Osteosarcoma-Derived Extracellular Vesicles Induce Lung Fibroblast Reprogramming.

Tumor-secreted extracellular vesicles (EVs) have been identified as mediators of cancer-host intercellular communication and shown to support pre-metastatic niche formation by modulating stromal cells at future metastatic sites. While osteosarcoma, the most common primary malignant bone tumor in children and adolescents, has a high propensity for pulmonary metastases, the interaction of osteosarcoma cells with resident lung cells remains poorly understood. Here, we deliver foundational in vitro evidence that osteosarcoma cell-derived EVs drive myofibroblast/cancer-associated fibroblast differentiation. Human lung fibroblasts displayed increased invasive competence, in addition to increased α-smooth muscle actin expression and fibronectin production upon EV treatment. Furthermore, we demonstrate, through the use of transforming growth factor beta receptor 1 (TGFBR1) inhibitors and CRISPR-Cas9-mediated knockouts, that TGFß1 present in osteosarcoma cell-derived EVs is responsible for lung fibroblast differentiation. Overall, our study highlights osteosarcoma-derived EVs as novel regulators of lung fibroblast activation and provides mechanistic insight into how osteosarcoma cells can modulate distant cells to potentially support metastatic progression.

Involvement and Clinical Aspects of MicroRNA in Osteosarcoma.

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents, but its pathogenesis has been difficult to establish because of its well-known heterogeneous nature. OS has been associated with genetic and cytogenetic abnormalities, which include function-impairing mutations in tumor suppressors and the activation of oncogenes. OS tumorigenesis has been linked to alterations of several genes characterized by a high level of genetic instability and recurrent DNA amplifications and deletions. MicroRNAs (miRNAs), 18-25-nucleotide noncoding RNAs, are critical for various biological processes like differentiation, cell growth and cell death. Dysregulation of miRNA expression leads to phenotypic and genotypic changes in cells, which leads to cancer. Studies on miRNAs have initiated a significant effect in both diagnosis and treatment of cancer. This review focuses on the current knowledge of clinical applications of miRNAs for the better diagnosis and management of OS.

Small-molecule screen identifies modulators of EWS/FLI1 target gene expression and cell survival in Ewing's sarcoma.

Ewing's sarcoma family of tumors (EFT) is characterized by the presence of chromosomal translocations leading to the expression of oncogenic transcription factors such as, in the majority of cases, EWS/FLI1. Because of its key role in Ewing's sarcoma development and maintenance, EWS/FLI1 represents an attractive therapeutic target. Here, we characterize PHLDA1 as a novel direct target gene whose expression is repressed by EWS/FLI1. Using this gene and additional specific well-characterized target genes such as NROB1, NKX2.2 and CAV1, all activated by EWS/FLI1, as a read-out system, we screened a small-molecule compound library enriched for FDA-approved drugs that modulated the expression of EWS/FLI1 target genes. Among a hit-list of nine well-known drugs such as camptothecin, fenretinide, etoposide and doxorubicin, we also identified the kinase inhibitor midostaurin (PKC412). Subsequent experiments demonstrated that midostaurin is able to induce apoptosis in a panel of six Ewing's sarcoma cell lines in vitro and can significantly suppress xenograft tumor growth in vivo. These results suggest that midostaurin might be a novel drug that is active against Ewing's cells, which might act by modulating the expression of EWS/FLI1 target genes.

Exploring the Role of Osteosarcoma-Derived Extracellular Vesicles in Pre-Metastatic Niche Formation and Metastasis in the 143-B Xenograft Mouse Osteosarcoma Model.

