hypoxia-induced wsb1 promotes the metastatic potential of ...molecular and cellular pathobiology...

Molecular and Cellular Pathobiology

Hypoxia-Induced WSB1 Promotes the MetastaticPotential of Osteosarcoma CellsJi Cao1,Yijie Wang1, Rong Dong1, Guanyu Lin1, Ning Zhang2, Jing Wang3, Nengming Lin4,Yongchuan Gu5, Ling Ding1, Meidan Ying1, Qiaojun He1, and Bo Yang1

Abstract

Intratumoral hypoxia occurs in many solid tumors, where it isassociated with the development of metastatic character. How-ever, the connections between these phenomena are not fullyunderstood. In this study, we define an integrative role for the E3ubiquitin ligase subunit WSB1. In primary osteosarcomas,increased levels of WSB1 correlated with pulmonary metastaticpotential. RNAi-mediated attenuation of WSB1 or disruption ofits E3 ligase activity potently suppressed tumormetastasis. Quan-

titative proteomic and functional analyses revealed that WSB1ubiquitylates the Rho-binding protein RhoGDI2 andpromotes itsproteasomal degradation, thereby activating Rac1 to stimulatetumor cell motility and invasion. Our findings show how WSB1regulates key steps of the metastatic cascade in hypoxia-drivenosteosarcoma, and they highlight a candidate therapeutic targetto potentially improve the survival of patients with metastaticdisease. Cancer Res; 75(22); 4839–51. �2015 AACR.

IntroductionOsteosarcoma is the most common type of bone cancer that

usually occurs in juvenile groups (1,2). Despite remarkableadvances in the combined used of chemotherapy, radiotherapyand surgical ablation of the primary tumor, survival rates haveyet to improve over those of the 1970s (3). Approximately 40%to 50% of patients develop metastasis, often pulmonary metas-tasis, even after curative resection of the primary tumor, and the5-year survival rate of osteosarcoma patients with metastases iseven lower than 30% (4-6). Accordingly, the leading cause offatal outcomes in relapsed osteosarcoma is tumor metastasis.Targeting tumor metastasis may thus represent a promisingstrategy to intervene into human osteosarcoma. However, littleprogress has been made in this area as the molecular mechan-isms underlying osteosarcoma invasion and metastasis remainpoorly understood.

Meanwhile, intratumoral hypoxia contributes to increasedrisk of invasion, metastasis, treatment failure, and patient

mortality in most types of solid tumors, including osteosarco-ma (7-10). Hypoxia-inducible factor-1 alpha (HIF1a), a tran-scription factor that is stabilized in hypoxia, regulates theexpression of multiple target genes to help tumor cells adaptto and survive in hypoxic conditions, contributing to poorchemotherapy responses, as well as decreased overall survivaland disease-free survival in osteosarcoma patients (11-13).Recently, exposure of osteosarcoma cells to hypoxic conditionswas reported to increase the invasive potential of the tumorcells in vitro (14), suggesting that hypoxia might promote themetastasis of osteosarcoma cells. Considerable effort has beendirected at understanding the potential mechanism of hypoxia-promoted cancer metastasis (7,15), but most of these studieswere focused on angiogenesis, chemokines, and stromal–tumorcell interactions. Previous work also demonstrated that HIF1aadapts tumor cells to hypoxic conditions via context and oftencell-type specific mechanisms. It is intriguing to ask whetherand how hypoxia could promote the metastasis of humanosteosarcoma.

WD repeat and SOCS box containing 1 (WSB1) were recentlyidentified as a new member of the suppressor of cytokinesignaling (SOCS) box protein family, that is upregulated inmultiple types of human cancers (16). WSB1, in complex withelongin B/C-Cullin5-Rbx1, forms an E3 ubiquitin ligase forthyroid-hormone–activating type 2-iodothyronine deiodinase(D2; ref. 17) and homeodomain-interaction protein kinase 2(HIPK2; ref. 18), thus negatively regulating tumor progressionand chemoresistance. More recent evidence suggested that WSB1might be a direct target of HIF1 in a human hepatocellularcarcinoma model, with a proposed function of WSB1 in hypox-ia-promoted chemoresistance. Given that WSB1 is a substrate-recognizing subunit of E3 ubiquitin ligase, it is intriguing to askwhether and howWSB1might function in hypoxia-drivenmetas-tasis in human osteosarcoma, possibly through mechanismsinvolving substrates other than D2 and HPIK2.

In this study, we first examined the potential role of WSB1 inregulating osteosarcoma cell metastasis in vitro and in vivo, as wellas its association with hypoxia. Quantitative proteomic and

1Institute of Pharmacology and Toxicology, Zhejiang Province KeyLaboratory of Anti-Cancer Drug Research, College of PharmaceuticalSciences, Zhejiang University, Hangzhou, China. 2Department ofOrthopedics, The Second Affiliated Hospital of Zhejiang University,Zhejiang University, Hangzhou, China. 3Zhejiang Province Key Labo-ratoryofMedical Molecular Imaging,The SecondAffiliatedHospital ofZhejiang University, Zhejiang University, Hangzhou, China. 4Institutefor Individualized Medicine, Hangzhou First People's Hospital, Hang-zhou, China. 5Auckland Cancer Society Research Centre, The Univer-sity of Auckland, Auckland, New Zealand.

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

J. Cao and Y. Wang contributed equally to this article.

Corrected online June 2, 2020.

Corresponding Author: Bo Yang, College of Pharmaceutical Sciences, ZhejiangUniversity, Room 113, Hangzhou, China, 310058. Phone: 86-571-88208400; Fax:86-571-88208400; E-mail: [email protected].

doi: 10.1158/0008-5472.CAN-15-0711

�2015 American Association for Cancer Research.

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functional analyses revealed that Rho guanosine diphosphatedissociation inhibitor 2 (RhoGDI2) as a novel downstreamprotein involved in WSB1-mediated cell migration.

Materials and MethodsCell lines and plasmids

The U2OS and MG63 cell lines were purchased from the CellBank of Type Culture Collection of the Chinese Academy ofSciences (Shanghai, China). The KHOS/NP cell line was kindlyprovided by Dr. Lingtao Wu (University of Southern California,CA). All the cell lines have been tested and authenticatedutilizing short tandem repeat (STR) profiling every 6 months.All primary osteosarcoma blasts were from fresh tissue sectionsfrom biopsies of osteosarcoma patients as described previously(19). All cells were cultured in DMEM or RPMI-1640 mediumsupplemented with 10% FBS in a humidified atmosphere of 5%CO2 at 37�C.

The full-length coding sequence for WSB1 and RhoGDI2 wasamplified from the U2OS cDNA library and subsequently sub-cloned into the PCMV6 plasmid (Origene). The WSB1-SOCSdeletion was produced based on full-length WSB1 using a pairof primers (forward: 50-GGAATTCATGGCCAGCTTTCCC-30;reverse: 50-GGAATTCTTAAACCTTATCGTCGTCATCCTT-30). Theindicated WSB1-SOCSmutation was produced by the GenescriptCompany.

