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‐EphA2S897), 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‐EphA2S897. 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‐EphA2S897 correlates with aggressiveness in ES, so blocking its function may be a promising treatment.
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Ewing sarcoma (ES) tumors, the second most frequent bone tumors among children and adolescents, are very aggressive and tend to recur and metastasize. Lack of knowledge regarding the molecular mechanisms that regulate this metastatic process is the main reason for the lack of efficient therapeutics. Here, by using gain‐ and loss‐of‐function experiments and in vitro and in vivo assays, the authors established a correlation between ES aggressiveness and phosphorylation of the tyrosine kinase receptor EphA2. Altogether, the findings suggest that blocking EphA2 expression or its function with drugs or genetic tools may be of use for the treatment of ES.
Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. ...Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients.
Orthotopic models are based on the implantation of tumor cells directly into the organ of origin, which allows interaction between the cells and the surrounding host tissues.Here we describe a ...modified version of an orthotopic model that closely recapitulates the steps required for metastasis development in Ewing sarcoma: tumor cells are injected into the calf muscles of the mouse, and once the tumor reaches a certain volume, the muscles containing the tumor are surgically resected. This procedure involves a nonaggressive surgery of the muscle which allows for the survival of the mouse during a period of time that is long enough to enable the development of distant metastases. This spreading of tumor cells to metastatic sites in other organs takes place by a physiological mechanism similar to what occurs in human Ewing sarcoma.
Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence. Patients with the most aggressive histological variant have an unfavorable prognosis due to a high ...metastasis incidence. Lysyl oxidase-like 2 (LOXL2) is a lysyl oxidase, member of a family of extracellular matrix (ECM) crosslinking enzymes that recently have emerged as important regulators of tumor progression and metastasis. We report that LOXL2 is overexpressed in RMS, suggesting a potential role for LOXL2 in RMS oncogenic progression. Consistently, transient and stable LOXL2 knockdown decreased cell migratory and invasive capabilities in two ARMS cell lines. Furthermore, introduction of LOXL2 in RMS non-expressing cells using wild type or mutated (catalytically inactive) constructs resulted in increased cell migration, cell invasion and number and incidence of spontaneous lung metastasis in vivo, independently of its catalytic activity. To further study the molecular mechanism associated with LOXL2 expression, a pull-down assay on LOXL2-transfected cells was performed and analyzed by mass spectrometry. The intermediated filament protein vimentin was validated as a LOXL2-interactor. Thus, our results suggest an oncogenic role of LOXL2 in RMS by regulating cytoskeleton dynamics and cell motility capabilities.
LOXL2 emerges as a key participant in the malignant phenotype of metastatic ARMS. Graphical abstract showing the proposed role of LOXL2 in ARMS tumor phenotype. LOXL2 increases clonogenic, viability, migratory and invasive capabilities of ARMS cells, contributing to the metastatic process independently of its catalytic activity. Intracellular non-glycosylated LOXL2 is enough to increase cell migration and invasion, however, it does not affect cell viability or colony formation. Mechanistically, LOXL2 interacts with vimentin and increases its cleavage, which is partially dependent on calpains. Calpain-2, another novel LOXL2-partner, could have a role in this cleavage. Altogether, we propose a regulation of cytoskeleton dynamics by LOXL2, and therefore, a modulation on cell motility. Display omitted
•LOXL2 increases cell migration, invasion and metastasis in alveolar rhabdomyosarcoma.•Oncogenic LOXL2 functions are independent of its enzymatic activity.•Intracellular and non-glycosylated LOXL2 is sufficient to increase cell migration and invasion.•LOXL2 may be regulating cell motility by interacting with vimentin and calpain-2.
