Metastatic Potential Of Osteosarcoma Research Articles

Osteosarcoma patient-derived orthotopic xenograft (PDOX) models for identification of novel and effective therapeutics.

228 Background: Osteosarcoma is the most common malignant primary tumor of bone and mainly occurs in young generations. Due to the heterogeneity, rarity, poor response rate to systemic therapy, and metastatic potential of osteosarcoma, individualized precision medicine and novel drug discovery are greatly needed. Toward this goal, we have established the patient-derived orthotopic xenograft (PDOX) mouse model with surgical orthotopic implantation for all major cancers. The PDOX models recapitulate human tumors better than subcutaneous-transplanted xenografts including patient-derived xenograft (PDX). Metastasis is observed to a greater extent in PDOX models due to the intact histology and correct-organ tumor micro-environment of the orthotopically implanted tissue. Therefore, the PDOX model is highly predictive of drug efficacy and useful for identifying effective drugs for tumors in which new drugs are difficult to develop. Methods: We have reported 18 osteosarcoma PDOX studies evaluating approved and experimental drugs since 2017. The present report reviews our research group’s experience with the osteosarcoma-PDOX model, and the power of the PDOX models to identify effective therapeutics. Results: Effective treatment for drug-resistant osteosarcoma includes regorafenib, as monotherapy, and temozolomide-irinotecan, trabectedin-irinotecan, sorafenib-everolimus, sorafenib-palbociclib, and olaratumab-doxorubicin-cisplatin, as combinations. Experimental therapy with recombinant methioninase, tumor-targeting S. typhimurium A1-R, or combinations of these agents and other drugs have shown surprising efficacy in the recalcitrant-osteosarcoma PDOX models. Conclusions: Owing to the high concordance of drug efficacy between patients and their corresponding PDOX models, these models provide improved and personalized treatment options for patients with osteosarcoma. The patient does not need to suffer from the potential drug toxicity and morbidity of ineffective chemotherapies. In an era of growing promise of new treatment and precision medicine, PDOX models can offer a unique opportunity to provide specific and individualized therapy and novel therapeutic options for osteosarcoma patients.

M6A regulators are associated with osteosarcoma metastasis and have prognostic significance: A study based on public databases.

Background:Osteosarcoma represents the most common malignant bone tumor with high metastatic potential and inferior prognosis. RNA methylation (N6-methyladenosine [m6A]) is a prevalent RNA modification that epigenetically influences numerous biological processes including tumorigenesis. This study aims to determine that m6A regulators are significant biomarkers for osteosarcoma, and establish a prognostic model to predict the survival of patients.Methods:In this study, we comprehensively analyzed the underlying associations between m6A regulators’ mRNA expressions and metastasis as well as prognosis of osteosarcoma patients in the Cancer Genome Atlas. Multivariate Cox-regression analysis was used to screen regulators that were significantly associated with overall survival of osteosarcoma patients. Least absolute shrinkage and selection operator (LASSO) Cox-regression analysis was used for constructing m6A regulator-based osteosarcoma prognostic signature.Results:Some of the regulators exhibited aberrant mRNA levels between osteosarcoma samples with and without metastasis. Multivariate Cox-regression analysis identified several regulators with potential prognostic significance. A risk score formula consisted of methyltransferase-like 3, YTH domains of Homo sapiens, and fat mass and obesity-associated protein was obtained through which patients could be prognostically stratified independently of potential confounding factors. The signature was also significantly associated with the metastatic potential of osteosarcoma. All the analyses could be well reproduced in another independent osteosarcoma cohort from the Gene Expression Omnibus.Conclusions:In conclusion, this study first revealed potential roles of m6A regulators in osteosarcoma metastasis and prognosis, which should be helpful for its clinical decision-making.

Sorafenib Induces Apoptosis and Inhibits NF-κB-mediated Anti-apoptotic and Metastatic Potential in Osteosarcoma Cells.