Clinical human tissue specimenThe clinical samples of osteosarcoma patients were obtained

from the Second AffiliatedHospital of ZhejiangUniversity Schoolof Medicine (Hangzhou, China) and Hangzhou First People'sHospital (Hangzhou, China). Written informed consents frompatients and approval from the Institutional Research EthicsCommittee of the hospital were obtained before the use of theseclinical materials for research purposes.

Lentivirus transductionThe shRNA-expressing lentiviral vector pGFP-V-RS against the

WSB1 gene was obtained from Origene. pCCL-WSB1 was con-structed by the Genescript Company according to PCMV6-WSB1.The virus particles were harvested 48 hours after transfection of293FT cells. For stable transfection, cells were grown in 6-wellplates at 60% to 70% confluency, and 1 mL of viral supernatantwas added with 1 mL polybrene.

Western blotting, real-time PCR, and immunoprecipitationWestern blotting, real-time PCR, and immunoprecipitation

were performed as described (20), with details in Supplementaryinformation.

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) was performed using

the EZ ChIP kit (EMDMillipore). Briefly, KHOS/NP cells culturedunder hypoxia conditions were collected and cross-linked withformaldehyde. Chromatin was sonicated on ice to an averagelength of 200 to 300 bp in an ultrasound bath. The chromatinwasthen incubated and precipitated with anti-IgG or anti-HIF1aantibodies. The immunoprecipitated DNA fragments weredetected by PCR analysis using primers specific for PM1, PM2,and PM3.

SILAC assayCells were cultured in DMEM and supplemented with either

[U-12C6]-L-lysine (light) or [U-13C6]-L-lysine (heavy) for at leasteight generations. The heavy labeling efficiency was measuredby mass spectrometer analysis. Then, cells were continuouslymaintained in SILAC medium until they reached the desiredconfluence and were harvested by trypsinization, and combinedin equal amounts. The enriched fractions were analyzed by massspectrometry.

IHC and immunofluorescence assayTumor processing and immunofluorescence assays were per-

formed as described previously (20), with details provided in theSupplementary information.

F-actin staining assay and confocal microscopyCells were grown on 35-mm glass-bottom dishes and fixed

with 4% paraformaldehyde for 15 minutes and then permea-bilized with 0.1% Triton X-100 in PBS for 10 minutes. The cellswere then blocked with 1% BSA for 20 minutes. Cells wereincubated with Texas Red-X phalloidin (Life Technologies)diluted in 5 mL/200mL in PBS for 20 minutes and then stainedwith DAPI (100 mg/mL) for 3 minutes. The glasses weremounted with anti-fade reagent. The cells were observed undera confocal microscope.

Cell mobility assayCells were seeded into 35-mmdishes coated with 0.5% gelatin.

After 24 hours, dishes were placed onto an inverted microscopeand imaged every 10 minutes up to 210 minutes. Temperaturewas maintained at 37�C.

Cell migration assayThe cell migration assay was performed in a 24-well Transwell

plate with 8 mmpolycarbonate sterile membrane (Corning Incor-porated). Cells were plated in the upper chamber at 2�104 cellsper insert in 200 mL of serum-free medium. Inserts were placed inwells containing 600 mL ofmedium supplemented with 10%FBS.Twenty-four hours later, cells on the upper surface were detachedwith a cotton swab. Filters were fixed and cells in the lower filterwere stained with 0.1% crystal violet for 15minutes and counted.The quantified results were calculated by counting three randomfields of migrated cells.

Measurement of in vivo activityTumors were established via by intravenous injection of

lentivirus-transfected KHOS/NP cells (1�106 cells/animal)into the tails of 3- to 4-week-old female BALB/c (nu/nu)mice (National Rodent Laboratory Animal Resource, Shanghai,China). At 14 days after injection, the mice were sent to theSecond Affiliated Hospital of Zhejiang University School ofMedicine and subjected to PET scanning. For the orthotopictransplantation model, 4-week-old BALB/c nude mice wereanesthetized with a mixture of xylazine and ketamine. A 26-gauge needle was used to perforate the femur head, after whicha second needle, coupled to a 1-mL syringe loaded with 1�105cells, was introduced through the puncture hole into themedullary cavity of the femur. After the mice were sacrificed,all lungs were dissected or further imaged by fluorescencestereomicroscopy and then fixed with formalin. Tissue sections

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Figure 1.WSB1 is a direct target gene of HIF1a in osteosarcoma that is highly associated with the progression and metastasis of human osteosarcoma. A, IHC staining ofWSB1 and HIF1a in osteosarcoma specimens. B, statistical analysis of IHC results in 40 human osteosarcoma specimens. C, Western blotting of HIF1a, WSB1,and BNIP3 in osteosarcoma cell lines and primary blasts cultured under normoxia or hypoxia for 24 hours. D, relative mRNA levels of WSB1 in KHOS/NP cells eithercultured under normoxia or hypoxia for 24 hours (left) or transfectedwith the indicated plasmids for 48 hours (right). Bars, mean� SD (n¼ 3). E,Western blotting ofHIF1a, WSB1, and BNIP3 in KHOS/NP cells transfected with siRNA-HIF1a under hypoxia. F, Western blotting of HIF1a, Myc-tag, WSB1, and BNIP3 in KHOS/NPcells transfected with Myc-tagged HIF1a under normoxia for 48 hours. G, schematic representation of the human WSB1 promoter sequence spanning 2.0kbupstream of the transcriptional start site. Three potential hypoxia-responsive elements are indicated. H, a dual luciferase analysis in KHOS/NP cells.HIF1a plasmid or control vector was cotransfected with empty pGL4 control (pGL), pGL4-WSB1-promoter (P), pGL4-WSB1-promoter-mutation 1 (PM1),pGL4-WSB1-promoter-mutation 2 (PM2), or pGL4-WSB1-promoter-mutation 3 (PM3). Transcriptional activity was determined 48 hours post-transfection as aratio relative to the control cells after normalization to Renilla activity. Bars, mean � SD (n ¼ 3). I, ChIP assay. Anti-IgG and anti-HIF1a antibodies were used inthe ChIP assay. J, Kaplan–Meier analysis of metastasis-free survival of 28 patients with osteosarcoma stratified by WSB1 protein levels in primary tumor.K and L, migration assay of primary blasts under normoxia or hypoxia for 24 hours. K, representative images of migrated cells. L, the number of migratedcells per field was quantified after normalization to normoxia (n ¼ 3). � , P < 0.05; �� , P < 0.01.

