RESUMEN
The subversion of endocytic routes leads to malignant transformation and has been implicated in human cancers. However, there is scarce evidence for genetic alterations of endocytic proteins as causative in high incidence human cancers. Here, we report that Epsin 3 (EPN3) is an oncogene with prognostic and therapeutic relevance in breast cancer. Mechanistically, EPN3 drives breast tumorigenesis by increasing E-cadherin endocytosis, followed by the activation of a ß-catenin/TCF4-dependent partial epithelial-to-mesenchymal transition (EMT), followed by the establishment of a TGFß-dependent autocrine loop that sustains EMT. EPN3-induced partial EMT is instrumental for the transition from in situ to invasive breast carcinoma, and, accordingly, high EPN3 levels are detected at the invasive front of human breast cancers and independently predict metastatic rather than loco-regional recurrence. Thus, we uncover an endocytic-based mechanism able to generate TGFß-dependent regulatory loops conferring cellular plasticity and invasive behavior.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Neoplasias de la Mama/fisiopatología , Endocitosis , Proteínas Adaptadoras del Transporte Vesicular/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cadherinas/genética , Cadherinas/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia , Transducción de Señal , Factor de Transcripción 4/genética , Factor de Transcripción 4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Rab5 is a small GTPase known to regulate vesicular trafficking during interphase. Here, we show that Rab5 also plays an unexpected role during mitotic progression. RNAi-mediated silencing of Rab5 caused defects in chromosome congression and extensive prometaphase delay, and it correlated with a severe reduction in the localization of the centromere-associated protein CENP-F to kinetochores. CENP-F is a component of the nuclear matrix required for chromosome congression that, at mitotic entry, localizes to the nuclear envelope and assembles on kinetochores, contributing to the establishment of kinetochore microtubule interactions. We found that Rab5 forms a complex with a subset of CENP-F in mitotic cells and regulates the kinetics of release of CENP-F from the nuclear envelope and its accumulation on kinetochores. Simultaneous depletion of both Rab5 and CENP-F recapitulated the mitotic defects caused by silencing of either Rab5 or CENP-F alone, indicating epistatic roles for these two proteins in the pathway that orchestrates chromosome congression. These results reveal the involvement of Rab5 in the proper execution of mitotic programs whose deregulation can undermine chromosomal stability.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/fisiología , Cinetocoros/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Línea Celular , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Cromosomas Humanos/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Microfilamentos/antagonistas & inhibidores , Proteínas de Microfilamentos/genética , Microscopía Confocal , Microtúbulos/metabolismo , Mitosis/fisiología , Complejos Multiproteicos/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al GTP rab5/antagonistas & inhibidores , Proteínas de Unión al GTP rab5/genéticaRESUMEN
The tandem repeated sea urchin alpha-histone genes are developmentally regulated by gene-specific promoter elements. Coordinate transcription of the five genes begins after meiotic maturation of the oocyte, continues through cleavage, and reaches its maximum at morula stage, after which these genes are shut off and maintained in a silenced state for the life cycle of the animal. Although cis regulative sequences affecting the timing and the level of expression of these genes have been characterized, much less is known about the mechanism of their repression. Here we report the results of a functional analysis that allowed the identification of the sequence elements needed for the silencing of the alpha-H2A gene at gastrula stage. We found that important negative regulative sequences are located in the 462 bp sns 5 fragment located in the 3' region. Remarkably, sns 5 contains the sns enhancer blocking element and the most 3' H2A codons. In addition, we made the striking observation that inhibition of the anti-enhancer activity of sns, by titration of the binding proteins in microinjected embryos, also affected the capability of sns 5 to down-regulate transgene expression at gastrula stage. A further sequence element essential for repression of the H2A gene was identified upstream of the enhancer, in the 5' region, and contains four GAGA repeats. Altogether these findings suggest that down-regulation of the alpha-H2A gene occurs by the functional interaction of the 5' and 3' cis sequence elements. These results demonstrate the involvement of a genomic insulator in the silencing of gene expression.