RESUMEN
Stress has been proposed to be a tumor promoting factor through the secretion of specific neuromediators, such as Urocortin2 and 3 (Ucn2/3), however its role in colorectal cancer (CRC) remains elusive. We observed that Ucn2/3 and their receptor the Corticotropin Releasing Factor receptor 2 (CRF2) were up-regulated in high grade and poorly differentiated CRC. This suggests a role for CRF2 in the loss of cellular organization and tumor progression. Using HT-29 and SW620 cells, two CRC cell lines differing in their abilities to perform cell-cell contacts, we found that CRF2 signals through Src/ERK pathway to induce the alteration of cell-cell junctions and the shuttle of p120ctn and Kaiso in the nucleus. In HT-29 cells, this signaling pathway also leads to the remodeling of cell adhesion by i) the phosphorylation of Focal Adhesion Kinase and ii) a modification of actin cytoskeleton and focal adhesion complexes. These events stimulate cell migration and invasion. In conclusion, our findings indicate that CRF2 signaling controls cellular organization and may promote metastatic potential of human CRC cells through an epithelial-mesenchymal transition like process. This contributes to the comprehension of the tumor-promoting effects of stress molecules and designates Ucn2/3-CRF2 tandem as a target to prevent CRC progression and aggressiveness.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Receptores de Hormona Liberadora de Corticotropina/metabolismo , Adhesión Celular/genética , Línea Celular , Movimiento Celular/genética , Neoplasias Colorrectales/genética , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Células HT29 , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Reacción en Cadena de la Polimerasa , Receptores de Hormona Liberadora de Corticotropina/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Integrity of the epithelial barrier is determined by apical junctional complexes which also participate in the signalling pathways inducing intestinal cell differentiation. Lipid rafts (LR) have been proposed to play a role in the organization and the function of these intercellular complexes. This study investigated potential mechanisms by which LR could participate in the establishment of adherens junctions (AJ) and the initiation of enterocytic differentiation. We showed that the differentiation of epithelial cells in rat colons correlates with the emergence of LR. Using HT-29 cells we demonstrated that during the differentiation process, LR are required for the recruitment and the association of p120ctn to E-cadherin. Silencing of flotillin-1, a LR component, alters the recruitment of AJ proteins in LR and delays the expression of differentiation markers. Furthermore, the ability of p120ctn/E-cadherin complexes to support cell differentiation is altered in HT-29 Rac1N17 cells. These results show a contributory role of LR in the enterocytic differentiation process, which serve as signalling platforms for Rac1-mediated organization of AJ. A better understanding of the mechanism involved in the establishment of junctional complex and their role in enterocytic differentiation provides new insights into the regulation of intestinal homeostasis.
Asunto(s)
Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Diferenciación Celular , Enterocitos/citología , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Apoptosis , Western Blotting , Proliferación Celular , Técnica del Anticuerpo Fluorescente , Células HT29 , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Ratas , Proteína Activadora de GTPasa p120/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Depending on its cellular localization, p120 catenin (p120ctn) can participate in various processes, such as cadherin-dependent cell-cell adhesion, actin cytoskeleton remodeling, and intracellular trafficking. Recent studies also indicate that p120ctn could regulate cell proliferation and contact inhibition. This report describes a new function of p120ctn in the regulation of cell cycle progression. Overexpression of the p120ctn isoform 3A in human colon adenocarcinoma cells (HT-29) results in cytoplasmic accumulation of the protein, as observed in many tumors. This cytoplasmic increase is correlated with a reduction in proliferation and inhibition of DNA synthesis. Under these conditions, experiments on synchronized cells revealed a prolonged S phase associated with cyclin E stabilization. Both confocal microscopy and biochemical analysis showed that cyclin E and cyclin-dependent kinase 2 colocalized with p120ctn in centrosomes during mitosis. These proteins are associated in a functional complex evidenced by coimmunoprecipitation experiments and the emergence of Thr199-phosphorylated nucleophosmin/B23. Such post-translational modification of this centrosomal target has been shown to trigger the initiation of centrosome duplication. Therefore, p120ctn-mediated accumulation of cyclin E in centrosomes may participate in abnormal amplification of centrosomes and the inhibition of DNA replication, thus leading to aberrant mitosis and polyploidy. Because these modifications are often observed in cancer, p120ctn may represent a new therapeutic target for future therapy.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Fosfoproteínas/metabolismo , Cateninas , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Ciclo Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Centrosoma/metabolismo , Neoplasias del Colon/genética , Citoplasma/metabolismo , Progresión de la Enfermedad , Amplificación de Genes , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Células HT29 , Humanos , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Fosforilación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Regulación hacia Arriba , Catenina deltaRESUMEN
Human intestinal cell differentiation is mediated by signaling pathways that remain largely undefined. We and others have shown that cell migration and differentiation along the crypt-villus axis is associated with temporal and spatial modulations of the repertoire, as well as with the function of integrins and E-cadherins and their substrates. Cross-talk between integrin and cadherin signaling was previously described and seems to coordinate this differentiation process. Here, we report that engagement of alpha6 and, to a lesser extent, alpha3 integrin subunits after HT-29 cell adhesion on laminin 5 increases the expression of E-cadherin, which then organizes into nascent adherens junctions. We further identify that phosphoinositide 3-kinase (PI 3-kinase) activation plays a key role in this cross-talk. Indeed, integrin-dependent adhesion on laminin 5 stimulates PI 3-kinase activity. Immunofluorescence and immunoprecipitation experiments revealed that activated PI 3-kinase is recruited at cell-cell contacts. Using LY294002, an inhibitor of PI 3-kinase activity, we found that this activation is essential for E-cadherin connection with the cytoskeleton and for biogenesis of adherens junctions. Finally, we demonstrated that PI 3-kinase could signal through Rac1b activation to control adherens junction assembly. Our results provide a mechanistic insight into integrin-cadherin cross-talk and identify a novel role for PI 3-kinase in the establishment of adherens junctions.