Ewing sarcoma (ES) is the second most common bone malignancy affecting children and young adults with poor prognosis due to high metastasis incidence. Our group previously described that EphA2, a tyrosine kinase receptor, promotes angiogenesis in Ewing sarcoma (ES) cells via ligand-dependent signaling. Now we wanted to explore EphA2 ligand-independent activity, controlled upon phosphorylation at S897 (p-EphA2 ), as it has been linked to metastasis in several malignancies. By reverse genetic engineering we explored the phenotypic changes after EphA2 removal or reintroduction. Gene expression microarray was used to identify key players in EphA2 signaling. Mice were employed to reproduce metastatic processes from orthotopically implanted engineered cells. We established a correlation between ES cells aggressiveness and p-EphA2 . Moreover, stable overexpression of EphA2 in low EphA2 expression ES cells enhanced proliferation and migration, but not a non-phosphorylable mutant (S987A). Consistently, silencing of EphA2 reduced tumorigenicity, migration and invasion in vitro, and lung metastasis incidence in experimental and spontaneous metastasis assays in vivo. A gene expression microarray revealed the implication of EphA2 in cell signaling, cellular movement and survival. ADAM19 knockdown by siRNA technology strongly reproduced the negative effects on cell migration observed after EphA2 silencing. Altogether, our results suggest that p-EphA2 correlates with aggressiveness in ES, so blocking its function may be a promising treatment.
Angiogenesis is the result of the combined activity of the tumor microenvironment and signaling molecules. The angiogenic switch is represented as an imbalance between pro- and anti-angiogenic factors and is a rate-limiting step in the development of tumors. Eph receptor tyrosine kinases and their membrane-anchored ligands, known as ephrins, constitute the largest receptor tyrosine kinase (RTK) subfamily and are considered a major family of pro-angiogenic RTKs. Ewing sarcoma (EWS) is a highly aggressive bone and soft tissue tumor affecting children and young adults. As other solid tumors, EWS are reliant on a functional vascular network for the delivery of nutrients and oxygen and for the removal of waste. Based on the biological roles of EphA2 in promoting angiogenesis, we explored the functional role of this receptor and its relationship with caveolin-1 (CAV1) in EWS angiogenesis. We demonstrated that lack of CAV1 results in a significant reduction in micro vascular density (MVD) on 3 different in vivo models. In vitro, this phenomenon correlated with inactivation of EphA2 receptor, lack of AKT response and downregulation of bFGF. We also demonstrated that secreted bFGF from EWS cells acted as chemoattractant for endothelial cells. Furthermore, interaction between EphA2 and CAV1 was necessary for the right localization and signaling of the receptor to produce bFGF through AKT and promote migration of endothelial cells. Finally, introduction of a dominant-negative form of EphA2 into EWS cells mostly reproduced the effects occurred by CAV1 silencing, strongly suggesting that the axis EphA2-CAV1 participates in the promotion of endothelial cell migration toward the tumors favoring EWS angiogenesis.
Ewing sarcoma (ES) is a bone and soft tissue sarcoma affecting mostly children and young adults. Caveolin-1 (CAV1) is a well-known target of EWS/FLI1, the main driver of ES, with an oncogenic role in ES. We have previously described how CAV1 is able to induce metastasis in ES via matrix metalloproteinase-9 (MMP-9). In the present study we showed how CAV1 silencing in ES reduced MEK1/2 and ERK1/2 phosphorylation. Accordingly, chemical inhibition of MEK1/2 resulted in reduction in MMP-9 expression and activity that correlated with reduced migration and invasion. IQ Motif Containing GTPase Activating Protein 1 (IQGAP1) silencing reduced MEK1/2 and ERK1/2 phosphorylation and MMP-9 expression. Furthermore, IQGAP1 silenced cells showed a marked decrease in their migratory and invasive capacity. We demonstrated that CAV1 and IQGAP1 localize in close proximity at the cellular edge, thus IQGAP1 could be the connecting node between CAV1 and MEK/ERK in ES metastatic phenotype. Analysis of the phosphorylation profile of CAV1-silenced cells showed a decrease of p-ribosomal protein S6 (RPS6). RPS6 can be phosphorylated by p90 ribosomal S6 kinases (RSK) proteins. CAV1-silenced cells showed reduced levels of p-RSK1 and treatment with U0126 provoked the same effect. Despite not affecting ERK1/2 and RPS6 phosphorylation status neither MMP-9 expression nor activity, RSK1 silencing resulted in a reduced migratory and invasive capacity in vitro and reduced incidence of metastases in vivo in a novel orthotopic model. The present work provides new insights into CAV1-driven metastatic process in ES unveiling novel key nodes.
Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence. Despite advances in therapy, patients with histological variant of rhabdomyosarcoma known as alveolar rhabdomyosarcoma (ARMS) have a 5-year survival of less than 30%. Caveolin-1 (CAV1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signaling. In the present study we report that compared to other forms of rhabdomyosarcoma (RMS) CAV1 expression is either undetectable or very low in ARMS cell lines and tumor samples. DNA methylation analysis of the promoter region and azacytidine-induced re-expression suggest the involvement of epigenetic mechanisms in the silencing of CAV1. Reintroduction of CAV1 in three of these cell lines impairs their clonogenic capacity and promotes features of muscular differentiation. In vitro, CAV1-expressing cells show high expression of Caveolin-3 (CAV3), a muscular differentiation marker. Blockade of MAPK signaling is also observed. In vivo, CAV1-expressing xenografts show growth delay, features of muscular differentiation and increased cell death. In summary, our results suggest that CAV1 could function as a potent tumor suppressor in ARMS tumors. Inhibition of CAV1 function therefore, could contribute to aberrant cell proliferation, leading to ARMS development.
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