Sorafenib, an oral multi-kinase inhibitor, has been shown to improve the outcome of patients with osteosarcoma (OS). However, the anti-OS effect and mechanism of sorafenib has not yet been fully understood. The main purpose of this study was to investigate the effect of sorafenib on apoptotic signaling and Nuclear Factor-κB (NF-κB)-mediated anti-apoptotic and metastatic potential in OS in vitro. The effect of sorafenib on apoptotic signaling transduction, anti-apoptotic, and metastatic potential of OS U-2 cells was verified with flow cytometry, trans-well invasion/migration, and western blotting assay. Sorafenib induced the extrinsic and intrinsic apoptotic pathways. In addition, sorafenib reduced the invasion and migration ability of OS cells, induced NF-κB activation, and the expression of anti-apoptotic proteins and metastasis-associated proteins encoded by NF-κB target genes. Sorafenib led to stimulation of extrinsic/intrinsic apoptotic pathways and NF-κB inactivation in U-2 OS cells.

Transcriptional activators YAP/TAZ and AXL orchestrate dedifferentiation, cell fate, and metastasis in human osteosarcoma

Osteosarcoma (OS) is a molecularly heterogeneous, aggressive, poorly differentiated pediatric bone cancer that frequently spreads to the lung. Relatively little is known about phenotypic and epigenetic changes that promote lung metastases. To identify key drivers of metastasis, we studied human CCH-OS-D OS cells within a previously described rat acellular lung (ACL) model that preserves the native lung architecture, extracellular matrix, and capillary network. This system identified a subset of cells—termed derived circulating tumor cells (dCTCs)—that can migrate, intravasate, and spread within a bioreactor-perfused capillary network. Remarkably, dCTCs highly expressed epithelial-to-mesenchymal transition (EMT)-associated transcription factors (EMT-TFs), such as ZEB1, TWIST, and SOX9, which suggests that they undergo cellular reprogramming toward a less differentiated state by coopting the same epigenetic machinery used by carcinomas. Since YAP/TAZ and AXL tightly regulate the fate and plasticity of normal mesenchymal cells in response to microenvironmental cues, we explored whether these proteins contributed to OS metastatic potential using an isogenic pair of human OS cell lines that differ in AXL expression. We show that AXL inhibition significantly reduced the number of MG63.2 pulmonary metastases in murine models. Collectively, we present a laboratory-based method to detect and characterize a pure population of dCTCs, which provides a unique opportunity to study how OS cell fate and differentiation contributes to metastatic potential. Though the important step of clinical validation remains, our identification of AXL, ZEB1, and TWIST upregulation raises the tantalizing prospect that EMT-TF-directed therapies might expand the arsenal of therapies used to combat advanced-stage OS.

Abstract B44: Role of EMT transcription factors in the metastatic potential of osteosarcoma

Abstract Objective: The main culprit for mortality in osteosarcoma (OS) is metastatic pulmonary disease, which originates from a highly selected group of tumor cells that acquire a heightened ability to migrate from the primary tumor, intravasate, and survive in the bloodstream while circulating to remote sites. The early steps of this process are thought to be regulated by epithelial-to-mesenchymal translation (EMT)-related transcription factors (EMT-TFs), which can surprisingly occur in nonepithelial malignancies such as glioma, leukemia, and sarcoma. To determine how EMT-TFs and other mediators of stemness contribute to the early steps of tumor metastasis, drug resistance, and ultimately to the shedding of circulating tumor cells (CTC), we explored the presence and role of these EMT-TFs such as SNAIL, ZEB1, TWIST, and AXL in OS. Methods: The acellularized rat lung (ACL) model used in our research provided a unique ability to isolate and characterize thousands of lab-derived circulating tumor cells (dCTCs) and compare them to the primary tumors formed in rat lung by injecting OS-D OS cell line. Expression of EMT-TFs (SNAIL, ZEB1, TWIST, AXL) in cells cultured in 2D monolayer, primary tumors (PT) formed in acellularized lung, and the derived CTCs were evaluated by immune-fluorescence (IF) staining using confocal microscopy and quantified by IMARIS software. We also compared the level of expression of EMT-TFs in parental OS cell line MG63, and its derivative metastatic cell lines MG63.2 and MG63.3 by IF and Western blotting. Circulating tumor cells were also isolated from OS patients for IF analysis of EMT-TFs and compared with their paired primary tumor. Results: The dCTCs collected from ACL OS model showed substantially higher expression of SNAIL, TWIST, and AXL, as well as considerably higher expression of ZEB1 compared with cells from 2D culture and PTs. AXL showed significantly increased expression in metastatic cell lines MG63.2 and MG63.3 compared with the parental cell line MG63. ZEB1, TWIST, or SNAIL did not replicate this difference in expression, likely due to the phenotypic changes that cells undergo when grown on the 2D monolayer. Results from the ACL experiments were validated by comparing the expression of AXL, TWIST, and ZEB1 in CTCs collected from patients with a limited set of paired primary patient tumors. Conclusion: An ACL model of the lung microenvironment yielded an opportunity to characterize lab-derived CTCs, using techniques that cannot readily be achieved from clinical specimens. Surprisingly, EMT-TFs were enriched in dCTCs, an important finding that suggests a small subset of OS cells—which are already high grade—can become even more stem-like as they navigate the initial steps required of early metastasis. Though we have just begun to validate this result using paired tumor/CTC clinical samples, early evidence confirms our lab findings. The identification of EMT-TF in OS CTCs suggests that antagonists of AXL, ZEB, or TWIST might impede the earliest steps in the metastatic cascade. Citation Format: Sana Mohiuddin, Salah-Eddine Lamhamedi-Cherradi, Dhruva K. Mishra, Kristi Pence, Sandhya Krishnan, Brian A. Mnegaz, Alejandra Ruiz Velasco, Danh Dinh Troung, Branko Cuglievan, Amelia Vetter, Eric R. Molina, Min P. Kim, Joseph A. Ludwig. Role of EMT transcription factors in the metastatic potential of osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B44.

