Ewing sarcoma from molecular biology to the clinic.

In Europe, with an incidence of 7.5 cases per million, Ewing sarcoma (ES) is the second most common primary malignant bone tumor in children, adolescents and young adults, after osteosarcoma. Since the 1980s, conventional treatment has been based on the use of neoadjuvant and adjuvant chemotherapeutic agents combined with surgical resection of the tumor when possible. These treatments have increased the patient survival rate to 70% for localized forms, which drops drastically to less than 30% when patients are resistant to chemotherapy or when pulmonary metastases are present at diagnosis. However, the lack of improvement in these survival rates over the last decades points to the urgent need for new therapies. Genetically, ES is characterized by a chromosomal translocation between a member of the FET family and a member of the ETS family. In 85% of cases, the chromosomal translocation found is (11; 22) (q24; q12), between the EWS RNA-binding protein and the FLI1 transcription factor, leading to the EWS-FLI1 fusion protein. This chimeric protein acts as an oncogenic factor playing a crucial role in the development of ES. This review provides a non-exhaustive overview of ES from a clinical and biological point of view, describing its main clinical, cellular and molecular aspects.

LIM Kinases in Osteosarcoma Development.

Tumorigenesis is a long-term and multistage process that often leads to the formation of metastases. During this pathological course, two major events appear to be crucial: primary tumour growth and metastatic expansion. In this context, despite research and clinical advances during the past decades, bone cancers remain a leading cause of death worldwide among paediatric cancer patients. Osteosarcomas are the most common malignant bone tumours in children and adolescents. Notwithstanding advances in therapeutic treatments, many patients succumb to these diseases. In particular, less than 30% of patients who demonstrate metastases at diagnosis or are poor responders to chemotherapy survive 5 years after initial diagnosis. LIM kinases (LIMKs), comprising LIMK1 and LIMK2, are common downstream effectors of several signalization pathways, and function as a signalling node that controls cytoskeleton dynamics through the phosphorylation of the cofilin family proteins. In recent decades, several reports have indicated that the functions of LIMKs are mainly implicated in the regulation of actin microfilament and the control of microtubule dynamics. Previous studies have thus identified LIMKs as cancer-promoting regulators in multiple organ cancers, such as breast cancer or prostate cancer. This review updates the current understanding of LIMK involvement in osteosarcoma progression.

Involvement of the TGF-ß Signaling Pathway in the Development of YAP-Driven Osteosarcoma Lung Metastasis.

BACKGROUND: The poor survival rate of patients with osteosarcoma (OS), specifically with metastases at diagnosis, undergoes the urgency to develop new therapeutic strategies. Although we recently demonstrated the key role of YAP/TEAD signaling in the growth of OS primary tumor, the molecular mechanisms by which YAP regulates metastases development remain poorly understood. METHODS: The molecular mechanisms by which YAP regulates metastases development were studied using an overexpression of mutated forms of YAP able or not able to interact with TEAD. Molecular signatures were identified using RNA-sequencing analysis and gene set enrichment. Interactions between YAP and Smad3 were studied using proximity ligation assay (PLA), immunoprecipitation, and promoter/specific gene assays. The involvement of the TGF-ß pathway in the ability of YAP to stimulate metastatic development in vivo was studied using an inhibitor of the TGF-ß cascade in a preclinical model of OS and in vitro on the ability of OS cells to migrate and invade. RESULTS: Our work shows that a high YAP expression is associated with the presence of lung metastases which predicts a poor prognosis. Molecular analysis indicates that TGF-ß signaling is involved in YAP-driven osteosarcoma cell pro-migratory phenotype, epithelial mesenchymal transition, cell migration, and in vivo lung metastasis development. Regardless of its ability to bind to TEAD, YAP interacts with Smad3 and stimulates the transcriptional activity of TGF-ß/Smad3, thereby enhancing the ability of TGF-ß to stimulate lung metastasis development. CONCLUSIONS: We demonstrated the crucial involvement of the TGF-ß/Smad3 signaling pathway in YAP-driven lung metastasis development in OS.

Ubiquitin-specific proteases as therapeutic targets in paediatric primary bone tumours?

In children and young adults, primary malignant bone tumours are mainly composed of osteosarcoma and Ewing's sarcoma. Despite advances in treatments, nearly 40% of patients succumb to these diseases. In particular, the clinical outcome of metastatic osteosarcoma or Ewing's sarcoma remains poor, with less than 30% of patients who develop metastases surviving five years after initial diagnosis. Over the last decade, the cancer research community has shown considerable interest in the processes of protein ubiquitination and deubiquitination. In particular, a growing number of studies show the relevance to target the ubiquitin-specific protease (USP) family in various cancers. This review provides an update on the current knowledge regarding the implication of these USPs in the progression of bone sarcoma: osteosarcoma and Ewing's sarcoma.

Overexpression of the Ubiquitin Specific Proteases USP43, USP41, USP27x and USP6 in Osteosarcoma Cell Lines: Inhibition of Osteosarcoma Tumor Growth and Lung Metastasis Development by the USP Antagonist PR619.

