Osteosarcoma Clinical Samples Research Articles

MiR-1-3p Inhibits Osteosarcoma Cell Proliferation and Cell Cycle Progression While Promoting Cell Apoptosis by Targeting CDK14 to Inactivate Wnt/Beta-Catenin Signaling.

Osteosarcoma (OS) is a common bone malignancy and is diagnosed frequently in children and young adults. According to previous RNA sequencing, miR-1-3p is downregulated in OS clinical samples. Nevertheless, the functions of miR-1-3p in OS cell process and the related mechanism have not been revealed yet. In the current study,miR-1-3p expression in OS tissues and cells were evaluated using quantitative polymerase chain reaction. CCK-8 assays were conducted to measure OS cell viability in response to miR-1-3p overexpression. Colony forming assays and EdU staining were conducted for measurement of cell proliferation, and flow cytometry analysis was performed to determine cell apoptosis and cell cycle progression. Protein levels of apoptotic markers, beta-catenin, and Wnt downstream targets were quantified using western blotting. The binding relation between miR-1-3p and cyclin dependent kinase 14 (CDK14) was validated utilizing luciferase reporter assays. Experimental results revealed thatmiR-1-3p expression was decreased in OS tissues and cells. Additionally,miR-1-3p inhibited cell proliferation and cell cycle progression while enhancing OS cell apoptosis. Moreover, miR-1-3p directly targeted CDK14 and inversely regulated CDK14 expression in OS cells. Furthermore, miR-1-3p inactivated the Wnt/beta-catenin signaling. CDK14 overexpression partially rescued the inhibitory impact of miR-1-3p on OS cell growth. Overall,miR-1-3p inhibits OS cell proliferation and cell cycle progression while promoting cell apoptosis by targeting CDK14 and inactivating the Wnt/beta-catenin signaling.

A biocatalytic peptidobiosensing molecular bridge for detecting osteosarcoma marker protein.

A biosensing scheme requiring only one-step sample incubation before signal collection, and using a compact "three-in-one" probe of target-binding, signal conversion, and amplification, may greatly simplify the design of biosensors. Therefore, sparing the multi-step addition of enzymes, protein, and nanomaterial, as well as the associated complexity and non-specific interactions. In this work, a peptide probe aimed at such compact features has been designed, based on protein-triggered, conformation-driven, and Cu (II) facilitated side-chain di-tyrosine cyclization. This design can use target-probe recognition to induce discriminated cross-linking and self-cleavage of the probe, resulting in retention or dissociation of a signal amplification motif from the search and consequently quantitative detection performance. The method has also been tested preliminarily in fractioned osteosarcoma clinical samples, showing an acceptable coherence between signal readout and clinical diagnosis. On the basis of these early findings, it is reasonable to assume that the proposed probe will be beneficial for the next development of tumor screening and prognosis sensors.

Impact of Complement C1qA and Complement C1qB on the Prognosis of Osteosarcoma

In order to obtain the target gene that affects the prognosis of osteosarcoma and to establish the risk ratio and prognosis of prediction model was the objective of the study. We obtained messenger ribonucleic acid transcriptome data from gene expression Omnibus database and clinical data from therapeutically applicable research to generate effective treatments database. Through intersection of differential genes, we obtained key genes in gene expression Omnibus database and then we used them to conduct survival analysis and establish risk ratio in therapeutically applicable research to generate effective treatments database. We obtained complement C1qA and complement C1qB as the differential key genes through GSE14359, GSE28424, GSE33382 and GSE36001 from gene expression Omnibus database. The expression of complement C1qA and complement C1qB were significantly different in 4 series data (p<0.01). Meanwhile, complement C1qA and complement C1qB have significant correlation in osteosarcoma clinical samples (p=8.61e-64, ρSpearman=0.97, 95 % confidence interval (0.96-0.98). The survival time of overexpression of complement C1qA and complement C1qB samples were significantly prolonged (p=0.0026 and p=0.006), area under the curve=0.689 (95 % confidence interval, 0.575-0.804), 0.702 (95 % confidence interval, 0.595-0.809), 0.641 (95 % confidence interval, 0.513-0.768) respectively. Complement C1qA and complement C1qB over-expression can significantly prolong survival time and can be used as predictors of survival in osteosarcoma.

