Suppressed Osteosarcoma Cell Migration Research Articles

Asiatic acid inhibits osteosarcoma cell migration and invasion via the AKT/Sp1/MMP1 axis.

Osteosarcoma is a malignant bone tumor affecting adolescents and children. No effective treatment is currently available. Asiatic acid (AA), a triterpenoid compound found in Centella asiatica, possesses anti-tumor, anti-inflammatory, and anti-oxidant properties in various types of tumor cells. This study aims to determine whether AA exerts antitumor effects in human osteosarcoma cells. Our results indicate that AA does not influence the viability, proliferative rate, or cell cycle phase of human osteosarcoma cells under non-toxic conditions. AA suppressed osteosarcoma cell migration and invasion by down-regulating matrix metalloproteinase 1 (MMP1) expression. Data in the TNMplot database suggested MMP1 expression was higher in osteosarcoma than in normal tissues, with associated clinical significance observed in osteosarcoma patients. Overexpression of MMP1 in osteosarcoma cells reversed the AA-induced suppression of cell migration and invasion. AA treatment decreased the expression of specificity protein 1 (Sp1), while Sp1 overexpression abolished the effect of AA on MMP1 expression and cell migration and invasion. AA inhibited AKT phosphorylation, and treatment with a PI3K inhibitor (wortmannin) increased the anti-invasive effect of AA on osteosarcoma cells via the p-AKT/Sp1/MMP1 axis. Thus, AA exhibits the potential for use as an anticancer drug against human osteosarcoma.

Sulfatinib, a novel multi-targeted tyrosine kinase inhibitor of FGFR1, CSF1R, and VEGFR1-3, suppresses osteosarcoma proliferation and invasion via dual role in tumor cells and tumor microenvironment.

Tumor progression is driven by intrinsic malignant behaviors caused by gene mutation or epigenetic modulation, as well as crosstalk with the components in the tumor microenvironment (TME). Considering the current understanding of the tumor microenvironment, targeting the immunomodulatory stromal cells such as cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) could provide a potential therapeutic strategy. Here, we investigated the effect of sulfatinib, a multi-targeted tyrosine kinase inhibitor (TKI) of FGFR1, CSF1R, and VEGFR1-3, on the treatment of osteosarcoma (OS). In vitro, the antitumor effect was tested by clony formation assay and apoptosis assay.The inhibition of tumor migration and invasion was detected by Transwell assay, and the de-polarization of macrophage was detected by flow cytometry.In vivo, subcutaneous and orthotopic tumor models were established to verify antitumor effect, and the underlying mechanism was verified by immunohistochemistry(IHC), immunofluorescence(IF) and flow cytometry. Sulfatinib suppressed OS cell migration and invasion by inhibiting epithelial-mesenchymal transition (EMT) by blocking the secretion of basic fibroblast growth factor (bFGF) in an autocrine manner. In addition, it regulated immune TME via inhibition of the migration of skeletal stem cells (SSCs) to the TME and the differentiation from SSCs to CAFs. Moreover, sulfatinib can suppress OS by modulation of the TME by inhibiting M2 polarization of macrophages. Systemic treatment of sulfatinib can reduce immunosuppression cells M2-TAMs, Tregs, and myeloid-derived suppressor cells (MDSCs) and increase cytotoxic T-cell infiltration in tumors, the lungs, and the spleens. Our preclinical experiments have shown that sulfatinib can inhibit the proliferation, migration, and invasion of OS by playing a dual role on tumor cells and the tumor microenvironment simultaneously and systematically reverse immunosuppression to immune activation status, which could be translated into clinical trials.

KMT5A Knockdown Suppresses Osteosarcoma Cell Proliferation and Metastasis Through Ꞵ-Catenin Signalling.

