average p-CHK2 levels in the HMGB3 siRNA–treated samples, indicating no clear relationship betweenHMGB3depletion and the ATM/p-ATM and CHK2/p-CHK2 damage sig-naling pathway. These data indicated that HMGB3 depletion ledto the attenuation of the ATR-CHK1-p-CHK1 DNA damage sig-naling pathway after cisplatin treatment.

Cisplatin treatment has been shown to modulate geneexpression profiles in ovarian cancer cells (17). We observeda decrease in the HMGB3 expression levels over time inA2780 cells following cisplatin treatment and was significantlylower at 96 hours in A2780 cells, but not in the chemoresistantA2780/CP70 cells (Fig. 3A and Fig. 4A). Subsequently, wemeasured the mRNA levels of the ATR and CHK1 kinases asa function of HMGB3 depletion and determined that the totalmRNA levels were significantly lower in both A2780 andA2780/CP70 cells (Fig. 4B). These results indicated that ATRand CHK1 expression levels were, to an extent, associated withthe HMGB3 levels in the ovarian cancer cells. Furthermore, viachromatin immunoprecipitation assays, we found that HMGB3was associated with the promoter/enhancer regions of ATR andCHK1 in the human genome, while the association of HMGB3with the CHK1 promoter appeared to be stronger than that ofthe ATR promoter in this assay (Fig. 4C). Consistent with itsrole in modulating gene expression, we observed significantreduction of luciferase expression in both ovarian cancer celltypes when HMGB3 was depleted (Supplementary Fig. S4),

suggesting that HMGB3 may be involved in modulating tran-scription in the ovarian cancer cells.

DiscussionThe HMGB proteins are architectural proteins that, among

other things, regulate chromatin structure, facilitate transcription-al regulation, bind preferentially to alternative DNA structures ordamaged DNA, and play a role in multiple DNA repair pathways.The data presented here demonstrate a role of HMGB3 in sensi-tizing cisplatin-resistant ovarian cancer cells to cisplatin treat-ment, possibly via the transcriptional repression and deregulationof the ATR-CHK1 damage signaling pathway.

The occurrence of cisplatin resistance is currently a therapeuticlimitation in the course of ovarian cancer treatment, as well as inthe treatment of other cancers. To counter the increased efflux ofdrugs in these resistant cells (5, 18), multiple approaches havebeen explored. For example, small-molecule chemosensitizerssuch as colchicine, genistein, and rapamycin were shown toincrease the intracellular accumulation of cisplatin in ovariancancer cells in vitro, resulting in reduced cell survival followingcisplatin treatment (19). Other smallmolecules have been shownto improve responses to cisplatin by reducing the expressionof themultidrug resistance associated protein 2, ultimately increasingintracellular cisplatin concentrations. Alternatively, studies haveshown that inhibition of various signaling pathways, includingthe IGF signaling pathway and colony-stimulating-factor 1

Figure 3.

Analysis of DNA damage checkpoint signaling kinases following treatment with cisplatin as a function of HMGB3 depletion. A, Untreated (NTC), nontargetingsiRNA (NT siRNA), or HMGB3 siRNA-treated A2780 ovarian cancer cells were exposed to cisplatin (2 mmol/L, the LD50 concentration) and cells were collected24, 48, 72, and 96 hours after treatment. Twenty to forty micrograms of total protein was loaded per lane and resolved by SDS-PAGE, probed with indicatedantibodies, and visualized viaWestern blot analysis. B, The cisplatin-resistant A2780/CP70 cells were treated as described above with siRNA and then with10 mmol/L cisplatin (the LD50 concentration) to analyze the levels of the DNA damage response proteins as above. All experiments were repeated at least threetimes. Representative blots are shown. C and D, Densitometric quantification of checkpoint kinases from experiments represented in Fig. 2A and B. All sampleswere normalized against the loading control, b-actin. Furthermore, the HMGB3 siRNA–treated samples (orange bars) were normalized against nontargetingsiRNA (NT siRNA)-treated samples (blue bars) to determine the effects of HMGB3 depletion on DNA damage responses to cisplatin treatment over time (aslisted in the figure). The solid bars represent the average amount of protein detected from at least three experiments, and the bars with the striped patternrepresent the phosphorylated forms of the proteins. ATR and CHK1/pCHK1 protein levels were consistently lower in the cisplatin-treated A2780 and A2780/CP70 cells when HMGB3 was depleted. Error bars,� SD. The differences in the distributions of the samples in the NT siRNA–treated control groups and HMGB3siRNA-treated groups were determined using the Bonferroni Mann–Whitney U test method and P < 0.05 was considered significant. � , P < 0.05. The distributionsof the averages of the ATR/p-ATR and CHK1/p-CHK1 samples were significantly lower (P < 0.0001) in the HMGB3 siRNA–treated groups compared with the NTsiRNA–treated groups in both the cell lines. The distributions of the averages in the p-ATM samples were significantly different in A2780 (�� , P < 0.005) and inA2780/CP70 cells (P < 0.0048) in the HMGB3 siRNA–treated samples compared with NT siRNA–treated samples. No such significant difference was observed inthe p-CHK2 samples.

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receptor, may improve responses to cisplatin treatment in resis-tant tumor cells (20, 21). Upregulation of proapoptotic pro-teins (22) and/or the inhibition of the expression of PARP-1, aprotein involved in DNA repair, have also shown potential forovercoming cisplatin resistance (23).

Targeting the HMGB proteins has shown some promise incancer therapy, although targeting HMGB1 has been a matterof debate due to the conflict between its intracellular DNA-associated functions and extracellular cytokine functions (24).Nevertheless, HMGB1 has been shown to be a promising thera-peutic target for prostate cancer (25). siRNA-mediated depletionof HMGB2 has been shown to increase chemo- and radiosensi-tivity of head and neck squamous cell carcinomas (26), breastcancer cells (27), and colorectal cancer cells (28). Similarly,HMGB3 depletion has been shown to lower the proliferativepotential of colorectal cancer cells (29).

Our novel findings indicating the upregulation of HMGB3 incancer cells, and its role in transcriptional repression of the DNAdamage signaling kinases ATR and CHK1, suggest that HMGB3may represent a target in ovarian cancer for therapeutic interven-tion to overcome cisplatin resistance. Toward this goal, we haveidentified a potential small-molecule interaction site within oneof the DNA-binding domains of HMGB3 and we are currentlyscreening for small-molecule inhibitors of HMGB3. Such endea-

vors may assist in the development of novel approaches toimprove the outcome for patients with ovarian cancer.

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

Authors' ContributionsConception and design: A. Mukherjee, W.A. Reh, K.M. VasquezDevelopment of methodology: A. Mukherjee, W.A. Reh, K.M. VasquezAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): W.A. RehAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Mukherjee, K. Gaines, W.A. Reh, K.M. VasquezWriting, review, and/or revision of the manuscript: A. Mukherjee, V. Huynh,K. Gaines, K.M. VasquezAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Mukherjee, V. Huynh, K.M. VasquezStudy supervision: A. Mukherjee, K.M. Vasquez

AcknowledgmentsWe would like to thank the DPRI core facility for assistance with the FACS

analysis and would like to thank the Vasquez lab members for helpful discus-sions. This work was supported byNIH/NCI grants (CA193124 and CA093729;to K.M. Vasquez).

Received February 12, 2019; revised April 26, 2019; accepted May 1, 2019;published first May 6, 2019.

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HMGB3 positively influences ATR and CHK1 transcription. A, Change in HMGB3 expression in A2780 and A2780/CP70 cells as a function of cisplatin treatment. B,Reduced ATR and CHK1 total mRNA levels in A2780 and CP70 cells as a function of HMGB3 depletion. Error bars,� SD from aminimum of three experiments.� , P < 0.05; �� , P <0.005, determined by the paired t test. C, Chromatin immunoprecipitation assay showing the association of HMGB3 with the ATR promoters,ATR1 and ATR2, and the CHK1 promoter expressed as a percentage of input. Control indicates a region 2.5-kb upstream of the ATR1 promoter and RPL30indicates the amplification of ribosomal protein L30 exon3 as a negative control. IgG, immunoglobulin G; H3, histone 3; HMGB3, high-mobility group box 3 IP. Thevalues represented are the averages of two experiments.

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www.aacrjournals.org Cancer Res; 79(13) July 1, 2019 3191

HMGB3 Modulates Cisplatin Sensitivity

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2019;79:3185-3191. Published OnlineFirst May 6, 2019.Cancer Res   Anirban Mukherjee, Van Huynh, Kailee Gaines, et al.   Chemoresistant Ovarian Cancer Cells to CisplatinTargeting the High-Mobility Group Box 3 Protein Sensitizes

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