The Mechanism Study Of WSB1 Protein Involved In Hypoxia-Driven Osteosarcoma Metastasis

ObjectiveOsteosarcoma is the most common type of bone cancer with high metastasis rate and poor outcomes. Despite remarkable advances in combined used of chemotherapy and surgical ablation of the primay tumor,5-year survival rate has been improved over the past years, however the overall cure rate has not been improved. Accordingly, Hypoxia-induced tumor metastasis is the leading cause of fatal outcomes in osteosarcoma. Studying the molecular mechanisms underlying osteosarcoma invasion and metastasis, and then targeting tumor metastasis may thus represent a promising strategy to intervene into human osteosarcoma.MethodsPart 1 Hypoxia-induced WSB1 is highly associated with progression and metastasis of human osteosarcomaHuman osteosarcoma cell lines U2OS, KHOS/NP, MG63 and primary osteosarcoma cells were used in this study. (1) Immunohistochemistry staining was used to analyze protein expression in tissue sections. (2) Immunofluorescence was used to detect the colocalization of HIF-la and WSB1. (3) Western blotting assay was used to detect the expression levels of proteins. (4) Real-time PCR assay was used to analyze mRNA levels of WSB 1 in hypoxia condition. (5) Dual Luciferase reporter assay combined with chromatin immunoprecipitation were employed to measure the transcriptional activity of WSB1 and the binding site between HIF-la and WSB1. (6) Transwell assay was used to determine primary osteosarcoma cells migration under hypoxia. (7) Molecular cloning techniques were applied to construct WSB1 and produce lentivirus for the overexpression of the correlated protein in cells. (8) Transwell assay and wound healing assay were used to determine cells migration. (9) Tail-vein xenografts in BALB/C mice were performed to study the metastatic potential in vivo.Part 2 The mechanism study of WSB1 driving osteosarcoma cell metastatic poteintial(1) Molecular cloning techniques were applied to construct SOCS box deletion or mutation plasmid, and to produce lentivirus for the overexpression of the correlated protein in cells. (2) Transwell assay and wound healing assay were used to determine cells migration. (3) SILAC assay was applied to find out the target proteins of WSB1. (4) Immunohistochemistry staining was used to analyze WSB1 and RhoGDI2 expression in tissue sections. (5) Immunoprecipitation was used for the detection of the binding of WSB1 and RhoGDI2. (6) Immunofluorescence was used to detect the ability of F-Actin polymerization. (8) Time-lapse image was employed to determine the cell mobility. (9) H&E staining was used to determine the metastatic nodes of lung.ResultsPart 1 Hypoxia-induced WSB1 is highly associated with progression and metastasis of human osteosarcoma(1) The expression level of WSB1 is significant correlated with HIF-laWe first explored WSB1 expression in 40 primary osteosarcoma tissues by IHC staining. High expression levels of both the hypoxia marker protein HIF-la and the WSB1 protein were detected in most tumor specimens. Three osteosarcoma cell lines(U2OS, KHOS/NP, MG63) and two primary osteosarcoma blasts(MDOS15, MDOS20) were cultured under hypoxic conditions (1% O2) for 24 hours. The protein expression of WSB1 was significantly induced by hypoxia in all five osteosarcoma cells. Because WSB1 expression is significantly increased at the mRNA level under hypoxia, it is reasonable to question whether HIF-la mediates hypoxia-induced WSB1 transcription. By targeting HIF-1? using two specific siRNAs, we found that HIF-1? depletion significantly downregulated its target, BNIP3, and also significantly blocked the hypoxia-induced WSB1 expression. In addition, both the mRNA and protein levels of WSB1 were upregulated, along with the expression of HIF-1? and BNIP3, in cells that overexpressed HIF-la.(2) HIF-la transactivates WSB1 under hypoxia conditionsMoreover, based on WSB 1-promoter luciferase reporter assays and site-directed mutagenesis, we confirmed that HIF-la was able to transactivate the WSB1 promoter via direct binding to its-339 bp region. ChIP assays also validated that HIF-1? bound to the-339 bp genomic region of WSB 1 under hypoxia. These results not only suggest that WSB1 is a direct target of HIF-1?, but also imply that WSB1 might be involved in hypoxia-triggered tumorigenesis.(3) The expression level of WSB 1 is highly associated with progression and metastasis of human osteosarcomaGiven that hypoxia has been reported to play a crucial role in promoting tumor progression and metastasis, we further hypothesized that hypoxia-induced WSB1 expression might be pathologically relevant to the progression of osteosarcoma. Importantly, WSB1 protein levels were not only higher in tumors with metastasis than without it, but were also prognostic for metastasis-free survival. Further more, hypoxia could promote the migration of primary osteosarcoma blasts, where WSB1 expression levels were significantly upregulated under hypoxic conditions. Thus, hypoxia-induced WSB1 is clearly associated with the metastatic potential of human osteosarcoma.