MiR-186-5p Functions as a Tumor Suppressor in Human Osteosarcoma by Targeting FOXK1.

Aberrantly expressed miRNAs play a vital role in the development of some cancers, such as human osteosarcoma (OS). However, the detailed molecular mechanisms underlying miR-186-5p-involved osteosarcoma are unclear. qRT-PCR and western blot analysis were employed to measure the expressions of miR-186-5p and forkhead box k1 (FOXK1). CCK-8 assay evaluated the effect of miR-186-5p and FOXK1 on cell proliferation. Transwell assay confirmed cell migration and invasion. Eventually, the dual-luciferase reporter assay validated 3'-untranslated region (3'-UTR) of FOXK1 as a direct target of miR-186-5p. Down-regulation of miR-186-5p was identified in OS tissues and cell lines, and negatively correlated with distant metastasis, Enneking stage and poor 5-year prognosis as well as the expression of forkhead box k1 (FOXK1) protein. Further assays demonstrated that miR-186-5p overexpression had inhibitory effects on in-vitro cell proliferation, cell cycle, and in-vivo tumor growth. miR-186-5p overexpression also inhibited the epithelial-tomesenchymal transition (EMT), migration and invasion of OS cells. Importantly, miR-186-5p directly targeted FOXK1 3'-UTR and negatively regulated its expression. Silencing of FOXK1 expression enhanced the inhibitory effects of miR-186-5p on OS cell proliferation, migration and invasion. These findings highlighted miR-186-5p as a tumor suppressor in the regulation of progression and metastatic potential of OS, and may benefit the development of therapies targeting miR-186-5p in patients with OS.

The nuclear-cytoplasmic trafficking of a chromatin-modifying and remodelling protein (KMT2C), in osteosarcoma.

Osteosarcoma is the most common paediatric primary non-hematopoietic bone tumor; the survival is related to the response to chemotherapy and development of metastases. KMT2C is a chromatin-modifying and remodelling protein and its expression has never been studied in osteosarcoma. The aim of this study was to understand the role of KMT2C in the osteosarcoma carcinogenesis and metastatic progression to identify a new molecular target and to provide new therapeutic approach.We performed the immunohistochemical and gene expression analysis of KMT2C in 32 samples of patients with diagnosis of osteosarcoma with known clinic-pathological data and we analysed the expression of genes involved in the metastatic pathway in four osteosarcoma cell lines by blocking the KMT2C expression using siRNA.We found a nuclear-cytoplamic trafficking of KMT2C and the cytoplasmic localization was higher than the nuclear localization (p < 0.0001). Moreover, the percentage of cells with cytoplasmic positivity increased from low grade primary tissue to metastatic tissues. The cytoplasmic localization of KMT2C could lead to a change in its function supporting osteosarcoma carcinogenesis and progression. Our hypothesis is that KMT2C could affect the enhancer activity of genes influencing the invasive properties and metastatic potential of osteosarcoma.

