Hypoxia-Induced WSB1 Promotes the Metastatic Potential Of

Published OnlineFirst September 30, 2015; DOI: 10.1158/0008-5472.CAN-15-0711 Cancer Molecular and Cellular Pathobiology Research

Hypoxia-Induced WSB1 Promotes the Metastatic Potential of Osteosarcoma Cells Ji 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 is titative proteomic and functional analyses revealed that WSB1 associated with the development of metastatic character. How- ubiquitylates the Rho-binding protein RhoGDI2 and promotes its ever, the connections between these phenomena are not fully proteasomal degradation, thereby activating Rac1 to stimulate understood. In this study, we deﬁne an integrative role for the E3 tumor cell motility and invasion. Our ﬁndings show how WSB1 ubiquitin ligase subunit WSB1. In primary osteosarcomas, regulates key steps of the metastatic cascade in hypoxia-driven increased levels of WSB1 correlated with pulmonary metastatic osteosarcoma, and they highlight a candidate therapeutic target potential. RNAi-mediated attenuation of WSB1 or disruption of to potentially improve the survival of patients with metastatic its E3 ligase activity potently suppressed tumor metastasis. Quan- disease. Cancer Res; 75(22); 1–13. 2015 AACR.

Introduction most types of solid tumors, including osteosarcoma (7-10). Hypoxia-inducible factor-1 alpha (HIF1a), a transcription factor Osteosarcomaisthemostcommontypeofbonecancerthat that is stabilized in hypoxia, regulates the expression of multiple usually occurs in juvenile groups (1,2). Despite remarkable target genes to help tumor cells adapt to and survive in hypoxic advances in the combined used of chemotherapy, radiotherapy conditions, contributing to poor chemotherapy responses, as well and surgical ablation of the primary tumor, survival rates have as decreased overall survival and disease-free survival in osteo- yet to improve over those of the 1970s (3). Approximately 40% sarcoma patients (11-13). Recently, exposure of osteosarcoma to 50% of patients develop metastasis, often pulmonary metas- cells to hypoxic conditions was reported to increase the invasive tasis, even after curative resection of the primary tumor, and the potential of the tumor cells in vitro (14), suggesting that hypoxia 5-year survival rate of osteosarcoma patients with metastases is might promote the metastasis of osteosarcoma cells. Consider- even lower than 30% (4-6). Accordingly, the leading cause of able effort has been directed at understanding the potential fatal outcomes in relapsed osteosarcoma is tumor metastasis. mechanism of hypoxia-promoted cancer metastasis (7,15), but Targeting tumor metastasis may thus represent a promising most of these studies were focused on angiogenesis, chemokines, strategy to intervene into human osteosarcoma. However, little and stromal–tumor cell interactions. Previous work also demon- progress has been made in this area as the molecular mechan- strated that HIF1a adapts tumor cells to hypoxic conditions via isms underlying osteosarcoma invasion and metastasis remain context and often cell-type specific mechanisms. It is intriguing to poorly understood. ask whether and how hypoxia could promote the metastasis of Meanwhile, intratumoral hypoxia contributes to increased risk human osteosarcoma. of invasion, metastasis, treatment failure, and patient mortality in WD repeat and SOCS box containing 1 (WSB1) were recently identified as a new member of the suppressor of cytokine signaling (SOCS) box protein family, that is upregulated in 1Institute of Pharmacology and Toxicology, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical multiple types of human cancers (16). WSB1, in complex with Sciences, Zhejiang University, Hangzhou, China. 2Department of elongin B/C-Cullin5-Rbx1, forms an E3 ubiquitin ligase for Orthopedics, The Second Affiliated Hospital of Zhejiang University, thyroid-hormone–activating type 2-iodothyronine deiodinase Zhejiang University, Hangzhou, China. 3Zhejiang Province Key Labo- ratory of Medical Molecular Imaging,The Second Affiliated Hospital of (D2; ref. 17) and homeodomain-interaction protein kinase 2 Zhejiang University, Zhejiang University, Hangzhou, China. 4Institute (HIPK2; ref. 18), thus negatively regulating tumor progression for Individualized Medicine, Hangzhou First People's Hospital, Hang- and chemoresistance. More recent evidence suggested that WSB1 zhou, China. 5Auckland Cancer Society Research Centre, The Univer- sity of Auckland, Auckland, New Zealand. might be a direct target of HIF1 in a human hepatocellular carcinoma model, with a proposed function of WSB1 in hypox- Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). ia-promoted chemoresistance. Given that WSB1 is a substrate- recognizing subunit of E3 ubiquitin ligase, it is intriguing to ask J. Cao and Y. Wang contributed equally to this article. whether and how WSB1 might function in hypoxia-driven metas- Corresponding Author: Bo Yang, College of Pharmaceutical Sciences, Zhejiang tasis in human osteosarcoma, possibly through mechanisms University, Room 113, Hangzhou, China, 310058. Phone: 86-571-88208400; Fax: involving substrates other than D2 and HPIK2. 86-571-88208400; E-mail: [email protected]. In this study, we first examined the potential role of WSB1 in doi: 10.1158/0008-5472.CAN-15-0711 regulating osteosarcoma cell metastasis in vitro and in vivo, as well 2015 American Association for Cancer Research. as its association with hypoxia. Quantitative proteomic and

