Motility Of Osteosarcoma Cells Research Articles

Identification of TNFRSF21 as an inhibitory factor of osteosarcoma based on a necroptosis-related prognostic gene signature and molecular experiments

BackgroundOsteosarcoma is one of the most common malignant bone tumors with bad prognosis. Necroptosis is a form of programmed cell death. Recent studies showed that targeting necroptosis was a new promising approach for tumor therapy. This study aimed to establish a necroptosis-related gene signature to evaluated prognosis and explore the relationship between necroptosis and osteosarcoma.MethodsData from The Cancer Genome Atlas was used for developing the signature and the derived necroptosis score (NS). Data from Gene Expression Omnibus served as validation. Principal component analysis (PCA), Cox regression, receiver operating characteristic (ROC) curves and Kaplan-Meier survival analysis were used to assess the performance of signature. The association between the NS and osteosarcoma was analyzed via gene set enrichment analysis, gene set variation analysis and Pearson test. Single-cell data was used for further exploration. Among the genes that constituted the signature, the role of TNFRSF21 in osteosarcoma was unclear. Molecular experiments were used to explore TNFRSF21 function.ResultsOur data revealed that lower NS indicated more active necroptosis in osteosarcoma. Patients with lower NS had a better prognosis. PCA and ROC curves demonstrated NS was effective to predict prognosis. NS was negatively associated with immune infiltration levels and tumor microenvironment scores and positively associated with tumor purity and stemness index. Single-cell data showed necroptosis heterogeneity in osteosarcoma. The cell communication pattern of malignant cells with high NS was positively correlated with tumor progression. The expression of TNFRSF21 was down-regulated in osteosarcoma cell lines. Overexpression of TNFRSF21 inhibited proliferation and motility of osteosarcoma cells. Mechanically, TNFRSF21 upregulated the phosphorylation levels of RIPK1, RIPK3 and MLKL to promote necroptosis in osteosarcoma.ConclusionsThe necroptosis prognostic signature and NS established in this study could be used as an independent prognostic factor, TNFRSF21 may be a necroptosis target in osteosarcoma therapy.

METTL3-mediated m6A modification of circRNF220 modulates miR-330-5p/survivin axis to promote osteosarcoma progression

BackgroundCircular RNAs (circRNAs) play a crucial role in regulating various physiological processes. However, the precise regulatory mechanisms of circRNF220s in osteosarcoma (OS) are not well understood.MethodsThe abundances of circRNF220, miR-330-5p, and survivin were determined using qRT-PCR. To assess the m6A accumulation in circRNF220, a methylated RNA immunoprecipitation (Me-RIP) assay was conducted. Cellular multiplication, motility, and invasion were examined using the cell Counting Kit-8 (CCK-8), EdU, colony formation, Transwell, and wound-healing assays. The binding relationships were measured through RNA immunoprecipitation (RIP) and luciferase reporter assays. In vivo functionality was assessed using xenograft models.ResultsCircRNF220 was identified as being overexpressed in both OS cells and tissues. In vitro experiments demonstrated that silencing circRNF220 impeded the proliferation, invasion, and motility of OS cells. Similarly, in vivo studies confirmed that downregulating circRNF220 inhibited the growth of OS. Further mechanistic investigations unveiled that METTL3-modulated circRNF220 regulated the progression of OS by upregulating survivin expression through acting as a sponge for miR-330-5p.ConclusionThe modulation of METTL3-regulated circRNF220 has been found to promote the progression of OS by modulating the miR-330-5p/survivin axis. This novel finding suggests a potentially unique approach to managing OS.

IGFBP-3 stimulates human osteosarcoma cell migration by upregulating VCAM-1 expression

AimsInsulin-like growth factor (IGF) signaling has been documented in several human malignancies and is thought to contribute to cellular differentiation and migration, as well as malignant progression. A major binding molecule of IGF, IGF-binding protein 3 (IGFBP-3), regulates multiple IGF effects. Here, we focused on the effect of IGFBP-3 in the motility of osteosarcoma cells and examined signaling regulation. Materials and methodsUsing a human osteosarcoma tissue array, immunohistochemical staining determined levels of IGFBP-3 expression in osteosarcoma tissue and in normal tissue. The wound healing migration assay, Transwell migration assay, luciferase reporter assay, immunofluorescence staining, Western blot and real-time quantitative PCR were performed to examine whether IGFBP-3 facilitates VCAM-1-dependent migration of osteosarcoma cells. Key findingsIn this study, we found significantly higher IGFBP-3 levels in osteosarcoma tissue compared with normal healthy tissue. IGFBP-3 treatment of two human osteosarcoma cell lines promoted cell migration and upregulated levels of VCAM-1 expression via PI3K/Akt and AP-1 signaling. SignificanceIGFBP-3 appears to be a novel therapeutic target in metastatic osteosarcoma.

