NOX1 and NOX2: Two enzymes that promote endothelial-to-mesenchymal transition induced by melanoma conditioned media.

Tumor microenvironment plays an important role in melanoma progression. Recent studies reported endothelial cells (EC) are involved in endothelial-to-mesenchymal transition (EndMT). During this phenotypic switch, EC progressively lose their endothelial markers and acquire mesenchymal properties. Depending on their concentration, reactive oxygen species (ROS) can control tumor growth. In EC, ROS are mainly produced by NAPDH oxidases (NOX) such as NOX1 and NOX2. The aim of the present study was to determine the role of these enzymes in EndMT induced by conditioned media (CM) from SK-MEL 28 melanoma cells. The capacity of CM to induce EndMT in HUVEC after 24 h, 48 h or 72 h has been evaluated by following endothelial HUVECs proliferation, migration and their capacity to form capillary on ECMgel®. Furthermore, EndMT was confirmed by western blot and flow cytometry. To determine the role of NOX in EndMT, specific NOX1 and/or NOX2 inhibitors has been tested. TGF-ß2 + /- IL-1ß was used as positive control. ROS production was determined through DCFDA assay. An altered endothelial phenotype was found in CM-treated HUVECs. This phenotypic modification was correlated with a decrease in both capillary formation on ECMgel® and cell proliferation and an increase in cell migration. Exposure to CM for 48 h significantly enhanced intracellular HUVECs ROS production and this increase was prevented by the dual pharmacological inhibition of NOX1 and NOX2. Furthermore, inhibition of NOX1/2 also leads to a partial reversion of CM-induced EndMT. These data confirmed the role of NOX1 and NOX2 in EndMT induced by melanoma cancer cell secretome.

Pyridazinone Derivatives Limit Osteosarcoma-Cells Growth In Vitro and In Vivo.

Osteosarcoma is a rare primary bone cancer that mostly affects children and young adults. Current therapeutic approaches consist of combining surgery and chemotherapy but remain unfortunately insufficient to avoid relapse and metastases. Progress in terms of patient survival has remained the same for 30 years. In this study, novel pyridazinone derivatives have been evaluated as potential anti-osteosarcoma therapeutics because of their anti-type 4 phosphodiesterase activity, which modulates the survival of several other cancer cells. By using five-four human and one murine osteosarcoma-cell lines, we demonstrated differential cytotoxic effects of four pyridazinone scaffold-based compounds (mitochondrial activity and DNA quantification). Proapoptotic (annexin V positive cells and caspase-3 activity), anti-proliferative (EdU integration) and anti-migratory effects (scratch test assay) were also observed. Owing to their cytotoxic activity in in vitro conditions and their ability to limit tumor growth in a murine orthotopic osteosarcoma model, our data suggest that these pyridazinone derivatives might be hit-candidates to develop new therapeutic strategies against osteosarcoma.

ICG-001, an Inhibitor of the ß-Catenin and cAMP Response Element-Binding Protein Dependent Gene Transcription, Decreases Proliferation but Enhances Migration of Osteosarcoma Cells.

High-grade osteosarcomas are the most frequent malignant bone tumors in the pediatric population, with 150 patients diagnosed every year in France. Osteosarcomas are associated with low survival rates for high risk patients (metastatic and relapsed diseases). Knowing that the canonical Wnt signaling pathway (Wnt/ß-catenin) plays a complex but a key role in primary and metastatic development of osteosarcoma, the aim of this work was to analyze the effects of ICG-001, a CBP/ß-catenin inhibitor blocking the ß-catenin dependent gene transcription, in three human osteosarcoma cell lines (KHOS, MG63 and 143B). The cell proliferation and migration were first evaluated in vitro after ICG-001 treatment. Secondly, a mouse model of osteosarcoma was used to establish the in vivo biological effect of ICG-001 on osteosarcoma growth and metastatic dissemination. In vitro, ICG-001 treatment strongly inhibits osteosarcoma cell proliferation through a cell cycle blockade in the G0/G1 phase, but surprisingly, increases cell migration of the three cell lines. Moreover, ICG-001 does not modulate tumor growth in the osteosarcoma mouse model but, rather significantly increases the metastatic dissemination to lungs. Taken together, these results highlight, despite an anti-proliferative effect, a deleterious pro-migratory role of ICG-001 in osteosarcoma.

Sonic Hedgehog Signature in Pediatric Primary Bone Tumors: Effects of the GLI Antagonist GANT61 on Ewing's Sarcoma Tumor Growth.