The role of MT1-MMP in the progression and metastasis of osteosarcoma

The dysregulation of Membrane - type 1 matrix metalloproteinase (MT1-MMP) has been extensively studied in numerous cancer types, and plays key roles in angiogenesis, cancer progression, and metastasis. MT1-MMP is a predictor of poor prognosis in osteosarcoma (OS), yet the molecular mechanisms of disease progression are unclear. This review provides a summary of the literature relating to the gene and protein expression of MT1-MMP (MMP-14) in OS clinical samples, evaluates the expression in cell lines and experimental models, and analyses its potential role in the progression and metastasis of OS. In addition, the therapeutic potential of MT1-MMP as a drug target has been assessed. Due to the biological complexity of MMPs, inhibition has proven to be challenging. However, exploiting the expression and proteolytic capacity of MT1-MMP could open new avenues in the search for novel, safer and selective drugs for use in OS.

MicroRNA-545-5p regulates apoptosis, migration and invasion of osteosarcoma by targeting dimethyladenosine transferase 1.

The metastasis of osteosarcoma is a major threat to both adolescents and young adults. Identifying novel targets that may prevent osteosarcoma metastasis is critical in developing advanced clinical therapies for treating this cancer. The present study aimed to explore the mechanism of microRNA (miR)-545-5p in the metastasis of osteosarcoma. The present study identified miR-545-5p as a potential target that was downregulated in both osteosarcoma clinical samples and cell lines, and in the latter, ectopically expressed miR-545-5p caused apoptosis. In addition, miR-545-5p exerted inhibitory effects in osteosarcoma migration and invasion. Overexpression of miR-545-5p induced xenograft growth inhibition in vivo. In addition, miR-545-5p targeted dimethyladenosine transferase 1 (DIMT1), an oncogenic protein that facilitates osteosarcoma proliferation, migration and invasion. Taken together, the results of the present study suggest that miR-545-5p functions as a tumor suppressor in osteosarcoma that promotes apoptosis, while inhibiting migration and invasion by targeting DIMT1. Taken together, the results of the present study suggest two potential novel targets for osteosarcoma treatment and metastasis prevention.

Circular RNA circVRK1 suppresses the proliferation, migration and invasion of osteosarcoma cells by regulating zinc finger protein ZNF652 expression via microRNA miR-337-3p

ABSTRACT Circular RNA is an innovative kind of endogenous non-coding RNA, which could take part in tumorigenesis. Nonetheless, the potential molecular mechanisms of circVRK1 in the progression of osteosarcoma remain unresolved. In the current study, we initially investigated circVRK1 levels in osteosarcoma clinical samples and cell lines by qRT-PCR analysis and northern blot assay. RNase R treatments, RNA stability assay and nucleoplasmic separation assay were conducted to identify the characteristics of circVRK1. We adopted CCK-8, colony formation, wound-healing, and transwell assays to assess the biological effects of circVRK1 on the proliferation, migration, and invasiveness of osteosarcoma cells in vitro. We then constructed a xenograft model in nude mice to confirm the suppressive role of circVRK1 in vivo. Moreover, dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays were utilized to elucidate the underlying molecular mechanisms mediated by circVRK1. We demonstrated that circVRK1 was a stable circular transcript localized in the cytoplasm of osteosarcoma cells, and the down-regulation of circVRK1 in osteosarcoma tissues was related to poor outcome of patients. Meanwhile, over-expressed circVRK1 obviously restrained the growth, migration, and invasion of osteosarcoma in vitro and in vivo. Mechanistically, circVRK1 was assumed to be a microRNA sponge for miR-337-3p, and ZNF652 was the downstream gene of miR-337-3p. CircVRK1 overexpression or miR-337-3p knockdown accelerated ZNF652 expression, and up-regulated miR-337-3p efficiently abolished the promotion of ZNF652 induced by circVRK1. Moreover, rescue experiments have proved that circVRK1 inhibits the progression of osteosarcoma by modulating the miR-337-3p/ZNF652 axis. Therefore, we conclude that circVRK1 promotes ZNF652 expression by sponging miR-337-3p. CircVRK1 serves as a molecule sponge for miR-337-3p and mediates the ceRNA network to promote the expression of ZNF652, thus suppresses osteosarcoma proliferation, migration and invasion.