WSB1 Promotes Osteosarcoma Metastasis

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Figure 2.WSB1 drives osteosarcoma cell metastatic potential both in vitro and in vivo. A, Western blotting of WSB1 and Myc-tag. B, migration assay of theindicated cells, along with representative images. The number of migrated cells per field was quantified (n ¼ 3) after normalization. Bars, mean � SD (n ¼ 3).C–F, tail-vein injection of KHOS/NP cells was performed in nude mice (n ¼ 4), and the formation of metastatic node was determined on day 14. C,representative micro-PET image. Color scale is indicated. D, the intensity of micro-PET was evaluated (n ¼ 4). E, lung tissues were examined under afluorescence stereo microscope. A representative image is shown. F, the number of metastatic nodes per lung was determined. Bars, mean � SD (n ¼ 4).G and H, orthotopic transplantation of KHOS/NP cells was performed in nude mice (n ¼ 3), and the metastatic nodes were determined. G, representativeimages of lungs. Yellow arrow, a metastatic node. (Continued on the following page.)

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(3 mm) were stained with hematoxylin/eosin. The AnimalResearch Committee at Zhejiang University approved all ani-mal studies and animal care was provided in accordance withinstitutional guidelines.

Statistical analysisThe relationship between HIF1a and WSB1 expression was

analyzed using the Spearman rank correlation test. Survivalcurves were plotted via the Kaplan–Meier method. The valuesfor all samples in the different experimental conditions wereaveraged, and the standard error or SD of the mean wascalculated. Differences between means were determined usingthe unpaired Student t tests and were considered significantat P < 0.05.

ResultsHypoxia-induced WSB1 is highly associated with progressionand metastasis of human osteosarcoma

We first exploredWSB1 expression in 40 primary osteosarcomatissues by IHC staining. Interestingly, high expression levels ofboth the hypoxia marker protein HIF1a and the WSB1 proteinwere detected in most tumor specimens (Fig. 1A and B). More-over, a statistically significant correlation was also found betweenthese two proteins (R ¼ 0.825, P < 0.01; Fig. 1B), suggesting aconnection between high WSB1 levels and hypoxic microenvir-onments. To better understand the effect of hypoxia on WSB1expression, three osteosarcoma cell lines and two primary oste-osarcoma blasts (19) were cultured under hypoxic conditions(1%O2) for 24 hours. The protein expression of WSB1, as well asHIF1a and its downstream target BNIP3, was significantlyinduced by hypoxia in all five osteosarcoma cells (Fig. 1C).Because WSB1 expression is significantly increased at the mRNAlevel under hypoxia (Fig. 1D), it is reasonable to questionwhetherHIF1a mediates hypoxia-induced WSB1 transcription. By target-ing HIF1a using two specific siRNAs, we found that HIF1a-depletion significantly downregulated its target, BNIP3, and alsosignificantly blocked the hypoxia-induced WSB1 expression (Fig.1E). In addition, both themRNA and protein levels ofWSB1wereupregulated, along with the expression of HIF1a and BNIP3, incells that overexpressed HIF1a (Fig. 1D and F). Moreover, basedonWSB1�promoter¼ luciferase reporter assays and site-directedmutagenesis, we confirmed that HIF1a was able to transactivatethe WSB1 promoter via direct binding to its -339 bp region (Fig.1G and H). ChIP assays also validated that HIF1a bound to the-339 bp genomic region of WSB1 under hypoxia (Fig. 1I). Theseresults not only suggest that WSB1 is a direct target of HIF1a, butalso imply that WSB1 might be involved in hypoxia-triggeredtumorigenesis.

Given that hypoxia has been reported to play a crucial role inpromoting tumor progression and metastasis (7,8), we furtherhypothesized that hypoxia-induced WSB1 expression might be

pathologically relevant to the progression of osteosarcoma.Importantly, WSB1 protein levels were not only higher in tumorswith metastasis than without it (Supplementary Fig. S1A), butwere also prognostic for metastasis-free survival (Fig. 1J). Further-more, hypoxia could promote the migration of primary osteo-sarcomablasts (Fig. 1K and L),whereWSB1 expression levelsweresignificantly upregulated under hypoxic conditions (Fig. 1C).Thus, hypoxia-induced WSB1 is clearly associated with the met-astatic potential of human osteosarcoma.

WSB1drives osteosarcoma cellmetastatic potential both in vitroand in vivo

To further address the effect of WSB1 on osteosarcoma cellmetastasis, we next examined the effects of manipulating WSB1levels. Lentiviral transduction enabled the stable overexpressionof WSB1 compared with vector-transduced cells, at levels com-parable with those observed under hypoxia (Fig. 2A and Supple-mentary Fig. S1B). The migration assays showed that, comparedwith the vector-transduced cells, WSB1-overexpressing KHOS/NPand U2OS cells traversed more efficiently into the lower chamberof the Transwell under normoxia (Fig. 2B). Consistent with thisfinding, we observed that WSB1 overexpression enhanced thewound-closure capability of KHOS/NP cells (Supplementary Fig.S2). Therefore, WSB1 may modulate the migration capacity ofosteosarcoma cells in vitro.

To further quantify metastatic potential in vivo, we performedtail-vein xenografts in BALB/c (nu/nu) mice and examined therates of lung colonization bymicro-PET scan. As shown in Fig. 2Cand D, increased signal intensities in lungs were observed on day14 in mice transplanted with WSB1-overexpressing KHOS/NPcells compared with control mice, indicating the formation ofmore metastatic nodes in mice injected with WSB1-overexpres-sing cells. The lentiviral vector pCCL expresses a GFP tag (Sup-plementary Fig. S1C), allowing us to visualizemetastatic nodes inlungs via fluorescence imaging. As shown in Fig. 2E, WSB1-over-expressing cells formed more metastatic nodules in the lung thancontrol cells. Histologic examination confirmed that the numberof micrometastatic lesions was markedly increased in the lungsof mice transplanted with WSB1-overexpressing cells (Fig. 2F andSupplementary Fig. S3). Moreover, we further examined themetastasis phenotype of those two cell lines using an orthotopictransplantationmodel. Consistent with our intravenous injectionmodel, WSB1-overexpressing cells induced more lung metastasisnodes than control cells (Fig. 2G and H).

