Santiago Rello-Varona

Caveolin-1 promotes Ewing sarcoma metastasis regulating MMP-9 expression through MAPK/ERK pathway

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.

“(Not) All (Dead) Things Share the Same Breath”: Identification of Cell Death Mechanisms in Anticancer Therapy

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 importance of being dead: cell death mechanisms assessment in anti-sarcoma therapy

Cell death can occur through different mechanisms, defined by their nature and physiological implications. Correct assessment of cell death is crucial for cancer therapy success. Sarcomas are a large and diverse group of neoplasias from mesenchymal origin. Among cell death types, apoptosis is by far the most studied in sarcomas. Albeit very promising in other fields, regulated necrosis and other cell death circumstances (as so-called “autophagic cell death” or “mitotic catastrophe”) have not been yet properly addressed in sarcomas. Cell death is usually quantified in sarcomas by unspecific assays and in most cases the precise sequence of events remains poorly characterized. In this review, our main objective is to put into context the most recent sarcoma cell death findings in the more general landscape of different cell death modalities.

PO-477 Bcl-xl inhibition enhances dinaciclib-induced cell death in soft-tissue sarcoma cell lines

Introduction Metastatic disease in soft-tissue sarcomas (STSs) lack successful treatments. Mortality rates are indeed quite high. Dinaciclib is a representative of the new CDK inhibitor class of drugs. It preferentially targets CDK1 and CDK9, involved respectively in cell cycle and transcription regulation. Data in literature shows that Dinaciclib is a good candidate for combinatorial therapies. Material and methods We analysed Dinaciclib apoptotic induction (visualised by Flow Cytometry) in a series of different STSs established cell lines. Cell lines were thus categorised as Dinaciclib-sensitive or Dinaciclib-tolerant. Differences in relevant protein expression behaviour during treatment led to hypothesis proposal for key regulators. Validation of targets was performed by siRNA technology prior to engage in drug combination testing. Drugs safety and efficiency was finally addressed by in vivo experiments. Results and discussions Relevant differences in apoptotic extent and timing among cell lines were found. Responses varied from more that 75% of cell death (72 hour treatment) in liposarcoma 402–91 to a mere 25% in leiomyosarcoma SK-LMS-1. The inhibition status of anti-apoptotic protein Bcl-xL was identified as the main determinant of the rhythm and extent of apoptotic demise. Dinaciclib-tolerant cells kept Bcl-xL active for longer times and get rid of BIM faster than sensitive cells. Both Bcl-xL knock-down and chemical Bcl-xL inhibitors (BH3-mimetics) overcame Dinaciclib tolerance and triggered complete annihilation of cell cultures (95% of cell death after 24 hour). Once safely escalated, drug combination effectiveness was tested on mice engrafted tumours. Conclusion The group of CDK inhibitors can be employed as therapeutic agents for STSs. Levels of the Bcl-2 family of proteins inform about cell proneness to trigger apoptotic cell death. This information can be used to design combination approaches involving BH3-mimetics that enhance efficiency and reduce treatment resistance.

Abstract B19: Tumor-suppressive activities associated to MIR10A-5p expression in developmental sarcomas

Ewing sarcoma (ES) and alveolar rhabdomyosarcoma (ARMS) are pediatric sarcomas characterized by tumor-specific translocations. Besides acting as a direct modulator of transcription, fusion proteins appear to exert its oncogenic functions by epigenetic modifications on the transcriptome. Therefore, the identification of specific DNA methylation markers would be helpful for understanding their pathogenetic mechanism as well as for developing new therapeutic strategies. By using the Illumina Infinium HumanMethylation450 we have analyzed the methylome of ES and ARMS tumors and cell lines. ES and ARMS samples showed, among other epigenetic alterations, differential hypermethylation in the promoter of MIR10A-5p, thus suggesting an inhibition of its expression. Expression of MIR10A-5p is effectively low in ES and ARMS cell lines and patient samples and the treatment with the epigenetic modifier 5-aza restores its expression. MIR10A-5p stable overexpression in two ES (A673 and TC252) and two ARMS (Rh4 and RMS13) cell lines reduces proliferation, diminishes clonogenic growth, and decreases cellular migration. Cell death induced by MIR10A-5p reintroduction is only achieved in the p53wt ES cell line TC252. Proteomic profile of A673 MIR10A-5p cells is altered as assessed by iTRAQ. Protein kinase CRKL is one of the proteins downregulated due to MIR10A-5p reintroduction. Indeed, CRKL expression is also reduced in the other three MIR10A-5p stable overexpression models. Doxycycline-induced silencing of CRKL in ARMS cell line Rh30 provokes cell growth reduction in vitro and in vivo, G0/G1 arrest, and a decrease in the clonogenic capacity (Yeung CL et al., 2013). Interestingly, the migratory ability of Rh30 and A673 CRKL-silenced cells is also impaired. Thus, our results uncover MIR10A-5p as a putative tumor suppressor in ES and ARMS, suggesting a link between MIR10A-5p and CRKL. Further characterization of the oncogenic role of CRKL and the molecular mechanisms associated to MIR10A-5p tumor-suppressor activities in ES and ARMS is ongoing. Citation Format: David Herrero-Martin, Santiago Rello-Varona, Silvia Garcia-Monclus, Juan Huertas-Martinez, Olga Almacellas-Rabaiget, Lee J. Helman, Oscar M. Tirado. Tumor-suppressive activities associated to MIR10A-5p expression in developmental sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B19.

Cell Death Identification in Anticancer Therapy-Response.

We would like to thank Brown and colleagues ([1][1]) for their effort and their valuable comments on our review about cell death mechanisms in anticancer therapy. We understand their view on assessing the persistence of clonogenic survival from quantification of death records in anticancer research