MiR-487a performs oncogenic functions in osteosarcoma by targeting BTG2 mRNA.

Aberrant microRNA (miRNA) expression plays a critical role in osteosarcoma (OS) pathogenesis. In this study, we elucidated the involvement of miR-487a in OS and the underlying molecular mechanisms. We found that miR-487a was upregulated in OS clinical samples and cell lines. Knockdown of miR-487a suppressed OS cell growth and invasion and induced apoptosis; however, overexpression of miR-487a promoted OS cell growth and invasion. Accordingly, downregulation of miR-487a significantly suppressed tumor growth of OS xenografts in vivo. Furthermore, B-cell translocation gene 2 (BTG2) mRNA was found to be a novel target of miR-487a. Knockdown of BTG2 using small interfering RNA (siRNA) recapitulated the oncogenic effects of miR-487a, whereas BTG2 overexpression partially reversed these effects. Finally, miR-487a levels were found to be negatively correlated with BTG2 expression in OS clinical samples. Collectively, our data suggest that miR-487a is an oncogenic miRNA in OS and it lowers BTG2 expression.

Leptin acts on mesenchymal stem cells to promote chemoresistance in osteosarcoma cells.

Leptin signaling influences osteoblastogenesis and modulates the fate of mesenchymal stem cells (MSCs) during bone and cartilage regeneration. Although MSCs abound in the osteosarcoma (OS) microenvironment, and leptin exhibits pro-tumorigenic properties, leptin’s influence on OS progression and chemoresistant signaling in MSCs remains unclear. Using cell viability and apoptosis assays, we showed that medium conditioned by leptin-treated human MSCs promotes cisplatin resistance in cultured human OS cells. Moreover, GFP-LC3 expression and chloroquine treatment experiments showed that this effect is mediated by stimulation of autophagy in OS cells. TGF-β expression in MSCs was upregulated by leptin and suppressed by leptin receptor knockdown. Silencing TGF-β in MSCs also abolished OS cell chemoresistance induced by leptin-conditioned medium. Cisplatin resistance was also induced when leptin-conditioned MSCs were co-injected with MG-63 OS cells to generate subcutaneous xenografts in nude mice. Finally, we observed a significant correlation between autophagy-associated gene expression in OS clinical samples and patient prognosis. We conclude that leptin upregulates TGF-β in MSCs, which promotes autophagy-mediated chemoresistance in OS cells.

ATF4 destabilizes RET through nonclassical GRP78 inhibition to enhance chemosensitivity to bortezomib in human osteosarcoma

Rationale: Activating transcription factor 4 (ATF4) is a central regulator of the cellular stress response and reduces tumor burden by controlling the expression of target genes implicated in the induction of apoptosis. Evidence shows ATF4 activation is responsible for proteasome inhibitor bortezomib (BTZ)-induced osteosarcoma (OS) cell death. However, it remains unclear how such suppressive function is impaired during prolonged therapeutic interventions.Methods: Stable cells and in vivo xenograft models were generated to reveal the essential role of ATF4 in cell apoptosis and tumor growth. Fluorescence in situ hybridization (FISH) and immunohistochemistry were employed to detect the expression and significance of ATF4 in the specimens from osteosarcoma patients. Biochemical differences between chemoresistant and chemosensitive cancer cells were determined by proliferation, apoptosis, real-time PCR, immunoblotting and immunofluorescence. Promoter activity was analysed using the luciferase reporter assay. Immunoprecipitation was used to explore the interaction of proteins with other proteins or DNAs.Results: ATF4 significantly inhibited OS tumorigenesis, whereas knockdown of ATF4 prevented the antitumor effects of BTZ. Normal osteoblasts are supposed to preferentially express ATF4, but ATF4 silencing was detected in both OS clinical samples and BTZ-resistant sublines (OS/BTZ). We found that ATF4 downregulation was tightly linked to the aberrant expression of RET, primarily due to RET stabilization in OS/BTZ cells. Loss of RET upregulated ATF4 and potentiated the apoptotic response to BTZ. ATF4 recognized the TK domain of RET by recruiting its transactivated E3 ligase Cbl-c to accelerate RET proteasomal turnover, which in turn prevented BTZ resistance. In contrast, the chaperone GRP78 bound to RET and interfered with ATF4/RET interactions, promoted RET stabilization. Intriguingly, ATF4 repressed GRP78 transcription in OS/BTZ cells via the first ERSE, instead of transactivating GRP78 in wild-type OS via classical CRE element, revealing a dual targeting of RET and GRP78 to overcome chemoresistance.Conclusion: The results uncover a crucial role for ATF4 in blocking the progression and resistance response in RET/GRP78-positive human osteosarcoma.