CXCL13/CXCR5 Interaction Facilitates VCAM-1-Dependent Migration in Human Osteosarcoma.

Osteosarcoma is the most common primary tumor of the skeletal system and is well-known to have an aggressive clinical outcome and high metastatic potential. The chemokine (C-X-C motif) ligand 13 (CXCL13) plays a vital role in the development of several cancers. However, the effect of CXCL13 in the motility of osteosarcoma cells remains uncertain. Here, we found that CXCL13 increases the migration and invasion potential of three osteosarcoma cell lines. In addition, CXCL13 expression was upregulated in migration-prone MG-63 cells. Vascular cell adhesion molecule 1 (VCAM-1) siRNA and antibody demonstrated that CXCL13 promotes migration via increasing VCAM-1 production. We also show that CXCR5 receptor controls CXCL13-mediated VCAM-1 expression and cell migration. Our study identified that CXCL13/CXCR5 axis facilitate VCAM-1 production and cell migration in human osteosarcoma via the phospholipase C beta (PLCβ), protein kinase C α (PKCα), c-Src, and nuclear factor-κB (NF-κB) signaling pathways. CXCL13 and CXCR5 appear to be a novel therapeutic target in metastatic osteosarcoma.

Prazosin inhibits the growth and mobility of osteosarcoma cells

Background: Osteosarcoma is a primary malignant bone tumor that frequently occurs in adolescents and children, its high aggressiveness and rapid metastasis often resulting in poor prognoses. In previous studies, Prazosin has been shown to possess anti-proliferative properties against prostate cancer and glioblastoma cells. In our study, we investigated Prazosin’s underlying mechanisms and its effects on the biological behaviors of osteosarcoma cells. Methods: Osteosarcoma cell lines MG63 and 143B were treated with different concentrations of Prazosin, and a CCK8 assay assessed its effect on cell viability. Colony formation, Transwell and flow cytometry assays were used to examine its effects on cell proliferation, cell migration, and cell invasion and apoptosis, respectively. The expression of relevant proteins was then examined using western blotting. Results: Our data showed that Prazosin dose-dependently reduced the viability of MG63 and 143B cells and significantly inhibited their clonogenic ability. Moreover, Prazosin attenuated the cell migration and invasion abilities of MG63 and 143B cells when compared with the NC group. It also accelerated cell apoptosis in mitochondrial pathways by regulating Bcl-2/Bax axis and caspase 3. Furthermore, Prazosin treatment inactivated the Akt/mTOR pathway by down-regulating Akt and mTOR phosphorylation (p-Akt, p-mTOR) and the expression of P70 and cyclin D1. Conclusions: Our data highlights the fact that Prazosin inhibits cell growth, inhibits the motility of osteosarcoma cells, and promotes apoptosis, suggesting that Prazosin is a potential anti-cancer agent in osteosarcoma therapy.

Prazosin inhibits the growth and mobility of osteosarcoma cells.

BackgroundOsteosarcoma is a primary malignant bone tumor that frequently occurs in adolescents and children, its high aggressiveness and rapid metastasis often resulting in poor prognoses. In previous studies, Prazosin has been shown to possess anti-proliferative properties against prostate cancer and glioblastoma cells. In our study, we investigated Prazosin’s underlying mechanisms and its effects on the biological behaviors of osteosarcoma cells.MethodsOsteosarcoma cell lines MG63 and 143B were treated with different concentrations of Prazosin, and a CCK8 assay assessed its effect on cell viability. Colony formation, Transwell and flow cytometry assays were used to examine its effects on cell proliferation, cell migration, and cell invasion and apoptosis, respectively. The expression of relevant proteins was then examined using western blotting.ResultsOur data showed that Prazosin dose-dependently reduced the viability of MG63 and 143B cells and significantly inhibited their clonogenic ability. Moreover, Prazosin attenuated the cell migration and invasion abilities of MG63 and 143B cells when compared with the NC group. It also accelerated cell apoptosis in mitochondrial pathways by regulating Bcl-2/Bax axis and caspase 3. Furthermore, Prazosin treatment inactivated the Akt/mTOR pathway by down-regulating Akt and mTOR phosphorylation (p-Akt, p-mTOR) and the expression of P70 and cyclin D1.ConclusionsOur data highlights the fact that Prazosin inhibits cell growth, inhibits the motility of osteosarcoma cells, and promotes apoptosis, suggesting that Prazosin is a potential anti-cancer agent in osteosarcoma therapy.