Ras-Association Domain Family 1 Isoform A (RASSF1A) Gene Polymorphism rs1989839 is Associated with Risk and Metastatic Potential of Osteosarcoma in Young Chinese Individuals: A Multi-Center, Case-Control Study.

BackgroundThe ras-association domain family 1 isoform A (RASSF1A) gene serves as a bona fide tumor suppressor gene. The polymorphisms in RASSF1A were previously reported to be associated with the risk of solid malignant tumors. We hypothesized herein that RASSF1A gene polymorphisms are involved in the risk and prognosis of osteosarcoma (OS).Material/MethodsWe recruited 279 young OS cases and 286 tumor-free controls from the east Chinese population. Five tagSNPs of RASSF1A gene (rs2236947A/C, rs2073497A/C, rs1989839C/T, rs72932987C/T, and rs4688728G/T) were genotyped. DNA was isolated from blood samples and then underwent PCR analysis for genotyping.Resultsrs1989839C/T is an important predictor of osteosarcoma risk and outcome. The CT genotype of rs1989839 is highly related to elevated risk of osteosarcoma. Furthermore, rs1989839C/T is also associated with the Enneking stage of osteosarcoma and risk of lung metastasis. One of the other 4 SNPs, rs2236947A/C, shows a borderline significance in predicting osteosarcoma risk.ConclusionsOur study is the first to prove that RASSF1A gene polymorphisms may potentially be predictive for osteosarcoma risk and prognosis.

Amphiregulin enhances intercellular adhesion molecule-1 expression and promotes tumor metastasis in human osteosarcoma.

Osteosarcoma is a common, high malignant, and metastatic bone cancer. Amphiregulin (AREG) has been associated with cancer cellular activities. However, the effect of AREG on metastasis activity in human osteosarcoma cells has yet to be determined. We determined that AREG increases the expression of intercellular adhesion molecule-1 (ICAM-1) through PI3K/Akt signaling pathway via its interaction with the epidermal growth factor receptor, thus resulting in the enhanced cell migration of osteosarcoma. Furthermore, AREG stimulation increased the association of NF-κB to ICAM-1 promoter which then up-regulated ICAM-1 expression. Finally, we observed that shRNA silencing of AREG decreased osteosarcoma metastasis in vivo. Our findings revealed a relationship between osteosarcoma metastatic potential and AREG expression and the modulating effect of AREG on ICAM-1 expression.

Abstract 5378: Epigenetic regugulation of osteosarcoma metastatic phenotype via histone deacetylace inhibition

Abstract Introduction: The epigenetic modification of cancer cells regulates tumor development and metastasis. Epigenetic changes include DNA methylation and histone modification, both of which regulate gene expression but do not alter the genetic code. We have demonstrated differential DNA methylation status of tumor suppressor genes between highly metastatic K7M2 osteosarcoma (OS) cells and less metastatic K12 OS cells. The demethylation of tumor suppressor genes repressed the metastatic phenotype of K7M2 cells. Histone deacetylase inhibitors (HDAC inhibitors, HDIs) have been evaluated for epigenetic regulation of various malignancies, but the role of HDAC inhibitors in OS is unclear. Wehave investigated the effect of the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), on highly metastatic K7M2 OS cells. Methods: 1. Osteosarcoma cells: K7M2 and K12 are murineOS cell populations with differing metastatic potentials. K7M2 is highly metastatic to the lung but K12 is much less metastatic. 2. SAHA treatment: SAHA (25, 50, or 100 μM) in proliferation medium (10% FBS in DMEM) was used to culture the K7M2 cells. Cells were incubated with SAHA for 48 hours prior to fixation for observation, harvesting for mRNA isolation, or plating for invasion assays. Results: 1. SAHA treatment (25, 50, or 100 μM for 48 hours) significantly decreased cell proliferation compared with untreated cells. 2. SAHA treatment of K7M2 cells modified cell morphology. K7M2 cells were cultured with or without SAHA (25, 50, 100 μM) for 48 hours. Staining of the actin cytoskeleton revealed structural differences in SAHA-treated cells: treated cells displayed fewer invadopodia and their shape was more polygonal, resembling less metastatic K12 cells. 3. SAHA treatment of K7M2 cells modified the expression tumorigenesis and metastasis-related genes. Semi-quantitative polymerase chain reaction showed that the expressions of mammalian target of rapamycin (mTOR), aldehyde dehydrogenase-1 (ALDH-1), and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) were down-regulated in SAHA-treated K7M2 cells. The expression of microtubule-associated protein 1A/1B-light chain (LC3) was up-regulated. 4. K7M2 cells were cultured with or without SAHA (50 μM) for 2 days, and their invasion capacity through 2.5% matrigel was analyzed. SAHA treatment significantly reduced the cells’ invasion capacity. Conclusion: These data demonstrate that highly metastatic OS cells can be epigenetically modified with HDAC inhibition. SAHA treatment of K7M2 cells reduced both their proliferation and invasiveness, indicating that HDAC inhibition may potentially reduce tumor development and metastasis. The effect of SAHA on OS cells could be related to cytoskeletal changes and/or diminished expression of genes regulating tumor development and metastasis. In the future we will test the ability of SAHA to affect OS metastatic potential in vivo. Citation Format: Kurt R. Weiss, Xiaodong Mu, Daniel Brynien. Epigenetic regugulation of osteosarcoma metastatic phenotype via histone deacetylace inhibition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5378. doi:10.1158/1538-7445.AM2015-5378