Osteosarcoma (OS) is the most common malignant solid bone tumor in children and young adults. We aimed to investigate the effects and cellular mechanisms of KMT5A on OS cell activity. The protein expression was evaluated in the clinical normal, adjacent and OS osteogenic tissues. Knockdown of KMT5A was achieved by KMT5A siRNAs in a human OS cell line, MG63, to detect cell proliferation and metastasis. KMT5A expression was upregulated in clinical OS tissues. Knockdown of KMT5A inhibited cell proliferation but enhanced cell death, with significantly reduced cyclinD1 and Bcl2 and increased cleaved-caspase9 levels. KMT5A knockdown also suppressed OS cell migration and invasion capacity and deceased MMP3 and vimentin expression. β-catenin levels were upregulated in OS tissues and blocking KMT5A resulted in a significant decline in β-catenin expression in the OS cells. Further administration of β-catenin activator remarkably increased protein levels of KMT5A, cyclinD1, Bcl2, MMP3, and vimentin, which showed reversed effects of KMT5A knockdown on OS cell activity. KMT5A knockdown plays an inhibitory role in OS cell proliferation and metastasis through β-catenin signalling, which provides basic evidence and suggests potential targets for OS therapeutic research.

The Thioredoxin-1 Inhibitor, PX-12, Suppresses Local Osteosarcoma Progression.

The thioredoxin-1 (Trx-1) inhibitor, PX-12, is active against several cancer types. This study aimed to evaluate its effects on local osteosarcoma (OS) progression and to describe PX-12-related signal transduction pathways. Publicly available expression cohort data were analyzed to determine the relationship between the expression levels of TXN, which codes for the Trx protein, and survival in patients with OS. Murine LM8 OS cells were stimulated with PX-12. Apoptosis-related protein levels, cell viability, caspase activity, and wound healing were evaluated. PX-12 efficacy in suppressing tumor progression was evaluated in C3H mice injected with LM8 cells. High TXN expression was a negative prognostic factor for metastasis and overall survival in OS patients. PX-12 induced apoptosis in OS cells via the oxidative stress-MAPK-caspase 3 pathway and suppressed OS cell migration. PX-12 suppressed local OS progression. PX-12 is a potential therapeutic agent for use in suppressing local OS progression.

Erythrocyte membrane protein band 4.1-like 3 inhibits osteosarcoma cell invasion through regulation of Snai1-induced epithelial-to-mesenchymal transition.

Erythrocyte membrane protein band 4.1-like 3 (EPB41L3) is an important membrane skeletal protein that may interact with numerous membrane proteins. Loss of EPB41L3 is reported in multiple cancer types, and it is originally identified as a tumor suppressor. In this study, through analyzing expression profiling retrieved from the Gene Expression Omnibus (GEO) dataset, we find that EPB41L3 is upregulated in primary osteosarcoma (OS) and osteosarcoma cell lines. Importantly, EPB41L3 may promote osteosarcoma cell proliferation and suppress osteosarcoma cell migration and invasion. Reduced EPB41L3 leads to a decrease of E-cadherin as well as an increase of N-cadherin and Vimentin, implying a prominent epithelial-to-mesenchymal transition. Furthermore, we demonstrate that EPB41L3 inhibits the epithelial-to-mesenchymal transition through destabilizing the Snai1 protein, one of the most important transcription factors of the epithelial-to-mesenchymal transition process. Collectively, our study has first established the complex and vital roles of EPB41L3 and implicated EPB41L3 as a potential biomarker in osteosarcoma.

Up-Regulation of Hsa_circ_0008792 Inhibits Osteosarcoma Cell Invasion and Migration and Promotes Apoptosis by Regulating Hsa-miR-711/ZFP1.

ObjectiveCircular RNA is a newly discovered non-coding RNA. It plays an important role in regulating gene expression, and may take part in tumor progression. This study aimed to investigate the functions of hsa_circ_0008792 in osteosarcoma regulation.MethodsWe identified a circular RNA, hsa_circ_0008792, by using bioinformatics to analyze the GSE96962 dataset. The capacities of migration and invasion were assessed by wound-healing assay and transwell Matrigel assay. The ratios of G0/G1, S, and G2/M phases in cell cycle and apoptosis were measured using flow cytometry.ResultsHsa_circ_0008792 is expressed at low levels in osteosarcoma cells, and up-regulation of hsa_circ_0008792 could suppress osteosarcoma cell migration and invasion and promote apoptosis. This regulation is mediated by hsa-miR-711/ZFP1. The expression level of hsa_circ_0008792 showed no influence on cell cycle of osteosarcoma cells.ConclusionOsteosarcoma is suppressed by hsa_circ_0008792/hsa-miR-711/ZFP1 axis.