(4) WSB1 drives osteosarcoma cell metastatic potential in vitroLentiviral transduction enabled the stable overexpression of WSB1 compared with vector-transduced cells, at levels comparable with those observed under hypoxia. The migration assays showed that, compared with the vector-transduced cells, WSBl-overexpressing KHOS/NP and U2OS cells traversed more efficiently into the lower chamber of the Transwell under normoxia. Consistent with this finding, we observed that WSB1 overexpression enhanced the wound-closure capability of KHOS/NP cells. We next verified the effects of WSB1 depletion on the migration and metastasis of KHOS/NP cells using shRNA. In contrast with overexpression, WSB1 depletion markedly inhibited the hypoxia-driven migration of KHOS/NP cells.(5) WSB1 drives osteosarcoma cell metastatic potential in vivoTo further quantify metastatic potential in vivo, we performed tail-vein xenografts in B ALB/c (nu/nu) mice and examined the rates of lung colonization by micro-PET scan. Increased signal intensities in lungs were observed on day 14 in mice transplanted with WSB1-overexpressing KHOS/NP cells compared with control mice, indicating the formation of more metastatic nodes in mice injected with WSB1-overexpressing cells. Histologic examination confirmed that the number of micrometastatic lesions was markedly increased in the lungs of mice transplanted with WSBl-overexpressing cells. Moreover, we further examined the metastasis phenotype of those two cell lines using an orthotopic transplantation model. Consistent with our intravenous injection model, WSB1-overexpressing cells induced more lung metastasis nodes than control cells. In agreement with the in vitro results, WSB1 depletion significantly decreased the metastatic nodules in vivo.Part 2 The mechanism study of WSB1 driving osteosarcoma cell metastatic poteintial(1) E3 ubiquitin ligase activity is required for WSB1-driven cell migrationWe constructed a mutant WSB1 that lacks the SOCS box to determine whether E3 ubiquitin ligase activity is required for WSB1-promoted cell migration. SOCS box deletion completely lost its migration-promoting activity, as did a WSB1 point-mutant lacking E3 ligase activity.(2) SILAC quantitative proteomic profiling identifiesTo further understand the downstream pathways induced by WSB1 in the metastatic process of osteosarcoma, a large-scale proteomic analysis was subsequently performed. RhoGDI2, a critical and potent inhibitor of Rho proteins, was the top candidate from the SILAC analysis. Forced WSB1 overexpression significantly inhibited RhoGDI2 protein expression.(3) WSB1 promotes the proteasome-dependent degradation of RhoGDI2We are encouraged to further address whether WSB1 mediates the degradation of RhoGDI2. We exposed WSB1 overexpressed cells and its control cells (pCCL) to CHX at different time points and estimated the expression levels of RhoGDI2. The degradation rates of RhoGDI2 were remarkably increased in WSB1-overexpressed cells. Furthermore, MG132 (a specific proteasome inhibitor) treatment totally reverses the WSB1 overexpression-triggered decrease of RhoGDI2 protein levels. Collectively, our data further suggest that hypoxia-drvien WSB1 promotes proteasome-dependent degradation of RhoGDI2. We next examined whether WSB1 regulates RhoGDI2 pro-tein degradation through a direct interaction. Immunofluorescence results demonstrated that exogenous WSB1 colocalizes with exogenous RhoGDI2 in 293T cells transfected with FLAG-WSB1 and RhoGDI2-HA plasmids. Furthermore, this interaction was further validated by immunoprecipitation. To confirm the role of WSB 1 as an E3 ligase for RhoGDI2 polyubiquitination, we performed ubiquitination assays. Polyubiquitinated RhoGDI2 only accumulated in cells that both overexpressed WSB1 and were treated with MG132. Collectively, our data suggest that hypoxia-driven WSB1 promotes the proteasome-dependent degradation of RhoGDI2.(4) WSB1 activates Rho signaling pathwayMoreover, WSB1 overexpression significantly enhanced F-actin expression, and the membrane ruffles formation. Dynamic monitoring of the random motility of KHOS/NP cells revealed an increased displacement of WSB1-overexpressing cells from their origin sites compared with control cells. These data clearly demonstrate that WSB1 activates Rho signaling pathway and leads to enhanced cell motility and migration ability.(5) RhoGDI2 overexpression could reverse WSB1-driven metastasis of osteosarcoma cellsTo determine whether overexpression of RhoGDI2 could reverse the WSB1-driven metastasis of osteosarcoma cells, we first established a stable ectopic RhoGDI2 expressing cell line using lentivirus system on the basis of WSB1 overexpression. Notably, RhoGDI2 overexpression almost completely abrogated WSB1-promoted cell migration. In consistent with our in vitro data, WSB1 overexpression markedly increased the pulmonary metastatic nodules (GFP-positive) in lung.ConclusionHypoxia induces WSB1 expression, which leads to Rho pathway activation via RhoGDI2-depletion and activation of the GTP-bound form of Rho proteins and actin polymerization, and consequently promotes osteosarcoma cell motility, migration, and metastasis. These results delineate a novel HIF-1?-WSB1-RhoGDI2 signaling axis in hypoxia triggered cell mobility, enrich the system info of hypoxia-driven metastasis. The discovery of WSB1 as a novel hypoxia-driven osteosarcoma metastasis-supporting protein provides new oppprtunities of targeting E3 ubiquitin ligase to prevent and treat cancer metastasis.