We next verified the effects ofWSB1 depletion on themigrationand metastasis of KHOS/NP cells using shRNA. Two specificsequences against WSB1 significantly inhibited the hypoxia-dependent expression of WSB1 (Fig. 2I). Meanwhile, in contrastwith overexpression (Fig. 2B), WSB1 depletion markedly inhib-ited the hypoxia-driven migration of KHOS/NP cells (Fig. 2J). Inagreement with the in vitro results, WSB1 depletion significantlydecreased the signal intensity in the lungs of tail-vein-injected

(Continued.) H, the number of metastatic nodes per lung was determined. Bars, mean � SD (n ¼ 3). I, Western blotting of WSB1 in KHOS/NP cells infectedwith the indicated lentivirus under normoxia or hypoxia for 24 hours. Relative WSB1 levels were normalized to b-actin as indicated. J, migration assay ofKHOS/NP cells infected with the indicated lentivirus. Representative images of migrated cells are shown. The number of migrated cells per field was quantifiedafter normalization. Bars, mean � SD (n ¼ 3). K–N, tail-vein injection of KHOS/NP cells infected with the indicated lentivirus was performed in nude mice (n ¼4), and the formation of metastatic nodes was determined on day 14. K, representative micro-PET images of mice 14 days after injection. Color scale isindicated. L, the intensity of micro-PET was evaluated (n ¼ 4). M, H&E staining of lung tissues. N, the number of metastatic nodes per lung was determined.Bars, mean � SD (n ¼ 4). � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






nude mice in micro-PET scans (Fig. 2K and L). Importantly,histologic examination of lung tissues revealed that in some casesdepletingWSB1 totally blocked themetastatic nodules in vivo (1/4in shRNA-#1 group and 2/4 in shRNA-#2 group), whereas miceinjected with control cells formed 8 to 18 metastatic nodulesper lung in all four mice (Fig. 2M and N). Our data indicate thatWSB1 is necessary for the aggressive, highly metastatic phenotypeof osteosarcoma cells.

Collectively, these overexpression and knockdown results gen-erated from in vitro and in vivo studies, together with the resultsfromosteosarcoma biopsies and primary cell lines, demonstrate anew phenotype of WSB1 wherein the hypoxia-dependent induc-tion ofWSB1 is necessary and sufficient to promote themetastaticpotential of osteosarcoma cells.

E3 ubiquitin ligase activity is required for WSB1-driven cellmigration

Previous reports have revealed that WSB1 is an E3 ubiquitinligase (17, 18); therefore, it is interesting to know whether E3ubiquitin ligase activity is critical for WSB1 to promote cellmigration. WSB1 is composed of seven WD40 repeats and aC-terminal SOCS box (Fig. 3A), and the SOCS box is known tobe involved in protein degradation via the ECS ubiquitin ligasecomplex (21). We therefore constructed a mutant WSB1 that

lacks the SOCS box (Fig. 3B, DSOCS) to determine whether E3ubiquitin ligase activity is required for WSB1-promoted cellmigration. As illustrated in Fig. 3C and D, infection with wild-type WSB1 lentivirus significantly promoted migration com-pared with the vector control. In contrast, DSOCS completelylost its migration-promoting activity, as did a WSB1 point-mutant (339-421, AAA) lacking E3 ligase activity (Fig. E–H).Taken together, these data further suggest that E3 ubiquitinligase activity is required for the WSB1-driven migration ofosteosarcoma cells.

SILAC quantitative proteomic profiling identifies theproteasome-dependent degradation of RhoGDI2 caused byWSB1 overexpression

To further understand the downstream pathways induced byWSB1 in the metastatic process of osteosarcoma, a large-scaleproteomic analysis was subsequently performed. The pro-teomes of ectopic WSB1-overexpressing and control vector-infected KHOS/NP cells were quantified using SILAC followedby mass spectrometry analysis (Fig. 4A). A total of 4,163proteins were identified from both cell lysates, and 4,094proteins were further quantified, of which 1,078 proteins weresignificantly perturbed by WSB1 overexpression, with 560

Figure 3.E3 ubiquitin ligase activity is required for WSB1-promoted cell migration. A, schematic representation of Myc-tagged wild-type and DSCOS WSB1 constructs.B, Western blotting of Myc-tag in KHOS/NP cells infection with the indicated lentivirus. C and D, migration assay of KHOS/NP cells infected with theindicated lentivirus. C, representative images of migrated cells. D, the number of migrated cells per field was quantified. Bars, mean � SD (n ¼ 3). E, schematicrepresentation of Myc-tagged wild-type and SOCS-box-site-mutant (Mut-WSB1) WSB1 constructs. F, Western blotting of Myc-tag in KHOS/NP cells infectionwith the indicated lentivirus. G and H, migration assay of KHOS/NP cells infected with the indicated lentivirus. G, representative images of migrated cells. H, thenumber of migrated cells per field was quantified. Bars, mean � SD (n ¼ 3). ��, P < 0.001.

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proteins being upregulated and 518 proteins downregulated(>3.0-fold, Fig. 4B and Supplementary Table S1). Bioinformat-ics analysis further revealed that these altered proteins aremainly involved in protein binding (50%) and catalytic activity(29%) and also participated in both cellular (23%) and met-abolic processes (18%; Fig. 4C). The expression of metastasis-associated proteins was further analyzed. Twenty-four proteinsinvolved in cell metastasis were identified, and 7 of these 24proteins changed by more than 3.0-fold in response to WSB1overexpression (Supplementary Fig. S4). These quantitativeproteomic data provide additional confirmation that WSB1promotes osteosarcoma cell metastasis.

Interestingly, RhoGDI2, a critical and potent inhibitor of Rhoproteins (22),was the top candidate from the SILAC analysis, withthe most significant decrease in protein abundance (Fig. 4D).Because our data suggest that E3 ubiquitin ligase activity isrequired for the WSB1-driven migration of osteosarcoma cells,we hypothesized that the downregulation of RhoGDI2 may serveas the downstream target of WSB1. To confirm this, the RhoGDI2protein expression level in WSB1-overexpressing KHOS/NP cellswas determined. ForcedWSB1 overexpression significantly inhib-ited RhoGDI2 protein expression, whereas RhoGDI1, anothermember of the RhoGDI family, did not change (Fig. 4E), indi-cating the selective inhibition of RhoGDI2 by WSB1. Because

Figure 4.SILAC quantitative proteomic profiling identifies the RhoGDI2 protein as the downstream target of WSB1 signaling. A, schematic representation of the SILACassay. B, the number of proteins identified and quantified using the SILAC assay. C, bioinformatics analysis of differentially expressed proteins with at least a 3-foldchange. D, RhoGDI2 and RhoGDI1 protein expression in the SILAC assay were quantified. E, Western blotting of WSB1, Myc-tag, RhoGDI2, and RhoGDI1 inKHOS/NP cells. F, Western blotting of HIF1a, WSB1, and RhoGDI2 in KHOS/NP cells cultured under normoxia or hypoxia for 24 hours. G, Western blotting ofWSB1 in KHOS/NP cells infection with the indicated lentivirus under normoxia or hypoxia for 24 hours. H, IHC staining of WSB1 and RhoGDI2 in osteosarcomaspecimens. I, statistical analysis of IHC results in 40 human osteosarcoma specimens.