The pre-metastatic niche (PMN) is a tumor-driven microenvironment in distant organs that can foster and support the survival and growth of disseminated tumor cells. This facilitates the establishment of secondary lesions that eventually form overt metastasis, the main cause of cancer-related death. In recent years, tumor-derived extracellular-vesicles (EVs) have emerged as potentially key drivers of the PMN. The role of the PMN in osteosarcoma metastasis is poorly understood and the potential contribution of osteosarcoma cell-derived EVs to PMN formation has not been investigated so far. Here, we characterize pulmonary PMN development using the spontaneously metastasizing 143-B xenograft osteosarcoma mouse model. We demonstrate the accumulation of CD11b+ myeloid cells in the pre-metastatic lungs of tumor-bearing mice. We also establish that highly metastatic 143-B and poorly metastatic SAOS-2 osteosarcoma cell-derived EV education in naïve mice can recapitulate the recruitment of myeloid cells to the lungs. Surprisingly, despite EV-induced myeloid cell infiltration in the pre-metastatic lungs, 143-B and SAOS-2 EVs do not contribute towards the 143-B metastatic burden in the context of both spontaneous as well as experimental metastasis in severe-combined immunodeficient (SCID) mice. Taken together, OS-derived EVs alone may not be able to form a functional PMN, and may perhaps require a combination of tumor-secreted factors along with EVs to do so. Additionally, our study gives a valuable insight into the PMN complexity by providing the transcriptomic signature of the premetastatic lungs in an osteosarcoma xenograft model for the first time. In conclusion, identification of regulators of cellular and molecular changes in the pre-metastatic lungs might lead to the development of a combination therapies in the future that interrupt PMN formation and combat osteosarcoma metastasis.

The miR-143/145 Cluster, a Novel Diagnostic Biomarker in Chondrosarcoma, Acts as a Tumor Suppressor and Directly Inhibits Fascin-1.

Chondrosarcoma is the second most frequent bone sarcoma. Due to the inherent chemotherapy and radiotherapy resistance and absence of known therapeutic targets, clinical management is limited to surgical resection. Consequently, patients with advanced disease face a poor prognosis. Hence, elucidating regulatory networks governing chondrosarcoma pathogenesis is vital for development of effective therapeutic strategies. Here, miRNA and mRNA next generation sequencing of different subtypes of human chondrogenic tumors in combination with in silico bioinformatics tools were performed with the aim to identify key molecular factors. We identified miR-143/145 cluster levels to inversely correlate with tumor grade. This deregulation was echoed in the miRNA plasma levels of patients and we provided the first evidence that circulating miR-145 is a potential noninvasive diagnostic biomarker and can be valuable as an indicator to improve the currently challenging diagnosis of cartilaginous bone tumors. Additionally, artificial upregulation of both miRNAs impelled a potent tumor suppressor effect in vitro and in vivo in an orthotopic xenograft mouse model. A combined in silico/sequencing approach revealed FSCN1 as a direct target of miR-143/145, and its depletion phenotypically resembled miR-143/145 upregulation in vitro. Last, FSCN1 is a malignancy-promoting factor associated with aggressive chondrosarcoma progression. Our findings underscore miR-143/145/FSCN1 as important players in chondrosarcoma and may potentially open new avenues for specific therapeutic intervention options. © 2020 American Society for Bone and Mineral Research.

A bispecific antibody targeting IGF-IR and EGFR has tumor and metastasis suppressive activity in an orthotopic xenograft osteosarcoma mouse model.

Osteosarcoma is a highly aggressive bone cancer and the second most frequent cause of cancer-associated death in childhood and adolescence. Pulmonary metastases account for the high mortality rate in osteosarcoma patients. Therefore, novel therapeutic approaches, efficiently restraining the metastatic disease, are mandatory for a significant improvement of the currently poor patients' survival. Although initial studies with antibodies targeting insulin-like growth factor receptor (IGF-IR) showed promising potential for the treatment of patients with bone and soft tissue sarcomas, phase II clinical trials revealed variable results, which implied activation of alternative signaling pathways leading to therapy resistance. Since a cross-talk between IGF-IR and the epidermal growth factor receptor (EGFR) has been demonstrated in several cancer types, co-targeting of these two receptors was considered in the present study as a valuable therapeutic strategy to overcome single-agent treatment resistance in osteosarcoma. The effects of IGF-IR and/or EGFR targeting by intraperitoneal administration of the monospecific IGF-IR antibody R1507 or the EGFR antibody Cetuximab or the bispecific IGF-IR/EGFR antibody XGFR* on primary tumor growth and pulmonary metastasis were investigated in an intratibial human xenograft osteosarcoma mouse model. In vitro functional assays demonstrated that targeting IGF-IR and EGFR didn't affect osteosarcoma cell viability, but inhibited ligand-activated intracellular signaling and cell migratory capacity. The blocking potential of ligand-induced signaling in vitro was similar for all antibodies, but, in vivo, only XGFR* treatment significantly inhibited intratibial primary tumor growth and pulmonary metastasis. The therapeutic response to XGFR* was associated with an infiltration of innate immune system effector cells into the tumor microenvironment. Taken together, our study highlights the bispecific anti-IGF-IR/EGFR antibody XGFR* as an innovative promising effective candidate for the treatment of metastatic osteosarcoma and provides the rationale for future clinical studies.