LncRNA FAL1 is a negative prognostic biomarker and exhibits pro-oncogenic function in osteosarcoma.

Long noncoding RNA focally amplified lncRNA on chromosome 1 (FAL1) plays an important role in the development of several human malignancy entities. However, the expression and function of FAL1 in the carcinogenesis and development of osteosarcoma remain unknown. In this study, we detected the FAL1 levels in human osteosarcoma tissues and cell lines by quantitative real-time polymerase chain reaction and investigated its role in osteosarcoma using in vitro assays. Our results showed that FAL1 expression was significantly upregulated in human osteosarcoma tissues and cell lines compared with their normal controls. FAL1 expression level was positively correlated with the distance metastasis (P = .008) and tumor stage ( P = .013). Higher expression of FAL1 conferred a significantly poorer survival, as determined by the Kaplan-Meier method with the log-rank test. Multivariate Cox proportional hazards analysis revealed that FAL1 was probably an independent risk of overall survival. Additionally, the serum FAL1 expression level was associated with osteosarcoma status in patients. Receiver operating characteristic curve analysis demonstrated that FAL1 expression was different between patients with osteosarcoma and healthy cohorts (area under the curve, 0.839; P < .001). Furthermore, knockdown of FAL1 by small interfering RNA significantly inhibited cell proliferation via inducing G2/M arrest in osteosarcoma cells. Depletion of FAL1 also suppressed osteosarcoma cell migration and invasion, which might be mediated by inhibiting the epithelial-mesenchymal transition (EMT) program. Taken together, our study showed that lncRNA FAL1 exhibits an important pro-oncogenic effect on osteosarcoma progression by targeting EMT, which may serve as a potential therapeutic target for osteosarcoma.

MiR-302b Suppresses Osteosarcoma Cell Migration and Invasion by Targeting Runx2

Osteosarcoma patients with lung metastasis and local invasion remain challenging to treat despite the significant contribution of the combination of surgery and neo-adjuvant chemotherapy. Our previous microarray study demonstrated that miR-302b had significantly lower expression in osteosarcoma cell lines than in osteoblast cell lines. In the present study, we further elucidated the role of miR-302b in regulating the migration and invasiveness of osteosarcoma. MiR-302b expression was markedly down-regulated in osteosarcoma cell lines and clinical tumour tissues. Lower levels of miR-302b expression were significantly associated with metastasis and high pathological grades. A functional study demonstrated that over-expression of miR-302b suppressed tumour cell proliferation, invasion and migration in vitro and in vivo. Runx2 was identified as a direct target gene for miR-302b by bioinformatics analysis and dual-luciferase reporter gene assay. Moreover, over-expression of miR-302b induced down-regulation of Runx2, OPN, MMP-2, MMP-9, MMP-12, MMP-14, and VEGF in 143B cells. Exogenous expression of Runx2 partially rescued the inhibitory effect of miR-302b on the invasion and migration activity of 143B osteosarcoma cells. Taken together, our results indicate that miR-302b functions as a tumour repressor in the invasion and migration of osteosarcoma by directly downregulating Runx2 expression and may be a potential therapeutic target for osteosarcoma.

Paris saponin VII suppresses osteosarcoma cell migration and invasion by inhibiting MMP‑2/9 production via the p38 MAPK signaling pathway.