GAPLINC is a predictor of poor prognosis and regulates cell migration and invasion in osteosarcoma

Gastric adenocarcinoma predictive long intergenic non-coding (GAPLINC) is a novel long non-coding RNA (lncRNA) and has been found to function as an oncogenic lncRNA in gastric cancer, colorectal cancer, and bladder cancer. The expression status and biological function of GAPLINC in osteosarcoma are still unknown. Thus, we analyzed the association between GAPLINC expression and clinicopathological characteristics in osteosarcoma clinical samples, and conducted loss-of-function study in osteosarcoma cell lines. In our results, GAPLINC expression is elevated in osteosarcoma tissues and cell lines, and correlated with advanced Enneking stage, present distant metastasis, and poor histological grade. Survival analyses indicated that GAPLINC expression was negatively associated with overall survival, and GAPLINC high-expression was an independent risk factor in osteosarcoma patients. The in vitro studies showed knockdown of GAPLINC depressed osteosarcoma cell migration and invasion via inhibiting CD44 expression, but no effect on cell proliferation. In conclusion, GAPLINC may serve as a potential biomarker for predicting prognosis and developing therapy for osteosarcoma.

MiR-149 promotes human osteocarcinoma progression via targeting bone morphogenetic protein 9 (BMP9).

To investigate the roles of miR-149 in the progression of human osteosarcoma (OS). miR-149 level was upregulated in tissues from OS patients more than in normal subjects. Cell proliferation and apoptosis assays revealed that miR-149 increased cell proliferation and inhibited cell apoptosis in OS cell line (MG63). An increase of Bcl-2 gene expression and a decrease of cleaved-caspase-3, and cleaved-PARP expression were observed in MG63 cells with transfection of miR-149. Additionally, bone morphogenetic protein 9 (BMP9) was identified as a target of miR-149 in MG63 cells, and BMP9 expression was negatively correlated with miR149 level in OS clinical samples. Co-overexpression of BMP9 with miR-149 in MG63 cells prohibited miR-149-mediated promotive effects on OS progression. Importantly, overexpression of miR-149 conferred chemoresistance in MG63 cells. miR-149 promotes OS progression via targeting BMP9.

MiR-200bc/429 Inhibits Osteosarcoma Cell Proliferation and Invasion by Targeting PMP22.

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs which play a crucial role in diverse biological processes and could contribute to cancer development and progression. MiR-200bc/429 have been found to be aberrantly expressed in osteosarcoma (OS). However, the features of miR-200bc/429 in the tumorigenesis and progress of OS remain poorly understood.Material/MethodsThe miR-200bc/429 expression was firstly identified in human OS clinical samples and cell lines by quantitative real-time PCR (qRT-PCR). After transfection with miR-200bc/429 mimics or negative control in U2OS or MG63 cells, cell proliferation was measured by CCK-8 assay. Following that, wound-healing assay and Transwell invasion assay were performed to evaluate cell migration and invasion, respectively. Finally, luciferase reporter assay and Western blot analysis were performed to determine if peripheral myelin protein-22 (PMP22) is a direct target of miR-200bc/429.ResultsResults revealed that miR-200bc/429 were significantly depressed in human OS tissues and cell lines by qRT-PCR. Then, restoration of miR-200bc/429 significantly inhibited cell proliferation (P<0.05) and invasion (P<0.05) in vitro. Luciferase reporter assay and Western blot analysis revealed that miR-200bc/429 could directly target PMP22 3′ untranslated region (UTR) and inhibit its expression in U2OS and MG63 cells.ConclusionsThese findings suggest that miR-200bc/429 inhibit OS cells proliferation and invasion by targeting PMP22, and function as a tumor suppressor and may be a patent molecular marker as well as a potential target for OS therapy.