Evaluation of F8-TNF-α in Models of Early and Progressive Metastatic Osteosarcoma.

The targeted delivery of tumor necrosis factor-α (TNF-α) with antibodies specific to splice isoforms of fibronectin [e.g., F8-TNF, specific to the extra-domain A (EDA) domain of fibronectin] has already shown efficacy against experimental sarcomas but has not yet been investigated in orthotopic sarcomas. Here, we investigated F8-TNF in a syngeneic K7 M2-derived orthotopic model of osteosarcoma as a treatment against pulmonary metastases, the most frequent cause of osteosarcoma-related death. Immunofluorescence on human osteosarcoma tissue confirmed the presence of EDA in primary tumors (PTs) as well as metastases. In mice, the efficacy of F8-TNF against PTs and early pulmonary metastases was evaluated. Intratibial PT growth was not affected by F8-TNF, yet early micrometastases were reduced possibly due to an F8-TNF-dependent attraction of pulmonary CD4+, CD8+, and natural killer cells. Furthermore, immunofluorescence revealed stronger expression of EDA in early pulmonary metastases compared with PT tissue. To study progressing pulmonary metastases, a hind limb amputation model was established, and the efficacy of F8-TNF, alone or combined with doxorubicin, was investigated. Despite the presence of EDA in metastases, no inhibition of progressive metastatic growth was detected. No significant differences in numbers of CD4+ or CD8+ cells or F4/80+ and Ly6G+ myeloid-derived cells were observed, although a strong association between metastatic growth and presence of pulmonary Ly6G+ myeloid-derived cells was detected. In summary, these findings demonstrate the potential of F8-TNF in activating the immune system and reducing early metastatic growth yet suggest a lack of efficacy of F8-TNF alone or combined with doxorubicin against progressing osteosarcoma metastases.

Plasma levels of miRNA-155 as a powerful diagnostic marker for dedifferentiated liposarcoma.

Atypic lipomatous tumors (ALT) and dedifferentiated liposarcomas (DDLS) are closely related liposarcoma subtypes, often difficult to distinguish but they exhibit an entirely different clinical outcome. Recently discovered regulatory functions of miRNAs in liposarcoma progression prompted us to investigate miRNAs as potential diagnostic biomarkers in liposarcoma with a main focus on circulating miRNAs for fast and reliable differential diagnosis. Tumor and blood samples of 35 patients with lipomatous lesions collected between June 2011 and September 2014 were analyzed by qRT-PCR. They included 10 lipomas, 7 ALT, 5 DDLS and 13 myxoid liposarcomas (MLS). Ten samples of normal fat tissue and blood from 20 healthy volunteers were used as controls. A meta-analysis of public data on miRNA expression in liposarcoma revealed 9 miRNAs with potential diagnostic power. Out of these, miRNA-155 was found significantly elevated in the circulation of DDLS patients as compared to the plasma levels detected in all other liposarcoma subtypes and in healthy subjects. miRNA-155 levels in the plasma samples correlated significantly (r=0.41, p=0.02) with those in corresponding tumor extracts. This correlation was even more pronounced in an analysis of plasma and tumor extracts of malignant liposarcoma subtypes alone (r=0.51, p=0.02). Receiver operating characteristic analysis indicated that plasma miRNA-155 levels have a high diagnostic accuracy for distinguishing DDLS from healthy subjects (AUC=0.91, p=0.005) and from lipomas (AUC=0.86, p=0.02), MLS (AUC=0.92, p=0.006) and most importantly ALT (AUC=0.91, p=0.01) patients. In conclusion, this study identified miRNA-155 as a first blood biomarker for the differential diagnosis of DDLS.

