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Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 24-10-2016, El sarcoma de Ewing se caracteriza por la presencia de translocaciones cromosómicas que originan genes de fusión entre el gen EWSR1 y diferentes miembros de la familia de factores de transcripción ETS, principalmente FLI1. El gen de fusión resultante, EWS-FLI1, es un factor de transcripción aberrante que desempeña un papel central en el origen del sarcoma de Ewing a través de la regulación transcripcional de otros genes diana. En esta Tesis mostramos que Sprouty 1 (SPRY1), un inhibidor downstream de los receptores del factor de crecimiento fibroblástico (FGFRs) y otros receptores activadores de Ras, es un gen regulado negativamente por EWS-FLI1 en células de sarcoma de Ewing A673. El análisis de la expresión de SPRY1 en una selección de células de sarcoma de Ewing mostró que SPRY1 no se expresa en estas líneas celulares, lo que sugiere que SPRY1 podría actuar como un supresor tumoral en células de sarcoma de Ewing. En concordancia con estos resultados, la re-expresión de SPRY1 inhibió la proliferación, el crecimiento clonogénico y la migración en tres líneas celulares diferentes de sarcoma de Ewing. Además, la re-expresión de SPRY1 inhibió la ruta de señalización de Ras/MAPK/ERK inducida por suero o FGFb. Por otro lado, al tratar las células de sarcoma de Ewing con el inhibidor de FGFR PD173074, se redujo la proliferación inducida por FGFb, la formación de colonias y el crecimiento de tumores in vivo de forma dependiente de dosis. Aunque la expresión de SPRY1 en células de sarcoma de Ewing es baja, SPRY1 se expresa de forma variable en las muestras de tumores primarios. El análisis de una cohorte grande de pacientes indica que los pacientes con tumores que presentaron una expresión mayor de SPRY1 tuvieron un mejor pronóstico. En resumen, los resultados mostrados en esta Tesis indican que la regulación negativa de SPRY1 mediada por EWS-FLI1 produce una desregulación de la proliferación celular inducida por FGFb, lo que sugiere que la ruta de señalización de FGFR/Ras/MAPK/ERK podría ser una diana interesante para el desarrollo de nuevas aproximaciones terapéuticas para esta devastadora enfermedad., Ewing sarcoma is characterized by chromosomal translocations fusing the EWSR1 gene with various members of the ETS family of transcription factors, most commonly FLI1. EWS-FLI1 is an aberrant transcription factor driving Ewing sarcoma tumorigenesis by either transcriptionally inducing or repressing specific target genes. Herein, we showed that Sprouty 1 (SPRY1), which is a physiological negative feedback inhibitor downstream of fibroblast growth factor (FGF) receptors (FGFRs) and other Ras-activating receptors, is an EWS-FLI1 repressed gene. EWS-FLI1 knock-down specifically increased the expression of SPRY1 while other Sprouty family members remained unaffected. Analysis of SPRY1 expression in a panel of Ewing sarcoma cells showed that SPRY1 was not expressed in Ewing sarcoma cell lines, suggesting that it could act as a tumor suppressor gene in these cells. In agreement, induction of SPRY1 in three different Ewing sarcoma cell lines functionally impaired proliferation, clonogenic growth and migration. In addition, SPRY1 expression inhibited ERK/MAPK signalling induced by serum and basic FGF (bFGF). Moreover, treatment of Ewing sarcoma cells with the potent FGFR inhibitor PD173074 reduced bFGF-induced proliferation, colony formation and in vivo tumor growth in a dose-dependent manner, thus mimicking SPRY1 activity in Ewing sarcoma cells. Although the expression of SPRY1 was low when compared to other tumors, SPRY1 was variably expressed in primary Ewing sarcoma tumors and higher expression levels were significantly associated with improved outcome in a large patient cohort. Taken together, our data indicate that EWS-FLI1-mediated repression of SPRY1 leads to unrestrained bFGF-induced cell proliferation, suggesting that targeting the FGFR/Ras/MAPK/ERK pathway can constitute a promising therapeutic approach for this devastating disease., A la Red Temática de Investigación Cooperativa en Cáncer (RTICC) por la financiación concedida (RD12/0036/0027). Me gustaría agradecer en especial a la Asociación Pablo Ugarte por su fantástica labor, por haber depositado su confianza en nosotros y por haber financiado de forma sostenida este proyecto (TPY-M 1149/13)

Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second “European interdisciplinary Ewing sarcoma research summit” assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intratumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits. Unión Europea 261743(ENCCA) Unión Europea 259348 (ASSET)

International audience; Deciphering the ways in which somatic mutations and germline susceptibility variants cooperate to promote cancer is challenging. Ewing sarcoma is characterized by fusions between EWSR1 and members of the ETS gene family, usually EWSR1-FLI1, leading to the generation of oncogenic transcription factors that bind DNA at GGAA motifs. A recent genome-wide association study identified susceptibility variants near EGR2. Here we found that EGR2 knockdown inhibited proliferation, clonogenicity and spheroidal growth in vitro and induced regression of Ewing sarcoma xenografts. Targeted germline deep sequencing of the EGR2 locus in affected subjects and controls identified 291 Ewing-associated SNPs. At rs79965208, the A risk allele connected adjacent GGAA repeats by converting an interspaced GGAT motif into a GGAA motif, thereby increasing the number of consecutive GGAA motifs and thus the EWSR1-FLI1-dependent enhancer activity of this sequence, with epigenetic characteristics of an active regulatory element. EWSR1-FLI1 preferentially bound to the A risk allele, which increased global and allele-specific EGR2 expression. Collectively, our findings establish cooperation between a dominant oncogene and a susceptibility variant that regulates a major driver of Ewing sarcomagenesis

Purpose The relevance of the subfamily A members of ATP-binding cassette (ABCA) transporters as biomarkers of risk and response is emerging in different tumors, but their mechanisms of action have only been partially defined. In this work, we investigated their role in Ewing sarcoma (EWS), a pediatric cancer with unmet clinical issues. Methods The expression of ABC members was evaluated by RT-qPCR in patients with localized EWS. The correlation with clinical outcome was established in different datasets using univariate and multivariate statistical methods. Functional studies were conducted in cell lines from patient-derived xenografts (PDXs) using gain- or loss-of-function approaches. The impact of intracellular cholesterol levels and cholesterol lowering drugs on malignant parameters was considered. Results We found that ABCA6, which is usually poorly expressed in EWS, when upregulated became a prognostic factor of a favorable outcome in patients. Mechanistically, high expression of ABCA6 impaired cell migration and increased cell chemosensitivity by diminishing the intracellular levels of cholesterol and by constitutive IGF1R/AKT/mTOR expression/activation. Accordingly, while exposure of cells to exogenous cholesterol increased AKT/mTOR activation, the cholesterol lowering drug simvastatin inhibited IGF1R/AKT/mTOR signaling and prevented Ser166 phosphorylation of MDM2. This, in turn, favored p53 activation and enhanced pro-apoptotic effects of doxorubicin. Conclusions Our study reveals that ABCA6 acts as tumor suppressor in EWS cells via cholesterol-mediated inhibition of IGF1R/AKT/MDM2 signaling, which promotes the pro-apoptotic effects of doxorubicin and reduces cell migration. Our findings also support a role of ABCA6 as biomarker of EWS progression and sustains its assessment for a more rational use of statins as adjuvant drugs.

In this article an incorrect affiliation had inadvertently been added. The original article has been updated.