During the last decades, the knowledge of cell death mechanisms involved in anticancer therapy has grown exponentially. However, in many studies, cell death is still described in an incomplete ...manner. The frequent use of indirect proliferation assays, unspecific probes, or bulk analyses leads too often to misunderstandings regarding cell death events. There is a trend to focus on molecular or genetic regulations of cell demise without a proper characterization of the phenotype that is the object of this study. Sometimes, cancer researchers can feel overwhelmed or confused when faced with such a corpus of detailed insights, nomenclature rules, and debates about the accuracy of a particular probe or assay. On the basis of the information available, we propose a simple guide to distinguish forms of cell death in experimental settings using cancer cell lines.
The EphA2 receptor tyrosine kinase is overexpressed in most solid tumors and acts as the major driver of tumorigenesis. In this study, we developed a novel approach for targeting the EphA2 receptor ...using a 2′-fluoro-modified pyrimidine RNA aptamer termed ATOP. We identified the ATOP EphA2 aptamer using a novel bioinformatics strategy that compared aptamers enriched during a protein SELEX using recombinant human EphA2 and a cell-internalization SELEX using EphA2-expressing MDA231 tumor cells. When applied to EphA2-expressing tumor cell lines, the ATOP EphA2 aptamer attenuated tumor cell migration and clonogenicity. In a mouse model of spontaneous metastasis, the ATOP EphA2 aptamer slowed primary tumor growth and significantly reduced the number of lung metastases. The EphA2 ATOP aptamer represents a promising candidate for the development of next-generation targeted therapies that provide safer and more effective treatment of EphA2-overexpressing tumors.
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Santana-Viera and colleagues developed a novel 2′-F-Py RNA aptamer against EphA2 receptor using a combinatorial SELEX strategy. Its antitumorigenic properties shown in vitro and in vivo put forward this aptamer as a promising candidate for the development of aptamer-based therapeutics toward the many tumors overexpressing EphA2.
Abstract
Soft-tissue sarcomas (STS) are an uncommon and heterogeneous group of malignancies that result in high mortality. Metastatic STS have very bad prognosis due to the lack of effective ...treatments. Dinaciclib is a model drug for the family of CDK inhibitors. Its main targets are cell cycle regulator CDK1 and protein synthesis controller CDK9. We present data supporting Dinaciclib ability to inactivate
in vitro
different STS models at nanomolar concentrations. Moreover, the different rhythms of cell death induction allow us to further study into the mechanism of action of the drug. Cell death was found to respond to the mitochondrial pathway of apoptosis. Anti-apoptotic Bcl-x
L
was identified as the key regulator of this process. Already natural low levels of pro-apoptotic proteins BIM and PUMA in tolerant cell lines were insufficient to inhibit Bcl-x
L
as this anti-apoptotic protein showed a slow decay curve after Dinaciclib-induced protein synthesis disruption. Combination of Dinaciclib with BH3-mimetics led to quick and massive apoptosis induction
in vitro
, but
in vivo
assessment was prevented due to liver toxicity. Additionally, Bcl-x
L
inhibitor A-1331852 also synergized with conventional chemotherapy drugs as Gemcitabine. Thus, Bcl-x
L
targeted therapy arises as a major opportunity to the treatment of STS.
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.
Duchenne muscular dystrophy (DMD) is a fatal and incurable muscle degenerative disorder. We identify a function of the protease urokinase plasminogen activator (uPA) in mdx mice, a mouse model of ...DMD. The expression of uPA is induced in mdx dystrophic muscle, and the genetic loss of uPA in mdx mice exacerbated muscle dystrophy and reduced muscular function. Bone marrow (BM) transplantation experiments revealed a critical function for BM-derived uPA in mdx muscle repair via three mechanisms: (1) by promoting the infiltration of BM-derived inflammatory cells; (2) by preventing the excessive deposition of fibrin; and (3) by promoting myoblast migration. Interestingly, genetic loss of the uPA receptor in mdx mice did not exacerbate muscular dystrophy in mdx mice, suggesting that uPA exerts its effects independently of its receptor. These findings underscore the importance of uPA in muscular dystrophy.
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