WSB1 is specifically induced by hypoxia, we examined whetherthe downregulation of RhoGDI2 could also be achieved underhypoxic conditions. As expected, anobvious decrease inRhoGDI2was observed to accompany the induction of WSB1 under hyp-oxia (Fig. 4F). In contrast, WSB1 knockdown effectively reversed

the hypoxia-dependent decrease in RhoGDI2 levels (Fig. 4G).Moreover, a negative correlation between WSB1 and RhoGDI2was also found in 40 primary osteosarcoma tissues by IHCstaining (R ¼ �0.65, P < 0.01; Fig. 4H and I). In addition,RhoGDI2 protein levels were inversely correlated with metastasis

Figure 5.WSB1 is an E3 ligase for RhoGDI2 and promotes RhoGDI2 ubiquitination and degradation. A,Western blotting ofWSB1, Myc-tag, RhoGDI2, and RhoGDI1 in KHOS/NPcells infected with the indicated lentivirus. B and C, Western blotting of RhoGDI2 in KHOS/NP cells infected with the indicated lentivirus under 10 mg/mLcycloheximide (CHX) treatment (B) or 10 mmol/L MG132 treatment (C). D, colocalization of exogenous WSB1 and RhoGDI2. 293T cells were transfected withFLAG-WSB1 and RhoGDI2-HA plasmids followed by MG132 treatment and then subjected to immunofluorescence staining with anti-Flag and anti-HA antibodies.E, interaction between ectopic WSB1 and RhoGDI2. 293T cells transfected with FLAG-WSB1 and RhoGDI2-HA plasmids, followed by MG132 treatment.Cell lysates were immunoprecipitated with anti-FLAG or anti-HA antibody, followed by immunoblotting with anti-HA or anti-FLAG antibody. F, interaction betweenendogenous WSB1 and RhoGDI2. KHOS/NP cells treated with MG132 for 8h under hypoxia. Cell lysates were immunoprecipitated with anti-RhoGDI2 antibody,followed by immunoblotting with anti-WSB1 antibody. G, WSB1 promotes the ubiquitination of RhoGDI2. KHOS/NP cells were transfected with empty vector orWSB1, followed by MG132 treatment. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-ubiquitin antibody.

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of osteosarcoma (Supplementary Fig. S5A). These data clearlysupport the idea that RhoGDI2 is the downstream target ofWSB1 signaling.

Given that our data further demonstrated that RhoGDI2 mRNAlevels were not significantly decreased in WSB1-overexpressingcells and under hypoxia (Supplementary Fig. S5B), as well as thefact that DSOCS failed to decrease the amount of RhoGDI2(Fig. 5A), we are encouraged to further address whether WSB1mediates the degradation of RhoGDI2. To this end, cycloheximide(CHX)wasused toprevent de novoprotein synthesis; thus, RhoGDI2levels would primarily reflect the protein degradation process. Weexposed WSB1-overexpressing cells and control cells to CHX andestimated the RhoGDI2 expression. As shown in Fig. 5B, RhoGDI2degradation rates were remarkably increased in WSB1-overexpres-sing cells. Furthermore, MG132 (a specific proteasome inhibitor)treatment totally reverses the WSB1 overexpression-triggereddecrease in RhoGDI2 protein levels (Fig. 5C). In keeping with theseresults, MG132 also inhibited RhoGDI2 protein degradation underhypoxic condition (Supplementary Fig. S5C).

We next examined whether WSB1 regulates RhoGDI2 pro-tein degradation through a direct interaction. Immunofluores-cence results demonstrated that exogenous WSB1 colocalizeswith exogenous RhoGDI2 in 293T cells transfected with FLAG-WSB1 and RhoGDI2-HA plasmids (Fig. 5D). Furthermore, thisinteraction was further validated by immunoprecipitation.Ectopically expressed WSB1 and RhoGDI2 were coprecipitatedfrom 293T cells overexpressing both proteins (Fig. 5E). Con-sistently, endogenous WSB1 also interacts with endogenousRhoGDI2 as confirmed by immunoprecipitation (Fig. 5F) andimmunofluorescence assays (Supplementary Fig. S5D). There-fore, our data clearly demonstrate that WSB1 directly interactswith RhoGDI2. To confirm the role of WSB1 as an E3 ligase forRhoGDI2 polyubiquitination, we performed ubiquitinationassays. Polyubiquitinated RhoGDI2 only accumulated in cellsthat both overexpressed WSB1 and were treated with MG132(Fig. 5G). Collectively, our data suggest that hypoxia-drivenWSB1 promotes the proteasome-dependent degradation ofRhoGDI2.

Figure 6.RhoGDI2 acts downstream ofWSB1 tomediate cell motility and migration. A,endogenous active GTP-bound formof Rac1 was enriched by a pull-downassay and detected by Westernblotting. Total Rac1 was detectedusing anti-Rac1 antibody. B and C,quantification of the F-actinfluorescence intensity andmeasurement of ruffles-positive cellsinfected with the indicated lentivirus.B, representative images of eachgroup. C, fluorescence intensities of F-actin stained with phalloidin werenormalized to control cells.Percentages of ruffle-positive cells indifferent groups were calculatedbased on the immunofluorescence.Bars,mean�SD (n¼ 3). �� ,P

Figure 7.RhoGDI2 overexpression reverses the WSB1-driven metastasis of osteosarcoma. A, Western blotting of RhoGDI2 in KHOS/NP cells infected with the indicatedlentivirus. B and C, migration assay of KHOS/NP cells infected with the indicated lentivirus. B, representative images of migrated cells. C, the number ofmigrated cells per field was quantified. Bars, mean � SD (n ¼ 3). (Continued on the following page.)


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RhoGDI2 acts downstream of WSB1 to mediate cell motilityand migration

RhoGDIs modulate the cycling of Rho GTPases between activeGTP-bound and inactive GDP-bound states (23). Our data indi-cate that ectopic WSB1 expression is associated with a markeddecrease in RhoGDI2; thus, the activation of Rho GTPases shouldbe expected in WSB1-overexpressing cells. By specifically pullingdown the active GTP-bound form of Rho GTPases, we detected asubstantial level of active Rac1 protein, a member of the RhoGTPases, in the wild-type WSB1-overexpression group but not inthe vector or DSOCS groups (Fig. 6A). Our findings indicate thatWSB1-triggereed RhoGDI2 suppression activates Rho GTPases.

Rho GTPases are well known as regulator of actin cytoskeletalorganization and cellmotility.We therefore examined the effect ofWSB1 overexpression on the status of actin filament organizationand cell motility. As illustrated in Fig. 6B and C, WSB1 over-expression significantly enhanced the fluorescence intensity ofpolymerized actin (F-actin), which suggests abundant F-actinexpression, compared with the vector or mutant WSB1 groups.In addition to F-actin expression, the appearance of membraneruffles and the formation of lamellipodia were also observed inWSB1-overexpressing cells. Dynamic monitoring of the randommotility of KHOS/NP cells revealed an increased displacement ofWSB1-overexpressing cells from their origin sites compared withcontrol cells (Fig. 6D and Supplementary Figs S6 and S7). Thesedata clearly demonstrate that WSB1 increases the amount of F-actin, promotes the formation of lamellipodia, and consequentlyleads to enhanced cell motility and migration ability.