Downregulation of Herg1 suppresses osteosarcoma proliferation and invasion by targeting Hippo signaling pathway

Objective: To detect the effect and regulatory mechanism of human ether à go-go related gene 1 (Herg 1) knockdown on the proliferation and invasion of osteosarcoma (OS). Methods: We constructed a recombinant adenovirus vector (Ad5-Herg1-shRNA) expressing short hair RNA (shRNA) against Herg1 and tested the knockdown efficiency. Then, the effects of Herg 1 knockdown on the proliferation, growth and invasion of osteosarcoma were measured by using cell counting kit-8 (CCK-8), wound healing assay, Transwell assay and xenograft model of nude mice, respectively. Tandem affinity purification, mass spectrometry and dual luciferase reporter assay were used to find out the molecules interacted with Herg1. Western blot was used to detect the expressions of large tumor suppressor gene (LATS1), p-LATS1, Yes-associated protein (YAP) and p-YAP in cells after infection of Ad5-Herg1-shRNA. Results: Compared to Ad5-control-shRNA, Ad5-Herg1-shRNA dramatically inhibited the expression of Herg1 in OS cells. The result of CCK8 array demonstrated that 143B cell vitalities of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (65.47±3.90)% and (79.90±1.52)%, significantly lower than (100.00±6.14)% of Ad5-control-shRNA group. Meanwhile, U2OS cell vitality of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (69.69±1.36)% and (76.72±2.75)%, significantly lower than (100.00±3.01)% of Ad5-control-shRNA group (all P<0.001). The results of wound healing array showed that 143B cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (33.03±2.88)% and (36.47±4.16)%, significantly lower than (97.78±2.28)% of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were (68.07±0.90)% and (73.97±1.25)%, significantly lower than (96.50±1.12)% of Ad5-control-shRNA group (all P<0.001). The results of Transwell showed that 143B cell invasion numbers of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 36.50±12.15 and 44.83±7.62, significantly lower than 195.33±19.68 of Ad5-control-shRNA group. Meanwhile, U2OS cell migration rates of Ad5-Herg1-shRNA1 and Ad5-Herg1-shRNA2 group were 21.83±7.99 and 22.85±7.08, significantly lower than 83.33±12.36 of Ad5-control-shRNA group (all P<0.001). The results of xenograft model of OS showed that the tumor volume and weight of Ad5-Herg1-shRNA group were significantly smaller than of Ad5-control-shRNA group after 14 days and 5 weeks of inoculation, respectively (P<0.001). Moreover, knockdown of Herg1 inhibited the metastasis of OS cells. In mechanism, Herg1 protein interacted with NF2 protein. Knockdown of Herg1 significantly suppressed the expression levels of LATS1 and YAP protein, and promoted the phosphorylation of LATS1 and YAP in OS cells (all P<0.001). Conclusion: Our findings suggest that Herg1 participates in the proliferation and motility of OS cells and may serve as a potential therapeutic target for osteosarcoma patients.