Expression of c‐FLIP in pulmonary metastases in osteosarcoma patients and human xenografts

We have previously shown that Fas expression inversely correlates with the metastatic potential of osteosarcoma (OS) to the lung. FasL is constitutively expressed in the lung microenvironment and eliminates Fas(+) OS cells leaving Fas(-) cells to form metastases. Absence of FasL in the lung epithelium or blocking the Fas-signaling pathway interfered with this clearance mechanism allowing Fas(+) cells to remain and form lung metastases. We also demonstrated that while the majority of patient OS lung metastases were Fas(-), 10-20% of the lesions contained Fas(+) cells, suggesting that these cells were not sensitive to FasL-induced apoptosis. The expression of c-FLIP, an inhibitor of the Fas pathway, has been associated with tumor development, progression, and resistance to chemotherapy. We therefore evaluated the expression of c-FLIP in OS patient tumor specimens and human xenograft lung metastases. OS patient tissues, which included both primary and metastatic lesions, were evaluated for the expression of c-FLIP. In addition, tumors from human OS xenografts were examined for c-FLIP expression. c-FLIP expression was significantly higher in the lung metastases than in the primary tumors. c-FLIP may play an important role in the metastatic potential of OS to the lung. Inhibition of c-FLIP may be a future therapeutic target.

MS-275 sensitizes osteosarcoma cells to Fas ligand-induced cell death by increasing the localization of Fas in membrane lipid rafts

Fas expression is inversely correlated with the metastatic potential of osteosarcoma (OS) cells to the lungs. Fas+ cells are rapidly eliminated when they enter the lungs via their interaction with constitutive Fas ligand (FasL) on the lung epithelium, whereas Fas− OS cells escape this FasL-induced apoptosis and survive in the lung microenvironment. Upregulation of Fas expression in established OS lung metastases results in tumor regression. Here, we demonstrate that treatment of Fas− OS cells with the histone deacetylase inhibitor MS-275 results in the upregulation of Fas mRNA and sensitizes these cells to FasL-induced apoptosis. However, flow cytometry analysis revealed that Fas cell surface protein expression was not significantly increased. Rather, we observed increased levels of Fas within the membrane lipid rafts, as demonstrated by an increase in Fas expression in detergent-insoluble lipid raft fractions and colocalization with GM1+ lipid rafts. We had previously shown that MS-275 treatment inhibited expression of the anti-apoptotic cellular FLICE-inhibitory protein (c-FLIP). Here, we demonstrated that transfection of cells with short hairpin RNA to c-FLIP also resulted in the localization of Fas to lipid rafts. Overall, our studies indicate that MS-275 sensitizes OS cells to FasL by upregulating the expression of Fas in membrane lipid rafts, which correlates with the c-FLIP-dependent distribution of Fas to lipid rafts.