RhoGDI2 overexpression reverses the WSB1-driven metastasisof osteosarcoma cells

To determine whether RhoGDI2 overexpression could reverseWSB1-drivenmetastasis of osteosarcoma cells, wefirst establisheda stable cell line ectopically expressing RhoGDI2 (Fig. 7A). Nota-bly, RhoGDI2 overexpression almost completely abrogatedWSB1-promoted cell migration (Fig. 7B andC). Similar responseswere also observed via immunofluorescence staining of F-actin, asRhoGDI2 overexpression decreased the WSB1-enhanced fluores-cence intensity of F-actin and the formation of membrane ruffles(Fig. 7D and E). Moreover, RhoGDI2 overexpression blockedWSB1-enhanced wound-closure capability of KHOS/NP cells(Supplementary Fig. S8). These data suggest thatWSB1-enhancedcell motility and migration can be completely abrogated byRhoGDI2 overexpression in vitro.

To test whether this effect could be reproduced in vivo, wegenerated an osteosarcoma pulmonary metastasis model usingnude mice intravenously injected with the indicated cells. Con-sistent with our in vitro data, WSB1 overexpression markedlyincreased the number of pulmonary metastatic nodules (GFPpositive). However, the additional ectopic expression ofRhoGDI2 impaired the effect of WSB1 overexpression as dem-onstrated by the decrease in pulmonary metastatic nodules (Fig.7F and G). Taken together, these results not only indicate that

RhoGDI2-overexpression reverses WSB1-driven metastasis, butalso further support a pivotal role for theWSB1–RhoGDI2 axis inthe metastasis of osteosarcoma cells.

DiscussionPulmonarymetastasis has been recognized as themain cause of

fatal outcomes in osteosarcoma patients, but its molecular mech-anism is rarely discussed (1,24). Accumulating evidence suggeststhat tumor progression requires adaptation to hypoxic microen-vironments, andmolecules andproteins induced byhypoxia havethus attracted extensive attention. As a major regulator of thecellular response to hypoxia, HIF1a has been previously detectedin osteosarcoma specimens (11,12,14,25). In line with previouswork (26), our data also confirm that hypoxia induces WSB1expression in aHIF1a-dependentmanner,which further supportsthe notion that high expression levels of theHIF1a–WSB1 axis areassociated with poor prognosis in osteosarcoma patients. Thereare increasing reports connecting hypoxia-promoted cancermetastasis with poor prognosis. Nevertheless, most of thesestudies focus on angiogenesis, chemokine, and stromal–tumorcell interactions,with little evidenceof adirect effect of hypoxia oncell migration (7,15). More recently, HIF1a has been shown toactivate the transcription of the RhoA and Rho kinase 1 (ROCK1)genes, indicating the activation of cell motility (27). Our findingssuggest that hypoxia-driven WSB1 can promote the proteasomaldegradation of RhoGDI2, leading to cytoskeletal changes thatunderlie the invasive cancer cell phenotype. In this regard, ourresults elucidate a novel branch of HIF1a signaling that directlymediates cell motility.

Despite evidence that WSB1 is expressed in some types ofcancer, its role in cancer progression is still controversial. A geneexpression analysis of 37 neuroblastoma patients revealed thatincreased WSB1 copy number correlates with good prognosis(28). In contrast, reduced cell proliferation and enhanced resis-tance to apoptosis are observed in both pancreatic cancer cells andneuroblastoma cells after WSB1 overexpression (16,29). Morerecently, data from hepatocellular carcinoma cells further suggestthat WSB1 plays a critical role in hypoxia-induced chemoresis-tance (26). Our study shows that WSB1 controls RhoGDI2 toenhance the activity of Rho proteins and promotes osteosarcomacellmigration. In addition toosteosarcoma cells, we alsoobservedthe enhancement of WSB1-mediatedmigration in non–small celllung cancer cell line A549 and cervical cancer cell line HeLa (datanot shown), implying that the function ofWSB1 inpromoting cellmigration may not be cell line specific. To our knowledge, ourstudy is the first to demonstrate that WSB1 promotes the motilityand metastatic potential of cancer cells.

Here, we show that WSB1 directly binds to RhoGDI2 andpromotes the degradation of RhoGDI2. However, their bindingdomain is unknown. Previous studies reported that RhoGDI2 iscomposed of two domains: a flexible N-terminal domain ofapproximately 70 residues and a folded 132-residue C-terminaldomain. The N-terminal domain has two incipient helical

(Continued.) D and E, quantification of the F-actin fluorescence intensity and measurement of ruffles-positive cells infected with the indicated lentivirus.D, representative image of each group. E, fluorescence intensities of F-actin stained with phalloidin were normalized to control cells. Percentages of ruffle-positivecells in different groups were calculated. Bars, mean � SD (n ¼ 3). F and G, tail-vein injection of KHOS/NP cells infected with the indicated lentivirus wasperformed in nude mice (n ¼ 4), and the formation of metastatic nodes was determined on day 14. After the mice were sacrificed, the lung tissues wereexamined under a fluorescence stereo microscope. F, two representative images. G, the number of metastatic nodes per lung was determined. Bars, mean � SD(n ¼ 4). � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






structures (residues 36-57 and 20-25), which adopts a helix-turn-helix structure in the complex and plays an essential role for thebinding (30). Therefore, further studies are needed to confirmwhether RhoGDI20s N-terminal domain is also critical for WSB1binding.

Metastasis is a complex process that leads to the disseminationof cancer cells from the primary tumor to distant organs. A crucialstep is cytoskeletal reprogramming, which transforms rigid,immobile epithelial cells to motile, invasive cancer cells(31,32). The small Rho GTPase family members Rho, Rac, andCdc42 play critical roles in cell migration by regulating thedynamic assembly of actin filaments (33). RhoGDIs inhibit RhoGTPases by direct interaction and sequester Rho proteins in thecytoplasm to restrain them from activation at the membrane(23, 34). In our study, we found that the degradation of RhoGDI2in WSB1-overexpressing cells led to Rac1 activation, and subse-quently increased both the expression of polymerized actin andthe formation of membrane ruffles, consistent with previousreports that the preferential binding of RhoGDI2 to Rac1 is relatedto a distinctive regulation of Rho proteins by RhoGDI2 (35, 36).