Nitidine Chloride Inhibits SIN1 Expression in Osteosarcoma Cells

Nitidine chloride (NC) has been demonstrated to exert a tumor-suppressive function in various types of human cancers. However, the detailed mechanism of NC-mediated anti-tumor effects remains elusive. It has been reported that SIN1, a component of mTORC2 (mammalian target of rapamycin complex C2), plays an oncogenic role in a variety of human cancers. Therefore, the inhibition of SIN1 could be useful for the treatment of human cancers. In this study, we explored whether NC triggered an anti-cancer function via the inhibition of SIN1 in osteosarcoma (OS) cells. An MTT assay was performed to measure the effect of NC on the cell growth of osteosarcoma cells, and flow cytometry was used to detect the apoptotic rate of the cells after NC treatment. The expression of SIN1 was detected by western blotting. Wound-healing assay and Transwell chamber invasion assay were conducted to analyze the motility of osteosarcoma cells following NC exposure. We found that exposure to NC led to the inhibition of cell growth, migration, and invasion and the induction of apoptosis. Mechanistically, we found that NC inhibited the expression of SIN1 in osteosarcoma cells. Overexpression of SIN1 abrogated the inhibition of cell growth and motility induced by NC in osteosarcoma cells. Our results indicate that NC exhibits its tumor-suppressive activity via the inhibition of SIN1 in osteosarcoma cells, suggesting that NC could be a potential inhibitor of SIN1 in osteosarcoma.

LncRNA MEG3 negatively modified osteosarcoma development through regulation of miR-361-5p and FoxM1.

This study was aimed to figure out whether long noncoding RNA MEG3/miR-361-5p/FoxM1 signaling would contribute to improved proliferation and metastasis of osteosarcoma cells. We altogether collected 204 pairs of osteosarcoma tissues and adjacent normal tissues, and obtained four human osteosarcoma cell lines. Then pcDNA3.1-MEG3, si-MEG3, miR-361-5p mimic, miR-361-5p inhibitor, pcDNA3.1-FoxM1, si-FoxM1, and negative control (NC) were, respectively, transfected into the osteosarcoma cells. Furthermore, real time polymerase chain reaction was utilized to determine the mRNA expressions of maternally expressed gene 3 (MEG3) and miR-361-5p, and western blot analysis was applied for determining the FoxM1 expression. Besides, dual luciferase reporter gene assay was adopted to verify if MEG3 can be directly targeted by miR-361-5p. Finally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, colony formation assay, flow cytometry, wound healing assay, and transwell assay were conducted to investigate the influence of MEG3, miR-361-5p, and FoxM1 expressions on the viability, proliferation, apoptosis, migration, and invasion of osteosarcoma cells. MEG3 and miR-361-5p were observed to be significantly downregulated within both osteosarcoma tissues and cell lines, whereas FoxM1 was upregulated in osteosarcoma tissues and cell lines (p < 0.05). MEG3 directly bound to miR-361-5p, and significantly upgraded its expression (p < 0.05). The upregulated MEG3 and miR-361-5p or the downregulated FoxM1 appeared to substantially inhibit proliferation, migration, and invasion of osteosarcoma cells (p < 0.05). Finally, the proliferation, migration, invasion, and motility of osteosarcoma cells within the miR-NC + pcDNA3.1-FoxM1 group and pcDNA + pcDNA-FoxM1 group were markedly promoted when compared with the miR-361-5p mimic group and pcDNA3.1-MEG3 group (p < 0.05). The MEG3/miR-361-5p/FoxM1 axis could potentially serve as therapeutic targets or diagnostic biomarkers for osteosarcoma.

Melatonin attenuates osteosarcoma cell invasion by suppression of C-C motif chemokine ligand 24 through inhibition of the c-Jun N-terminal kinase pathway.

Osteosarcoma, with its high metastatic potential, is the most prevalent malignant bone tumor in children and adolescents. Melatonin possesses multiple tumor-suppressing properties for a myriad of tumors, but little is known about the effects of melatonin on osteosarcoma metastasis. In this study, we demonstrated that melatonin elicited very low cytotoxicity and significantly inhibited cellular motility, migration, and invasion in human osteosarcoma U2OS and HOS cells. Moreover, using RNA sequencing technology, we revealed that melatonin repressed C-C motif chemokine ligand 24 (CCL24) gene expression in U2OS cells. Manipulation of CCL24 levels influenced the motility of osteosarcoma cells as cell migration and invasion were enhanced by the addition of recombinant human CCL24 and attenuated by the silencing of CCL24. Moreover, melatonin increased and decreased the activation of extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) 1/2, respectively, in a dose-dependent manner in U2OS and HOS cells while exerting no evident influence on the level and activation of p38, Akt, FAK, steroid receptor coactivator, or Raf. In further functional experiments, the use of JNK inhibitors (SP600125 and DN-JNK) confirmed that the pharmaceutic inhibition of JNK augmented the melatonin-mediated CCL24 suppression and migration of U2OS cells. Overall, our results revealed that melatonin attenuated chemokine CCL24 levels through inhibition of the JNK pathway to hinder human osteosarcoma cell invasion, thereby highlighting the therapeutic potential of melatonin for osteosarcoma metastasis.