Osteosarcoma (OS) and Ewing's sarcoma (ES) are the most common malignant bone tumors in children and adolescents. In many cases, the prognosis remains very poor. The Sonic hedgehog (SHH) signaling pathway, strongly involved in the development of many cancers, regulate transcription via the transcriptional factors Gli1-3. In this context, RNAseq analysis of OS and ES cell lines reveals an increase of some major compounds of the SHH signaling cascade in ES cells, such as the transcriptional factor Gli1. This increase leads to an augmentation of the transcriptional response of Gli1 in ES cell lines, demonstrating a dysregulation of Gli1 signaling in ES cells and thus the rationale for targeting Gli1 in ES. The use of a preclinical model of ES demonstrates that GANT61, an inhibitor of the transcriptional factor Gli1, reduces ES primary tumor growth. In vitro experiments show that GANT61 decreases the viability of ES cell, mainly through its ability to induce caspase-3/7-dependent cell apoptosis. Taken together, these results demonstrates that GANT61 may be a promising therapeutic strategy for inhibiting the progression of primary ES tumors.

Endothelial-to-Mesenchymal Transition in Cancer.

Cancer is one of the most important causes of morbidity and mortality worldwide. Tumor cells grow in a complex microenvironment constituted of immune, stromal, and vascular cells that supports growth, angiogenesis, and metastasis. Endothelial cells (ECs) are major components of the vascular microenvironment. These cells have been described for their plasticity and potential to transdifferentiate into mesenchymal cells through a process known as endothelial-to-mesenchymal transition (EndMT). This complex process is controlled by various factors, by which ECs convert into a phenotype characterized by mesenchymal protein expression and motile, contractile morphology. Initially described in normal heart development, EndMT is now identified in several pathologies, and especially in cancer. In this review, we highlight the process of EndMT in the context of cancer and we discuss it as an important adaptive process of the tumor microenvironment that favors tumor growth and dissemination but also resistance to treatment. Thus, we underline targeting of EndMT as a potential therapeutic strategy.

The YAP/TEAD Axis as a New Therapeutic Target in Osteosarcoma: Effect of Verteporfin and CA3 on Primary Tumor Growth.

Although some studies suggested that disruption of the Hippo signaling pathway is associated with osteosarcoma progression, the molecular mechanisms by which YAP regulates primary tumor growth is not fully clarified. In addition, the validation of YAP as a therapeutic target through the use of inhibitors in a preclinical model must be demonstrated. RNA-seq analysis and Kaplan-Meier assays identified a YAP signature in osteosarcoma patients and a correlation with patients' outcomes. Molecular and cellular analysis (RNAseq, PLA, immunoprecipitation, promoter/specific gene, proliferation, cell cycle assays) using overexpression of mutated forms of YAP able or unable to interact with TEAD, indicate that TEAD is crucial for YAP-driven cell proliferation and in vivo tumor growth. In addition, in vivo experiments using an orthotopic mice model of osteosarcoma show that two YAP/TEAD inhibitors, verteporfin and CA3, reduce primary tumor growth. In this context, in vitro experiments demonstrate that these inhibitors decrease YAP expression, YAP/TEAD transcriptional activity and cell viability mainly by their ability to induce cell apoptosis. We thus demonstrate that the YAP/TEAD signaling axis is a central actor in mediating primary tumor growth of osteosarcoma, and that the use of YAP inhibitors may be a promising therapeutic strategy against osteosarcoma tumor growth.

In vitro and in vivo discrepancy in inducing apoptosis by mesenchymal stromal cells delivering membrane-bound tumor necrosis factor-related apoptosis inducing ligand in osteosarcoma pre-clinical models.

BACKGROUND: Osteosarcoma (OS) is the most frequent pediatric malignant bone tumor. OS patients have not seen any major therapeutic progress in the last 30 years, in particular in the case of metastatic disease, which requires new therapeutic strategies. The pro-apoptotic cytokine Tumor necrosis factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL) can selectively kill tumor cells while sparing normal cells, making it a promising therapeutic tool in several types of cancer. However, many OS cell lines appear resistant to recombinant human (rh)TRAIL-induced apoptosis. We, therefore, hypothesized that TRAIL presentation at the membrane level of carrier cells might overcome this resistance and trigger apoptosis. METHODS: To address this, human adipose mesenchymal stromal cells (MSCs) transfected in a stable manner to express membrane-bound full-length human TRAIL (mbTRAIL) were co-cultured with several human OS cell lines. RESULTS: This induced apoptosis by cell-to-cell contact even in cell lines initially resistant to rhTRAIL. In contrast, mbTRAIL delivered by MSCs was not able to counteract tumor progression in this OS orthotopic model. DISCUSSION: This was partly due to the fact that MSCs showed a potential to support tumor development. Moreover, the expression of mbTRAIL did not show caspase activation in adjacent tumor cells.