To date, three members (RhoGDI1, 2, and 3) of the RhoGDIfamily have been identified (23). Of these proteins, RhoGDI1 iswell characterized as a ubiquitously expressed member of thisfamily, whereas the expression of RhoGDI2 and RhoGDI3 istissue specific (23,34). Although RhoGDI2 is highly expressedparticularly in lymphocytes, the widespread tissue distribution ofits mRNA has been recently reported (37). The loss of RhoGDI2 isassociated with multiple types of metastatic cancers (22,38-40),which is also consistent with our finding that the WSB1-triggereddepletion of RhoGDI2 promotes the metastatic potential ofosteosarcoma cells. Of interest, our data show that WSB1selectively inhibits RhoGDI2 but not RhoGDI1. Although itremains to be determined how WSB1 carries out this distinctiveregulation, a similar phenomenon has also been observed inrictor-triggered RhoGDI2 expression, as revealed by theincrease in RhoGDI2 rather than RhoGDI1 in rictor-null MEFs(41,42). Because only the loss of RhoGDI2, but not RhoGDI1,has been reported to be associated with metastasis, and becausethe functional distinction between RhoGDI1 and RhoGDI2remains elusive, these clues further suggest the existence ofhighly organized intracellular network that coordinates indi-vidual signaling to mediate metastasis.

Colonization by intravenously injected tumor cells is widelyused as a model for detecting metastasis. This model primarilyleads to the formation of metastatic nodules in the lungs. Giventhat themainmetastasis site of osteosarcoma is lung, intravenousinjection model is a reasonable assay to explore the role of WSB1in osteosarcoma metastasis. In our case, WSB1 overexpressionmarkedly increased the pulmonary metastatic nodules, whereasRhoGDI2 blocked WSB1-enhanced metastatic ability. However,

the intravenous injection model usually depicts only the latephases of the invasion–metastasis cascade, such as cancer celldissemination and organ colonization, and fails to represent theearlier stages of the metastatic process, such as local invasion andintravasation. Therefore, we also examined spontaneous metas-tasis using an orthotopic transplantationmodel, which involves amore comprehensive process. Our results further confirmed thatWSB1-overexpressing osteosarcoma cells lead to more lung met-astatic nodes than control cells.

In summary, our current study shows that hypoxia inducesWSB1 expression, which leads to Rho pathway activation viaRhoGDI2-depletion and activation of the GTP-bound form ofRho proteins and actin polymerization, and consequently pro-motes osteosarcoma cell motility, migration, and metastasis.These results not only reveal a novel physiologic function forWSB1, but also delineate, for the first time, a critical molecularmechanism wherein hypoxia induces a WSB1–RhoGDI2 signal-ing axis to trigger cancer cell motility. The discovery of WSB1 as anovel hypoxia-driven osteosarcoma metastasis-supporting pro-tein provides new opportunities to prevent and treat osteosarco-ma with metastases.

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

Authors' ContributionsConception and design: J. Cao, Y. Wang, M. Ying, Q. He, B. YangDevelopment of methodology: Y. Wang, R. Dong, G. LinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Cao, Y. Wang, R. Dong, G. Lin, N. Zhang, J. Wang,N. Lin, L. DingAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Cao, Y. Wang, R. Dong, G. Lin, N. Zhang, J. Wang,L. Ding, M. Ying, Q. HeWriting, review, and/or revision of the manuscript: J. Cao, N. Zhang, Y. Gu,M. Ying, B. YangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): N. Lin, Y. Gu, Q. HeStudy supervision: N. Lin, Q. He, B. Yang

Grant SupportThis work was supported by grants from the National Natural Science

Foundation of China (No. 81373440 to L. Ding; No. 81273535 to B. Yang),the China Postdoctoral Science Foundation (No. 2014M550330 to J. Cao), andthe Fundamental Research Funds for the Central Universities (J. Cao,Q.He, andNo. 2014XZZX004 to B. Yang).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 12, 2015; revised July 27, 2015; accepted August 11, 2015;published OnlineFirst September 30, 2015.

References1. Ritter J, Bielack SS. Osteosarcoma. AnnOncol 2010;21(Suppl 7):vii320–5.2. Bakhshi S, Radhakrishnan V. Prognostic markers in osteosarcoma. Expert

Rev Anticancer Ther 2010;10:271–87.3. Dai X, Ma W, He X, Jha RK. Review of therapeutic strategies for osteosar-

coma, chondrosarcoma, and Ewing's sarcoma. Med Sci Monit 2011;17:RA177–90.

4. Kager L, Zoubek A, Potschger U, Kastner U, Flege S, Kempf-Bielack B, et al.Primary metastatic osteosarcoma: presentation and outcome of patients

treated on neoadjuvant Cooperative Osteosarcoma Study Group proto-cols. J Clin Oncol 2003;21:2011–8.

5. Miller BJ, Cram P, Lynch CF, Buckwalter JA. Risk factors for metastaticdisease at presentation with osteosarcoma: an analysis of the SEER data-base. J Bone Joint Surg Am 2013;95:e89.

6. Longhi A, Errani C, De Paolis M, Mercuri M, Bacci G. Primary boneosteosarcoma in the pediatric age: state of the art. Cancer Treat Rev2006;32:423–36.

Cao et al.





7. Semenza GL. Oxygen sensing, homeostasis, and disease. N Engl J Med2011;365:537–47.

8. Semenza GL. Oxygen sensing, hypoxia-inducible factors, and diseasepathophysiology. Annu Rev Pathol 2014;9:47–71.

9. Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer2011;11:393–410.

10. Keith B, Johnson RS, SimonMC. HIF1alpha and HIF2alpha: sibling rivalryin hypoxic tumour growth and progression. Nat Rev Cancer 2012;12:9–22.

11. Yang QC, Zeng BF, Dong Y, Shi ZM, Jiang ZM, Huang J. Overexpression ofhypoxia-inducible factor-1alpha in human osteosarcoma: correlationwithclinicopathological parameters and survival outcome. Jpn J Clin Oncol2007;37:127–34.

12. Chen Y, Yang Y, Yuan Z, Wang C, Shi Y. Predicting chemosensitivity inosteosarcoma prior to chemotherapy: An investigational study of biomar-kers with immunohistochemistry. Oncol Lett 2012;3:1011–16.

13. El Naggar A, Clarkson P, Zhang F, Mathers J, Tognon C, Sorensen PH.Expression and stability of hypoxia inducible factor 1alpha in osteosar-coma. Pediatr Blood Cancer 2012;59:1215–22.

14. Guo M, Cai C, Zhao G, Qiu X, Zhao H, Ma Q, et al. Hypoxia promotesmigration and induces CXCR4 expression via HIF-1alpha activation inhuman osteosarcoma. PLoS One 2014;9:e90518.

15. Majmundar AJ, Wong WJ, Simon MC. Hypoxia-inducible factors and theresponse to hypoxic stress. Mol Cell 2010;40:294–309.

16. Archange C, Nowak J, Garcia S, Moutardier V, Calvo EL, Dagorn JC, et al.The WSB1 gene is involved in pancreatic cancer progression. PLoS ONE2008;3:e2475.

17. Dentice M, Bandyopadhyay A, Gereben B, Callebaut I, Christoffolete MA,Kim BW, et al. The Hedgehog-inducible ubiquitin ligase subunit WSB-1modulates thyroid hormone activation and PTHrP secretion in the devel-oping growth plate. Nat Cell Biol 2005;7:698–705.