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functional analyses revealed that Rho guanosine diphosphate SILAC assay dissociation inhibitor 2 (RhoGDI2) as a novel downstream Cells were cultured in DMEM and supplemented with either protein involved in WSB1-mediated cell migration. [U-12C6]-L-lysine (light) or [U-13C6]-L-lysine (heavy) for at least eight generations. The heavy labeling efficiency was measured Materials and Methods by mass spectrometer analysis. Then, cells were continuously maintained in SILAC medium until they reached the desired Cell lines and plasmids confluence and were harvested by trypsinization, and combined The U2OS and MG63 cell lines were purchased from the Cell in equal amounts. The enriched fractions were analyzed by mass Bank of Type Culture Collection of the Chinese Academy of spectrometry. Sciences (Shanghai, China). The KHOS/NP cell line was kindly provided by Dr. Lingtao Wu (University of Southern California, fl CA). All the cell lines have been tested and authenticated IHC and immuno uorescence assay fl utilizing short tandem repeat (STR) profiling every 6 months. Tumor processing and immuno uorescence assays were per- All primary osteosarcoma blasts were from fresh tissue sections formed as described previously (20), with details provided in the from biopsies of osteosarcoma patients as described previously Supplementary information. (19). All cells were cultured in DMEM or RPMI-1640 medium supplemented with 10% FBS in a humidified atmosphere of 5% F-actin staining assay and confocal microscopy fi CO2 at 37 C. Cells were grown on 35-mm glass-bottom dishes and xed The full-length coding sequence for WSB1 and RhoGDI2 was with 4% paraformaldehyde for 15 minutes and then permea- amplified from the U2OS cDNA library and subsequently sub- bilized with 0.1% Triton X-100 in PBS for 10 minutes. The cells cloned into the PCMV6 plasmid (Origene). The WSB1-SOCS were then blocked with 1% BSA for 20 minutes. Cells were deletion was produced based on full-length WSB1 using a pair incubated with Texas Red-X phalloidin (Life Technologies) of primers (forward: 50-GGAATTCATGGCCAGCTTTCCC-30; diluted in 5 mL/200mL in PBS for 20 minutes and then stained reverse: 50-GGAATTCTTAAACCTTATCGTCGTCATCCTT-30). The with DAPI (100 mg/mL) for 3 minutes. The glasses were indicated WSB1-SOCS mutation was produced by the Genescript mounted with anti-fade reagent. The cells were observed under Company. a confocal microscope.

Clinical human tissue specimen Cell mobility assay The clinical samples of osteosarcoma patients were obtained Cells were seeded into 35-mm dishes coated with 0.5% gelatin. from the Second Affiliated Hospital of Zhejiang University School After 24 hours, dishes were placed onto an inverted microscope and imaged every 10 minutes up to 210 minutes. Temperature of Medicine (Hangzhou, China) and Hangzhou First People's Hospital (Hangzhou, China). Written informed consents from was maintained at 37 C. patients and approval from the Institutional Research Ethics Committee of the hospital were obtained before the use of these Cell migration assay clinical materials for research purposes. The cell migration assay was performed in a 24-well Transwell plate with 8 mm polycarbonate sterile membrane (Corning Incor- porated). Cells were plated in the upper chamber at 2104 cells Lentivirus transduction per insert in 200 mL of serum-free medium. Inserts were placed in The shRNA-expressing lentiviral vector pGFP-V-RS against the wells containing 600 mL of medium supplemented with 10% FBS. WSB1 gene was obtained from Origene. pCCL-WSB1 was con- Twenty-four hours later, cells on the upper surface were detached structed by the Genescript Company according to PCMV6-WSB1. with a cotton swab. Filters were fixed and cells in the lower filter The virus particles were harvested 48 hours after transfection of were stained with 0.1% crystal violet for 15 minutes and counted. 293FT cells. For stable transfection, cells were grown in 6-well The quantified results were calculated by counting three random plates at 60% to 70% confluency, and 1 mL of viral supernatant fields of migrated cells. was added with 1 mL polybrene.