Overexpression of X-Box Binding Protein 1 (XBP1) Correlates to Poor Prognosis and Up-Regulation of PI3K/mTOR in Human Osteosarcoma

Increasing evidence demonstrates that dysregulation of XBP1 function contributes to tumorigenesis in some cancers. However, little is known about the role of XBP1 in the progression of osteosarcoma (OS). The expression of XBP1 in OS samples was measured by quantitative RT-PCR and Western blotting assays. Cell cycle analysis and cell counting kit 8 (CCK8) assays were performed to determine the effects of XBP1 expression on cells growth capacity. Cell apoptosis coassay was applied to determine cell survival. The expression of genes affected by XBP1 was examined by quantitative RT-RCR and validated by Western blotting assays. XBP1 was overexpressed in OS clinical samples compared with corresponding non-cancerous tissues. Overexpression of XBP1 was significantly associated with advanced clinical stages, high degree of malignancy and low tumor necrosis rate. Furthermore, hypoxia activated XBP1, and silencing XBP1 significantly enhanced OS cell apoptosis. Knock-down of XBP1 resulted in inhibition of OS growth. Most importantly, knockdown of XBP1 led to down-regulation of PIK3R3 and mTOR. Taken together, XBP1 is up-regulated and has a pro-tumor effect in OS with activation of PI3K/mTOR signaling. Thus, targeting XBP1 may provide a new potential therapeutic method for OS.

Targeting polo-like kinase 1 by NMS-P937 in osteosarcoma cell lines inhibits tumor cell growth and partially overcomes drug resistance.

Polo-like kinase 1 (PLK1) has emerged as a prognostic factor in various neoplasms, but only scarce data have been reported for high-grade osteosarcoma (OS). In this study, we assessed PLK1 expression and the efficacy of PLK1 inhibitor NMS-P937 in OS. PLK1 expression was assessed on 21 OS clinical samples and on a panel of human OS cell lines. In vitro efficacy of NMS-P937 was evaluated on nine drug-sensitive and six drug-resistant human OS cell lines, either as single agent or in combination with the drugs used in chemotherapy for OS. PLK1 expression was higher in OS clinical samples and cell lines compared to normal human tissue. A higher PLK1 expression at diagnosis appeared to be associated with an unfavourable clinical outcome. PLK1 silencing produced growth inhibition, cell cycle retardation and apoptosis induction in human OS cell lines. NMS-P937 proved to be highly active in both drug-sensitive and drug-resistant cell lines, with the only exception of ABCB1-overexpressing, Doxorubicin (DX)-resistant variants. However, in these cells, the association of NMS-P937 with DX was able to revert DX-resistance by negatively interfering with ABCB1 transport activity. NMS-P937 was also able to decrease clonogenic and migration ability of human OS cell lines. PLK1 can be proposed as a new candidate target for OS. Targeting PLK1 in OS with NMS-P937 in association with conventional chemotherapeutic drugs may be a new interesting therapeutic option, since this approach has proved to be active against drug resistant cells.

