RESUMEN
Rab40b is a SOCS box-containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b-Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b-Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b-Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.
Asunto(s)
Citoesqueleto de Actina/genética , Actinas/genética , Proteínas del Citoesqueleto/genética , Movimiento Celular/genética , Matriz Extracelular/genética , Adhesiones Focales/genética , Humanos , Fibras de Estrés/genéticaRESUMEN
Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca2+ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca2+ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca2+ signaling, respectively.
Asunto(s)
Señalización del Calcio/fisiología , Canales de Potasio/metabolismo , Animales , Calcio/metabolismo , Adhesión Celular/genética , Línea Celular , Movimiento Celular/genética , Adhesiones Focales/genética , Humanos , Ratones , Canales de Potasio/fisiología , Transducción de Señal , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Cell migration initiates by traction generation through reciprocal actomyosin tension and focal adhesion reinforcement, but continued motility requires adaptive cytoskeletal remodeling and adhesion release. Here, we asked whether de novo gene expression contributes to this cytoskeletal feedback. We found that global inhibition of transcription or translation does not impair initial cell polarization or migration initiation, but causes eventual migratory arrest through excessive cytoskeletal tension and over-maturation of focal adhesions, tethering cells to their matrix. The transcriptional coactivators YAP and TAZ mediate this feedback response, modulating cell mechanics by limiting cytoskeletal and focal adhesion maturation to enable persistent cell motility and 3D vasculogenesis. Motile arrest after YAP/TAZ ablation was partially rescued by depletion of the YAP/TAZ-dependent myosin phosphatase regulator, NUAK2, or by inhibition of Rho-ROCK-myosin II. Together, these data establish a transcriptional feedback axis necessary to maintain a responsive cytoskeletal equilibrium and persistent migration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Citoesqueleto/metabolismo , Células Progenitoras Endoteliales/metabolismo , Adhesiones Focales/metabolismo , Mecanotransducción Celular , Neovascularización Fisiológica , Transactivadores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Citoesqueleto/genética , Retroalimentación Fisiológica , Adhesiones Focales/genética , Cinética , Ratones Endogámicos C57BL , Ratones Noqueados , Miosina Tipo II/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/deficiencia , Transactivadores/genética , Transcripción Genética , Proteínas Señalizadoras YAP , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
miRNAs have been identified as key regulators of almost all cellular processes, therefore, their dysregulation is involved with several diseases, including cancer. miRNAs specifically related to the metastastic cascade are called metastamiRs and can be involved with different steps of this process, including loss of adhesion. Osteosarcoma (OS) is the most common primary malignant pediatric bone tumor that often presents metastatic disease at diagnosis; therefore, a deeper study of adhesion-associated miRNAs could shed light on its pathophysiology. Online databases were used to select four miRNAs (miR-139; miR-181b; miR-584; miR-708) predicted or validated to target proteins related to adherent junctions and focal adhesion pathways, and their expression levels and possible associations with clinical features evaluated in primary OS samples. Our results showed downregulation of miR-139-5p and miR-708-5p in OS samples compared to non-neoplastic controls. Moreover, lower expression of miR-708-5p was associated with poor overall survival and higher expression of miR-181b-5p related to worst chemotherapy response (low HUVOS level). Based on these results, we selected miR-139-5p and miR-708-5p for further functional testing. Inducing the expression of miR-139-5p diminished the clonogenic capacity of the HOS cell line, while upregulation of miR-708-5p was related to a lower cellular adhesion. In summary, this work identified new signatures of microRNA dysregulation that may serve as useful prognostic markers in this aggressive pediatric bone tumor.
