bortezomib inhibits giant cell tumor of bone...

Small Molecule Therapeutics

Bortezomib Inhibits Giant Cell Tumor of Bonethrough Induction of Cell Apoptosis and Inhibitionof Osteoclast Recruitment, Giant Cell Formation,and Bone ResorptionLeqin Xu1,2,3, Jian Luo1,2, Rongrong Jin1, Zhiying Yue1, Peng Sun1,4, Zhengfeng Yang1,Xinghai Yang2,WeiWan2, Jishen Zhang2, Shichang Li4, Mingyao Liu1,2,5, and Jianru Xiao1,2

Abstract

Giant cell tumor of bone (GCTB) is a rare and highlyosteolytic bone tumor that usually leads to an extensive bonelesion. The purpose of this study was to discover novel ther-apeutic targets and identify potential agents for treating GCTB.After screening the serum cytokine profiles in 52 GCTB patientsand 10 normal individuals using the ELISA assay, we found thatNF-kB signaling–related cytokines, including TNFa, MCP-1,IL1a, and IL17A, were significantly increased in GCTB patients.The results were confirmed by IHC that the expression andactivity of p65 were significantly increased in GCTB patients.Moreover, all of the NF-kB inhibitors tested suppressed GCTBcell growth, and bortezomib (Velcade), a well-known protea-some inhibitor, was the most potent inhibitor in blockingGCTB cells growth. Our results showed that bortezomib notonly induced GCTB neoplastic stromal cell (NSC) apoptosis,

but also suppressed GCTB NSC–induced giant cell differenti-ation, formation, and resorption. Moreover, bortezomib spe-cifically suppressed GCTB NSC–induced preosteoclast recruit-ment. Furthermore, bortezomib ameliorated GCTB cell–induced bone destruction in vivo. As a result, bortezomibsuppressed NF-kB–regulated gene expression in GCTB NSCapoptosis, monocyte migration, angiogenesis, and osteoclas-togenesis. Particularly, the inhibitory effects of bortezomibwere much better than zoledronic acid, a drug currently usedin treating GCTB, in our in vitro experimental paradigms.Together, our results demonstrated that NF-kB signaling path-way is highly activated in GCTB, and bortezomib could sup-press GCTB and osteolysis in vivo and in vitro, indicatingthat bortezomib is a potential agent in the treatment of GCTB.Mol Cancer Ther; 15(5); 854–65. �2016 AACR.

IntroductionGiant cell tumor of bone (GCTB) is a rare, benign, highly

osteolytic bone tumor with unpredictable behavior, whichaccounts for approximately 5% of primary bone tumors and20% of benign bone tumors (1, 2). Although deemed histo-logically benign, it induces serious bone and surrounding softtissue destruction and is locally aggressive (3). As many as 6%of patients develop metastases, most frequently in the pulmo-nary parenchyma (3). The cellular elements of GCTB includethree major cell types: the multinucleated osteoclast–like giantcells, the round-shaped monocyte/macrophage origin mono-nuclear cells, and the spindle-shaped, fibroblast-like neoplasticstromal cells (NSC; ref. 4). The NSCs are the only proliferatingpopulation and represent the neoplastic component, whichalso express high concentrations of receptor activator of nuclearfactor-k B ligand (RANKL), and attract and activate monocyte/macrophage mononuclear cells to differentiate into osteoclast-like giant cells (5–10).

Surgery is the preferred treatment option currently and can beeffective if it is possible to achieve adequate resection. However,27% to 65%of patients following primary surgical treatments stilldevelop local recurrences or metastasis (11). The tumor recur-rence ratewill be higher if the resection is not clean (12). To reduce

1Shanghai Key Laboratory of Regulatory Biology, Shanghai Changz-hengHospital andEastChinaNormalUniversityJointResearchCenterforOrthopedicOncology, Institute ofBiomedical Sciences andSchoolof Life Sciences, East China Normal University, Shanghai, P.R. China.2Department of Orthopedic Oncology, Shanghai Changzheng Hospi-tal and East ChinaNormal University Joint Research Center for Ortho-pedic Oncology, Shanghai Changzheng Hospital, The Second MilitaryMedical University, Shanghai, P.R. China. 3Xiamen Hospital of Tradi-tional Chinese Medicine, Fujian University of Traditional Chinese Med-icine. Xiamen, P.R. China. 4The Key Laboratory of Adolescent HealthAssessment and Exercise Intervention of Ministry of Education, EastChina Normal University, Shanghai, P.R. China. 5Department of Molec-ular and Cellular Medicine, Institute of Biosciences and Technology,Texas A&M University Health Science Center, Houston, Texas.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

J. Luo, M. Liu, and J. Xiao share senior authorship.

L. Xu, J. Luo, and R. Jin contributed equally to this article.

Corresponding Authors: Jian Luo, East China Normal University, 500 Dong-chuan Road, Shanghai 200241, China. Phone: 8621-2420-6947; Fax: 8621-5434-4922; E-mail: [email protected]; and Jianru Xiao, Department of OrthopedicOncology, Shanghai Changzheng Hospital, The Second Military Medical Univer-sity, 415 Fengyang Road, Shanghai 200003, China. E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0669

�2016 American Association for Cancer Research.

MolecularCancerTherapeutics

Mol Cancer Ther; 15(5) May 2016854

on April 28, 2019. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst February 9, 2016; DOI: 10.1158/1535-7163.MCT-15-0669

http://mct.aacrjournals.org/

the risk of recurrence, aggressive surgical approaches (joint resec-tion, amputation, hemipelvectomy) are often applied. For unre-sectable GCTB patients, radiotherapy and serial embolization canrelieve the symptoms for a short period, but the duration of effectis limited (13). Moreover, malignant transformation can occurafter radiation (14). Therefore, there are considerable scientificand public interests in finding safe and effective pharmacologicagents for GCTB.

Bisphosphonates, which are standard drugs for treating oste-oporosis, have been used for treating GCTB in China (15, 16) andFrance (17). Recently, denosumab has been approved by the FDAfor the treatment of unresectable GCTB (18). However, bothbisphosphonates and denosumab mainly target osteoclasts byinducing osteoclast apoptosis and suppressing osteoclast differ-entiation (19). The obvious limitation of these drugs for GCTB isthat these drugs show very mild inhibitory effect on the prolif-erative NSCs (20). It has been speculated that GCTB may recurwhen these drugs are withdrawn (20), suggesting that NSCs are avital target for curing GCTB. Therefore, suppression of bothosteoclast formation and NSC growth could be a promisingstrategy for curing GCTB.

In this study, after screening the serum cytokine profiles of 52GCTB patients and 10 normal individuals, we found that NF-kBsignaling–related cytokines were significantly increased in GCTBpatients. The results were confirmed by IHC that the expressionand activity of p65 were significantly increased in GCTB patients.Moreover, we examined all of the NF-kB inhibitors suppressingGCTB cell growth and found that bortezomib (Velcade), thefrontline drug for multiple myeloma, had the most potent effectin blocking GCTB NSC proliferation. Our results showed thatbortezomib not only inducedNSC apoptosis, but also suppressedGCTB NSC–induced monocyte migration, giant cell differentia-tion, formation, and resorption. Specifically, bortezomib sup-pressed GCTB NSC–induced preosteoclast recruitment, but notnormal osteoblast–induced cell recruitment. To further investi-gate the potential therapeutic effects of bortezomib in vivo, weestablished a GCTB-induced osteolysis mouse model by intrati-bial injection of primary GCTB cells. Our data showed thatbortezomib inhibited GCTB-induced bone destruction. Ourresults, for the first time, indicate that bortezomib could be anew promising agent for treating GCTB.

Materials and MethodsReagents

Bortezomib (Velcade, MG-341, PS-341) and zoledronic acid(zoledronate, CGP-4244)were purchased fromSelleckChemicalsLLC. Tartrate-resistant acidic phosphatase (TRAP) staining kit,pyrrolidine dithiocarbamate (PDTC), parthenolide, and Bay 11-7085 (BAY) were purchased from Sigma. Antibodies anti-p65,anti-STAT3, anti-IkBa, anti-actin, and anti-PARP were purchasedfrom Cell Signaling Technology; anti-human mitochondria waspurchased from Chemicon.

