RESUMEN
Notch has been linked to beta-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/beta-catenin (down-regulated when blocking Wnt/beta-catenin) that are directly regulated by Notch (repressed by gamma-secretase inhibitors and up-regulated by active Notch1 in the absence of beta-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through beta-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/beta-catenin pathway in tumors implanted s.c. in nude mice. Crossing APC(Min/+) with Jagged1(+/Delta) mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear beta-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by beta-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Proteínas Wnt/metabolismo , Alelos , Animales , Proteínas de Unión al Calcio/genética , Línea Celular , Núcleo Celular/metabolismo , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1 , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Proteínas Serrate-Jagged , Factores de Transcripción TCF/metabolismo , Transcripción Genética/genética , beta Catenina/metabolismoRESUMEN
Nuclear IKKα regulates gene transcription by phosphorylating specific substrates and has been linked to cancer progression and metastasis. However, the mechanistic connection between tumorigenesis and IKKα activity remains poorly understood. We have now analyzed 288 human colorectal cancer samples and found a significant association between the presence of nuclear IKK and malignancy. Importantly, the nucleus of tumor cells contains an active IKKα isoform with a predicted molecular weight of 45 kDa (p45-IKKα) that includes the kinase domain but lacks several regulatory regions. Active nuclear p45-IKKα forms a complex with nonactive IKKα and NEMO that mediates phosphorylation of SMRT and histone H3. Proteolytic cleavage of FL-IKKα into p45-IKKα is required for preventing the apoptosis of CRC cells in vitro and sustaining tumor growth in vivo. Our findings identify a potentially druggable target for treating patients with advance refractory CRC.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Quinasa I-kappa B/metabolismo , Animales , Catepsinas/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorrectales/patología , Células HCT116 , Células HEK293 , Células HT29 , Histonas/metabolismo , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Ratones , Ratones Desnudos , Co-Represor 2 de Receptor Nuclear/metabolismo , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Trasplante HeterólogoRESUMEN
We have previously shown that IKKs are aberrantly activated in colon cancer cells leading to SMRT phosphorylation and its release from the chromatin. We now show that IKKalpha phosphorylates the homologous N-CoR corepressor in serines 2345 and 2348 creating a functional 14-3-3 binding domain (RK(p)S(2348)KSP). Moreover, we have analyzed the subcellular localization of N-CoR in 43 colorectal cancer samples and we have found that aberrant cytoplasmic distribution of N-CoR is a general trait of these tumors.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Represoras/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Colon/citología , Colon/metabolismo , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/genética , Activación Enzimática , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Co-Represor 2 de Receptor Nuclear , Estructura Terciaria de Proteína , Recto/citología , Recto/metabolismo , Proteínas Represoras/genética , Alineación de Secuencia , Serina/metabolismoRESUMEN
IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.