Ezrin/NF-κB Pathway Regulates EGF-induced Epithelial-Mesenchymal Transition (EMT), Metastasis, and Progression of Osteosarcoma.

BackgroundEpithelial-mesenchymal transition (EMT) is responsible for metastasis of cancers, and NF-κB can promote tumor progression. Ezrin is an important molecule participating in EMT. However, whether Ezrin mediates NF-κB in EGF-induced osteosarcoma is unknown.Material/MethodsEzrin phosphorylation, NF-κB activation, and EGF-induced EMT were studied in MG63 and U20S cells with NF-κB inhibition, silencing, or over-expressing Ezrin. Cell morphology, proliferation, migration, and motility were analyzed. An osteosarcoma model was established in mice by injecting MG63 and U20S and reducing Ezrin.ResultsWith EGF induction in vitro, Ezrin Tyr353 and Thr567 were phosphorylated, and EMT, proliferation, migration, and motility of osteosarcoma cells were promoted. Silencing Ezrin suppressed and over-expressing Ezrin promoted the nuclear translocation of p65 and phosphorylated IκBα (p-IκBα) in EGF-induced osteosarcoma cells. NF-κB inhibitor blocked EGF-induced EMT in both cell types, as well as reserving cell morphology and suppressing proliferation, migration, and motility. In vivo, reducing Ezrin significantly suppressed metastasis of osteosarcoma xenografts, increased liver and lung weights, and activated NF-κB, which were both induced by EGF.ConclusionsEzrin/NF-κB regulated EGF-induced EMT, as well as progression and metastasis of osteosarcoma in vivo and in vitro. Ezrin/NF-κB may be a new therapeutic target to prevent osteosarcoma from deterioration.

Thrombospondin-1 promotes cell migration, invasion and lung metastasis of osteosarcoma through FAK dependent pathway.

Microenvironment at the metastatic locus usually differs greatly from that present in the site of primary tumor formation and it has a significant impact on the fate of the extravasated cancer cells. We compared gene expression signatures of primary tumors and lung metastatic tumors, and identified Thrombospondin-1 (TSP1) as highly up-regulated in the lung metastatic tumors. Immunohistochemical staining further indicated that TSP1 protein expression was higher in lung metastatic tumors compared to primary tumors in both osteosarcoma xenograft model and human clinical samples. TSP1 mRNA level is significantly associated with the Enneking stage of osteosarcoma and lung metastasis. TGF-β pathways could stimulate the TSP1 expression in osteosarcoma cells. Knockdown of TSP1 expression in osteosarcoma cells dramatically suppressed cell wound healing, migration and invasion. Treatment with recombinant TSP1 protein in osteosarcoma cells significantly promoted cell wound healing, migration and invasion. Meanwhile, suppression of TSP1 in osteosarcoma cells resulted in decreased pulmonary metastasis in vivo. Mechanistically, TSP1 increased expression of metastasis related genes, including MMP2, MMP9 and Fibronectin 1. TSP1 promoted osteosarcoma cell motility through the activation of FAK pathway. Taken together, our study provides evidence of the contributions of TSP1 to the lung metastasis of osteosarcoma and suggests that this protein may represent a potential therapeutic target for osteosarcoma lung metastasis.

PSMC2 is up-regulated in osteosarcoma and regulates osteosarcoma cell proliferation, apoptosis and migration.