Ethics statement and patient samplesThe use of all patient-derived tumor specimens was approved

by the Institutional Review Board and the research ethics com-mittee of Shanghai Changzheng Hospital (Shanghai, P.R. China)under reference of 2011/081, which appeared in the proceedingsof the meeting of the Ethics Committee on November 18, 2011.Written informed consent was obtained individually from each

patient. The clinical characteristics of all of the GCTB patients aresummarized in Supplementary Table S1. The progression of theGCTB was evaluated using the Campanacci grading (21) andEnneking staging systems (22).

Cytokine ELISA assay on GCTB patient serumThe amount of cytokines in the serum was measured using

ELISA kits, according to the manufacturer's instructions (SunnyELISA Kits, Mutisciences).

Cell growth inhibition assay, pit assay, and osteoclastogenesisassay

Cell viability was measured by the sulforhodamine B (SRB)assay as described previously (23). In the pit formation assay, 1�105 pooled GCTB cells were seeded onto FBS-coated dentin slices(SBA Sciences) in a 96-well plate. The number of resorption pitswas determined microscopically. For osteoclastogenesis assay,bone marrow cells isolated from C57BL/6 mice were cultured asdescribed previously (24).

Transwell migration assays of bone marrow monocytesThemigration of bonemarrowmonocytes was tested using the

cell BD Chamber Kit as described previously (24).

Detection of apoptosis via FITC-Annexin V/PI StainingFor Annexin V/propidium iodide (PI) assays, GCTB NSCs were

stained with Annexin V-FITC and PI and then evaluated forapoptosis by flow cytometry according to the manufacturer'sinstructions (BD Pharmingen).

Intratibial injection of primary GCTB cellsPrimary GCTB cells were resuspended in PBS at a working

concentration of 5 � 105 cells per 20 mL. The intratibial boneinjection was performed in 4- to 5-week-old BALB/c, male nudemice (nu/nu) anesthetized with pentobarbital (50 mg/kg) via apercutaneous approach (25). The procedures were approved bythe Animal Care and Use Committee of the East China NormalUniversity (ECNU; Shanghai, P.R. China).

X-radiography and micro-CTAll animal work was performed in accordance with accepted

standards of the Ethics Committee of ECNU (Shanghai, P.R.China). Animals were anesthetized and radiographs wereobtained using a Kodak DXS 4000 Pro System for 60 seconds at35 kVonday0 andweekly after intratibial injection.Micro-CTwasperformed using a SkyScan 1076 Desktop X-ray Microtomograph(SkyScan). The excised mouse legs were secured in polystyreneimaging tubes and scanned [50� magnification, 5.86 mm reso-lution, 7.5-second exposure time (40 kV and 258 UA), 0.45rotation step, 180�, and a 1 mm aluminum filter].

Hematoxylin and eosin, TRAP, IHC, and TUNEL stainingHematoxylin and eosin (H&E), TRAP, and immunohistochem-

ical staining were conducted as described previously (23). Briefly,the tumor specimens and normal bone tissues were processed byfixation with 4% paraformaldehyde in PBS for 24 hours at 4�C.The bone tissues were decalcified in 0.5 mol/L ethylenediaminetetraacetate (EDTA) for oneweek. Then, the tumor specimens andnormal bone tissues were embedded in paraffin. Four-micronparaffin-embedded sections were cut with a microtome and

Bortezomib Inhibits Giant Cell Tumor of Bone

www.aacrjournals.org Mol Cancer Ther; 15(5) May 2016 855




stained. The sections were imaged using the Leica DM 4000BMicroscope (Leica). For the terminal deoxynucleotidyl transfer-ase–mediated dUTP nick end labeling (TUNEL) assay, DeadEndColorimetric TUNEL System (Promega) was used. For quantifi-cation of apoptosis, stained cells were counted from 6 randomselected views using the IPP image analysis program.

Cell culture293T cells and the human fetal osteoblast cell line (hFOB1.19)

were obtained from the Institute of Biochemistry andCell Biologyof Shanghai (Shanghai, China) according to the standardprotocolin October 2009. The cell lines were authenticated and myco-plasma tested by the Chinese Academy of Sciences CommitteeType Culture Collection Cell Bank. No further cell line authen-tication has been performed by the authors. The pooled GCTBcells were isolated from tumor samples derived from tumorresections in Shanghai Changzheng Hospital (Shanghai, P.R.China). The tissue was mechanically cut into small pieces anddigested with 1.5 mg/mL collagenase B for 3 hours at 37�C inDMEM containing 4.5 g/L glucose and supplemented with 10%FBS, along with 100 U/mL penicillin and 100 mg/mL streptomy-cin. Cells were collected by filtration (100 mm diameter filter)centrifugation and washed twice in PBS. The cells were countedusing a hemocytometer and resuspended at a density of 5 � 105

cells per 20 mL. The pooled GCTB cell suspension was cultured in37�C humidified air with 5%CO2. Culture mediumwas changedevery 2 to 3 days until approximately 80% confluence. Confluentcells were subcultured after dissociating with trypsin and EDTA.The NSCs were obtained as described previously (7, 26, 27).Briefly, following several successive passages, the NSCs becamethehomogeneous cell type,whereas themultinucleated giant cellswere eliminated from the culture. Primary cultures obtained afterthe fifth or sixth passage (without any hematopoietic markers)andup to tenth passage, which represent the proliferating homog-enousNSCs population, were used for subsequent drug treatmentand experimental assays as follows.

Conditioned media collection from primary cells culturesWhen the primary GCTB cell cultures reached 80% confluence,

themediawere changed to serum-freemedia and incubated for 24hours at 37�C in a humidified atmosphere of 5% CO2 and 95%air. Conditioned media were subsequently collected. The condi-tioned media were stored at �80�C for subsequent studies.

Real-time PCRGCTB stromal cells were treated with different concentrations

of bortezomib for 24 hours, and total RNA was isolated using theTRIzol reagent according to the manufacturer's instructions. TheRNA concentrations were quantified with the Qubit Fluorometer.RT-PCRwas carried out using 1 ng of total RNA,whichwas reversetranscribed into complementary DNA using the Takara ReverseTranscription Kit according to the manufacturer's instructions.Quantitative RT-PCRwas performed using the Takara SYBRGreenRT-PCRKit. The PCRprotocol conditionswere as follows:HotStarTaq DNA polymerase activation step at 95�C for 2 minutes,followed by 40 cycles at various temperatures/times (i.e., 94�Cfor 15 seconds, 60�C for 30 seconds, and 72�C for 30 seconds). Atthe end of the amplification period, melting curve analysis wasdone to confirm the specificity of the amplicon. Fold changes ofgenes after treatment with bortezomib were calculated by nor-

malizing the Ct values to the b-actin internal control. Thesequences of the primer pairs in this experiment were shown inSupplementary Table S2.

Western blot analysisAfter treatment with various dosages of bortezomib for 48

hours, the GCTB stromal cells were lysed with RIPA buffer con-taining protease and phosphatase inhibitors. The protein con-centrations were measured with a BCA Kit (Beyotime). Equalamounts of protein were separated by SDS-PAGE and transferredto a polyvinylidene fluoride membrane. The membrane wasblocked with a solution containing 5% nonfat dry milk TBSTbuffer (20 mmol/L Tris–HCl, pH 7.4, 150 mmol/L NaCl, and0.1% Tween 20) for 1 hour. The indicated primary antibodieswere incubated overnight at 4�C, washed, and monitored byimmunoblotting using a DyLight 800–conjugated secondaryantibody. The membrane was scanned using a LI-COR InfraredImaged Odyssey (Gene Company Limited).