Upregulation of microRNA-17-92 cluster associates with tumor progression and prognosis in osteosarcoma

MicroRNA-17-92 (miR-17-92) cluster has been demonstrated to play a crucial role in various human cancers. However, its effects in osteosarcoma have not yet been elucidated. The purpose of this study was to investigate the clinical significance of miR-17-92 cluster in osteosarcoma. MiR-17-92 cluster expression in osteosarcoma clinical samples and cell lines was detected by real-time quantitative RT-PCR. Then, the association of miR-17-92 cluster level with survival of osteosarcoma patients was performed by the Kaplan-Meier and Cox proportional regression analyses. Furthermore, the effects of miR-17-92 cluster on tumorigenicity of osteosarcoma cell lines were evaluated by in vitro assays. The relative expression of miR-17-92 cluster in osteosarcoma tissues was significantly higher than those in adjacent normal tissues (P=0.001). And there was a relationship between miR-17-92 cluster upregulation and advanced TNM stage of osteosarcoma patients (P=0.037). Moreover, higher miR-17-92 cluster expression clearly predicted poorer Recurrence-free survival (P<0.001) and Overall survival (P=0.002). In the multivariate analysis, high miR-17-92 cluster expression was an independent prognostic factor for Recurrence-free survival (P<0.001) and Overall survival (P=0.002). Furthermore, the cellular proliferation, invasion, and migration of osteosarcoma cell lines were significantly accelerated by miR-17-92 cluster plasmid in vitro assays.Our findings showed that miR-17-92 cluster could serve as a promising marker for tumor recurrence and survival of osteosarcoma patients. Moreover, miR-17-92 cluster has been identified as a promoter for tumorigenicity of osteosarcoma cells, thus it might be a critical targeted therapy strategy for osteosarcoma.

Preclinical validation of Aurora kinases-targeting drugs in osteosarcoma

Background:Aurora kinases are key regulators of cell cycle and represent new promising therapeutic targets in several human tumours.Methods:Biological relevance of Aurora kinase-A and -B was assessed on osteosarcoma clinical samples and by silencing these genes with specific siRNA in three human osteosarcoma cell lines. In vitro efficacy of two Aurora kinases-targeting drugs (VX-680 and ZM447439) was evaluated on a panel of four drug-sensitive and six drug-resistant human osteosarcoma cell lines.Results:Human osteosarcoma cell lines proved to be highly sensitive to both drugs. A decreased drug sensitivity was observed in doxorubicin-resistant cell lines, most probably related to ABCB1/MDR1 overexpression. Both drugs variably induced hyperploidy and apoptosis in the majority of cell lines. VX-680 also reduced in vitro cell motility and soft-agar cloning efficiency. Drug association experiments showed that VX-680 positively interacts with all conventional drugs used in osteosarcoma chemotherapy, overcoming the cross-resistance observed in the single-drug treatments.Conclusion:Aurora kinase-A and -B represent new candidate therapeutic targets for osteosarcoma. In vitro analysis of the Aurora kinases inhibitors VX-680 and ZM447439 indicated in VX-680 a new promising drug of potential clinical usefulness in association with conventional osteosarcoma chemotherapeutic agents.

MiRNA expression profile in human osteosarcoma: Role of miR-1 and miR-133b in proliferation and cell cycle control

miRNA profile deregulation affecting downstream signaling pathways activates endpoints that represent potential biomarkers for prognosis and treatment of tumor patients. In the past 20 years conventional therapy for osteosarcoma (OS) reached a survival plateau, highlighting the need for new therapeutic approaches. In this study, microarray unsupervised and supervised analysis identified, respectively, 100 and 40 differentially expressed miRNAs in OS samples with different grades of malignancy compared to normal bone. When analyzing low-grade and high-grade OS by unsupervised analysis, 12 miRNAs were found to be differentially expressed. Real‑time PCR performed on a larger series of OS confirmed a significant lower expression of miR-1, miR‑133b and miR-378* in tumors with respect to control, also showing lower mRNA levels in 31 high-grade OS than in 25 low-grade and in metastatic versus non‑metastatic patients. We demonstrated that miR-1 and miR133b were downregulated in OS cell lines compared to normal osteoblasts. Secondly, by transfection with miRNA precursor molecules, we demonstrated that the ectopic expression of miR-1 and miR-133b in U2-OS cell lines significantly reduced cell proliferation and MET protein expression and negatively regulated cell invasiveness and motility in a short-term assay. Cell cycle distribution revealed block in G(1) and delay of cell cycle progression associated with increased apoptosis in miR-1- and miR‑133b‑transfected cells, respectively. Our data assessed specific miRNA profiling deregulation in OS clinical samples and suggest that the expression of miR-1 and miR-133b may control cell proliferation and cell cycle through MET protein expression modulation.