Proteasome 26S subunit ATPase 2 (PSMC2) is a recently identified gene potentially associated with certain human carcinogenesis. However, the expressional correlation and functional importance of PSMC2 in osteosarcoma is still unclear. Current study was focused on elucidating the significance of PSMC2 on malignant behaviors in osteosarcoma including proliferation, apoptosis, colony formation, migration as well as invasion. The high protein levels of PSMC2 in osteosarcoma samples were identified by tissue microarrays analysis. Besides, its expression in the levels of mRNA and protein was also detected in four different osteosarcoma cell lines by real-time PCR and western blotting separately. Silencing PSMC2 by RNA interference in osteosarcoma cell lines (SaoS-2 and MG-63) would significantly suppress cell proliferation, enhance apoptosis, accelerate G2/M phase and/or S phase arrest, and decrease single cell colony formation. Similarly, pharmaceutical inhibition of proteasome with MG132 would mimic the PSMC2 depletion induced defects in cell cycle arrest, apoptosis and colonies formation. Silencing of PSMC2 was able to inhibit osteosarcoma cell motility, invasion as well as tumorigenicity in nude mice. Moreover, the gene microarray indicated knockdown of PSMC2 notably changed a number of genes, especially some cancer related genes including ITGA6, FN1, CCND1, CCNE2 and TGFβR2, and whose expression changes were further confirmed by western blotting. Our data suggested that PSMC2 may work as an oncogene for osteosarcoma and that inhibition of PSMC2 may be a therapeutic strategy for osteosarcoma treatment.

Visfatin triggers the in vitro migration of osteosarcoma cells via activation of NF-κB/IL-6 signals

Pulmonary metastasis is the major challenge for clinical treatment of osteosarcoma patients. Recent studies indicated that visfatin, a 52kDa adipocytokine, can trigger the cell motility of various cancers, while its role in the progression of osteosarcoma remains not clear. Our present study revealed that visfatin can significantly promote the in vitro migration and invasion of osteosarcoma MG-63 and HOS cells and up regulate the expression of matrix metalloproteinase-2 (MMP-2) and fibronectin (FN). Furthermore, visfatin treatment also increased the expression of IL-6 in both MG-63 and HOS cells via a time dependent manner, while anti-IL-6 antibody can significantly attenuate visfatin induced cell invasion and up regulation of MMP-2 and FN. It suggested that up regulation of IL-6 mediated visfatin induced in vitro motility of osteosarcoma cells. Visfatin treatment can increase the phosphorylation of both p65 and ERK1/2 in MG-63 and HOS cells, while only the inhibitor of NF-κB, BAY 11–7082, can abolish visfatin induced up regulation of IL-6. BAY 11–7082 also attenuated visfatin induced upregulation of MMP-2 and FN in MG-63 cells. Western blot analysis revealed that visfatin treatment can significantly increase the phosphorylation of IκBα and IKKβ in MG-63 cells. ACHP, the inhibitor of IKK-β, blocked visfatin induced expression of IL-6 mRNA in both MG-63 and HOS cells. Collectively, our data suggested that visfatin can increase the motility of osteosarcoma cells via up regulation of NF-κB/IL-6 signals. It indicated that visfatin might be a potential therapeutic target of osteosarcoma treatment.

SPAG9 controls the cell motility, invasion and angiogenesis of human osteosarcoma cells.

Sperm-associated antigen 9 (SPAG9) is an oncoprotein involved in the progression of various human malignancies; however, its role in osteosarcoma (OS) remains poorly evaluated. The present study used Matrigel™ cell migration and invasion assays, tube formation assay, Cell Counting kit-8, quantitative polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay to investigate the role of SPAG9 in OS cell motility, invasion and angiogenesis. The results of the present study demonstrated that SPAG9 expression was upregulated in OS tissues, as compared with adjacent normal tissues, and knockdown of SPAG9 in an OS cell line inhibited cell motility and invasion via inactivation of metalloproteinase (MMP)-2 and MMP-9. Furthermore, the present study demonstrated that silencing of SPAG9 in OS cells inhibited tube formation, the proliferation of human umbilical vascular endothelial cells, and suppressed vascular endothelial growth factor (VEGF) expression and secretion, contributing to a reduction in angiogenesis. The results of the present study indicated that SPAG9 may be an important regulator in OS and may be involved in metastasis. Therefore SPAG9 may be a promising target for the treatment of metastatic OS.

Identification of Novel Ezrin Inhibitors Targeting Metastatic Osteosarcoma by Screening Open Access Malaria Box.