Measurement of in vivo activity Western blotting, real-time PCR, and immunoprecipitation Tumors were established via by intravenous injection of Western blotting, real-time PCR, and immunoprecipitation lentivirus-transfected KHOS/NP cells (1106 cells/animal) were performed as described (20), with details in Supplementary into the tails of 3- to 4-week-old female BALB/c (nu/nu) information. mice (National Rodent Laboratory Animal Resource, Shanghai, China). At 14 days after injection, the mice were sent to the Chromatin immunoprecipitation assay Second Affiliated Hospital of ZhejiangUniversitySchoolof Chromatin immunoprecipitation (ChIP) was performed using Medicine and subjected to PET scanning. For the orthotopic the EZ ChIP kit (EMD Millipore). Briefly, KHOS/NP cells cultured transplantation model, 4-week-old BALB/c nude mice were under hypoxia conditions were collected and cross-linked with anesthetized with a mixture of xylazine and ketamine. A 26- formaldehyde. Chromatin was sonicated on ice to an average gauge needle was used to perforate the femur head, after which length of 200 to 300 bp in an ultrasound bath. The chromatin was a second needle, coupled to a 1-mL syringe loaded with 1105 then incubated and precipitated with anti-IgG or anti-HIF1a cells, was introduced through the puncture hole into the antibodies. The immunoprecipitated DNA fragments were medullary cavity of the femur. After the mice were sacrificed, detected by PCR analysis using primers specific for PM1, PM2, all lungs were dissected or further imaged by fluorescence and PM3. stereomicroscopy 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 of WSB1 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 cellseither cultured under normoxia or hypoxia for 24 hours (left) or transfected with the indicated plasmids for 48 hours (right). Bars, mean SD (n ¼ 3). E, Western blotting of HIF1a, 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/NP cells transfected with Myc-tagged HIF1a under normoxia for 48 hours. G, schematic representation of the human WSB1 promoter sequence spanning 2.0kb upstream 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 a ratio 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 in the 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 migrated cells per field was quantified after normalization to normoxia (n ¼ 3). , P < 0.05; , P < 0.01.

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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 the indicated 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 a fluorescence 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, representative images of lungs. Yellow arrow, a metastatic node. (Continued on the following page.)

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(3 mm) were stained with hematoxylin/eosin. The Animal pathologically relevant to the progression of osteosarcoma. Research Committee at Zhejiang University approved all ani- Importantly, WSB1 protein levels were not only higher in tumors mal studies and animal care was provided in accordance with with metastasis than without it (Supplementary Fig. S1A), but institutional guidelines. were also prognostic for metastasis-free survival (Fig. 1J). Further- more, hypoxia could promote the migration of primary osteosarcoma blasts (Fig. 1K and L), where WSB1 expression levels were Statistical analysis significantly upregulated under hypoxic conditions (Fig. 1C). a The relationship between HIF1 and WSB1 expression was Thus, hypoxia-induced WSB1 is clearly associated with the met- analyzed using the Spearman rank correlation test. Survival astatic potential of human osteosarcoma. curves were plotted via the Kaplan–Meier method. The values for all samples in the different experimental conditions were averaged, and the standard error or SD of the mean was WSB1 drives osteosarcoma cell metastatic potential both in vitro calculated. Differences between means were determined using and in vivo the unpaired Student t tests and were considered significant To further address the effect of WSB1 on osteosarcoma cell at P < 0.05. metastasis, we next examined the effects of manipulating WSB1 levels. Lentiviral transduction enabled the stable overexpression of WSB1 compared with vector-transduced cells, at levels com- Results parable with those observed under hypoxia (Fig. 2A and Supple- Hypoxia-induced WSB1 is highly associated with progression mentary Fig. S1B). The migration assays showed that, compared and metastasis of human osteosarcoma with the vector-transduced cells, WSB1-overexpressing KHOS/NP We first explored WSB1 expression in 40 primary osteosarcoma and U2OS cells traversed more efficiently into the lower chamber tissues by IHC staining. Interestingly, high expression levels of of the Transwell under normoxia (Fig. 2B). Consistent with this both the hypoxia marker protein HIF1a and the WSB1 protein finding, we observed that WSB1 overexpression enhanced the were detected in most tumor specimens (Fig. 1A and B). More- wound-closure capability of KHOS/NP cells (Supplementary Fig. over, a statistically significant correlation was also found between S2). Therefore, WSB1 may modulate the migration capacity of these two proteins (R ¼ 0.825, P < 0.01; Fig. 1B), suggesting a osteosarcoma cells in vitro. connection between high WSB1 levels and hypoxic microenvir- To further quantify metastatic potential in vivo, we performed onments. To better understand the effect of hypoxia on WSB1 tail-vein xenografts in BALB/c (nu/nu) mice and examined the expression, three osteosarcoma cell lines and two primary oste- rates of lung colonization by micro-PET scan. As shown in Fig. 2C osarcoma blasts (19) were cultured under hypoxic conditions and D, increased signal intensities in lungs were observed on day (1% O2) for 24 hours. The protein expression of WSB1, as well as 14 in mice transplanted with WSB1-overexpressing KHOS/NP HIF1a and its downstream target BNIP3, was significantly cells compared with control mice, indicating the formation of induced by hypoxia in all five osteosarcoma cells (Fig. 1C). more metastatic nodes in mice injected with WSB1-overexpres- Because WSB1 expression is significantly increased at the mRNA sing cells. The lentiviral vector pCCL expresses a GFP tag (Sup- level under hypoxia (Fig. 1D), it is reasonable to question whether plementary Fig. S1C), allowing us to visualize metastatic nodes in HIF1a mediates hypoxia-induced WSB1 transcription. By target- lungs via fluorescence imaging. As shown in Fig. 2E, WSB1-over- ing HIF1a using two specific siRNAs, we found that HIF1a- expressing cells formed more metastatic nodules in the lung than depletion significantly downregulated its target, BNIP3, and also control cells. Histologic examination confirmed that the number significantly blocked the hypoxia-induced WSB1 expression (Fig. of micrometastatic lesions was markedly increased in the lungs 1E). In addition, both the mRNA and protein levels of WSB1 were of mice transplanted with WSB1-overexpressing cells (Fig. 2F and upregulated, along with the expression of HIF1a and BNIP3, in Supplementary Fig. S3). Moreover, we further examined the cells that overexpressed HIF1a (Fig. 1D and F). Moreover, based metastasis phenotype of those two cell lines using an orthotopic on WSB1promoter ¼ luciferase reporter assays and site-directed transplantation model. Consistent with our intravenous injection mutagenesis, we confirmed that HIF1a was able to transactivate model, WSB1-overexpressing cells induced more lung metastasis the WSB1 promoter via direct binding to its -339 bp region (Fig. nodes than control cells (Fig. 2G and H). 1G and H). ChIP assays also validated that HIF1a bound to the We next verified the effects of WSB1 depletion on the migration -339 bp genomic region of WSB1 under hypoxia (Fig. 1I). These and metastasis of KHOS/NP cells using shRNA. Two specific results not only suggest that WSB1 is a direct target of HIF1a, but sequences against WSB1 significantly inhibited the hypoxia- also imply that WSB1 might be involved in hypoxia-triggered dependent expression of WSB1 (Fig. 2I). Meanwhile, in contrast tumorigenesis. with overexpression (Fig. 2B), WSB1 depletion markedly inhib- Given that hypoxia has been reported to play a crucial role in ited the hypoxia-driven migration of KHOS/NP cells (Fig. 2J). In promoting tumor progression and metastasis (7,8), we further agreement with the in vitro results, WSB1 depletion significantly hypothesized that hypoxia-induced WSB1 expression might be decreased 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,WesternblottingofWSB1 in KHOS/NP cells infected with the indicated lentivirus under normoxia or hypoxia for 24 hours. Relative WSB1 levels were normalized to b-actin as indicated. J, migration assay of KHOS/NP cells infected with the indicated lentivirus. Representative images of migrated cells are shown. The number of migrated cells per ﬁeld was quantiﬁed after 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 ¼ 4), and the formation of metastatic nodes was determined on day 14. K, representative micro-PET images of mice 14 days after injection. Color scale is indicated. 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.

