RESUMEN
Ezrin is a key regulator of cancer metastasis that links the extracellular matrix to the actin cytoskeleton and regulates cell morphology and motility. We discovered a small-molecule inhibitor, NSC305787, that directly binds to ezrin and inhibits its function. In this study, we used a nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS-MS)-based proteomic approach to identify ezrin-interacting proteins that are competed away by NSC305787. A large number of the proteins that interact with ezrin were implicated in protein translation and stress granule dynamics. We validated direct interaction between ezrin and the RNA helicase DDX3, and NSC305787 blocked this interaction. Downregulation or long-term pharmacological inhibition of ezrin led to reduced DDX3 protein levels without changes in DDX3 mRNA. Ectopic overexpression of ezrin in low-ezrin-expressing osteosarcoma cells caused a notable increase in DDX3 protein levels. Ezrin inhibited the RNA helicase activity of DDX3 but increased its ATPase activity. Our data suggest that ezrin controls the translation of mRNAs preferentially with a structured 5' untranslated region, at least in part, by sustaining the protein level of DDX3 and/or regulating its function. Therefore, our findings suggest a novel function for ezrin in regulation of gene translation that is distinct from its canonical role as a cytoskeletal scaffold at the cell membrane.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , ARN Helicasas DEAD-box/metabolismo , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Adamantano/análogos & derivados , Adamantano/farmacología , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Línea Celular Tumoral , Cromatografía Liquida , Proteínas del Citoesqueleto/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , Humanos , Ratones , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , Unión Proteica/efectos de los fármacos , Proteómica , Quinolinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Resonancia por Plasmón de Superficie , Espectrometría de Masas en TándemRESUMEN
Ewing tumor is driven by the oncogenic EWS-FLI1 fusion protein that functions as an aberrant transcription factor. The identification of EWS-FLI1 protein partners is essential to enhance its vulnerability as a therapeutic target. We utilized phage display library screening against recombinant EWS-FLI1 protein. We identified 27 unique Ewing sarcoma binding peptides. The cytotoxicity evaluation of these peptides with in EWS-FLI1 containing cell lines yielded one potent peptide called ESAP1 (TMRGKKKRTRAN). ESAP1 binds EWS-FLI1 with 0.202 micromolar affinity as measured in surface plasmon resonance. The minimal interaction region of ESAP1 is characterized and found that the lysine residues are critical for cellular cytotoxicity. ESAP1 reduces the transcriptional activity of EWS-FLI1 as well as disrupts cell cycle kinetics in Ewing Tumor cells. These findings provide both a novel experimental probe and a potential therapeutic scaffold for Ewing Tumor.
Asunto(s)
Proteínas de Fusión Oncogénica/metabolismo , Péptidos/metabolismo , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Sarcoma de Ewing/patología , Secuencia de Aminoácidos , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Humanos , Cinética , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/genética , Biblioteca de Péptidos , Unión Proteica , Proteína Proto-Oncogénica c-fli-1/antagonistas & inhibidores , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/antagonistas & inhibidores , Proteína EWS de Unión a ARN/genética , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de Superficie , Activación TranscripcionalRESUMEN
The Hedgehog (Hh) pathway is activated in some human cancers, including medulloblastoma. The glioma-associated oncogene homolog (GLI) transcription factors are critical mediators of the activated Hh pathway, and their expression may be elevated in some tumors independent of upstream Hh signaling. Thus, therapies targeting GLI transcription factors may benefit a wide spectrum of patients with mutations at different nodal points of the Hh pathway. In this study, we present evidence that arsenic trioxide (ATO) suppresses human cancer cell growth and tumor development in mice by inhibiting GLI1. Mechanistically, ATO directly bound to GLI1 protein, inhibited its transcriptional activity, and decreased expression of endogenous GLI target genes. Consistent with this, ATO inhibited the growth of human cancer cell lines that depended on upregulated GLI expression in vitro and in vivo in a xenograft model of Ewing sarcoma. Furthermore, ATO improved survival of a clinically relevant spontaneous mouse model of medulloblastoma with activated Hh pathway signaling. Our results establish ATO as a Hh pathway inhibitor acting at the level of GLI1 both in vitro and in vivo. These results warrant the clinical investigation of ATO for tumors with activated Hh/GLI signaling, in particular patients who develop resistance to current therapies targeting the Hh pathway upstream of GLI.