Ezrin is a member of the ERM (ezrin, radixin, moesin) family of proteins and functions as a linker between the plasma membrane and the actin cytoskeleton. Ezrin is a key driver of tumor progression and metastatic spread of osteosarcoma. We discovered a quinoline-based small molecule, NSC305787, that directly binds to ezrin and inhibits its functions in promoting invasive phenotype. NSC305787 possesses a very close structural similarity to commonly used quinoline-containing antimalarial drugs. On the basis of this similarity and of recent findings that ezrin has a likely role in the pathogenesis of malaria infection, we screened antimalarial compounds in an attempt to identify novel ezrin inhibitors with better efficacy and drug properties. Screening of Medicines for Malaria Venture (MMV) Malaria Box compounds for their ability to bind to recombinant ezrin protein yielded 12 primary hits with high selective binding activity. The specificity of the hits on ezrin function was confirmed by inhibition of the ezrin-mediated cell motility of osteosarcoma cells. Compounds were further tested for phenocopying the morphologic defects associated with ezrin suppression in zebrafish embryos as well as for inhibiting the lung metastasis of high ezrin-expressing osteosarcoma cells. The compound MMV667492 exhibited potent anti-ezrin activity in all biologic assays and had better physicochemical properties for drug-likeness than NSC305787. The drug-like compounds MMV020549 and MMV666069 also showed promising activities in functional assays. Thus, our study suggests further evaluation of antimalarial compounds as a novel class of antimetastatic agents for the treatment of metastatic osteosarcoma.

Spondin 1 promotes metastatic progression through Fak and Src dependent pathway in human osteosarcoma

Spondin 1 (SPON1) is cell adhesion protein that involved in attachment of sensory neuron cells and outgrowth of neurites. Its cellular functions and related mechanisms in cancers, however, remain largely unexplored. In this study, we first identified that SPON1 acts a critical factor in the metastatic progression of osteosarcoma through analysis of a GEO dataset. Then we demonstrated that SPON1 was significantly up-regulated in 72 osteosarcoma specimens compared with benign osteochondroma samples and elevated SPON1 was positively correlated with MMP9 expression. Knockdown of SPON1 expression in two metastatic osteosarcoma cell lines, HKOS and KRIB, dramatically suppressed cell migration and invasion. Treatment with recombinant SPON1 protein in two non-metastatic osteosarcoma cell lines, HOS and U2OS, significantly promoted cell migration and invasion in vitro. Meanwhile, suppression of SPON1 in KHOS cells resulted in decreased pulmonary metastasis in vivo. Mechanistically, we determined that the effects of SPON1 on osteosarcoma cell motility were primarily mediated through Fak and Src dependent pathway. Taken together, our study provides evidence of the contributions of SPON1 and the Fak and Src signaling to the progression of osteosarcoma and suggests that this axis may represent a potential therapeutic target for osteosarcoma.

Interleukin-32 stimulates osteosarcoma cell invasion and motility via AKT pathway-mediated MMP-13 expression.

As a pro-inflammatory cytokine, interleukin-32 (IL-32) is reported to play an important role in tumor development and progression. However, its effects on the invasion and motility of osteosarcoma cells remain elusive. The aim of the present study was to determine the molecular mechanisms of IL-32 in osteosarcoma cells using RT-PCR and western blot analysis. The results showed that IL-32 stimulation dose-dependently promoted the invasion and motility of osteosarcoma cells. Knockdown of endogenous IL-32 by siRNA inhibited osteosarcoma cell invasion and motility. Moreover, IL-32 induced the activation of AKT in a time-dependent manner. IL-32 stimulation was also capable of increasing the expression and secretion of matrix metalloproteinase (MMP)-13, which is involved in tumor invasion and metastasis. In addition, blockade of AKT activation suppressed IL-32-mediated invasion, motility and MMP-13 upregulation in osteosarcoma cells. Taken together, our results suggest that IL-32 stimulation promotes the invasion and motility of osteosarcoma cells, possibly via the activation of AKT and the upregulation of MMP-13 expression. Thus, IL-32 may serve as a marker for diagnosis, as well as for the treatment of osteosarcoma.