Osteosarcoma (OS) is the most common malignant bone tumor in children and teenagers. In many cases, such as poor response to treatment or the presence of metastases at diagnosis, the survival rate of patients remains very low. Although in the literature, more and more studies are emerging on the role of Ubiquitin-Specific Proteases (USPs) in the development of many cancers, few data exist regarding OS. In this context, RNA-sequencing analysis of OS cells and mesenchymal stem cells differentiated or not differentiated into osteoblasts reveals increased expression of four USPs in OS tumor cells: USP6, USP27x, USP41 and USP43. Tissue microarray analysis of patient biopsies demonstrates the nucleic and/or cytoplasmic expression of these four USPs at the protein level. Interestingly, Kaplan-Meyer analysis shows that the expression of two USPs, USP6 and USP41, is correlated with patient survival. In vivo experiments using a preclinical OS model, finally demonstrate that PR619, a USP inhibitor able to enhance protein ubiquitination in OS cell lines, reduces primary OS tumor growth and the development of lung metastases. In this context, in vitro experiments show that PR619 decreases the viability of OS cells, mainly by inducing a caspase3/7-dependent cell apoptosis. Overall, these results demonstrate the relevance of targeting USPs in OS.

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.

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.

Connexin43 intercellular communication drives the early differentiation of human bone marrow stromal cells into osteoblasts.

Although it has been demonstrated that human bone marrow stromal cells (hBMSCs) express the ubiquitous connexin43 (Cx43) and form functional gap junctions, their role in the early differentiation of hBMSCs into osteoblasts remains poorly documented. Using in vitro assays, we show that Cx43 expression and gap junctional intercellular communication (GJIC) are increased during the differentiation of hBMSCs into osteoblasts, both at the protein and mRNA levels. Two independent procedures to reduce GJIC, a pharmacological approach with GJIC inhibitors (18α-glycyrrhetinic acid and Gap27 peptide) and a molecular approach using small interfering RNA against Cx43, demonstrated that the presence of Cx43 and functional junctional channels are essential to the ability of hBMSCs to differentiate into osteoblasts in vitro. In addition, a reduced GJIC decreases the expression of Runx2, the major transcription factor implicated in the control of osteoblast commitment and early differentiation of hBMSCs into osteoblasts, suggesting that GJIC mediated by Cx43 is implicated in this process. Together our results demonstrate that GJIC mediated by the Cx43 channels plays a central role throughout the differentiation of hBMSC into osteoblasts, from the early stages to the process of mineralization.

Analysis of gap junctional intercellular communications using a dielectrophoresis-based microchip.

Gap junctions are transmembrane structures that directly connect the cytoplasm of adjacent cells, making intercellular communications possible. It has been shown that the behaviour of several tumours - such as bone tumours - is related to gap junction intercellular communications (GJIC). Several methodologies are available for studying GJIC, based on measuring different parameters that are useful for multiple applications, such as the study of carcinogenesis for example. These methods nevertheless have several limitations. The present manuscript describes the setting up of a dielectrophoresis (DEP)-based lab-on-a-chip platform for the real-time study of Gap Junctional Intercellular Communication between osteosarcoma cells and the main cells accessible to their microenvironment. We conclude that using the DEParray technology for the GJIC assessment has several advantages comparing to current techniques. This methodology is less harmful for cells integrity; cells can be recovered after interaction to make further molecular analysis; it is possible to study GJIC in real time; we can promote cell interactions using up to five different populations. The setting up of this new methodology overcomes several difficulties to perform experiments for solving questions about GJIC process that we are not able to do with current technics.

TGF-ß Signaling in Bone Remodeling and Osteosarcoma Progression.

Osteosarcomas are the most prevalent malignant primary bone tumors in children. Despite intensive efforts to improve both chemotherapeutics and surgical management, 40% of all osteosarcoma patients succumb to the disease. Specifically, the clinical outcome for metastatic osteosarcoma remains poor; less than 30% of patients who present metastases will survive five years after initial diagnosis. Treating metastatic osteosarcoma thus remains a challenge. One of the main characteristics of osteosarcomas is their ability to deregulate bone remodelling. The invasion of bone tissue by tumor cells indeed affects the balance between bone resorption and bone formation. This deregulation induces the release of cytokines or growth factors initially trapped in the bone matrix, such as transforming growth factor-ß (TGF-ß), which in turn promote tumor progression. Over the past years, there has been considerable interest in the TGF-ß pathway within the cancer research community. This review discusses the involvement of the TGF-ß signalling pathway in osteosarcoma development and in their metastatic progression.

Anticancer activity of halofuginone in a preclinical model of osteosarcoma: inhibition of tumor growth and lung metastases.

Osteosarcoma is the main malignant primary bone tumor in children and adolescents for whom the prognosis remains poor, especially when metastases are present at diagnosis. Because we recently demonstrated that TGF-ß/Smad cascade plays a crucial role in osteosarcoma metastatic progression, we investigated the effect of halofuginone, identified as an inhibitor of the TGF-ß/Smad3 cascade, on osteosarcoma progression. A preclinical model of osteosarcoma was used to evaluate the impact of halofuginone on tumor growth, tumor microenvironment and metastasis development. In vivo experiments showed that halofuginone reduces primary tumor growth and lung metastases development. In vitro experiments demonstrated that halofuginone decreases cell viability mainly by its ability to induce caspase-3 dependent cell apoptosis. Moreover, halofuginone inhibits the TGF-ß/Smad3 cascade and the response of TGF-ß key targets involved in the metastases dissemination process such as MMP-2. In addition, halofuginone treatment affects the "vicious cycle" established between tumor and bone cells, and therefore the tumor-associated bone osteolysis. Together, these results demonstrate that halofuginone decreased primary osteosarcoma development and associated lung metastases by targeting both the tumor cells and the tumor microenvironment. Using halofuginone may be a promising therapeutic strategy against tumor progression of osteosarcoma specifically against lung metastases dissemination.