RESUMEN
The Ewing sarcoma (ES) EWS-FLI1 chimeric oncoprotein is a prototypic aberrant ETS transcription factor with activating and repressive regulatory functions. We report that EWS-FLI1-repressed promoters are enriched in forkhead box (FOX) recognition motifs, and identify FOXO1 as a EWS-FLI1-suppressed regulator orchestrating a major subset of EWS-FLI1-repressed genes. In addition to FOXO1 regulation by direct promoter binding of EWS-FLI1, its subcellular localization and activity is regulated by cyclin-dependent kinase 2- and AKT-mediated phosphorylation downstream of EWS-FLI1. Restoration of nuclear FOXO1 expression in ES cells impaired proliferation and significantly reduced clonogenicity. Gene-expression profiling revealed a significant overlap between EWS-FLI1-repressed and FOXO1-activated genes. As a proof of principle for a potential therapeutic application of our findings, the treatment of ES cell lines with methylseleninic acid (MSA) reactivated endogenous FOXO1 in the presence of EWS-FLI1 in a dose- and time-dependent manner and induced massive cell death dependent on FOXO1. In an orthotopic xenograft mouse model, MSA increased FOXO1 expression in the tumor paralleled by a significant decrease in ES tumor growth. FOXO1 reactivation by small molecules may therefore serve as a promising strategy for a future ES-specific therapy.
Asunto(s)
Neoplasias Óseas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/metabolismo , Animales , Antineoplásicos/farmacología , Secuencia de Bases , Sitios de Unión , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Línea Celular Tumoral , Proliferación Celular , Secuencia de Consenso , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteína Forkhead Box O1 , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Silenciador del Gen , Humanos , Ratones , Proteínas de Fusión Oncogénica/genética , Compuestos de Organoselenio/farmacología , Fosforilación , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteína Proto-Oncogénica c-fli-1/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/genética , Transcripción Genética , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Though p53 mutations are rare in ES, there is a strong indication that p53 mutant tumours form a particularly bad prognostic group. As such, novel treatment strategies are warranted that would specifically target and eradicate tumour cells containing mutant p53 in this subset of ES patients. METHODS: PRIMA-1(Met), also known as APR-246, is a small organic molecule that has been shown to restore tumour-suppressor function primarily to mutant p53 and also to induce cell death in various cancer types. In this study, we interrogated the ability of APR-246 to induce apoptosis and inhibit tumour growth in ES cells with different p53 mutations. RESULTS: APR-246 variably induced apoptosis, associated with Noxa, Puma or p21(WAF1) upregulation, in both mutant and wild-type p53 harbouring cells. The apoptosis-inducing capability of APR-246 was markedly reduced in ES cell lines transfected with p53 siRNA. Three ES cell lines established from the same patient at different stages of the disease and two cell lines of different patients with identical p53 mutations all exhibited different sensitivities to APR-246, indicating cellular context dependency. Comparative transcriptome analysis on the three cell lines established from the same patient identified differential expression levels of several TP53 and apoptosis-associated genes such as APOL6, PENK, PCDH7 and MST4 in the APR-246-sensitive cell line relative to the less APR-246-sensitive cell lines. CONCLUSION: This is the first study reporting the biological response of Ewing sarcoma cells to APR-246 exposure and shows gross variability in responses. Our study also proposes candidate genes whose expression might be associated with ES cells' sensitivity to APR-246. With APR-246 currently in early-phase clinical trials, our findings call for caution in considering it as a potential adjuvant to conventional ES-specific chemotherapeutics.