18. Choi DW, Seo YM, Kim EA, Sung KS, Ahn JW, Park SJ, et al. Ubiquitinationand degradation of homeodomain-interacting protein kinase 2 by WD40repeat/SOCS box protein WSB-1. J Biol Chem 2008;283:4682–9.

19. Zhang L, Zhou Q, Zhang N, Li W, Ying M, Ding W, et al. E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma viapromoting ubiquitination-mediated degradation of RARalpha. Cell Cycle2014;13:1277–87.

20. Cao J, Ying M, Xie N, Lin G, Dong R, Zhang J, et al. The oxidation states ofDJ-1 dictate the cell fate in response to oxidative stress triggered by 4-hpr:autophagy or apoptosis? Antioxidants & redox signaling 2014;21:1443–59.

21. Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, Hilton DJ.The SOCS box: a tale of destruction and degradation. Trends Biochem Sci2002;27:235–41.

22. Said N, Theodorescu D. Pathways of metastasis suppression in bladdercancer. Cancer Metastasis Rev 2009;28:327–33.

23. Dovas A, Couchman JR. RhoGDI: multiple functions in the regulation ofRho family GTPase activities. Biochem J 2005;390(Pt 1):1–9.

24. Walkley CR, Qudsi R, Sankaran VG, Perry JA, Gostissa M, Roth SI, et al.Conditionalmouse osteosarcoma, dependent on p53 loss and potentiatedby loss of Rb, mimics the human disease. Genes Dev 2008;22:1662–76.

25. Z CZ, Luo C, Yang Z, Wang L. Heparanase participates in the growth andinvasion of human U-2OS osteosarcoma cells and its close relationship

with hypoxia-inducible factor-1alpha in osteosarcoma. Neoplasma 2010;57:562–71.

26. Tong Y, Li QG, Xing TY, Zhang M, Zhang JJ, Xia Q. HIF1 regulates WSB-1expression topromotehypoxia-induced chemoresistance inhepatocellularcarcinoma cells. FEBS Lett 2013;587:2530–5.

27. Gilkes DM, Xiang L, Lee SJ, Chaturvedi P, Hubbi ME, Wirtz D, et al.Hypoxia-inducible factors mediate coordinated RhoA-ROCK1 expressionand signaling in breast cancer cells. Proc Natl Acad Sci U S A 2014;111:E384–93.

28. Chen QR, Bilke S, Wei JS, Greer BT, Steinberg SM, Westermann F, et al.Increased WSB1 copy number correlates with its over-expression whichassociates with increased survival in neuroblastoma. Genes ChromosomesCancer 2006;45:856–62.

29. Shichrur K, Feinberg-Gorenshtein G, Luria D, Ash S, Yaniv I, Avigad S.Potential role of WSB1 isoforms in growth and survival of neuroblastomacells. Pediatr Res 2014;75:482–6.

30. Golovanov AP, Chuang TH, DerMardirossian C, Barsukov I, Hawkins D,Badii R, et al. Structure-activity relationships in flexible protein domains:regulation of rho GTPases by RhoGDI and D4 GDI. J Mol Biol 2001;305:121–35.

31. Plotnikov SV,WatermanCM.Guiding cellmigrationby tugging. CurrOpinCell Biol 2013;25:619–26.

32. Ogden A, Rida PC, Aneja R. Heading off with the herd: how cancer cellsmight maneuver supernumerary centrosomes for directional migration.Cancer Metastasis Rev 2013;32:269–87.

33. Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev CellDev Biol 2005;21:247–69.

34. Boulter E, Garcia-Mata R. RhoGDI: A rheostat for the Rho switch. SmallGTPases 2010;1:65–68.

35. Scheffzek K, Stephan I, Jensen ON, Illenberger D, Gierschik P. The Rac-RhoGDI complex and the structural basis for the regulation of Rhoproteinsby RhoGDI. Nat Struct Biol 2000;7:122–6.

36. Zhang Y, Rivera Rosado LA, Moon SY, Zhang B. Silencing of D4-GDIinhibits growth and invasive behavior in MDA-MB-231 cells by activationof Rac-dependent p38 and JNK signaling. J Biol Chem 2009;284:12956–65.

37. Theodorescu D, Sapinoso LM, Conaway MR, Oxford G, Hampton GM,Frierson HF Jr. Reduced expression of metastasis suppressor RhoGDI2 isassociated with decreased survival for patients with bladder cancer. ClinCancer Res 2004;10:3800–6.

38. Gildea JJ, Seraj MJ, Oxford G, Harding MA, Hampton GM, Moskaluk CA,et al. RhoGDI2 is an invasion and metastasis suppressor gene in humancancer. Cancer Res 2002;62:6418–23.

39. Stevens EV, Banet N, Onesto C, Plachco A, Alan JK, Nikolaishvili-FeinbergN, et al. RhoGDI2 antagonizes ovarian carcinoma growth, invasion andmetastasis. Small GTPases 2011;2:202–10.

40. HardingMA, TheodorescuD. RhoGDI2: a newmetastasis suppressor gene:discovery and clinical translation. Urol Oncol 2007;25:401–6.

41. Agarwal NK, Chen CH, Cho H, Boulbes DR, Spooner E, Sarbassov DD.Rictor regulates cell migration by suppressing RhoGDI2. Oncogene 2013;32:2521–6.

42. Agarwal NK, KazykenD, Sarbassov dosD. Rictor encounters RhoGDI2: thesecond pilot is taking a lead. Small GTPases 2013;4:102–5.






CANCER RESEARCH | CORRECTION

Correction: Hypoxia-Induced WSB1Promotes the Metastatic Potential ofOsteosarcoma CellsJi Cao, Yijie Wang, Rong Dong, Guanyu Lin, Ning Zhang, Jing Wang,Nengming Lin, Yongchuan Gu, Ling Ding, Meidan Ying, Qiaojun He, andBo Yang

In the original version of this article (1), thefirst and second lanes of the BNIP3Western blotanalysis image in Fig. 1E were inadvertently duplicated in the BNIP3Western blot analysisimage ofU2OS cells in Fig. 1C. The authors have provided the correct image for Fig. 1C, andthus the error has been corrected in the latest online HTML and PDF versions of the article.The authors regret this error.

Reference1. Cao J, Wang Y, Dong R, Lin G, Zhang N, Wang J, et al. Hypoxia-induced WSB1 promotes the metastatic

potential of osteosarcoma cells. Cancer Res 2015;75:4839–51.

Published online June 1, 2020.Cancer Res 2020;80:2421doi: 10.1158/0008-5472.CAN-20-1147�2020 American Association for Cancer Research.

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2015;75:4839-4851. Published OnlineFirst September 30, 2015.Cancer Res Ji Cao, Yijie Wang, Rong Dong, et al. Osteosarcoma CellsHypoxia-Induced WSB1 Promotes the Metastatic Potential of

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