Current concepts on pathogenesis and biology of metastatic osteosarcoma tumors

Osteosarcoma (OS) remains the most common malignancy among orthopaedic neoplasms. Despite advanced surgical techniques and attempts to use second-line chemotherapy, 5 year overall survival in OS patients is still reported to be as low as 60-70%. Progression to metastatic disease is the main cause of treatment failures. Broadening current knowledge on the pathogenesis and biology of metastatic OS tumors is a key element in improving treatment results, i.e. identifying potential therapeutic targets. Recent studies have brought new concepts into this field. This paper outlines the most important issues which may influence treatment methods in the near future. In a few sections, we discuss (1) a model of OS dissemination with special regard to proteins mediating the lysis of the extracellular matrix; (2) the mechanisms protecting circulating OS cells from programmed death; (3) the relationship between angiogenesis, its pathogenesis, and OS metastatic potential; (4) the role of cytokines in OS progression and site-specific metastasis formation; (5) an example of treatment resistance mechanism - the P glycoprotein efflux pump; and, finally, we theorize on (6) whether cancer stem cells may play a role in OS progression.

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 &amp;lt; 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 &amp;lt; 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

Abstract 4869: p27kip1 is a metastasis-associated antigen that promotes cell migration in osteosarcoma

Abstract Metastasis is the leading cause of death in osteosarcoma (OS). Despite the advent of cancer therapy, patients with metastasis still have a dismal outcome. Hence, identification and characterization of new cellular targets that are critical to the metastatic process are urgently needed in order to improve both the treatment and detection of metastatic patients. Previous evidence has indicated that cancer patients produce autoantibodies. Many of these tumor-associated autoantibodies target cellular proteins that are essential for tumor formation. In this study, we used a high-density Human Protein Array (HPA) that contains over 8,000 purified proteins, to identify tumor-associated antigens that are associated with metastatic OS at the time of diagnosis. Plasma samples from patients with metastatic (n=10) or localized (n=16) OS, as well as hospitalized children with non-cancerous diseases (n=21) were pooled separately and analyzed using HPA. Totally, 16 autoantibodies were found to be reactive in metastatic OS. Of those, 6 of them showed higher prevalence in the metastatic cases relative to the localized cases and the control cases (p&amp;lt;0.01). One of these autoantibodies targets a cell cycle inhibitor protein, p27kip1, which is involved in tumor suppression and invasion. The higher prevalence of p27kip1 autoantibody in metastatic OS was subsequently validated using a novel HaloLink assay and individual plasma samples. To test if p27kip1 plays a role in metastatic OS, immunohistochemistry was performed on three pairs of metastatic OS cell lines and their parental, non-metastatic cell lines. We found that the p27kip1 protein, which is normally localized in the nucleus, was mislocalized in the cytoplasm in all three metastatic OS cell lines. Functional studies demonstrated that the wild type or CDK- mutant of p27kip1 harboring a nuclear export signal significantly induced OS cell migration in vitro. In summary, our results indicate that mislocalization of p27kip1 can promote metastatic potential of OS. Further characterization of p27kip1 and its related pathways may lead to the development of a novel therapeutic strategy for treating metastatic OS and other sarcomas. 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 4869. doi:10.1158/1538-7445.AM2011-4869

Loss of Osteoclasts Contributes to Development of Osteosarcoma Pulmonary Metastases

We conducted a transcriptomic screen of osteosarcoma (OS) biopsies and found that expression of osteoclast-specific tartrate-resistant acid phosphatase 5 (ACP5/TRAP) is significantly downregulated in OS compared with nonmalignant bone (P < 0.0001). Moreover, lesions from OS patients with pulmonary metastases had 2-fold less ACP5/TRAP expression (P < 0.018) than lesions from patients without metastases. In addition, we found a direct correlation (P = 0.0166) between ACP5/TRAP expression and time to metastasis. Therefore, we examined whether metastasis-competent (MC) OS cells could induce loss of ACP5(+) osteoclasts and contribute to metastasis. We found that MC OS cell lines can inhibit osteoclastogenesis in vitro and in vivo. In addition, osteoclasts can inhibit the migration of MC OS cells in vitro. Finally, ablation of osteoclasts with zoledronic acid increases the number of metastatic lung lesions in an orthotopic OS model, whereas fulvestrant treatment increases osteoclast numbers and reduces metastatic lesions. These data indicate that the metastatic potential of OS is determined early in tumor development and that loss of osteoclasts in the primary lesion enhances OS metastasis.