Immunofluorescence analysisGCTB stromal cells on coverslips were incubated with various

concentrations of bortezomib for 4 hours. Then, cells were incu-bated with 0.2 nmol/L TNFa for 5 minutes. Cells were washedtwice with PBS and fixed by 4% paraformaldehyde at 4�C for 10minutes. Permeabilization of the cells was performed by incu-bating the cells with 0.1% saponin and 1% FBS in PBS at 4�C for10 minutes. The cells were blocked with 3% BSA at room tem-perature for 30 minutes and then incubated with primary anti-body against P65 at 4�C overnight (0.1% saponin and 1% FBS inPBS). Cells were washed twice in PBS and then incubated simul-taneously with FITC-labeled secondary antibody for 1 hour atroom temperature. Negative controls were prepared by incuba-tion of the cells with anti-IgG antibody. Nucleus was stained withDAPI for 2 minutes. After staining, cells were rinsed four timeswith PBS and prepared for microscopic analysis. The images werecaptured using an immunofluorescence microscope (Olympus).

Luciferase assay293T cells in 24-well plates were transfected with NF-kB lucif-

erase reporter construct using Lipofectamine 2000 (Invitrogen).Twenty-four hours posttransfection, cells were incubated withbortezomib for 4 hours and then lysed in 100 mL of Passive LysisBuffer (Promega). To assay for NF-kB activity, luciferase wasmeasured using the Dual-Luciferase Reporter Assay System (Pro-mega), with luminescence read in relative light units. Luciferasevalues were normalized to the Renilla transfection control.

Statistical analysisData were presented as the means � SD of three independent

experiments done in triplicate. Statistical analysis was performedby Student t test or one-way ANOVA. In all cases, P < 0.05 wasconsidered to be statistically significant.

ResultsNF-kB signaling pathway is highly activated in GCTB

To identify potential signaling pathways for drug discovery inGCTB, we screened the expression of various cancer-related cyto-kines in the serumofGCTBpatients andhealthy individuals usingELISA. Our data showed that NF-kB signaling–related cytokines,including TNFa, MCP-1, IL1a, and IL17A, were significantly

Xu et al.

Mol Cancer Ther; 15(5) May 2016 Molecular Cancer Therapeutics856




increased in GCTB patients (n ¼ 52) compared with healthyindividuals (n¼ 10),whereas the STAT signaling–related cytokineIFNg and the SMAD signaling–related cytokine TGFb had littledifference between GCTB patients (Supplementary Table S1) andhealthy controls (Fig. 1A). To further confirm whether the NF-kBsignaling pathway is activated in GCTB, we examined the expres-sion ofNF-kB/p65 by IHC. As expected, the expression of p65wasstrikingly increased in both GCTB multinucleated giant cells andNSCs (Fig. 1B and C), which is indicated by H&E and TRAPstaining (Fig. 1B). In particular, the expression of p65 wasenriched in nuclei (Fig. 1B), and the phosphorylation of p65was highly upregulated in GCTB tumor tissues (SupplementaryFig. S1), suggesting that NF-kB signaling was highly activated inGCTB. Similarly, the expression of STAT3 was weak and wasunchanged between GCTB and healthy controls (Fig. 1B). All ofthe data demonstrated that the NF-kB signaling pathway is highlyactivated in GCTB.

Bortezomib suppresses cell growth of GCTBWe next examined whether NF-kB inhibitors affect cell growth

of GCTB. We choose 4 NF-kB inhibitors, which have been suc-cessfully used in preclinical and clinical studies: the p65 inhibitorsPDTC and parthenolide, the IKK inhibitor BAY, and the IkBadegradation inhibitor bortezomib (28). Our results showed thatall of the inhibitors suppressed GCTB cell growth (Fig. 2A).However, the bisphosphonate, zoledronic acid, which has beenused for treating GCTB in China and France (15–17), had littleeffect on GCTB cell growth (Fig. 2A). Among the inhibitors,bortezomib had the most potent effect on cell growth, with amean IC50 of approximately 10 nmol/L. Therefore, we choosebortezomib for further investigation.

Bortezomib induced apoptosis of GCTB NSCsThere are three major cell types in GCTB, including the

multinucleated osteoclast–like giant cells, the monocyticround-shaped macrophage-like cells, and the spindle-shaped,fibroblast-like NSCs (4). The NSCs mainly contribute to GCTBgrowth (1). To examine whether bortezomib inhibits the NSCgrowth, we isolated the NSCs and evaluated their proliferationby the SRB assay. We found that bortezomib significantlysuppressed NSC growth in a dose-dependent manner, with anIC50 of 10 to 50 nmol/L in more than 20 different patientsamples (Fig. 2B). In contrast, the bisphosphonate zoledronicacid had little effect on NSC growth, with an IC50 of more than100 mmol/L (Fig. 2B). Similar results were obtained from cellnumber counts that bortezomib, but not zoledronic acid,suppressed cell growth in a dose-dependent manner (Fig.2C). Because the cells with bortezomib treatment exhibitedapoptotic morphology, we further performed the apoptoticassay in 4 patient samples. The data (Fig. 2D, left) showedthat bortezomib dose dependently induced NSC apoptosis at arelatively low concentration (10–40 nmol/L). In contrast, onlythe highest concentration of zoledronic acid (150 mmol/L)could mildly induce cell apoptosis (Fig. 2D, left). The caspaseapoptotic pathway assay also confirmed that the accumulationof cleaved PARP and caspase-3 was concentration dependentlyincreased when treated with bortezomib in two patient samples(Fig. 2D, right). Together, all of the data indicated that borte-zomib suppressed GCTB NSC growth by the induction of NSCapoptosis.

Bortezomib inhibits osteoclast-like giant cell formation andresorption

NSCs strongly attract and activate osteoclast-like giant cells andtheir precursors in GCTB (9). Therefore, we examined whetherbortezomib inhibits osteoclast-like giant cell formation in GCTB.Weused three in vitro giant cell formationmodels todetermine theroles of bortezomib. Thefirstmodel was primary culture of GCTB,in which the NSCs express high concentrations of RANKLand induce differentiation of RANK-positive osteoclast-like giantcells and their precursors (Fig. 3A; refs.8, 29). The second modelwas the induction of normal mouse bone marrow monocyte(BMM) differentiation by conditioned medium of NSC(Fig. 3B; ref. 30), and the third model was to investigate thestage at which bortezomib affected giant cell formation(Fig. 3C). In all three experimental paradigms, bortezomibinhibited giant cell differentiation and formation in a dose-dependent manner, with an IC50 of 2.5 to 5 nmol/L (Fig. 3A–C). Interesting, bortezomib inhibited giant cell formation atboth early (Fig. 3B) and late stages (Fig. 3C).

To further examine the effect of bortezomib on giant cellfunction, we performed the resorption pit assay in GCTB primarycultures. The results showed that bortezomib inhibited resorptionpit formation in a dose-dependent manner (Fig. 3A, left). Intrigu-ingly, bortezomib almost completely blocked pit formation andpit depth at 10 nmol/L (Fig. 3A, right). Taken together, bortezo-mib inhibited giant cell differentiation, formation, and resorptionfunction.

Bortezomib specifically abrogates NSC-inducedrecruitment of BMM

Next, we examined whether bortezomib had any effect onosteoclast precursor cell migration attracted by NSCs using theTranswell migration assay. Using the medium and normalosteoblast cells (hFOB1.19 cell line) as control, we seededNSCs in the bottom of the chamber and normal mouse bonemarrowmonocyte cells in the top of the chamber (Fig. 4A). Ourresults showed that, compared with medium and normalosteoblast cells (hFOB1.19), the NSC strikingly attracted thenormal bone marrow monocyte cells (Fig. 4B and C), andbortezomib inhibited NSC-attracted BMM migration in adose-dependent manner (Fig. 4B and C). Interestingly,although normal osteoblast cells (hFOB1.19) also inducedBMM migration, bortezomib had little effect on normal oste-oblast cell–induced migration at the same concentrations (Fig.4B and C). These results indicated that bortezomib specificallysuppressed NSC-induced BMM migration.