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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 the indicated lentivirus. C, representative images of migrated cells. D, the number of migrated cells per field was quantified. Bars, mean SD (n ¼ 3). E, schematic representation of Myc-tagged wild-type and SOCS-box-site-mutant (Mut-WSB1) WSB1 constructs. F, Western blotting of Myc-tag in KHOS/NP cells infection with the indicated lentivirus. G and H, migration assay of KHOS/NP cells infected with the indicated lentivirus. G, representative images of migrated cells. H, the number of migrated cells per field was quantified. Bars, mean SD (n ¼ 3). , P < 0.001.

nude mice in micro-PET scans (Fig. 2K and L). Importantly, lacks the SOCS box (Fig. 3B, DSOCS) to determine whether E3 histologic examination of lung tissues revealed that in some cases ubiquitin ligase activity is required for WSB1-promoted cell depleting WSB1 totally blocked the metastatic nodules in vivo (1/4 migration. As illustrated in Fig. 3C and D, infection with wild- in shRNA-#1 group and 2/4 in shRNA-#2 group), whereas mice type WSB1 lentivirus significantly promoted migration com- injected with control cells formed 8 to 18 metastatic nodules pared with the vector control. In contrast, DSOCS completely per lung in all four mice (Fig. 2M and N). Our data indicate that lost its migration-promoting activity, as did a WSB1 point- WSB1 is necessary for the aggressive, highly metastatic phenotype mutant (339-421, AAA) lacking E3 ligase activity (Fig. E–H). of osteosarcoma cells. Taken together, these data further suggest that E3 ubiquitin Collectively, these overexpression and knockdown results gen- ligase activity is required for the WSB1-driven migration of erated from in vitro and in vivo studies, together with the results osteosarcoma cells. from osteosarcoma biopsies and primary cell lines, demonstrate a new phenotype of WSB1 wherein the hypoxia-dependent induction of WSB1 is necessary and sufficient to promote the metastatic SILAC quantitative proteomic profiling identifies the potential of osteosarcoma cells. proteasome-dependent degradation of RhoGDI2 caused by WSB1 overexpression E3 ubiquitin ligase activity is required for WSB1-driven cell To further understand the downstream pathways induced by migration WSB1 in the metastatic process of osteosarcoma, a large-scale Previous reports have revealed that WSB1 is an E3 ubiquitin proteomic analysis was subsequently performed. The pro- ligase (17, 18); therefore, it is interesting to know whether E3 teomes of ectopic WSB1-overexpressing and control vector- ubiquitin ligase activity is critical for WSB1 to promote cell infected KHOS/NP cells were quantified using SILAC followed migration. WSB1 is composed of seven WD40 repeats and a by mass spectrometry analysis (Fig. 4A). A total of 4,163 C-terminal SOCS box (Fig. 3A), and the SOCS box is known to proteins were identified from both cell lysates, and 4,094 be involved in protein degradation via the ECS ubiquitin ligase proteins were further quantified, of which 1,078 proteins were complex (21). We therefore constructed a mutant WSB1 that significantly perturbed by WSB1 overexpression, with 560

