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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. Ó2015 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 Ó2015 American Association for Cancer Research. as its association with hypoxia. Quantitative proteomic and www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst September 30, 2015; DOI: 10.1158/0008-5472.CAN-15-0711 Cao et al. 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 2Â104 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
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