Abstract 3987: Evaluation of anti–malarial compounds as inhibitors of ezrin in osteosarcoma cells

Abstract Ezrin is a member of the ERM (Ezrin, Radixin, Moesin) family of proteins that functions as a cross-linker between the plasma membrane and actin cytoskeleton, thus allowing the cell to regulate cytoskeletal dynamics in response to external stimuli. Ezrin plays important roles in cell motility, membrane trafficking, morphogenesis, adhesion, survival and apoptosis. Ezrin also participates in crucial signal transduction pathways. Overexpression of ezrin contributes to the aggressive cancer behavior in various tumor types especially by enhancing the metastatic phenotype. Thus, ezrin represents a promising molecular target for cancer therapy. In our earlier studies, we have established that NSC305787 can directly bind to ezrin and inhibit its activity, which results in reduced metastasis. Since NSC305787 shares significant structural similarities with some anti-malarial drugs, we tested 400 anti-malarial compounds including 200 drug-like compounds and 200 probe-like compounds for their potential to directly bind to ezrin protein. We used surface plasmon resonance technology (Biacore) to screen direct binding. In this initial screen, 12 compounds demonstrated binding to ezrin over background values and selected as primary hits. Detailed SPR analysis of primary hits resulted in 7 compounds with comparable KD values to that obtained with our lead compound NSC305787. Primary hits were also evaluated for inhibition of the cell motility of murine osteosarcoma (OS) cells K7M2 and K12 that express high and low levels of ezrin, respectively. Cell motility and chemotaxis assays were conducted in parallel with viability assays to rule out general toxicity. Selected anti-malarial compounds were effective in inhibiting the motility of OS cells with high ezrin compared to cells with low ezrin. In conclusion, several anti-malarial drugs may have new indications in cancer therapy by inhibiting ezrin. Citation Format: Haydar Celik, Abraham Kallarakal, Daisy Colon-Lopez, Jeffrey Toretsky, Jurgen Bosch, Aykut Uren. Evaluation of anti–malarial compounds as inhibitors of ezrin in osteosarcoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2014-3987

Abstract 2683: Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma.

Abstract Osteosarcoma is a highly invasive bone malignancy in which metastasis accounts for the vast majority of death and morbidity in patients. Understanding the mechanisms controlling metastasis is essential for improving patient survival in this disease. Recent studies have shown that Metadherin (MTDH) plays an essential role in mediating tumorigenesis and metastasis in a variety of human cancers. Our study assessed the role of MTDH in osteosarcoma metastasis and elucidated the mechanisms underlying its metastasis-promoting activity. We used western blot, qPCR, and flow cytometry to measure the expression of MTDH in a panel of osteosarcoma cell lines, and used immunohistochemistry to confirm expression in patient-derived material. In parallel experiments we used MTDH-specific shRNA to reduce endogenous MTDH expression, and blocked cell surface MTDH by anti-MTDH antibodies. The impact of MTDH inhibition was assessed in vitro using transwell migration assays and matrigel invasion assays. We also developed an orthotopic xenograft mouse model to study the relationship between MTDH expression and osteosarcoma pulmonary metastasis. To investigate the role of MTDH in cell-extracellular matrix (ECM) interaction, a series of adhesion assays were performed to assess the adhesion ability of osteosarcoma cells with or without MTDH inhibition to major components of ECM. Compared to normal human osteoblasts, all osteosarcoma cell lines tested displayed increased MTDH expression. MTDH knockdown significantly reduced migration and invasion in osteosarcoma cells. Blockade of cell surface MTDH with an anti-MTDH antibody targeting the extracellular domain of MTDH also profoundly impeded osteosarcoma cell motility and invasiveness. In the in vivo experiments, down-regulation of MTDH in osteosarcoma cells delayed primary tumor growth and prohibited pulmonary metastasis. Immunohistochemical analysis of tumors samples isolated from the mice showed that lung metastases had elevated MTDH expression level compared to primary tumors. Altogether, these data indicated an important role for Metadherin in osteosarcoma metastasis. Further, knockdown of MTDH by shRNA and inhibition of cell surface MTDH by anti-MTDH antibodies both profoundly decreased cell adhesion to matrigel and specific ECM proteins, which suggested that MTDH, particularly the proportion localized at the cell surface, may be involved in the interaction between tumor cells and the non-cellular matrix proteins that constitute tissue stroma. The interaction will be confirmed by future experiments. By identifying the ECM protein binding partners for MTDH, we may establish a new paradigm for the function of MTDH in mediating tumor cell migration and invasion in osteosarcoma. Citation Format: Limin Zhu, Adrianna Buford, Yanwen Yang, Pingyu Zhang, Wei-Lien Wang, Dennis P. Hughes. Metadherin mediates tumor invasion and interaction with extracellular matrix proteins in osteosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2683. doi:10.1158/1538-7445.AM2013-2683