Bortezomib suppresses GCTB-induced osteolysis in vivoBecause of the lack of suitable animalmodels of GCTB (31), we

established a GCTB-induced osteolysis mouse model to evaluatethe potential therapeutic effects of bortezomib in vivo. After 2weeks of intratibial injection of GCTB cells in nude mice, theextent of osteolytic bone destruction in tibia was detected by X-rayradiographic imaging. Then, the mice with similar osteolytic areawere randomly divided into three groups for vehicle control orbortezomib treatment for another 70 days (n ¼ 6). We chosebortezomib dosages that were based on the clinical dosage scaleddown for mice and are widely used in mouse models (32). Ourresults showed that, compared with normal mice, the vehiclecontrol–treated mice displayed severe osteolytic lesions by X-ray






A

B

TGFb

IL1a

Normal GCTB 0 5

10 15 20

IL17ANormal GCTB

P = 0.023

TNFa

01

30

IFNg

Con

cent

ratio

n(

(¥1

01 pg

/mL)

Con

cent

ratio

n(¥

101

pg/m

L)

Con

cent

ratio

n(¥

101

pg/m

L)C

once

ntra

tion

(¥10

pg/m

L)

Con

cent

ratio

n(¥

102

pg/m

L)

Con

cent

ratio

n¥1

03 pg

/mL)

0 2 4 6 8

Normal GCTB

0 1 2 3

30

Normal GCTB

0

5

10

Normal GCTB 0 2 4 6 8

10

Normal GCTB

10 15

HE

TRA

P

Normal bone tissues GCTB tissues

STA

T3

Patient 1 Patient 2 Patient 3 Patient 4 Normal 1 Normal 2

C

2

20

34

20 10 10 P = 0.013

MCP-1 45 50

P = 0.001

P = 0.016 P = 0.279 P = 0.969

p65

GCTB

Bone

Figure 1.NF-kB signaling pathway is highly activated in GCTB. A, serum level of cytokines in GCTB patients and healthy controls as determined by ELISA assay. The serumconcentrations for TNFa, MCP-1, IL1a, and IL17 were significantly increased in the GCTB patient group (n ¼ 52) compared with the healthy control group (n ¼ 10;P < 0.05). There were no statistically significant differences between GCTB group and healthy group for the serum levels of TGFb and IFNg . Horizontal lines indicatethe mean. B, H&E (HE), TRAP, and immunohistochemical staining for p65 and STAT3 in GCTB and normal bone. From the enlarged image of the p65immunohistochemical staining (bottom), the p65 signal was highly enriched in both multinucleated giant cell and the NSC nuclei (scale bar, 50 mm). The 4 patientsamples were randomly chosen from 52 GCTB patients. C, immunohistochemical staining for p65 in the GCTB tumor/normal tissue interface. The dotted lineseparates the GCTB and the normal bone tissue (scale bar, 50 mm).

Xu et al.





imaging and micro-CT scanning (Fig. 5A). As expected, bortezo-mib significantly suppressed osteolytic bone destruction (Fig. 5Aand B, top). Similar results were obtained from pathologic sec-tioning and TRAP staining that bortezomib significantly sup-pressed osteolysis and the number and size of osteoclasts(Fig. 5A and B, middle). Apoptotic TUNEL staining also con-firmed that bortezomib markedly induced neoplastic cell apo-ptosis (Fig. 5A and B, middle). The results were confirmed bystaining for specific human protein (human mitochondrial pro-tein), showing that thehumanproteinwas largely decreased in thebortezomib-treated group, (Fig. 5A). All of the data suggested thatonly bortezomib had an inhibitory effect on both osteoclastdifferentiation and neoplastic cell apoptosis. Furthermore, body

weight results showed that there was no evidence of weight loss inbortezomib-treated mice (Fig. 5B, bottom).

Bortezomib inhibits NF-kB signaling pathway in GCTBTo determine the molecular mechanism of bortezomib in

treating GCTB NSCs, we investigated whether bortezomib inhi-bits the NF-kB signaling pathway using three differentapproaches. First, we found that bortezomib inhibited TNFa-induced IkBa degradation in a time-dependent manner by West-ern blot analysis (Fig. 6A, top left). Second, we verified thatnuclear translocation of p65 was time and dose dependentlyabrogated by bortezomib using Western blotting (Fig. 6A, topright) and immunofluorescence staining (Fig. 6A, bottom left),

0

Cel

l via

bilit

y (%

of c

ontr

ol)

0.20.40.60.81.01.21.4

BZABZB

0 0.01 0.1 mmol/L

ZAB

ZB

CZABZB

0

Cel

l num

ber (

¥103

)

5

10

15

20

25

Mea

n pe

rcen

tage

of

cell

apop

tosi

s (%

)

0

20

40

60

80

100

Ctrl 50 100 150 10 20 40

*

*

*** ***0 10 20 40

Patient 1

PARPCleaved PARPCleaved caspase-3

b-Actin

Patient 70 10 20 40 BZB (nmol/L)

A

2.37

1.61

2.40

1.69

1.38

2.17

3.51

5.54

13.10

18.29

21.79

31.17

21.89

42.01

Ctrl 50 100 150

BZB (nmol/L)10 20 40

ZA (mmol/L)

ZA (mmol/L)

BZB(nmol/L)

D

Annexin V

PI

Annexin V

PI

Annexin V

PI

Annexin V

PI

Annexin V

PI

Annexin V

PI

Annexin V

PI

ZABZBBAYPARPDTC

0

20

40

60

80

100

Inhi

bitio

n(%

of c

ontr

ol) IC50 (mmol/L)

= 419.4= 0.012= 13.41= 20.14= 28.13

1001010.10.01

Compound concentration (mmol/L) 0.00100.005

0.010.05 0.1 0.5 1 10

100mmol/L

mmol/L100100.50.10.05

0.010.005

0.001Ctrl 1

Figure 2.Bortezomib (BZB) inhibited the cell growth of GCTB and induced NSC apoptosis. A, inhibitory effects of NF-kB signaling pathway inhibitors on GCTB cell growth.Primary cultures of three GCTB patients (8� 103 cells) were treated with different concentrations (0, 0.01, 0.1, 1, 10, and 100 mmol/L) of NF-kB inhibitor [BZB, BAY,parthenolide (PAR), and PDTC] or zoledronic acid (ZA) for 48 hours. The inhibition curves and the one-half IC50 of the dose–response curves were generatedby the GraphPad Prism 5 software. The experimentswere performed in triplicate; scale bars, SD. B, inhibitory effect of bortezomib or zoledronic acid on NSC growth.Primary culturedNSCsof 20GCTBpatients (8� 103 cells)were treatedwith various concentrations (0, 0.01, 0.1, 1, 10, and 100mmol/L) of bortezomib (dashed lines) orzoledronic acid (solid lines) for 48hours (n¼ 20). Cell proliferationwasmeasuredby SRB.Dots show themeans of experiments performed in triplicate; scale bars, SD.The horizontal dashed line represents the concentration for which the compound is able to kill 50% of the cells (median lethal dose, LD50). The 20 patient sampleswere randomly chosen from 52 GCTB patients. C, inhibition of NSC growth by bortezomib. NSCs (8 � 103 cells) were treated with indicated concentrations ofbortezomib or zoledronic acid. After 48 hours, cells were photographed (left) using phase-contrast microscopy and counted (right). Columns show themeans of experiments performed in triplicate; scale bars, SD. Magnification, 40�. D, bortezomib induces NSC apoptosis. Bortezomib induces NSC apoptosis byAnnexin V staining using zoledronic acid as control (left). Primary cultured NSCs isolated from 10 patients (1 � 105 cells/sample) were treated with indicatedconcentrations of bortezomib or zoledronic acid for 48 hours and then stained with Annexin V-FITC and subjected to flow cytometry for apoptotic analysis asdescribed under Materials and Methods. The number indicates the percentage of apoptotic cells. Overall quantitation (left) and representative data (middle) fromthree concentrationswere shown (right; n¼ 10; � ,P <0.05; �� ,P <0.001); right, induction of PARP and caspase-3 cleavage by bortezomib inNSCs. GCTB stromal celllines, primary-cultured NSCs isolated from two randomly chosen patients (1 � 104 cells) were incubated with indicated bortezomib concentrations for24 hours. Whole-cell extracts were prepared and subjected to Western blot analysis using anti-PARP and anticleaved caspase-3 antibodies.