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Figure 4. SILAC quantitative proteomic profiling identifies the RhoGDI2 protein as the downstream target of WSB1 signaling. A, schematic representation of the SILAC assay. B, the number of proteins identified and quantified using the SILAC assay. C, bioinformatics analysis of differentially expressed proteins with at least a 3-fold change. D, RhoGDI2 and RhoGDI1 protein expression in the SILAC assay were quantified. E, Western blotting of WSB1, Myc-tag, RhoGDI2, and RhoGDI1 in KHOS/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 of WSB1 in KHOS/NP cells infection with the indicated lentivirus under normoxia or hypoxia for 24 hours. H, IHC staining of WSB1 and RhoGDI2 in osteosarcoma specimens. I, statistical analysis of IHC results in 40 human osteosarcoma specimens. proteins being upregulated and 518 proteins downregulated Interestingly, RhoGDI2, a critical and potent inhibitor of Rho (>3.0-fold, Fig. 4B and Supplementary Table S1). Bioinformat- proteins (22), was the top candidate from the SILAC analysis, with ics analysis further revealed that these altered proteins are the most significant decrease in protein abundance (Fig. 4D). mainly involved in protein binding (50%) and catalytic activity Because our data suggest that E3 ubiquitin ligase activity is (29%) and also participated in both cellular (23%) and met- required for the WSB1-driven migration of osteosarcoma cells, abolic processes (18%; Fig. 4C). The expression of metastasis- we hypothesized that the downregulation of RhoGDI2 may serve associated proteins was further analyzed. Twenty-four proteins as the downstream target of WSB1. To confirm this, the RhoGDI2 involved in cell metastasis were identified, and 7 of these 24 protein expression level in WSB1-overexpressing KHOS/NP cells proteins changed by more than 3.0-fold in response to WSB1 was determined. Forced WSB1 overexpression significantly inhib- overexpression (Supplementary Fig. S4). These quantitative ited RhoGDI2 protein expression, whereas RhoGDI1, another proteomic data provide additional confirmation that WSB1 member of the RhoGDI family, did not change (Fig. 4E), indi- promotes osteosarcoma cell metastasis. cating the selective inhibition of RhoGDI2 by WSB1. Because

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Figure 5. WSB1 is an E3 ligase for RhoGDI2 and promotes RhoGDI2 ubiquitination and degradation. A, Western blotting of WSB1, Myc-tag, RhoGDI2, and RhoGDI1 in KHOS/NP cells infected with the indicated lentivirus. B and C, Western blotting of RhoGDI2 in KHOS/NP cells infected with the indicated lentivirus under 10 mg/mL cycloheximide (CHX) treatment (B) or 10 mmol/L MG132 treatment (C). D, colocalization of exogenous WSB1 and RhoGDI2. 293T cells were transfected with FLAG-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 between endogenous 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 or WSB1, followed by MG132 treatment. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-ubiquitin antibody.

WSB1 is speciﬁcally induced by hypoxia, we examined whether the hypoxia-dependent decrease in RhoGDI2 levels (Fig. 4G). the downregulation of RhoGDI2 could also be achieved under Moreover, a negative correlation between WSB1 and RhoGDI2 hypoxic conditions. As expected, an obvious decrease in RhoGDI2 was also found in 40 primary osteosarcoma tissues by IHC was observed to accompany the induction of WSB1 under hyp- staining (R ¼0.65, P < 0.01; Fig. 4H and I). In addition, oxia (Fig. 4F). In contrast, WSB1 knockdown effectively reversed RhoGDI2 protein levels were inversely correlated with metastasis

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Figure 6. RhoGDI2 acts downstream of WSB1 to mediate cell motility and migration. A, endogenous active GTP-bound form of Rac1 was enriched by a pull-down assay and detected by Western blotting. Total Rac1 was detected using anti-Rac1 antibody. B and C, quantification of the F-actin fluorescence intensity and measurement of ruffles-positive cells infected with the indicated lentivirus. B, representative images of each group. C, fluorescence intensities of F- actin stained with phalloidin were normalized to control cells. Percentages of ruffle-positive cells in different groups were calculated based on the immunofluorescence. Bars, mean SD (n ¼ 3). , P < 0.01. D, time-lapse images of representative KHOS/NP cells infected with the indicated lentivirus.