Abstract 1434: EMMPRIN expression is associated with metastatic progression in osteosarcoma

Abstract Introduction: Despite the improvement of survival in localized osteosarcoma patients, patients with metastasis still carry a poor prognosis. Thus, identification of factors contributing to metastasis is needed. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell-surface glycoprotein which plays multiple roles in physiologic and pathologic conditions. EMMPRIN is highly expressed in several cancers and stimulates adjacent stromal cells or tumor cells to produce matrix metalloproteinase (MMP). EMMPRIN also stimulates expression of vascular endothelial growth factor (VEGF) which leads to angiogenesis. These findings have made EMMPRIN an attractive therapeutic target in many cancer types. The goals of this study were; to investigate the expression of EMMPRIN in osteosarcoma; to examine if EMMPRIN, via tumor-stroma interaction, is associated with metastatic potential in osteosarcoma. Methods: Level of EMMPRIN mRNA expression was evaluated by RT-PCR in 6 tumor-derived osteosarcoma cell lines and by immunohistochemistry prechemotherapy biopsies from 52 patients. Clinical data of these patients were reviewed to examine the association of EMMPRIN expression and clinical outcome. Transfection of EMMPRIN-targeting siRNA in SaOS-2 was performed to test the role of EMMPRIN in osteosarcoma progression. To study the role of EMMPRIN in tumor-stromal interaction, co-culture of SaOS-2 with osteoblast (hFOB) was experimented. Functional in vitro assays that reflect metastatic potential of osteosarcoma cells were performed. MMP production, VEGF production, cell invasion and proliferation were studied by gelatin zymography, VEGF ELISA, Matrigel invasion assay and WST-1 assay, respectively. Results: EMMPRIN was expressed in 90% by immunohistochemistry with strong accentuation along the cell membrane. Level of EMMRIN mRNA expression was significantly higher in 5 of 6 tumor-derived cell lines compared to MG63. Patients with high EMMPRIN expression had significantly worse metastasis-free survival. EMMPRIN mRNA levels was significantly down-regulated by siRNA transfection compared to control-siRNA transfected cell. Co-culture of osteoblast and SaOS-2 enhanced stimulation of pro-MMP2. This stimulation was reversed by transfection of SaOS-2 with EMMPRIN-targeting siRNA. Number of cells crossing the chamber was statistically lower in EMMPRIN-siRNA transfected cells. When osteoblast and SaOS-2 were co-cultured, VEGF expression was increased compared to SaOS-2 only culture. EMMPRIN-targeting siRNA transfection resulted in decrease of VEGF expression. SaOS-2 cells transfected with EMMPRIN-siRNA showed decreased proliferation potential compared to control-siRNA transfected cells. Conclusion: Our data suggest that highly expressed EMMPRIN plays an important role in metastatic potential of osteosarcoma. EMMPRIN could serve as a potential therapeutic target in osteosarcoma. 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 1434.

Abstract 2064: Down-regulation of miR-31 is associated with metastatic potential of human and canine osteosarcoma