A

Ba-MEM

a-MEM

CM BZB 2.5 nmol/L

BZB 5 nmol/L BZB 7.5 nmol/L BZB 10 nmol/L

BZB (nmol/L)

2.5 5 7.5Ctrl 10TR

AP

Pit a

ssay

Z se

ctio

nXY

sec

tion

2.5 5 7.5Ctrl 10BZB (nmol/L)

Dep

th (m

m)

40

30

20

10

0

******

******

No.

of T

RAP

+ce

lls >

3 n

ucle

i

2.5 5 7.5Ctrl 10BZB (nmol/L)

******

******

100

80

60

40

0

20

Ctrl

GCTB medium

BZB 0 nmol/L BZB 2.5 nmol/L

BZB 5 nmol/L BZB 7.5 nmol/L

C

***

No.

of T

RAP

+ ce

lls>

3 nu

clei

BZB(nmol/L)

2.5 5 7.50Ctrl

80

60

40

20

0

GCTB Medium

100

BZB (nmol/L)2.5 5 7.5CM 10

No.

of T

RAP

+ ce

lls>

3 nu

clei

80

60

40

20

0

******

**

*

***

***

*

**

Figure 3.Inhibitory effect of bortezomib (BZB) onGCTB-induced osteoclast-like giant cell formation and resorption. A, bortezomib dose dependently inhibited TRAP-positivecell formation and bone resorption in primary GCTB cell culture. For the TRAP staining assay, primary cultured GCTB cells (8 � 103 cells) were treated withor without bortezomib for 48 hours. Then, the cells were fixed and stained with TRAP. The cells were photographed (top, left), and the cell number was counted(top, right). Columns show the means of experiments performed in triplicate; scale bars, SD. Magnification, 40�. For the pit formation assay, primary-cultured GCTB cells (8 � 103 cells) were seeded on dentin slices and incubated with or without bortezomib for an additional two days. Then, the dentin sliceswere washed and stained with 0.5% toluidine blue for pit area (XY section) visualization (middle, left). Individual resorption pit depth was assessed byconfocal microscopy (Z section; bottom, left), and the mean depth (bottom, right) is shown (n ¼ 3; �� , P < 0.001). B, bortezomib suppressed NSC-inducedosteoclast formation. NSCs (1 � 104 cells) were incubated with indicated concentrations of bortezomib for 24 hours, and then the conditioned medium (CM) washarvested and diluted to treat BMM cells. Mouse BMMs (5 � 103 cells) were treated with the diluted NSC-conditioned medium (final concentration 10%) for4 days. Then, the cells were fixed and stained with TRAP solution. The cells were photographed (left), and the cell number was counted (right). Columns show themeans of experiments performed in triplicate (� , P < 0.05; �� , P < 0.01; �� , P < 0.001); scale bars, SD. Magnification, 40�. C, bortezomib suppressed late-stageNSC-induced osteoclast formation. NSCs (1 � 104 cells) were incubated with indicated concentrations of bortezomib for 24 hours, and then the conditionedmediumwas harvested and diluted to treat BMM cells. Themouse BMMs cells were incubated with RANKL (50 ng/mL) andM-CSF (10 ng/mL) for two days, and thenthe cell medium was changed to the diluted NSC-conditioned medium (final concentration 10%) for another two days, after which the cells were fixed andstained with TRAP solution. The cells were photographed (left), and the cell number was counted (right). Columns show the means of experiments performedin triplicate (� , P < 0.05; �� , P < 0.01; �� , P < 0.001); scale bars, SD. Magnification, 40�. a-MEM, minimum essential medium.

Xu et al.





respectively. Finally, using the luciferase reporter gene assay in293T cells, we examined the effect of bortezomib on the activity ofNF-kB. As shown in Fig. 6A (bottom right), the transcriptionalactivity of NF-kB was strikingly increased when exposed to TNFa.The increased NF-kB activity was inhibited by bortezomib in adose-dependent manner. As a result, bortezomib time depen-dently inhibited TNFa-induced NF-kB–regulated antiapoptoticgene product expression in NSCs, including Bcl-xl, Bcl-2, XIAP, c-IAP1, c-IAP2, and c-Flip (Fig. 6B). Bortezomib also dose depen-dently suppressed NF-kB–regulated monocyte migratory andangiogenic gene expression, includingmonocyte chemoattractantprotein-1 (MCP-1), stromal cell–derived factor 1 (SDF-1), VEGFand matrix metalloproteinase-9 (MMP-9; Fig. 6C). Together, ourresults indicate that bortezomib inhibits the NF-kB signalingpathway in GCTB.

DiscussionBortezomib, the frontline drug for multiple myeloma, has

drawn great interest as a potential therapeutic agent for thetreatment of a variety of cancer types, including breast, colon,kidney, lung, and brain (33). However, whether it has any

antitumor effect on GCTB and the molecular mechanisms isunknown. In this study, we demonstrated that the NF-kB signal-ing pathway was activated in GCTB. After screening NF-kB path-way inhibitors in GCTB, we found that bortezomib had the mostpotent inhibitory effect on GCTB cell growth. Moreover, borte-zomib induced NSC apoptosis, suppressed GCTBNSC–mediatedmonocyte migration, and inhibited GCTB-modulated giant celldifferentiation, formation, and resorption. Furthermore, borte-zomib ameliorated GCTB-induced bone destruction in vivo; par-ticularly, bortezomib inhibited bone destruction to a muchgreater extent than the bisphosphonate zoledronic acid in vivoand in vitro. At the molecular level, bortezomib suppressed IkBadegradation, p65 nuclear translocation, andNF-kB activation in adose-dependent manner. As a result, bortezomib suppressed NF-kB–regulated gene expression in GCTBNSC apoptosis, monocytemigration, angiogenesis, and osteoclastogenesis. To our knowl-edge, this is the first report of an investigation into the effects ofbortezomib on GCTB.

Bisphosphonates, the frequently used drugs for treating oste-oporosis by encouraging osteoclasts to undergo apoptosis, or celldeath, have already been used for treating GCTB in China andFrance (15–17). Denosumab, the human mAb against RANKL,also is a proven agent for treating osteoporosis and solid tumorbonemetastases by inhibiting osteoclast differentiation. Recently,denosumab has been approved by the FDA for the treatment ofunresectable GCTB (18). However, it has been speculated thatrecurrence may occur in GCTB patients when these drugs arewithdrawn (20). One of the reasons is that both bisphosphonatesand denosumab mainly target osteoclasts, but not proliferativeNSCs (20). On the other side, stress fracture is associated with thelong-term use of antiresorptive medications, such as denosumaband bisphosphonate (34, 35). Therefore, the suppression of bothosteoclast formation and NSC growth could be a promisingstrategy for curing GCTB. In our study, we found that bortezomibnot only induced NSC apoptosis with IC50 of 0.012 mmol/L, butalso suppressed NSCs-induced giant cell differentiation, forma-tion, and resorption in vivo and in vitro. As zoledronic acid, abisphosphonate, only has a mild inhibitory effect on NSCs withIC50 of 419.4 mmol/L, therefore, it is reasonable to speculate thatbortezomib would have a better effect on GCTB, including con-trolling tumor recurrence.