of osteosarcoma (Supplementary Fig. S5A). These data clearly We next examined whether WSB1 regulates RhoGDI2 pro- support the idea that RhoGDI2 is the downstream target of tein degradation through a direct interaction. Immunofluores- WSB1 signaling. cence results demonstrated that exogenous WSB1 colocalizes Given that our data further demonstrated that RhoGDI2 mRNA with exogenous RhoGDI2 in 293T cells transfected with FLAG- levels were not significantly decreased in WSB1-overexpressing WSB1 and RhoGDI2-HA plasmids (Fig. 5D). Furthermore, this cells and under hypoxia (Supplementary Fig. S5B), as well as the interaction was further validated by immunoprecipitation. fact that DSOCS failed to decrease the amount of RhoGDI2 Ectopically expressed WSB1 and RhoGDI2 were coprecipitated (Fig. 5A), we are encouraged to further address whether WSB1 from 293T cells overexpressing both proteins (Fig. 5E). Con- mediates the degradation of RhoGDI2. To this end, cycloheximide sistently, endogenous WSB1 also interacts with endogenous (CHX) was used to prevent de novo protein synthesis; thus, RhoGDI2 RhoGDI2 as confirmed by immunoprecipitation (Fig. 5F) and levels would primarily reflect the protein degradation process. We immunofluorescence assays (Supplementary Fig. S5D). There- exposed WSB1-overexpressing cells and control cells to CHX and fore, our data clearly demonstrate that WSB1 directly interacts estimated the RhoGDI2 expression. As shown in Fig. 5B, RhoGDI2 with RhoGDI2. To confirmtheroleofWSB1asanE3ligasefor degradation rates were remarkably increased in WSB1-overexpres- RhoGDI2 polyubiquitination, we performed ubiquitination sing cells. Furthermore, MG132 (a specific proteasome inhibitor) assays. Polyubiquitinated RhoGDI2 only accumulated in cells treatment totally reverses the WSB1 overexpression-triggered that both overexpressed WSB1 and were treated with MG132 decrease in RhoGDI2 protein levels (Fig. 5C). In keeping with these (Fig. 5G). Collectively, our data suggest that hypoxia-driven results, MG132 also inhibited RhoGDI2 protein degradation under WSB1 promotes the proteasome-dependent degradation of hypoxic condition (Supplementary Fig. S5C). RhoGDI2.

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

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WSB1 Promotes Osteosarcoma Metastasis