Metastasis is the primary cause of mortality in osteosarcoma. Targeting metastasis is a major strategy in osteosarcoma treatment. As a traditional Chinese medicine, Trillium tschonoskii Maxim has been widely used in the therapy of various diseases, including cancer. However, currently there is no evidence regarding the anti-metastasic effect of Paris saponin VII (PS VII), which is extracted from Trillium tschonoskii Maxim, on osteosarcoma cells and its underling mechanisms. The present study aimed to examine the effect of PS VII on the migration and invasion of osteosarcoma cells. Viability and proliferation of osteosarcoma cells were examined by MTT assay. Migration and invasion of osteosarcoma cells was then detected using scratch wound healing assays and Transwell assays, respectively. Additionally, the expression of matrix metalloproteinase (MMP)-2 and -9 was determined at the mRNA and protein level following treatment with PS VII. Mitogen-activated protein kinase (MAPK) expression was also detected by western blot analysis. Finally, an inhibitor of p38 MAPK was used to verify the effect of PS VII on the expression of MMP-2 and -9, as well as the migration and invasion osteosarcoma cells. This demonstrated that the proliferation, migration and invasion of the osteosarcoma cells were suppressed following treatment with PS VII. PS VII downregulated the expression of MMP-2 and -9 in a dose- and time-dependent manner. PS VII also exerted its ability to downregulate the phosphorylation of p38 MAPKs. Furthermore, by using a p38 inhibitor, SB203580, the role of PS VII in MMP-2 and -9 expression and osteosarcoma cell invasion was revealed. Taken together, these results demonstrated that PS VII suppresses the migration and invasion of osteosarcoma cells via the p38 MAPK signaling pathway.

MiR-193a-3p and miR-193a-5p suppress the metastasis of human osteosarcoma cells by down-regulating Rab27B and SRR, respectively.

MicroRNAs have been identified as key players in the development and progression of osteosarcoma, which is the most common primary malignancy of bone. Sequencing-based miR-omic and quantitative real-time PCR analyses suggested that the expression of miR-193a-3p and miR-193a-5p was decreased by DNA methylation at their promoter region in a highly metastatic osteosarcoma cell line (MG63.2) relative to their expression in the less metastatic MG63 cell line. Further wound-healing and invasion assays demonstrated that both miR-193a-3p and miR-193a-5p suppressed osteosarcoma cell migration and invasion. Moreover, introducing miR-193a-3p and miR-193a-5p mimics into MG63.2 cells or antagomiRs into MG63 cells confirmed their critical roles in osteosarcoma metastasis. Additionally, bioinformatics prediction along with biochemical assay results clearly suggested that the secretory small GTPase Rab27B and serine racemase (SRR) were direct targets of miR-193a-3p and miR-193a-5p, respectively. These two targets are indeed involved in the miR-193a-3p- and miR-193a-5p-induced suppression of osteosarcoma cell migration and invasion. MiR-193a-3p and miR-193a-5p play important roles in osteosarcoma metastasis through down-regulation of the Rab27B and SRR genes and therefore may serve as useful biomarkers for the diagnosis of osteosarcoma and as potential candidates for the treatment of metastatic osteosarcoma.

MicroRNA-128 inhibits EMT of human osteosarcoma cells by directly targeting integrin α2.

Deregulated expression of miRNAs contributes to the development of osteosarcoma. The present study was to evaluate the level of miR-128 and integrin α2 (ITGA2) in osteosarcoma tissues and cells. We further investigated the molecular mechanisms of miR-128 and ITGA2 in osteosarcoma cell lines. In the present study, we found that miR-128 expression was down-regulated in osteosarcoma tissues and MG-63, U2OS, and SAOS-2 cells (all p < 0.001). By contrast, ITGA2 was up-regulated. Furthermore, we found that miR-128 overexpression suppressed cell migration and invasion of MG-63 cells. Mechanically, miR-128 overexpression inhibited epithelial-mesenchymal transition (EMT) of MG-63 cells. Importantly, we identified that the 3'-untranslated region (3'-UTR) of ITGA2 was a direct target of miR-128. Luciferase reporter assays confirmed that miR-128 binding to the 3'-UTR regions of ITGA2 inhibited the expression of ITGA2 in MG-63 cells. At the same time, overexpressed ITGA2 also reversed EMT inhibited by miR-128. In conclusion, this study suggested that high miR-128 expression suppressed osteosarcoma cell migration, invasion, and EMT development through targeting ITGA2, which may be recommended as a therapeutic target for osteosarcoma.

Downregulation of connective tissue growth factor reduces migration and invasiveness of osteosarcoma cells.