Abstract Osteosarcoma is the most common primary bone tumor in humans and dogs. The primary cause for the poor survival of patients with osteosarcoma in both species is the emergence of metastases in organs distant from the primary tumor, especially in the lung. Therefore, identification of molecular determinants of osteosarcoma metastasis is highly warranted. In order to identify microRNAs (miRNAs) that regulate osteosarcoma metastasis, we examined, using qRT-PCR, levels of 10 miRNAs commonly associated with cancer metastasis (miR-10b, miR-146a, miR182, miR183, miR-206, miR-335, miR373, miR-520c-5p, miR-21 and miR-31) in total RNA samples prepared from canine parental osteosarcoma (POS) and highly metastatic POS (HMPOS) cells. Of the 10 miRNAs, only miR-31 showed differential expression between the two cell lines (reduced by 7.8-fold in HMPOS cells versus POS cells). Exposure of HMPOS cells to various chemopreventive agents (myo-inositol, indole-3-carbinol, diindolylmethane, deguelin, phenethyl isothiocyanate, cisplatin, and aza-2′-deoxycytidine) increased the level of miR-31 at least by two-fold, the most potent agents being deguelin and phenethyl isothiocyanate. Subsequently, we assessed the expression of miR-31 in additional canine osteosarcoma cell lines (three cell lines established from primary tumors and three cell lines developed from pulmonary metastases) and human osteosarcoma cell lines differing in metastatic potential (parental SaOS2, low- (LM2), and high- (LM7) metastatic cells). miR-31 levels decreased by 18-fold in canine cell lines derived from pulmonary metastatic tumors relative to that obtained from primary tumors and by 2-fold and 3-fold in LM2 and LM7 cells, respectively, compared to the expression in parental Saos-2 cells. Also, analysis of 3 primary human osteosarcoma tissues and 3 pulmonary metastatic tissues revealed a 13-fold reduction of miR-31 in metastatic tissues relative to primary tumor tissues. In preliminary experiments where attempts were made to identify potential targets of miR-31, exogenous over-expression of a synthetic miR-31 cassette in HMPOS cells negatively regulated a tight junction protein claudin-2, which is incriminated in cancer metastasis. In summary, our studies indicate that miR-31 is a promising marker for the metastatic potential of osteosarcoma and for disease prognosis. Moreover, reactivation of silenced miR-31 by chemopreventive/chemotherapeutic agents could reverse 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 2064.

Wnt10b induces chemotaxis of osteosarcoma and correlates with reduced survival

Osteosarcoma (OS) is a primary malignant tumor of the bone that typically presents in the second decade of life and has a poor prognosis, especially in metastatic cases. Wnt signaling contributes to the pathogenesis of tumors such as colon cancer and malignant melanoma. Wnt signaling controls normal bone formation during embryogenesis and homeostasis in adult organisms, thus we evaluated Wnt signaling in OS. We surveyed the expression of Wnts, their receptors, Frizzleds and LRPs, and soluble Wnt inhibitors (sFRPs) in four OS cell lines by RT-PCR. We also tested biological response of OS cell lines to exogenous Wnts by measuring beta-catenin stabilization, Dvl phosphorylation, TOPFLASH activity and chemotaxis. Human OS tumor microarrays were evaluated for expression of Wnt10b by immunohistochemistry. All cell lines tested showed expression of at least three Wnts and one Frizzled. Exogenous Wnt3a and Wnt10b treatment induced Dvl phosphorylation, beta-catenin stabilization and TCF4 transcriptional activity in both metastatic and non-metastatic murine OS cell lines. Metastatic OS cell lines showed better chemotaxis response to Wnts than the non-metastatic OS cell lines. Immunohistochemistry studies of 44 human OS samples demonstrated that Wnt10b expression correlated with decreased overall survival. These results further supports a possible autocrine or paracrine Wnt pathway in metastatic potential of OS.

Fas Expression in Lung Metastasis From Osteosarcoma Patients

The authors' animal studies have shown that the metastatic potential of osteosarcoma (OS) cells correlates inversely with Fas expression-that is, Fas-negative cells metastasize but Fas-positive cells do not. One reason for this in the context of OS lung metastases may be that Fas-positive cells are eliminated by engagement with the Fas ligand (FasL) constitutively expressed on the surface of pneumocytes, whereas Fas-negative tumor cells are not. The purpose of this study was to determine the status of Fas expression in OS lung metastases from patients. Specifically, archived paraffin-embedded specimens of lung metastases from 38 patients with OS were analyzed by immunohistochemistry. Lung nodules from 23 of the 38 patients (60%) were Fas negative, those from 12 patients (32%) were weakly positive, and that from only 1 patient (3%) was strongly positive. Findings in the samples from the remaining two patients (5%) could not be interpreted because of extensive necrosis. Most patients with the weakly positive tumors and the single patient with the strongly positive tumor received chemotherapy prior to lung resection. There was a significant correlation between Fas expression and the administration of preoperative salvage chemotherapy (P = 0.0013). These data indicate that loss of Fas may be one mechanism by which OS cells evade host resistance in the lung. Chemotherapy may induce regression by upregulating Fas.