RhoGDI2 acts downstream of WSB1 to mediate cell motility RhoGDI2-overexpression reverses WSB1-driven metastasis, but and migration also further support a pivotal role for the WSB1–RhoGDI2 axis in RhoGDIs modulate the cycling of Rho GTPases between active the metastasis of osteosarcoma cells. GTP-bound and inactive GDP-bound states (23). Our data indicate that ectopic WSB1 expression is associated with a marked Discussion decrease in RhoGDI2; thus, the activation of Rho GTPases should Pulmonary metastasis has been recognized as the main cause of be expected in WSB1-overexpressing cells. By specifically pulling fatal outcomes in osteosarcoma patients, but its molecular mech- down the active GTP-bound form of Rho GTPases, we detected a anism is rarely discussed (1,24). Accumulating evidence suggests substantial level of active Rac1 protein, a member of the Rho that tumor progression requires adaptation to hypoxic microen- GTPases, in the wild-type WSB1-overexpression group but not in vironments, and molecules and proteins induced by hypoxia have the vector or DSOCS groups (Fig. 6A). Our findings indicate that thus attracted extensive attention. As a major regulator of the WSB1-triggereed RhoGDI2 suppression activates Rho GTPases. cellular response to hypoxia, HIF1a has been previously detected Rho GTPases are well known as regulator of actin cytoskeletal in osteosarcoma specimens (11,12,14,25). In line with previous organization and cell motility. We therefore examined the effect of work (26), our data also confirm that hypoxia induces WSB1 WSB1 overexpression on the status of actin filament organization expression in a HIF1a-dependent manner, which further supports and cell motility. As illustrated in Fig. 6B and C, WSB1 over- the notion that high expression levels of the HIF1a–WSB1 axis are expression significantly enhanced the fluorescence intensity of associated with poor prognosis in osteosarcoma patients. There polymerized actin (F-actin), which suggests abundant F-actin are increasing reports connecting hypoxia-promoted cancer expression, compared with the vector or mutant WSB1 groups. metastasis with poor prognosis. Nevertheless, most of these In addition to F-actin expression, the appearance of membrane studies focus on angiogenesis, chemokine, and stromal–tumor ruffles and the formation of lamellipodia were also observed in cell interactions, with little evidence of a direct effect of hypoxia on WSB1-overexpressing cells. Dynamic monitoring of the random cell migration (7,15). More recently, HIF1a has been shown to motility of KHOS/NP cells revealed an increased displacement of activate the transcription of the RhoA and Rho kinase 1 (ROCK1) WSB1-overexpressing cells from their origin sites compared with genes, indicating the activation of cell motility (27). Our findings control cells (Fig. 6D and Supplementary Figs S6 and S7). These suggest that hypoxia-driven WSB1 can promote the proteasomal data clearly demonstrate that WSB1 increases the amount of F- degradation of RhoGDI2, leading to cytoskeletal changes that actin, promotes the formation of lamellipodia, and consequently underlie the invasive cancer cell phenotype. In this regard, our leads to enhanced cell motility and migration ability. results elucidate a novel branch of HIF1a signaling that directly mediates cell motility. RhoGDI2 overexpression reverses the WSB1-driven metastasis Despite evidence that WSB1 is expressed in some types of of osteosarcoma cells cancer, its role in cancer progression is still controversial. A gene To determine whether RhoGDI2 overexpression could reverse expression analysis of 37 neuroblastoma patients revealed that WSB1-driven metastasis of osteosarcoma cells, we first established increased WSB1 copy number correlates with good prognosis a stable cell line ectopically expressing RhoGDI2 (Fig. 7A). Nota- (28). In contrast, reduced cell proliferation and enhanced resis- bly, RhoGDI2 overexpression almost completely abrogated tance to apoptosis are observed in both pancreatic cancer cells and WSB1-promoted cell migration (Fig. 7B and C). Similar responses neuroblastoma cells after WSB1 overexpression (16,29). More were also observed via immunofluorescence staining of F-actin, as recently, data from hepatocellular carcinoma cells further suggest RhoGDI2 overexpression decreased the WSB1-enhanced fluores- that WSB1 plays a critical role in hypoxia-induced chemoresis- cence intensity of F-actin and the formation of membrane ruffles tance (26). Our study shows that WSB1 controls RhoGDI2 to (Fig. 7D and E). Moreover, RhoGDI2 overexpression blocked enhance the activity of Rho proteins and promotes osteosarcoma WSB1-enhanced wound-closure capability of KHOS/NP cells cell migration. In addition to osteosarcoma cells, we also observed (Supplementary Fig. S8). These data suggest that WSB1-enhanced the enhancement of WSB1-mediated migration in non–small cell cell motility and migration can be completely abrogated by lung cancer cell line A549 and cervical cancer cell line HeLa (data RhoGDI2 overexpression in vitro. not shown), implying that the function of WSB1 in promoting cell To test whether this effect could be reproduced in vivo,we migration may not be cell line specific. To our knowledge, our generated an osteosarcoma pulmonary metastasis model using study is the first to demonstrate that WSB1 promotes the motility nude mice intravenously injected with the indicated cells. Con- and metastatic potential of cancer cells. sistent with our in vitro data, WSB1 overexpression markedly Here, we show that WSB1 directly binds to RhoGDI2 and increased the number of pulmonary metastatic nodules (GFP promotes the degradation of RhoGDI2. However, their binding positive). However, the additional ectopic expression of domain is unknown. Previous studies reported that RhoGDI2 is RhoGDI2 impaired the effect of WSB1 overexpression as dem- composed of two domains: a flexible N-terminal domain of onstrated by the decrease in pulmonary metastatic nodules (Fig. approximately 70 residues and a folded 132-residue C-terminal 7F and G). Taken together, these results not only indicate that domain. 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-positive cells 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 was performed 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 were examined 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.

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Cao et al.