Targeting αvß3 and αvß5 integrins inhibits pulmonary metastasis in an intratibial xenograft osteosarcoma mouse model.

Osteosarcoma is an aggressive bone cancer that has a high propensity for metastasis to the lungs. Patients with metastatic disease face a very poor prognosis. Therefore, novel therapeutics, efficiently suppressing the metastatic process, are urgently needed. Integrins play a pivotal role in tumor cell adhesion, motility and metastasis. Here, we evaluated αvß3 and αvß5 integrin inhibition with cilengitide as a novel metastasis-suppressive therapeutic approach in osteosarcoma. Immunohistochemical analysis of αvß3 and αvß5 integrins expression in a tissue microarray of tumor specimens collected from osteosarcoma patients revealed that αvß5 integrin is mainly found on tumor cells, whereas αvß3 is predominantly expressed by stromal cells. In vitro functional assays demonstrated that cilengitide dose-dependently inhibited de novo adhesion, provoked detachment and inhibited migration of osteosarcoma cell lines. Cilengitide induced a decline in cell viability, blocked the cell cycle in the G1 phase and caused anoikis by activation of the Hippo pathway. In a xenograft orthotopic mouse model cilengitide minimally affected intratibial primary tumor growth but, importantly, suppressed pulmonary metastasis. The data demonstrate that targeting αvß3 and αvß5 integrins in osteosarcoma should be considered as a novel therapeutic option for patients with metastatic disease.

PI3K/AKT signaling modulates transcriptional expression of EWS/FLI1 through specificity protein 1.

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and is characterized by the presence of the balanced translocation t(11;22)(q24;q12) in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. This fusion protein is an essential oncogenic component of ES development which is necessary for tumor cell maintenance and represents an attractive therapeutic target. To search for modulators of EWS/FLI1 activity we screened a library of 153 targeted compounds and identified inhibitors of the PI3K pathway to directly modulate EWS/FLI1 transcription. Surprisingly, treatment of four different ES cell lines with BEZ235 resulted in down regulation of EWS/FLI1 mRNA and protein by ~50% with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14 bp minimal elements as being important for EWS/FLI1 transcription. We identified SP1 as modulator of EWS/FLI1 gene expression and demonstrated direct binding to one of these regions in the EWS/FLI1 promoter by EMSA and ChIP experiments. These results provide the first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer an additional molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.

Prognostic value and in vitro biological relevance of Neuropilin 1 and Neuropilin 2 in osteosarcoma.

Neoadjuvant chemotherapy in osteosarcoma increased the long-term survival of patients with localized disease considerably but metastasizing osteosarcoma remained largely treatment resistant. Neuropilins, transmembrane glycoproteins, are important receptors for VEGF dependent hyper-vascularization in tumor angiogenesis and their aberrant expression promotes tumorigenesis and metastasis in many solid tumors. Our analysis of Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2) immunostaining in a tissue microarray of 66 osteosarcoma patients identified NRP2 as an indicator of poor overall, metastasis-free and progression free survival while NRP1 had no predictive value. Patients with tumors that expressed NRP2 in the absence of NRP1 had a significantly worse prognosis than NRP1(-)/NRP2(-), NRP1(+) or NRP1(+)/NRP2(+) tumors. Moreover, patients with overt metastases and with NRP2-positive primary tumors had a significantly shorter survival rate than patients with metastases but NRP2-negative tumors. Furthermore, the expression of both NRP1 and NRP2 in osteosarcoma cell lines correlated to a variable degree with the metastatic potential of the respective cell line. To address the functional relevance of Neuropilins for VEGF signaling we used shRNA mediated down-regulation and blocking antibodies of NRP1 and NRP2 in the metastatic 143B and HuO9-M132 cell lines. In 143B cells, VEGFA signaling monitored by AKT phosphorylation was more inhibited by blocking of NRP1, whereas in HuO9-M132 cells NRP2 blocking was more effective indicating that NRP1 and NRP2 can substitute each other in the functional interaction with VEGFR1. Altogether, these data point to NRP2 as a powerful prognostic marker in osteosarcoma and together with NRP1 as a novel target for tumor-suppressive therapy.

