RESUMEN
Ewing sarcoma (EwS) is the second most common bone cancer in children and adolescents with a high metastatic potential. EwS development is driven by a specific chromosomal translocation resulting in the generation of a chimeric EWS-ETS transcription factor, most frequently EWS-FLI1.Nicotinamide adenine dinucleotide (NAD) is a key metabolite of energy metabolism involved in cellular redox reactions, DNA repair, and in the maintenance of genomic stability. This study describes targeting nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of NAD synthesis, by FK866 in EwS cells. Here we report that blocking NAMPT leads to exhaustive NAD depletion in EwS cells, followed by a metabolic collapse and cell death. Using conditional EWS-FLI1 knockdown by doxycycline-inducible shRNA revealed that EWS-FLI1 depletion significantly reduces the sensitivity of EwS cells to NAMPT inhibition. Consistent with this finding, a comparison of 7 EwS cell lines of different genotypes with 5 Non-EwS cell lines and mesenchymal stem cells revealed significantly higher FK866 sensitivity of EWS-ETS positive EwS cells, with IC50 values mostly below 1nM.Taken together, our data reveal evidence of an important role of the NAMPT-mediated NAD salvage pathway in the energy homeostasis of EwS cells and suggest NAMPT inhibition as a potential new treatment approach for Ewing sarcoma.
Asunto(s)
Acrilamidas/farmacología , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/metabolismo , Piperidinas/farmacología , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/enzimología , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Citocinas/metabolismo , Resistencia a Antineoplásicos , Células HeLa , Humanos , NAD/metabolismo , Nicotinamida Fosforribosiltransferasa/metabolismo , Sarcoma de Ewing/tratamiento farmacológico , Sarcoma de Ewing/enzimología , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologíaRESUMEN
MicroRNAs serve to fine-tune gene expression and play an important regulatory role in tissue specific gene networks. The identification and validation of miRNA target genes in a tissue still poses a significant problem since the presence of a seed sequence in the 3'UTR of an mRNA and its expression modulation upon ectopic expression of the miRNA do not reliably predict regulation under physiological conditions. The chimeric oncoprotein EWS-FLI1 is the driving pathogenic force in Ewing sarcoma. MiR-17-92, one of the most potent oncogenic miRNAs, was recently reported to be among the top EWS-FLI1 activated miRNAs. Using a combination of AGO2 pull-down experiments by PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) and of RNAseq upon miRNA depletion by ectopic sponge expression, we aimed to identify the targetome of miR-17-92 in Ewing sarcoma. Intersecting both datasets we found an enrichment of PAR-CLIP hits for members of the miR-17-92 cluster in the 3'UTRs of genes up-regulated in response to mir-17-92 specific sponge expression. Strikingly, approximately a quarter of these genes annotate to the TGFB/BMP pathway, the majority mapping downstream of SMAD signaling. Testing for SMAD phosphorylation, we identify quiet but activatable TGFB signaling and cell autonomous activity of the BMP pathway resulting in the activation of the stemness regulatory transcriptional repressors ID1 and ID3. Taken together, our findings shed light on the complex miRegulatory landscape of Ewing Sarcoma pointing miR-17-92 as a key node connected to TGFB/BMP pathway.
Asunto(s)
Regiones no Traducidas 3'/genética , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular Tumoral , Predisposición Genética a la Enfermedad/genética , Humanos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , ARN Largo no Codificante , Proteína EWS de Unión a ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Análisis de Secuencia de ARN , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Ewing sarcoma (ES) is an aggressive pediatric tumor driven by the fusion protein EWS-FLI1. We report that EWS-FLI1 suppresses TDO2-mediated tryptophan (TRP) breakdown in ES cells. Gene expression and metabolite analyses reveal an EWS-FLI1-dependent regulation of TRP metabolism. TRP consumption increased in the absence of EWS-FLI1, resulting in kynurenine and kynurenic acid accumulation, both aryl hydrocarbon receptor (AHR) ligands. Activated AHR binds to the promoter region of target genes. We demonstrate that EWS-FLI1 knockdown results in AHR nuclear translocation and activation. Our data suggest that EWS-FLI1 suppresses autocrine AHR signaling by inhibiting TDO2-catalyzed TRP breakdown.