structures (residues 36-57 and 20-25), which adopts a helix-turn- the intravenous injection model usually depicts only the late helix structure in the complex and plays an essential role for the phases of the invasion–metastasis cascade, such as cancer cell binding (30). Therefore, further studies are needed to confirm dissemination and organ colonization, and fails to represent the whether RhoGDI20s N-terminal domain is also critical for WSB1 earlier stages of the metastatic process, such as local invasion and binding. intravasation. Therefore, we also examined spontaneous metas- Metastasis is a complex process that leads to the dissemination tasis using an orthotopic transplantation model, which involves a of cancer cells from the primary tumor to distant organs. A crucial more comprehensive process. Our results further confirmed that step is cytoskeletal reprogramming, which transforms rigid, WSB1-overexpressing osteosarcoma cells lead to more lung met- immobile epithelial cells to motile, invasive cancer cells astatic nodes than control cells. (31,32). The small Rho GTPase family members Rho, Rac, and In summary, our current study shows that hypoxia induces Cdc42 play critical roles in cell migration by regulating the WSB1 expression, which leads to Rho pathway activation via dynamic assembly of actin filaments (33). RhoGDIs inhibit Rho RhoGDI2-depletion and activation of the GTP-bound form of GTPases by direct interaction and sequester Rho proteins in the Rho proteins and actin polymerization, and consequently pro- cytoplasm to restrain them from activation at the membrane motes osteosarcoma cell motility, migration, and metastasis. (23, 34). In our study, we found that the degradation of RhoGDI2 These results not only reveal a novel physiologic function for in WSB1-overexpressing cells led to Rac1 activation, and subse- WSB1, but also delineate, for the first time, a critical molecular quently increased both the expression of polymerized actin and mechanism wherein hypoxia induces a WSB1–RhoGDI2 signal- the formation of membrane ruffles, consistent with previous ing axis to trigger cancer cell motility. The discovery of WSB1 as a reports that the preferential binding of RhoGDI2 to Rac1 is related novel hypoxia-driven osteosarcoma metastasis-supporting pro- to a distinctive regulation of Rho proteins by RhoGDI2 (35, 36). tein provides new opportunities to prevent and treat osteosarco- To date, three members (RhoGDI1, 2, and 3) of the RhoGDI ma with metastases. family have been identified (23). Of these proteins, RhoGDI1 is well characterized as a ubiquitously expressed member of this Disclosure of Potential Conflicts of Interest family, whereas the expression of RhoGDI2 and RhoGDI3 is No potential conflicts of interest were disclosed. tissue specific (23,34). Although RhoGDI2 is highly expressed particularly in lymphocytes, the widespread tissue distribution of its mRNA has been recently reported (37). The loss of RhoGDI2 is Authors' Contributions associated with multiple types of metastatic cancers (22,38-40), Conception and design: J. Cao, Y. Wang, M. Ying, Q. He, B. Yang fi Development of methodology: Y. Wang, R. Dong, G. Lin which is also consistent with our nding that the WSB1-triggered Acquisition of data (provided animals, acquired and managed patients, depletion of RhoGDI2 promotes the metastatic potential of provided facilities, etc.): J. Cao, Y. Wang, R. Dong, G. Lin, N. Zhang, J. Wang, osteosarcoma cells. Of interest, our data show that WSB1 N. Lin, L. Ding selectively inhibits RhoGDI2 but not RhoGDI1. Although it Analysis and interpretation of data (e.g., statistical analysis, biostatistics, remainstobedeterminedhowWSB1 carries out this distinctive computational analysis): J. Cao, Y. Wang, R. Dong, G. Lin, N. Zhang, J. Wang, regulation, a similar phenomenon has also been observed in L. Ding, M. Ying, Q. He Writing, review, and/or revision of the manuscript: J. Cao, N. Zhang, Y. Gu, rictor-triggered RhoGDI2 expression, as revealed by the M. Ying, B. Yang increase in RhoGDI2 rather than RhoGDI1 in rictor-null MEFs Administrative, technical, or material support (i.e., reporting or organizing (41,42). Because only the loss of RhoGDI2, but not RhoGDI1, data, constructing databases): N. Lin, Y. Gu, Q. He hasbeenreportedtobeassociatedwithmetastasis,andbecause Study supervision: N. Lin, Q. He, B. Yang the functional distinction between RhoGDI1 and RhoGDI2 remains elusive, these clues further suggest the existence of Grant Support highly organized intracellular network that coordinates indi- This work was supported by grants from the National Natural Science vidual signaling to mediate metastasis. Foundation of China (No. 81373440 to L. Ding; No. 81273535 to B. Yang), Colonization by intravenously injected tumor cells is widely the China Postdoctoral Science Foundation (No. 2014M550330 to J. Cao), and used as a model for detecting metastasis. This model primarily the Fundamental Research Funds for the Central Universities (J. Cao, Q. He, and leads to the formation of metastatic nodules in the lungs. Given No. 2014XZZX004 to B. Yang). The costs of publication of this article were defrayed in part by the payment that the main metastasis site of osteosarcoma is lung, intravenous of page charges. This article must therefore be hereby marked advertisement injection model is a reasonable assay to explore the role of WSB1 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in osteosarcoma metastasis. In our case, WSB1 overexpression markedly increased the pulmonary metastatic nodules, whereas Received March 12, 2015; revised July 27, 2015; accepted August 11, 2015; RhoGDI2 blocked WSB1-enhanced metastatic ability. However, published OnlineFirst September 30, 2015.

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Correction: Hypoxia-Induced WSB1 Promotes the Metastatic Potential of Osteosarcoma Cells Ji Cao, Yijie Wang, Rong Dong, Guanyu Lin, Ning Zhang, Jing Wang, Nengming Lin, Yongchuan Gu, Ling Ding, Meidan Ying, Qiaojun He, and Bo Yang

In the original version of this article (1), the ﬁrst and second lanes of the BNIP3 Western blot analysis image in Fig. 1E were inadvertently duplicated in the BNIP3 Western blot analysis image of U2OS cells in Fig. 1C. The authors have provided the correct image for Fig. 1C, and thus the error has been corrected in the latest online HTML and PDF versions of the article. The authors regret this error.

Reference 1. 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;80:2421 doi: 10.1158/0008-5472.CAN-20-1147 Ó2020 American Association for Cancer Research.

AACRJournals.org | 2421 Published OnlineFirst September 30, 2015; DOI: 10.1158/0008-5472.CAN-15-0711

Hypoxia-Induced WSB1 Promotes the Metastatic Potential of Osteosarcoma Cells

Ji Cao, Yijie Wang, Rong Dong, et al.

Cancer Res Published OnlineFirst September 30, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-15-0711

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