RESUMEN
Mass spectrometry (MS) has become the tool of choice for the large scale identification and quantitation of proteins and their post-translational modifications (PTMs). This development has been enabled by powerful software packages for the automated analysis of MS data. While data on PTMs of thousands of proteins can nowadays be readily obtained, fully deciphering the complexity and combinatorics of modification patterns even on a single protein often remains challenging. Moreover, functional investigation of PTMs on a protein of interest requires validation of the localization and the accurate quantitation of its changes across several conditions, tasks that often still require human evaluation. Software tools for large scale analyses are highly efficient but are rarely conceived for interactive, in-depth exploration of data on individual proteins. We here describe MsViz, a web-based and interactive software tool that supports manual validation of PTMs and their relative quantitation in small- and medium-size experiments. The tool displays sequence coverage information, peptide-spectrum matches, tandem MS spectra and extracted ion chromatograms through a single, highly intuitive interface. We found that MsViz greatly facilitates manual data inspection to validate PTM location and quantitate modified species across multiple samples.
Asunto(s)
Procesamiento Proteico-Postraduccional , Programas Informáticos , Humanos , Estadística como Asunto/métodos , Espectrometría de Masas en Tándem/métodos , Interfaz Usuario-ComputadorRESUMEN
Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not ß-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking ß-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Proteínas de Fusión Oncogénica/genética , Factores de Transcripción/genética , Vía de Señalización Wnt/genética , Animales , Proteína Axina/genética , Proteína Axina/metabolismo , Western Blotting , Línea Celular , Línea Celular Tumoral , Proteínas Co-Represoras , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Ratones , Microscopía Confocal , Proteínas de Fusión Oncogénica/metabolismo , Interferencia de ARN , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma Sinovial/genética , Sarcoma Sinovial/metabolismo , Sarcoma Sinovial/patología , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Factores de Transcripción/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Cancer stem cells (CSCs) display plasticity and self-renewal properties reminiscent of normal tissue stem cells, but the events responsible for their emergence remain obscure. We recently identified CSCs in Ewing sarcoma family tumors (ESFTs) and showed that they retain mesenchymal stem cell (MSC) plasticity. In the present study, we addressed the mechanisms that underlie ESFT CSC development. We show that the EWS-FLI-1 fusion gene, associated with 85%-90% of ESFTs and believed to initiate their pathogenesis, induces expression of the embryonic stem cell (ESC) genes OCT4, SOX2, and NANOG in human pediatric MSCs (hpMSCs) but not in their adult counterparts. Moreover, under appropriate culture conditions, hpMSCs expressing EWS-FLI-1 generate a cell subpopulation displaying ESFT CSC features in vitro. We further demonstrate that induction of the ESFT CSC phenotype is the result of the combined effect of EWS-FLI-1 on its target gene expression and repression of microRNA-145 (miRNA145) promoter activity. Finally, we provide evidence that EWS-FLI-1 and miRNA-145 function in a mutually repressive feedback loop and identify their common target gene, SOX2, in addition to miRNA145 itself, as key players in ESFT cell differentiation and tumorigenicity. Our observations provide insight for the first time into the mechanisms whereby a single oncogene can reprogram primary cells to display a CSC phenotype.