As one of the most serious types of primary bone tumor, osteosarcoma (OSA) features metastatic lesions, and resistance to chemotherapy is common. The underlying mechanisms of these characteristics may account for the failure of treatments and the poor prognosis of patients with OSA. It has been reported that inhibition of Cyr61 suppresses OSA cell proliferation as it represents a target of statins. In addition to cystein‑rich protein 61 (Cyr61) and nephroblastoma overexpression, connective tissue growth factor (CTGF) is a member of the CCN family and may therefore exhibit effects on human OSA cells similar to those of Cyr61. In the current study, acridine orange/ethidium bromide staining were used to determine the rate of apoptosis. The present study demonstrated that small interfering RNA‑mediated silencing of CTGF promoted cell death and suppressed OSA cell migration and invasion, as indicated by wound healing and Transwell assays, while lentivirus‑mediated overexpression of CTGF reversed these effects. Furthermore, a colorimetric caspase assay demonstrated that CTGF knockdown enhanced the efficacy of chemotherapeutic drugs. The results of the present study provided a novel molecular target which may be utilized for the treatment of metastatic OSA.

Inhibition of Aurora-B suppresses osteosarcoma cell migration and invasion

Previous studies have suggested that Aurora-B may be involved in cancer metastasis. However, its role has been poorly evaluated in osteosarcoma (OS). The aim of this study was to investigate the correlation between Aurora-B expression and metastasis in human OS. The human OS cell line, U2-OS, and OS biopsy specimens were used in the study. The expression of Aurora-B protein was examined using immunohistochemistry and western blotting in OS tissues and U2-OS cells, respectively. AZD1152-hydroxyquinazoline-pyrazol-anilide, an inhibitor of Aurora-B, was used to inhibit Aurora-B expression in U2-OS cells. The effect of Aurora-B inhibition on U2-OS cell proliferation, invasion and migration was assessed using MTT, colony formation, wound healing and Transwell assays. The results showed that positive expression of the Aurora-B protein was observed in the nucleus, and that Aurora-B expression levels in the cases with pulmonary metastases were significantly higher than in those without metastasis. In vitro, the proliferation, invasion and migration of U2-OS cells were suppressed by the inhibition of Aurora-B. These results suggest that Aurora-B may be involved in OS metastasis, and may be a promising target in the treatment of OS metastasis.

CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and β-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and β-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.

Abstract 4325: CD99 suppresses osteosarcoma cell migration by favouring the oncosoppressor N-cadherin/beta-catenin signalling pathway in contrast to ezrin.

Abstract CD99 is a 32-kDa highly glycosylated transmembrane protein encoded by MIC2 gene which shares no homology with any known family of proteins except Xga and mouse CD99L2 proteins. Located in the pseudoautosomal region of chromosomes X and Y, MIC2 encodes two distinct products by alternative splicing of gene transcripts: a long form (32-kDa), corresponding to the full-length protein (CD99wt) and a short form harbouring a deletion in the intracytoplasmic domain (CD99 sh). CD99 is involved in multiple cellular events including cell adhesion, apoptosis, differentiation of T- cells and thymocytes, transendothelial migration of leukocytes, maintenance of cellular morphology and regulation of intracellular membrane protein trafficking either in physiology and in pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumor suppressor. The two alternative spliced isoforms are associated to distinct functional outcomes: CD99sh expression increases, whereas wtCD99 significantly inhibits migration, invasiveness and metastasis of osteosarcoma cells (Manara MC, 2006; Scotlandi K 2007). In this study, we specifically analyzed modulation of cell-cell contacts, reorganization of the actin cytoskeleton, and modulation of signalling pathways by comparing osteosarcoma cells characterized by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that expression of CD99wt induces recruitment of beta-catenin to adherens injunctions through N-cadherin up-regulation. In addition, forced expression of CD99wt inhibits the expression of several molecules crucial to remodeling the actin cytoskeleton, such as ACTR2 (also named ARP2), ARPC1A and ROCK II as well as ezrin, an ezrin/radixin/moesin (ERM) family member that has been clearly associated with tumor progression and metastatic spread either in experimental models and clinical samples of osteosarcoma. Functional studies by siRNA and/or specific inhibitors point to ROCKII as a crucial intracellular mediator regulating osteosarcoma metastasis capabilities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4325. doi:1538-7445.AM2012-4325