Modulation of the Osteosarcoma Expression Phenotype by MicroRNAs

BackgroundOsteosarcomas are the most common primary malignant tumors of bone and show multiple and complex genomic aberrations. miRNAs are non-coding RNAs capable of regulating gene expression at the post transcriptional level, and miRNAs and their target genes may represent novel therapeutic targets or biomarkers for osteosarcoma. In order to investigate the involvement of miRNAs in osteosarcoma development, global microarray analyses of a panel of 19 human osteosarcoma cell lines was performed.Principal findingsWe identified 177 miRNAs that were differentially expressed in osteosarcoma cell lines relative to normal bone. Among these, miR-126/miR-126*, miR-142-3p, miR-150, miR-223, miR-486-5p and members of the miR-1/miR-133a, miR-144/miR-451, miR-195/miR-497 and miR-206/miR-133b clusters were found to be downregulated in osteosarcoma cell lines. All miRNAs in the paralogous clusters miR-17-92, miR-106b-25 and miR-106a-92 were overexpressed. Furthermore, the upregulated miRNAs included miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-196a/miR-196b, miR-374a and members of the miR-29 and miR-130/301 families. The most interesting inversely correlated miRNA/mRNA pairs in osteosarcoma cell lines included miR-9/TGFBR2 and miR-29/p85α regulatory subunit of PI3K. PTEN mRNA correlated inversely with miR-92a and members of the miR-17 and miR-130/301 families. Expression profiles of selected miRNAs were confirmed in clinical samples. A set of miRNAs, miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-142-3p, miR-133b, miR-144, miR-195, miR-223, miR-451 and miR-497 was identified with an intermediate expression level in osteosarcoma clinical samples compared to osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone. Significance: This study provides an integrated analysis of miRNA and mRNA in osteosarcoma, and gives new insight into the complex genetic mechanisms of osteosarcoma development and progression.

Abstract 4000: Restoring LSAMP expression decreases the proliferation rate in osteosarcomas

Abstract Osteosarcomas are the most common primary malignant tumors of bone. The tumors are highly aggressive with poor prognosis, and display complex genomic aberrations. Previously, our group has identified a frequently deleted region in 3q13.31 in osteosarcoma clinical samples and cell lines [1]. The deleted region contains non-coding RNA genes, pseudogenes and the gene encoding the limbic-system associated membrane protein (LSAMP). The latter has previously been reported to be a candidate tumor suppressor gene in other cancer types, and more recently in osteosarcomas. Interestingly, our data show that low expression of LSAMP is statistically correlated with shorter patient survival. To examine the possible function of LSAMP in osteosarcomas, the expression was restored in an osteosarcoma cell line with a homozygous deletion of the gene. Characterization of the cell line with restored LSAMP expression showed decreased proliferation rate, which is consistent with its suggested role as a tumor suppressor gene. The migration rate and colony forming capability of the cells were unaffected. Interestingly, gene expression profiling of the restored lines, showed up-regulation of three genes, all proposed to have a role in cancer biology. The results indicate that the LSAMP protein might up-regulate the transcription of these three genes, and that LSAMP alone, or in conjunction with other genes, suppresses tumors by reducing their proliferation rate. Together, our data strengthens the hypothesis of LSAMP being a tumor suppressor gene in osteosarcomas. 1. Kresse, S.H., et al., LSAMP, a Novel Candidate Tumor Suppressor Gene in Human Osteosarcomas, Identified by Array Comparative Genomic Hybridization. Genes Chromosomes &amp; Cancer, 2009. 48(8): p. 679-693. 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 4000. doi:1538-7445.AM2012-4000

Frequent Attenuation of the WWOX Tumor Suppressor in Osteosarcoma Is Associated with Increased Tumorigenicity and Aberrant RUNX2 Expression

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P < 0.0001). Compared with the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorigenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas RUNX2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease.