OS Metastasis Research Articles

Abstract 3948: An inhibitor of uPA reduces osteosarcoma metastasis by blocking signaling in tumour cells and the bone marrow microenvironment .

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Pulmonary metastasis is the major complication of OS and results in just 10-20% long-term survival. We have shown that metastatic potential is an inherent characteristic of OS, and that the bone microenvironment contributes to OS metastasis (Endo-Munoz et al. Cancer Res 70:7063-72, 2010). Specifically, loss of osteoclasts increases the metastatic potential of OS, and bone marrow cells (BMC) increase the migration of OS cells. However, the factors regulating inherent and BMC-mediated metastasis have not been elucidated. We have used a multi-omics approach to identify genes and proteins that may mediate OS metastasis. We found very high levels of mRNA and protein expression and secretion of the urokinase plasminogen activator (uPA) and its receptor (uPAR) exclusively in metastatic OS. Addition of uPA-rich OS conditioned medium to metastatic OS cells significantly (P < 0.007) increased their basal in vitro migration to levels similar to those observed with recombinant human uPA. On the other hand, overall migration was significantly (P < 0.005) inhibited by addition of a neutralizing mAb against uPAR or an uPA inhibitor. Silencing of uPAR also abrogated the response to uPA and decreased metastasis in an orthotopic model, indicating that uPA/uPAR signaling acts in a positive feedback loop in the regulation of inherent OS metastasis. To investigate whether BMC could also be signaling through uPA/uPAR, we performed a comparative transcriptomic analysis of metastatic and non-metastatic OS cell lines before and after treatment with BMC conditioned medium, and found that uPA was the most significantly (FC = 7.97, B = 18.82) upregulated gene after BMC treatment. Moreover, immunohistochemistry on femurs of mice bearing OS tumours showed high expression of uPA in the bone marrow as well as in the leading edges of the tumour. In addition, BMC conditioned medium containing high levels of secreted uPA also significantly increased metastatic OS cell migration. Our data indicate that increased uPA gene expression induced by BMC could contribute to enhanced secretion of uPA by OS cells, which in turn could augment signaling through uPAR to increase migration. In addition, BMC secrete uPA into the microenvironment to further increase migration of OS tumour cells in close proximity. Finally, we used a therapeutic inhibitor of uPA in an orthotopic mouse model of OS. The uPA inhibitor reduced metastasis significantly (P = 0.0004) in the treated group. This opens the possibility that uPA inhibitors, which are already in clinical trials for a number of cancers, may also be a useful therapeutic in the treatment for OS metastasis. Citation Format: Liliana B. Endo-Munoz, Na Cai, Andrew Cumming, Dorothy Loo, Michelle Hill, Pamela Mukhopadhyay, Nicholas A. Saunders. An inhibitor of uPA reduces osteosarcoma metastasis by blocking signaling in tumour cells and the bone marrow microenvironment . [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 3948. doi:10.1158/1538-7445.AM2013-3948

Abstract 3092: Deregulation of miR-10b in osteosarcoma.

Abstract Osteosarcoma (OS) is the most common primary bone malignancy found in children and adolescents, but the underlying molecular pathogenesis is not yet well understood. Utilizing a genetically engineered mouse model of OS that faithfully recapitulates salient features of the human disease, we are able to examine potential molecular contributions, such as that of a class of small, regulatory RNAs called microRNAs (miRNAs), in the processes of tumor proliferation and tumor progression and/or metastasis. MiRNA expression profile of tumors isolated from primary and secondary (metastatic) tumor sites of the mouse model revealed significant differential expression of miRNAs between the two groups. MiR-10b was significantly down-regulated in metastatic tumors in comparison with their primary counterparts, and thus was hypothesized to play important roles in the process of metastatic progression and/or tumorigenesis. To determine its impact on invasiveness and/or proliferation of the tumor cells, miR-10b was re-expressed in metastatic cell lines and introduced through tail-vein or intramuscular injections into scid/nude mice. Quantitative analysis using a bioluminescence imaging system showed delayed metastatic progression, lessened pulmonary tumor burden, as well as decreased primary tumor size in animals injected with tumor cells over-expressing miR-10b as compared to the controls. These data suggest that miR-10b down-regulation contributes to the processes of metastasis and tumorigenesis in OS through altered interaction with its target genes. Currently, putative miR-10b gene targets generated by microarray and RNA-seq analysis are being validated to provide a better understanding of the molecular mechanisms underlying miR-10b regulation in OS, and to facilitate the discovery of pathways amenable to manipulation with novel therapeutics. Citation Format: K. Amy Chen, Jordan P. Sand, Katherine A. Janeway, Stuart H. Orkin. Deregulation of miR-10b 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 3092. doi:10.1158/1538-7445.AM2013-3092

Abstract LB-366: Everolimus (EV) potentiates Sorafenib (SOR)activity in osteosarcoma (OS) preclinical models: a combination targeting the crosstalk between ERK1/2 and mTORC1/2 signaling pathways

Abstract OS is the most frequent primary bone tumor in children and young adults.The mammalian Target of Rapamycin (mTOR) and the Extracellular Regulated Kinases (ERK)1/2 pathways have been shown to cooperate in survival advantage of OS. Inhibition of this signaling can be a rational pharmacological strategy to impinge on OS progression. This study pursued the dual blockage of ERK1/2 and mTOR pathways by combining the multikinase inhibitor SOR with the rapamycin analog EV. The synergistic anti-profilerative effect was found against 5 out 7 OS cell lines (combination index<1, based on Chou-Talalay method). The inhibition of OS colony growth, due to cell cycle arrest and apoptosis induction was obtained. Mechanistically SOR alone, but EV, inhibited phospho-ERK1/2. EV and SOR as single agent induced the down-regulation of P-S6, P-4EBP. EV alone induced an increase in phospho-AKT as a consequence of the release of negative feed back by MTORC1 on MTORC2. Nonetheless, the combined treatment completely abrogated both ERK1/2 and MTORC1/2. The crosstalk among these pathways was interrupted by AMPK activation due to sorafenib-induced ROS burst and MTORC2 disassembling. The anti-tumoral activity and the inhibition of angiogenesis and metastasis in vivo were demonstrated against OS xenografts in NOD/SCID mice and in chicken chorioallantoic membrane model. A 28 daily oral gavage of mice (6/group) with SOR 5mg/kg, EV 1mg/kg, or their combination reduced tumor growth to 34%, 46%, and 29% of control slowing down cell proliferation and inducing apoptosis. In vivo model of OS metastasis obtained by e.v injection of OS cells confirmed the high metastatic potential of human OS consistently with the clinical experience. However, daily treatment of mice induced a significant reduction in number and size of lung metastases (foci area 65%, 74% and 29% of control obtained after 14 daily SOR, EV, and combined treatment). Anti-migratory effect was evidenced in 7 OS cell lines and was attributable to SRC/FAK/ERM pathway inhibition. HUVEC branching morphogenesis inhibition and reduction of blood vessel density in xenografts confirmed a strong potentiation of anti-angiogenic activity of drug combination. These results straighten our understanding of the networking nature of oncogenic signal transduction circuits in OS, therefore sustaining the rational for testing combination of sorafenib and everolimus in clinical setting. 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 LB-366. doi:1538-7445.AM2012-LB-366

Wnt signaling pathway and osteosarcoma

The Wnt pathway plays a crucial role in skeletal development and is indispensable for determination of OS cell lines. In recent years, experimental evidences on Wnt signaling pathway in OS cell lines and OS animal models, and that of Wnt signaling pathway as a diagnosis and prognosis marker of OS suggested that Wnt signaling pathway plays an important role in the progression, invasion and metastasis of OS. In addition, the strategy and safety evaluation to target Wnt to treat OS are underway. Exploring the significant role of Wnt signaling pathway in OS may aid in personalizing therapeutics to increase patient survival. Key words: Osteosarcoma; Wnt signaling pathway

Abstract 1459: Mining for metastasis genes in osteosarcomas from p53 +/R172H mice

Abstract Osteosarcoma (OS) is the most frequent bone cancer in young adults. The 5-year relapse free survival rates in nonmetastatic osteosarcomas are up to 65%, while metastatic osteosarcomas are only 10-20%. Therefore, identifying genes involved in OS metastasis is important to risk-stratify patients and enable development of novel therapeutic targets. In mouse models, loss of one p53 allele causes 34% of mice to develop OS without obvious metastasis; however, mice carrying a p53 missense mutation (R172H) develop metastatic OS. To screen for gene expression differences in metastatic potential between p53+/− and p53R172H/+ osteosarcomas, we used Affymetrix Mouse Genome 430 2.0 chips and compared three p53+/− and eight p53R172H/+ primary OS tumors. Two hundred thirteen genes were identified that had a p value < 0.05 and an average fold difference of 2. Only 20 of these genes had reported gene functions that affected cell migration, invasion, cell-cell junctions, or tumor phenotypes, or were selected based not only on statistical significance but also because they were consistently up (or down) regulated in all 8 R172H/+ tumors analyzed. Of these 20 genes, eight were validated by q-PCR in a larger group of tumors; S100A8, S100A9, PLA2G16, Plac8, and MAP kinase-interacting serine/threonine kinase 2 (Mnk2) were elevated while FAT4, FAT3, and Brdw1 showed decreased expression in mutant p53 tumors. To further characterize the functions of these genes in OS metastasis, we chose a metastatic OS cell line (H318) derived from p53R172H/+ tumor that had lost the wild type p53 allele for short hairpin RNAs (shRNAs) knock-down experiments followed by screening for migration and invasion. Three of these genes, PLA2G16, Plac8, and Mnk2, showed effects on migration and invasion in vitro. Further studies led us to focus on PLAG2G16. Knock-down of PLA2G16 showed decreased migration, invasion and proliferation. Additionally, we also subcutaneously injected PLAG2G16 knock-down cells into congenic wild type mice. Remarkably these cells showed dramatic decrease in tumor progression and metastasis. These effects were reproducible in another cell line with a different p53 mutant, 4T1. To determine whether we could make a non-metastatic cell adopt metastatic properties by simply expressing PLA2G16, we expressed FLAG-tagged PLA2G16 in two p53-null cell lines that lacked metastatic potential. These cells showed a 2-fold and 7-fold increase in invasion in culture, respectively. These preliminary data suggest that the expression of PLA2G16 in mutant p53 cell lines contributes to migration, invasion, and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1459. doi:10.1158/1538-7445.AM2011-1459

Abstract 4917: PRKCE and other genetic networks in osteosarcoma metastasis

Abstract Osteosarcoma (OS) is the most common malignant bone cancer with approximately 1, 000 new cases reported annually in North America. Unfortunately OS has a poor 5-year survival rate (∼60%), and this is attributable largely to its propensity towards pulmonary metastasis: roughly a fifth (20%) of newly diagnosed cases are accompanied by macroscopic pulmonary metastases, and it is estimated that four fifths (80%) of all cases have undetectable microscopic metastases at diagnosis. The aim of the present study was to identify and characterize genetic networks that contribute to the metastasis of OS. Gene-expression profiling was performed on a cohort of 63 primary OS tumor samples: 46 with undetectable metastasis at diagnosis (‘localized’), and 17 that had detectable pulmonary metastasis at time of diagnosis (‘metastatic’). The relative abundance of transcripts from approximately 9, 000 genes was measured. This information was overlain on the Pathway Commons human interactome, and two independent methods of supervised network analysis were performed. Those networks with significantly differential gene expression, or ‘activity’ (Chuang et al. Mol. Sys. Biol. 2007), and those networks with significantly differential internal gene correlation, or ‘organization’ (Taylor et al. Nature Biotech. 2009) were discovered. Networks were assigned to biological processes using Gene Ontology Slim Generic. Real-time PCR was performed in vitro and in vivo to corroborate and validate the initial findings. The supervised network analysis has elucidated the 43 most significantly differentially activated, and 11 most significantly differentially organized networks in metastatic OS. One candidate differentially activated network, comprising RASGRP3, PRKCE and GNB2, was selected for follow-up. In vitro analysis in a panel of paired OS cell lines has corroborated the findings of the profiling screen: RASGRP3 was found to be under-expressed in some highly metastatic sub-lines, and PRKCE was found to be over-expressed in some highly metastatic sub-lines. Independent validation of PRKCE expression in the original tumor cohort has confirmed the increased expression in metastatic samples. A less stringent inspection of the network results reveals global trends in network aberrations: differentially activated networks are commonly found in transport, translation and protein modification processes, while dis-organized networks are commonly found in metabolism, signaling, transcription and organization processes. The study here described has revealed significant network aberrations in metastatic OS primary tumours. Additionally, several high-confidence networks have been identified for detailed follow-up. Subsequent in vitro and in vivo expression analysis has confirmed that one network is differentially expressed in metastatic OS. Knockdown assays are ongoing to demonstrate the impact of this network on OS metastatic progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4917. doi:10.1158/1538-7445.AM2011-4917

Abstract LB-298: The bone marrow microenvironment increases osteosarcoma tumour cell migration by signaling through uPA/uPAR

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Pulmonary metastasis is the major complication of OS and can occur in up to 50% of cases, resulting in just 10–20% long-term survival for these patients. We have shown that the bone microenvironment contributes to the metastatic potential of osteosarcoma (Endo-Munoz et al. Cancer Res 70:7063–72, 2010). Specifically, loss of osteoclasts increases the metastatic potential of OS in vitro and in vivo, and bone marrow cells (BMC) increase the migration of OS cells in vitro. However, the factors mediating this increase in migration has not been elucidated. Using a combined transcriptomic and proteomic (SILAC) approach, we found very high levels of mRNA and protein expression of the urokinase plasminogen activator (uPA) and its receptor (uPAR) in metastatic, but not in non-metastatic OS. Furthermore, examination of the cytokines secreted in the conditioned medium of metastatic and non-metastatic OS cells in an antibody array showed high and selective secretion of uPA by metastatic OS cells only. Addition of this conditioned medium to metastatic OS cells significantly (P < 0.007) increased their in vitro migration to levels similar to those observed with recombinant human uPA. No effect was observed for non-metastatic cells. Overall migration was significantly (P < 0.005) inhibited by addition of a neutralizing monoclonal antibody against uPAR, indicating that uPA/uPAR signaling acts in a positive feedback loop in the regulation of inherent OS cell migration. To investigate whether the uPA/uPAR system could also be involved in signaling by BMC, we performed a comparative transcriptomic analysis of metastatic and non-metastatic OS cell lines before and after treatment with BMC conditioned medium. We found uPA to be the most significantly upregulated gene after BMC treatment (FC = 7.97, B = 18.82), suggesting that increased uPA gene expression induced by BMC could contribute to enhanced secretion of uPA by OS cells, which in turn could augment signaling through uPAR to increase migration. Finally, we explored the possibility that BMC may also secrete uPA into the microenvironment to further increase migration of OS tumours in close proximity. Immunohistochemistry on femurs of mice bearing OS tumours showed high expression of uPA in the bone marrow as well as in the leading edges of the tumour. These data indicate that BMC increases OS cell migration not only by increasing uPA gene expression in the tumour, but also by secreting uPA which serves to amplify the migration-inducing signaling of tumour-secreted uPA. This opens the possibility that uPA inhibitors, which are already in clinical trials for a number of cancers, may also be a useful therapeutic in the treatment for OS metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-298. doi:10.1158/1538-7445.AM2011-LB-298

Prospective validation of the prognostic value of elevated serum vascular endothelial growth factor in patients with nasopharyngeal carcinoma: More distant metastases and shorter overall survival after treatment

The purpose of this prospective study was to investigate the prognostic value of serum vascular endothelial growth factor (sVEGF) in patients with nasopharyngeal carcinoma (NPC). sVEGF was prospectively detected in 306 patients with NPC with enzyme-linked immunosorbent assay before treatment. The correlations between sVEGF and the survival of these patients were evaluated. Patients were followed for at least 36 months. The mean sVEGF was 387.0 ng/L. sVEGF showed no difference in sex, age, and local recurrence. However, sVEGF was positively associated with histology, TNM classification, distant metastasis, and overall survival (OS; p < .05). The 4-year OS and distant metastasis-free survival (DMFS) of the high-sVEGF versus low-sVEGF groups were 68% versus 86% and 70% versus 89%, respectively (p < .05). Stratified analysis showed that patients with stage IV(a,b) , T(4) , N(1) , or N(positive) disease with high VEGF levels had worse 4-year OS and 4-year DMFS than those with low VEGF levels (p < .05). Multifactorial Cox regression confirmed sVEGF was among the independent prognostic factors. Elevation of sVEGF in patients with NPC predicts more posttreatment distant metastases and shorter OS and can be used as an independent poor prognostic indicator.

Abstract 1550: Small molecule inhibitors of ezrin as anti-metastatic agents in osteosarcoma

Abstract Osteosarcoma (OS) is the most common type of primary bone cancer and the third most common cancer in children and adolescents. Current combination modalities have improved OS treatment, but did not have a major improvement on patient survival. Thus targeting underlying molecular events can provide dramatic benefits especially for the treatment of patients with fatal metastatic disease. Recently it is shown in a mouse model of OS that high ezrin expression is linked to pulmonary metastasis, providing an early survival advantage for cancer cells reaching the lung. Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) family is conserved through evolution both structurally and functionally. By regulating membrane-cytoskeleton complexes, it plays key roles in normal cellular processes like maintenance of membrane dynamics, survival, adhesion, motility, cytokinesis, phagocytosis and integration of membrane transport with signaling pathways. Due to its critical role in these cellular functions and relevance to metastasis, ezrin is a validated molecular target for prevention of metastasis in OS. In order to identify small molecules that directly interact with recombinant ezrin protein, we screened a small molecule library by surface plasmon resonance technology (Biacore). Initial hits from this primary screen were then evaluated in functional assays. We tested our small molecules for inhibiting ezrin mediated chemotaxis in cell culture, mimicking ezrin morpholino phenotype in zebrafish development, inhibiting ezrin phosphorylation and ezrin-actin interaction, effecting xenopus embryo development and inhibiting lung metastasis of osteosarcoma cells in a mouse lung organ culture assay. Results from these secondary functional assays, binding kinetics values from Biacore experiments and drugability of the chemical structures allowed us to pick two lead compounds. In summary, we discovered two lead compounds that directly bind to ezrin protein with ∼10uM affinity and inhibit its function in multiple independent assays. These molecules form the foundation of new potential therapeutic agents for osteosarcoma metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1550.

Abstract 2348: Frzb/sFRP3 inhibits lung metastasis of osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignancy of bone. In spite of the recent advances in chemotherapy and surgery, 40-50% of the adolescent patients develop lung metastasis, 80% of patients with metastasis at diagnosis will relapse. Survival rate has shown no significant change over past 20 years. The molecular mechanism of metastasis of OS needs to be further explored and the novel anti-metastatic target needs to be developed. Our previous data showed that Frzb (also known as sFRP3), a secreted Fz-related protein, suppressed metastasis of fibrosarcoma. In this study, we examined the anti-metastatic effects of Frzb on OS, using the 143B and LM7 cell lines as a model. Frzb expression construct was stably transfected into 143B cells and LM7 cells. Over-expression of Frzb in OS cells was confirmed by real time PCR and western-blot. In a lung metastasis nude mice model, osteosarcoma LM7 cells expressing Frzb formed much fewer and much smaller lung metastatic nodules than control cells. Frzb also decreased in vitro cellular motility in a scratch wound assay. In order to determine the potential molecular mechanism underlying the anti-metastatic effect of Frzb on osteosarcoma, we examined the expression of c-Met, S100A4 and reversal of EMT (Epithelial Mesenchymal Transition). By real time PCR and western-blot assay, c-Met and S100A4, which were extensively reported to be involved in metastasis of different cancers, were both down-regulated by Frzb. However, most of epithelial markers (E-cadherin, keratin 8, keratin18, etc) and mesenchymal markers (N-cadherin, Vimentin, fibronectin) remained unchanged. Our study suggests that Frzb has strong anti-metastatic effect on osteosarcoma. The potential molecular mechanism may be through down-regulation of c-Met and S100A4 in OS cells by Frzb, instead of reversing the EMT. Therefore, strategies to over-express or deliver Frzb in osteosarcoma may lead to a reduction of lung metastasis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2348.

Osteosarcoma metastasis: Evidence for ACP5 downregulation and osteoclast involvement

nOsteosarcoma (OS) is the most common primary bone tumour innthe pediatric age group. Pulmonary metastasis, which can benpresent in up to 50% of cases, is the major fatal complicationnof osteosarcoma. While the long-term survival of patients withoutnmetastasis is 65-70%, the long-term survival of patients whondevelop metastasis is only 10-20%. Our gene expression profilingnstudy of primary osteosarcoma biopsies identified tartrate-resistantnacid phosphatase 5 (ACP5, TRAP), a marker of osteoclasts, whichnat the time of diagnosis, predicts with 93% accuracy those patientsnin our group that will progress to lung metastasis. These resultsnhave been confirmed by immunohistochemistry in the originalnpatient biopsies. Furthermore, within the metastatic patientngroup, the time for the development of detectable metastasisncorrelates directly with the level of ACP5 expression. We havenalso shown that the loss of ACP5 is associated with a loss ofnosteoclasts, and thatnin vitro, this loss of osteoclasts correlatesnwith enhanced osteosarcoma cell migration. We have used ninendifferent osteosarcoma cell lines to set up an orthotopic mousenmodel in which manipulation of osteoclast number and activity,nwith zoledronic acid or with a low calcium diet, is able tonmodulate the tumorigenicity and metastatic potential of osteosarcoma.nOur findings suggest that the loss of osteoclasts and concomitantndownregulation of ACP5 may contribute to OS metastasis, andnthat modulators of osteoclast number and activity may have ansignificant impact in patients at risk of metastasis.