The proinflammatory transcription factor NF-kB regulates theexpression of over 500 genes. Numerous NF-kB–regulated genesare involved in cancer cell survival, proliferation, invasion, angio-genesis, metastasis, and cytokine secretion (36). Therefore, theNF-kB signalingpathwayhas become an attractive potential targetfor drug discovery (37). A wide variety of agents, includinginhibitors of protein kinases, protein phosphatases, proteasomes,ubiquitination, acetylation, methylation, and the DNA-bindingsteps of NF-kB signaling pathway, have been identified as poten-tial NF-kB–targeting agents (36). Some of these are in clinicaltreatment or trials for certain cancer types (38, 39). Bortezomib,the most-studied proteasome inhibitor, has been approved fortreatingmultiplemyeloma andmantle cell lymphoma (40) and isalso in clinical trials for many liquid and solid tumors (33). It hasbeen reported that bortezomib-induced cytotoxicity ismostly dueto the inhibition of the NF-kB signaling pathway in head andneck, prostate, pancreatic, gastric, renal, ovarian, and breast can-cers (41–43). In our study, we demonstrated that bortezomib caninduce NSC apoptosis, and it concentration dependently inhib-ited the degradation of IkBa and the expression of NF-kB

B

C

hFO

Ba-

MEM

GC

TSC

0 nmol/L 2.5 nmol/L 5 nmol/L 7.5 nmol/L 10 nmol/L

BZB (nmol/L) a-MEM NSC

0 2.55 7.510hFOB1.19

Cel

l mig

ratio

n(¥

102 )

0

5

10

15

0 2.55 7.510 0 2.55 7.510

A

Monocyte

GCTB stromal cell/hFOB1.19

Figure 4.Specific inhibitory effect of bortezomib on NSC-induced BMM migration. A,schematic representation of the experiment. NSCs (2 � 104 cells) or normalhuman osteoblast cells hFOB1.19 (2 � 104 cells) or the medium control(a-MEM) were treated with indicated concentration of bortezomib for 24hours in the bottom of the chamber, and then normal mouse BMMs (5 � 104

cells) were seeded in the top of the chamber. Twenty-four hours after themonocytes migrated through the membrane, nonmigrated monocytes wereremoved, and migrated monocytes were stained, photographed, andcounted. B and C, bortezomib specifically inhibits NSC-induced BMMmigration but has little effect on normal human osteoblast cell hFOB1.19-induced monocyte migration. Migrated monocytes were photographed (B)and counted (C). Columns show the means of experiments performed intriplicate; scale bars, SD. Magnification, 40�. GCTSC, GCTB stromal cell;a-MEM, minimum essential medium.






downstream target genes. The data indicate that bortezomib, likein other cancer cells, induced NSC apoptosis through suppressingthe NF-kB signaling pathway.

Although several in vivo GCTB models are available, studieson tumor therapeutics are hampered because in vivo develop-ment was either incomplete or had short survival time or didnot induce osteolytic lesion. Stromal cells injected subcutane-ously into immunocompromised mice do not produce giantcells (44–46). Moreover, tumor tissues grown on chick chorio-allantoic membranes do not appear to recruit chicken mono-

cytes to synthesize new giant cells and are unsuitable for drugadministration, despite increased vascularization from themembrane, but only survive for 10 days (5, 31). In our study,we used patient-derived tumor cells isolated from GCTB toestablish an orthotopic murine model by intratibial injection.The intratibial injection method is one of the most widely usedmurine models to investigate cancer cell growth within thebone environment.

We found that bortezomib inhibits the expression of variousmigration regulators in NSCs, including MCP-1, SDF-1, VEGF,

Normal ControlBZB

0.3 mg/kg

A BH

ETR

APTU

NEL

Bod

y w

eigh

t (g)

0 2 4 6 8 10 12048

12162024

X-ra

yM

icro

-CT

Control BZB

0 2 4 6 8 10 120

2

4

6

8

10

Rat

io o

f bon

e le

sion

tono

rmal

bon

e (%

BL/

NB

)

*

Oc.

S/B

S (%

)

Nor Ctrl BZB0

5

10

15

20*** ***

N.O

c/B

.Pm

(N/m

m)

0

5

10

15

Nor Ctrl BZB

*** **

TUN

EL-p

ositi

ve c

ell

0

5

10

15

Nor Ctrl BZB

***

Weeks

Ant

i-HuM

i

Weeks

Control BZB

Figure 5.Bortezomib (BZB) suppresses GCTB-induced bone destruction in vivo. Twoweeks after GCTB cell intratibialinjection in nude mice, the osteolyticbone destruction in tibia was detectedby X-ray radiographic imaging. Themice with similar osteolytic area wererandomly divided into three groups forvehicle control or bortezomibtreatment (0.3 mg/kg, 3 times/week),respectively (n¼ 6). Seventy days later,the mouse tibia bones wereradiographed or scanned by micro-CTor sectioned. A, the mouse tibia boneswere analyzed by X-ray, micro-CT, H&E(HE) staining, TRAP staining, anti-human mitochondrial (Anti-HuMi)protein staining, and TUNEL staining.Arrowheads indicate TRAP-positiveosteoclasts in TRAP staining andTUNEL-positive apoptotic cells inTUNEL staining. Scale bars, 200 mm(H&E staining); 50 mm (TRAP staining);10 mm (TUNEL staining). B, bortezomibdecreased the area of GCTB cell–induced osteolytic bone destructionand induced cell apoptosis of GCTBNSCs. The area was quantified from theX-ray analysis by software (top). Scalebars, SD. � ,P<0.05. Bortezomib inhibitsthe GCTB-induced osteoclast numberand surface area (middle).Histomorphometric assessment ofosteoclast parameters includingosteoclast surface per bone surface (Oc.S/BS) and osteoclast numbers per boneperimeter (N.Oc/B.Pm). Scale bars, SD.�, P < 0.05; �� , P < 0.01; �� , P < 0.001.The TUNEL-positive cells were countedfrom TUNEL assay section (middle).Scale bars, SD. ��P < 0.001. The effectof bortezomib on mouse body weight(bottom).

Xu et al.





andMMP-9. All of these not only are potent chemoattractants forthe preosteoclast monocytes (47, 48), but also directly stimulateosteoclast differentiation (48, 49). These mechanisms were con-sistent with the phenotype that bortezomib inhibited the NSC-induced monocyte migration, osteoclast differentiation, forma-tion, and resorption. It has been reported that GCTB is stronglycorrelated to angiogenesis and aneurysmal bone cyst formation(50). Our results showed that bortezomib dose dependentlysuppressed the expression of VEGF, a growth factor critical fortumor angiogenesis. It is reasonable to speculate that bortezomibhas an inhibitory effect on angiogenesis in GCTB.

In conclusion, we demonstrate that bortezomib inhibitedGCTB growth through the induction of NSC apoptosis and theinhibition of monocyte recruitment, osteoclast formation, andbone resorption. Our results, for the first time, provide evidencethat bortezomib may represent a new antitumor therapy for thetreatment of GCTB.

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

Authors' ContributionsConception and design: J. Luo, R. Jin, Z. Yang, X. Yang, M. Liu, J. XiaoDevelopment of methodology: L. Xu, R. Jin, Z. YueAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Xu, R. Jin, Z. Yang, W. WanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Xu, J. Luo, R. Jin, Z. Yue, P. Sun, Z. Yang, J. Zhang,S. LiWriting, review, and/or revision of the manuscript: J. Luo, R. Jin, X. YangStudy supervision: J. Luo, J. Xiao

Grant SupportThis work is supported by grants from the National Basic Research Program

of China 2012CB910402 (to M. Liu and J. Luo), the National Natural ScienceFoundation of China 81272911 (to J. Luo), 81472048 (to J. Luo), 81330059

0 10 20 40TNFa

TNFaTNFa TNFa

TNFa

Ctrl

p65

DA

PIM

erge

N-PARPN-p65

A0 5 10 15 30 60

+ BZB0 5 10 15 30 60 (min) 0 5 10 15 30 60

TNFa + BZB0 5 10 15 30 60 (min)

BZB (nmol/L)

0

10

20

30

40

50

NF-

kB–d

epen

dent

lu

cife

rase

exp

ress

ion

(fold

s)

Ctrl 0 5 10 20 40 BZB (nmol/L)

b-Actin

b-ActinIkBa

Bcl-2

c-Flip

Bcl-xl

XIAP

c-IAP1c-IAP2

0 12 483624B

0 12 48 (h)3624TNFa TNFa + BZB

SDF-1 VEGFMCP-1 MMP-9

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

Rel

ativ

e ex

pres

sion

2.5 5 107.50BZB (nmol/L)

2.5 5 107.50BZB (nmol/L)

2.5 5 107.50BZB (nmol/L )

2.5 5 107.50BZB (nmol/L )

0

1

0

1

0

1

0

1

C

Figure 6.Inhibition of the NF-kB signaling pathway by bortezomib (BZB) in GCTB. A, bortezomib inhibits NF-kB signaling pathway. Bortezomib inhibits TNFa-induceddegradation of IkBa and nuclear translocation of p65 by Western blotting (top). NSCs were isolated and pretreated with or without bortezomib for4 hours and then stimulated with TNFa (0.2 nmol/L) for the indicated time. The cell lysates were collected and subjected to immunoblotting with antibodies asindicated (left). NSCs were isolated and pretreated with or without bortezomib for 4 hours and then stimulated with TNFa (0.2 nmol/L) for the indicated time.Nuclear fractions were collected and subjected to immunoblotting with antibodies as indicated (right). N-p65, nuclear p65; N-PARP, nuclear PARP.Bortezomib inhibits TNFa-induced p65 nuclear translocation by immunofluorescence staining (bottom left). NSCs were isolated and pretreated with or withoutbortezomib for 4 hours and then stimulated with TNFa (0.2 nmol/L) for 10 minutes. The localization of p65 was visualized by immunofluorescence analysis asdescribed under "Materials and Methods." Magnification, �40. Representative photos of n ¼ 3 experiments. Bortezomib suppresses TNFa-induced NF-kBluciferase reporter gene expression in a dose-dependentmanner (bottom right). After transfection of the NF-kB luciferase reporter gene in 293T cells, the cells wereincubated with the indicated concentration of bortezomib for 24 hours and then stimulated with TNFa (0.2 nmol/L) for another 4 hours. Cell lysatesupernatants were collected and assayed for luciferase activity. Columns show the means of experiments performed in triplicate; scale bars, SD. B, bortezomib timedependently inhibits TNFa-induced NF-kB–mediated antiapoptotic gene product expression in NSCs. NSCs were isolated and treated with or without bortezomib(20 nmol/L) and then stimulated with TNFa (0.2 nmol/L) for the indicated times. Cell lysates were collected and subjected to immunoblotting withantibodies as indicated. C, bortezomib dose dependently suppressed NF-kB–mediated migratory and angiogenic gene expression. NSCs were isolated and treatedwith or without bortezomib for 24 hours, and then total RNA was collected and subjected to real-time PCR. Columns show the means of experimentsperformed in triplicate; scale bars, SD.






(to J. Xiao), and 81330049 (to M. Liu), Innovation Program of ShanghaiMunicipal Education Commission 14ZZ051 (to J. Luo), and the Science andTechnology Commission of Shanghai Municipality 15140903600 (to J. Luo),09411950500 (to J. Xiao), and 12R21418300 (to J. Xiao).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 14, 2015; revised January 20, 2016; accepted February 1,2016; published OnlineFirst February 9, 2016.

References1. Cowan RW, Singh G. Giant cell tumor of bone: a basic science perspective.

Bone 2013;52:238–46.2. Niu X, Zhang Q, Hao L, Ding Y, Li Y, Xu H, et al. Giant cell tumor of the

extremity: retrospective analysis of 621 Chinese patients from one insti-tution. J Bone Joint Surg Am 2012;94:461–7.

3. Chawla S, Henshaw R, Seeger L, Choy E, Blay JY, Ferrari S, et al. Safety andefficacy of denosumab for adults and skeletally mature adolescents withgiant cell tumourof bone: interimanalysis of anopen-label, parallel-group,phase 2 study. Lancet Oncol 2013;14:901–8.

4. Zheng MH, Robbins P, Xu J, Huang L, Wood DJ, Papadimitriou JM. Thehistogenesis of giant cell tumour of bone: a model of interaction betweenneoplastic cells and osteoclasts. Histol Histopathol 2001;16:297–307.

5. Liu L, Aleksandrowicz E, Fan P, Schonsiegel F, Zhang Y, Sahr H, et al.Enrichment of c-Metþ tumorigenic stromal cells of giant cell tumor of boneand targeting by cabozantinib. Cell Death Dis 2014;5:e1471.

6. Wulling M, Delling G, Kaiser E. The origin of the neoplastic stromal cell ingiant cell tumor of bone. Hum Pathol 2003;34:983–93.

7. Huang L, Xu J, Wood DJ, Zheng MH. Gene expression of osteoprotegerinligand, osteoprotegerin, and receptor activator of NF-kappaB in giant celltumor of bone: possible involvement in tumor cell-induced osteoclast-likecell formation. Am J Pathol 2000;156:761–7.

8. Roux S, Amazit L, Meduri G, Guiochon-Mantel A, Milgrom E, MarietteX. RANK (receptor activator of nuclear factor kappa B) and RANK ligandare expressed in giant cell tumors of bone. Am J Clin Pathol2002;117:210–6.

9. Liao TS, Yurgelun MB, Chang SS, Zhang HZ, Murakami K, Blaine TA, et al.Recruitment of osteoclast precursors by stromal cell derived factor-1 (SDF-1) in giant cell tumor of bone. J Orthop Res 2005;23:203–9.

10. Ghert M, Simunovic N, Cowan RW, Colterjohn N, Singh G. Properties ofthe stromal cell in giant cell tumor of bone. Clin Orthop Relat Res2007;459:8–13.

11. van der Heijden L, Dijkstra PD, van de Sande MA, Kroep JR, Nout RA, vanRijswijk CS, et al. The clinical approach toward giant cell tumor of bone.Oncologist 2014;19:550–61.

12. Klenke FM, Wenger DE, Inwards CY, Rose PS, Sim FH. Giant cell tumor ofbone: risk factors for recurrence. Clin Orthop Relat Res 2011;469:591–9.

13. Bhatia S, Miszczyk L, Roelandts M, Nguyen TD, Boterberg T, Poortmans P,et al. Radiotherapy for marginally resected, unresectable or recurrent giantcell tumor of the bone: a rare cancer network study. Rare Tumors 2011;3:e48.

14. Sanerkin NG. Malignancy, aggressiveness, and recurrence in giant celltumor of bone. Cancer 1980;46:1641–9.

15. Yu X, Xu M, Xu S, Su Q. Clinical outcomes of giant cell tumor of bonetreated with bone cement filling and internal fixation, and oral bispho-sphonates. Oncol Lett 2013;5:447–51.

16. Tse LF, Wong KC, Kumta SM, Huang L, Chow TC, Griffith JF. Bispho-sphonates reduce local recurrence in extremity giant cell tumor of bone: acase-control study. Bone 2008;42:68–73.

17. Cornelis F, Truchetet ME, Amoretti N, Verdier D, Fournier C, Pillet O, et al.Bisphosphonate therapy for unresectable symptomatic benign bonetumors: a long-term prospective study of tolerance and efficacy. Bone2014;58:11–6.

18. Lewin J, Thomas D. Denosumab: a new treatment option for giant celltumor of bone. Drugs Today 2013;49:693–700.

19. Branstetter DG, Nelson SD, Manivel JC, Blay JY, Chawla S, Thomas DM,et al. Denosumab induces tumor reduction and bone formation in patientswith giant-cell tumor of bone. Clin Cancer Res 2012;18:4415–24.

20. Lau CP, Huang L, Chuen Wong K, Madhukar Kumta S. Comparison ofthe anti-tumor effects of denosumaband zoledronic acid on the neo-plastic stromal cells of giant cell tumor of bone. Connect Tissue Res2013;54:439–49.

21. CampanacciM, BaldiniN, Boriani S, Sudanese A.Giant-cell tumor of bone.J Bone Joint Surg Am 1987;69:106–14.

22. Enneking WF, Spanier SS, Goodman MA. A system for the surgical stagingof musculoskeletal sarcoma. Clin Orthop Relat Res 1980:106–20.

23. Tang X, Jin R, Qu G, Wang X, Li Z, Yuan Z, et al. GPR116, an adhesion G-protein-coupled receptor, promotes breast cancer metastasis viathe Galphaq-p63RhoGEF-Rho GTPase pathway. Cancer Res 2013;73:6206–18.

24. WuX, Li Z, Yang Z, ZhengC, Jing J, Chen Y, et al. Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator ofNF-kappaB ligand-inducedosteoclastogenesis and prevents ovariectomy-induced bone loss throughinhibition of mitogen-activated protein kinase/activator protein 1 andCa2þ-nuclear factor of activated T-cells cytoplasmic 1 signaling pathways.J Bone Miner Res 2012;27:1298–308.

25. Park SI, Kim SJ, McCauley LK, Gallick GE. Pre-clinical mouse models ofhuman prostate cancer and their utility in drug discovery. Curr ProtocPharmacol 2010;Chapter 14:Unit 14 5.

26. Mak IW, Cowan RW, Popovic S, Colterjohn N, Singh G, Ghert M. Upre-gulation of MMP-13 via Runx2 in the stromal cell of Giant Cell Tumor ofbone. Bone 2009;45:377–86.

27. Mak IW, Seidlitz EP, Cowan RW, Turcotte RE, Popovic S, Wu WC, et al.Evidence for the role ofmatrixmetalloproteinase-13 in bone resorption bygiant cell tumor of bone. Hum Pathol 2010;41:1320–9.

28. TangQL, Xie XB,Wang J, ChenQ,Han AJ, ZouCY, et al. Glycogen synthasekinase-3beta, NF-kappaB signaling, and tumorigenesis of human osteo-sarcoma. J Natl Cancer Inst 2012;104:749–63.

29. Roux S, Mariette X. RANK and RANKL expression in giant-cell tumour ofbone. Lancet Oncol 2010;11:514.

30. Zhou W, Yin H, Wang T, Liu T, Li Z, Yan W, et al. MiR-126–5p regulatesosteolysis formation and stromal cell proliferation in giant cell tumorthrough inhibition of PTHrP. Bone 2014;66:267–76.

31. Balke M, Neumann A, Szuhai K, Agelopoulos K, August C, Gosheger G,et al. A short-term in vivo model for giant cell tumor of bone. BMC Cancer2011;11:241.

32. Jones MD, Liu JC, Barthel TK, Hussain S, Lovria E, Cheng D, et al. Aproteasome inhibitor, bortezomib, inhibits breast cancer growth andreduces osteolysis by downregulating metastatic genes. Clin Cancer Res2010;16:4978–89.

33. Chen D, Frezza M, Schmitt S, Kanwar J, Q PD. Bortezomib as the firstproteasome inhibitor anticancer drug: current status and future perspec-tives. Curr Cancer Drug Targets 2011;11:239–53.

34. Schilcher J, Aspenberg P. Atypical fracture of the femur in a patient usingdenosumab–a case report. Acta Orthop 2014;85:6–7.

35. Agarwal S, Agarwal S, Gupta P, Agarwal PK, Agarwal G, Bansal A. Risk ofatypical femoral fracture with long-term use of alendronate (bispho-sphonates): a systemic review of literature. Acta Orthop Belg2010;76:567–71.

36. Gupta SC, Sundaram C, Reuter S, Aggarwal BB. Inhibiting NF-kappaBactivation by small molecules as a therapeutic strategy. Biochim BiophysActa 2010;1799:775–87.

37. Gupta SC, Kim JH, Kannappan R, Reuter S, Dougherty PM, Aggarwal BB.Role of nuclear factor kappaB-mediated inflammatory pathways in cancer-related symptoms and their regulation by nutritional agents. Exp Biol Med2011;236:658–71.

38. Kouroukis TC, Baldassarre FG, Haynes AE, Imrie K, Reece DE, CheungMC.Bortezomib in multiple myeloma: systematic review and clinical consid-erations. Curr Oncol 2014;21:e573–603.

39. ShahMA, Power DG, Kindler HL, Holen KD, KemenyMM, Ilson DH, et al.A multicenter, phase II study of bortezomib (PS-341) in patients withunresectable or metastatic gastric and gastroesophageal junction adeno-carcinoma. Invest New Drugs 2011;29:1475–81.

Xu et al.





40. Kane RC, Bross PF, Farrell AT, Pazdur R. Velcade: U.S. FDA approval for thetreatment of multiple myeloma progressing on prior therapy. Oncologist2003;8:508–13.

41. Thapa RJ, Chen P, Cheung M, Nogusa S, Pei J, Peri S, et al. NF-kappaBinhibition by bortezomib permits IFN-gamma-activated RIP1 kinase-dependent necrosis in renal cell carcinoma. Mol Cancer Ther 2013;12:1568–78.

42. Li C, Li R, Grandis JR, Johnson DE. Bortezomib induces apoptosis via Bimand Bik up-regulation and synergizes with cisplatin in the killing of headand neck squamous cell carcinoma cells. Mol Cancer Ther 2008;7:1647–55.

43. Codony-Servat J, Tapia MA, Bosch M, Oliva C, Domingo-Domenech J,MelladoB, et al. Differential cellular andmolecular effects of bortezomib, aproteasome inhibitor, in human breast cancer cells. Mol Cancer Ther2006;5:665–75.

44. Oreffo RO, Marshall GJ, Kirchen M, Garcia C, Gallwitz WE, Chavez J,et al. Characterization of a cell line derived from a human giant celltumor that stimulates osteoclastic bone resorption. Clin Orthop RelatRes 1993:229–41.

45. James IE, Dodds RA, Olivera DL, Nuttall ME, Gowen M. Human osteo-clastoma-derived stromal cells: correlation of the ability to form mineral-ized nodules in vitro with formation of bone in vivo. J Bone Miner Res1996;11:1453–60.

46. Singh S, SinghM,Mak I, GhertM. Expressional analysis of GFP-tagged cellsin an in vivo mouse model of giant cell tumor of bone. Open Orthop J2013;7:109–13.

47. Craig MJ, Loberg RD. CCL2 (Monocyte Chemoattractant Pro-tein-1) in cancer bone metastases. Cancer Metastasis Rev 2006;25:611–9.

48. Kim Y,Nizami S, GotoH, Lee FY.Modern interpretation of giant cell tumorof bone: predominantly osteoclastogenic stromal tumor. ClinOrthop Surg2012;4:107–16.

49. Taylor RM, Kashima TG, Knowles HJ, Athanasou NA. VEGF, FLT3 ligand,PlGF and HGF can substitute for M-CSF to induce human osteoclastformation: implications for giant cell tumour pathobiology. Lab Invest2012;92:1398–406.

50. Prando A, deSantos LA, Wallace S, Murray JA. Angiography in giant-cellbone tumors. Radiology 1979;130:323–31.






2016;15:854-865. Published OnlineFirst February 9, 2016.Mol Cancer Ther Leqin Xu, Jian Luo, Rongrong Jin, et al. Formation, and Bone ResorptionCell Apoptosis and Inhibition of Osteoclast Recruitment, Giant Cell Bortezomib Inhibits Giant Cell Tumor of Bone through Induction of

Updated version

10.1158/1535-7163.MCT-15-0669doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2016/02/09/1535-7163.MCT-15-0669.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/15/5/854.full#ref-list-1

This article cites 47 articles, 10 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://mct.aacrjournals.org/content/15/5/854To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-15-0669

http://mct.aacrjournals.org/content/suppl/2016/02/09/1535-7163.MCT-15-0669.DC1

http://mct.aacrjournals.org/content/15/5/854.full#ref-list-1

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/15/5/854


bortezomib inhibits giant cell tumor of bone...

Documents