Asunto(s)
Uniones Adherentes/genética , Biomarcadores de Tumor/genética , Neoplasias Óseas/secundario , Adhesiones Focales/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Osteosarcoma/patología , Adolescente , Adulto , Neoplasias Óseas/genética , Neoplasias Óseas/cirugía , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Masculino , Osteosarcoma/genética , Osteosarcoma/cirugía , Pronóstico , Tasa de Supervivencia , Adulto JovenRESUMEN
UNLABELLED: Background. This study aims to identify key genes and pathways involved in non-alcoholic fatty liver disease (NAFLD). MATERIAL AND METHODS: The dataset GSE48452 was downloaded from Gene Expression Omnibus, including 14 control liver samples, 27 healthy obese samples, 14 steatosis samples and 18 nonalcoholic steatohepatitis (NASH) samples. Differentially expressed genes (DEGs) between controls and other samples were screened through LIMMA package. Then pathway enrichment analysis for DEGs was performed by using DAVID, and alterations of enriched pathways were determined. Furthermore, protein-protein interaction (PPI) networks were constructed based on the PPI information from HPRD database, and then, networks were visualized through Cytoscape. Additionally, interactions between microRNAs (miRNAs) and pathways were analyzed via Fisher's exact test. RESULTS: A total of 505, 814 and 783 DEGs were identified for healthy obese, steatosis and NASH samples in comparison with controls, respectively. DEGs were enriched in ribosome (RPL36A, RPL14, etc.), ubiquitin mediated proteolysis (UBE2A, UBA7, etc.), focal adhesion (PRKCA, EGFR, CDC42, VEGFA, etc.), Fc?R-mediated phagocytosis (PRKCA, CDC42, etc.), and so on. The 27 enriched pathways gradually deviated from baseline (namely, controls) along with the changes of obese-steatosis-NASH. In PPI networks, PRKCA interacted with EGFR and CDC42. Besides, hsa-miR-330-3p and hsa-miR-126 modulated focal adhesion through targeting VEGFA and CDC42. CONCLUSIONS: The identified DEGs (PRKCA, EGFR, CDC42, VEGFA), disturbed pathways (ribosome, ubiquitin mediated proteolysis, focal adhesion, Fc?R-mediated phagocytosis, etc.) and miRNAs (hsa-miR-330-3p, hsa-miR-126, etc.) might be closely related to NAFLD progression. These results might contribute to understanding NAFLD mechanism, conducting experimental researches, and designing clinical practices.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico/genética , Obesidad Metabólica Benigna/genética , Adulto , Anciano , Estudios de Casos y Controles , Bases de Datos Genéticas , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Adhesiones Focales/genética , Perfilación de la Expresión Génica , Humanos , Masculino , Redes y Vías Metabólicas , MicroARNs/genética , MicroARNs/metabolismo , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad Metabólica Benigna/metabolismo , Fagocitosis/genética , Mapas de Interacción de Proteínas , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Proteolisis , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Adulto Joven , Proteína de Unión al GTP cdc42/genética , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Adhesiones Focales/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias Experimentales/metabolismo , Talina/biosíntesis , Neoplasias de la Lengua/metabolismo , Animales , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Movimiento Celular/genética , Adhesiones Focales/genética , Adhesiones Focales/patología , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Proteómica/métodos , Talina/genética , Neoplasias de la Lengua/genética , Neoplasias de la Lengua/patología , Regulación hacia Arriba/genéticaRESUMEN
The small GTPase Rab5 has been extensively studied in the context of endocytic trafficking because it is critical in the regulation of early endosome dynamics. In addition to this canonical role, evidence obtained in recent years implicates Rab5 in the regulation of cell migration. This novel role of Rab5 is based not only on an indirect relationship between cell migration and endosomal trafficking as separate processes, but also on the direct regulation of signaling proteins implicated in cell migration. However, the precise mechanisms underlying this connection have remained elusive. Recent studies have shown that the activation of Rab5 is a critical event for maintaining the dynamics of focal adhesions, which is fundamental in regulating not only cell migration but also tumor cell invasion.
Asunto(s)
Movimiento Celular/genética , Adhesiones Focales/genética , Invasividad Neoplásica/genética , Neoplasias/genética , Proteínas de Unión al GTP rab5/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Humanos , Neoplasias/patología , ARN Interferente Pequeño/genética , Transducción de Señal , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.