Cell-based small-molecule compound screen identifies fenretinide as potential therapeutic for translocation-positive rhabdomyosarcoma.

A subset of paediatric sarcomas are characterized by chromosomal translocations encoding specific oncogenic transcription factors. Such fusion proteins represent tumor specific therapeutic targets although so far it has not been possible to directly inhibit their activity by small-molecule compounds. In this study, we hypothesized that screening a small-molecule library might identify already existing drugs that are able to modulate the transcriptional activity of PAX3/FOXO1, the fusion protein specifically found in the pediatric tumor alveolar rhabdomyosarcoma (aRMS). Towards this end, we established a reporter cell line based on the well characterized PAX3/FOXO1 target gene AP2ß. A library enriched in mostly FDA approved drugs was screened using specific luciferase activity as read-out and normalized for cell viability. The most effective inhibitor identified from this screen was Fenretinide. Treatment with this compound resulted in down-regulation of PAX3/FOXO1 mRNA and protein levels as well as in reduced expression of several of its direct target genes, but not of wild-type FOXO1, in a dose- and time-dependent manner. Moreover, fenretinide induced reactive oxygen species and apoptosis as shown by caspase 9 and PARP cleavage and upregulated miR-9. Importantly, it demonstrated a significant anti-tumor effect in vivo. These results are similar to earlier reports for two other pediatric tumors, namely neuroblastoma and Ewing sarcoma, where fenretinide is under clinical development. Our results suggest that fenretinide might represent a novel treatment option also for translocation-positive rhabdomyosarcoma.

[Alterations of c-Myc and c-erbB-2 genes in ovarian tumours].

INTRODUCTION: According to clinical and epidemiological studies, ovarian cancer ranks fifth in cancer deaths among women. The causes of ovarian cancer remain largely unknown but various factors may increase the risk of developing it, such as age, family history of cancer, childbearing status etc. This cancer results from a succession of genetic alterations involving oncogenes and tumour suppressor genes, which have a critical role in normal cell growth regulation. Mutations and/or overexpression of three oncogenes, c-erbB-2, c-Myc and K-ras, and of the tumour suppressor gene p53, have been frequently observed in a sporadic ovarian cancer. OBJECTIVE: The aim of the present study was to analyse c-Myc and c-erbB-2 oncogene alterations, specifically amplification, as one of main mechanisms of their activation in ovarian cancers and to establish a possible association with the pathogenic process. METHODS: DNA was isolated from 15 samples of malignant and 5 benign ovarian tumours, using proteinase K digestion, followed by phenol-chloroform isoamyl extraction and ethanol precipitation. C-Myc and c-erbB-2 amplification were detected by differential PCR. The level of gene copy increase was measured using the Scion image software. RESULTS: The amplification of both c-Myc and c-erbB-2 was detected in 26.7% of ovarian epithelial carcinoma specimens. Only one tumour specimen concomitantly showed increased gene copy number for both studied genes. Interestingly, besides amplification, gene deletion was also detected (26.7% for c-erbB-2). Most of the ovarian carcinomas with alterations in c-Myc and c-erbB-2 belonged to advanced FIGO stages. CONCLUSION: The amplification of c-Myc and c-erbB-2 oncogenes in ovarian epithelial carcinomas is most probably a late event in the pathogenesis conferring these tumours a more aggressive biological behaviour. Similarly, gene deletions point to genomic instability in epithelial carcinomas in higher clinical stages as the result of clonal evolution and selection.