RESUMEN
Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/ß-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser(594), Thr(595), and Ser(597) of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates ß-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Regulación de la Expresión Génica , Fosfoproteínas/fisiología , Proteínas Wnt/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alanina/química , Animales , Medios de Cultivo Condicionados , Proteínas Dishevelled , Células HEK293 , Humanos , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Serina/química , Transducción de Señal , Treonina/química , Proteína Wnt-5a , Proteína Wnt3A/metabolismoRESUMEN
We evaluate whether 1,25(OH)(2)D(3) downregulates TP73 variants in colon and breast carcinomas, the role of survivin in this context, and the significance of this network in the clinic. Tumor cells were treated/untreated with 1,25(OH)(2)D(3) and transiently transfected with survivin. Levels of survivin and TP73 variants were evaluated by quantitative RT-PCR and Western blotting. In 75 colon and 60 breast cancer patients, the expressions of survivin and TP73 isoforms were determined. Tumor characteristics were examined in each patient. Survivin protein levels were also evaluated in a subgroup of patients and cell lines. Decrease in survivin and TAp73 transcripts and protein and ΔNp73 mRNA was detected after 1,25(OH)(2)D(3) treatment. Ectopic survivin expression led to an increase in the TAp73, ΔNp73, ΔEx2p73, and ΔEx2-3p73 transcripts. In cancer patients, direct correlations were observed between TP73 variants and survivin levels. 1,25(OH)(2)D(3) negatively regulate survivin and TP73 variants in colon and breast cancer cells. Positive regulation of TP73 isoforms by survivin may exist, which reinforces the possibility that the downregulation of TP73 forms by 1,25(OH)(2)D(3) is survivin-dependent.
Asunto(s)
Neoplasias de la Mama/genética , Calcitriol/metabolismo , Calcitriol/farmacología , Neoplasias del Colon/genética , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/genética , Proteínas Supresoras de Tumor/genética , Western Blotting , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/metabolismo , Reacción en Cadena de la Polimerasa , Pronóstico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Survivin , Proteína Tumoral p73 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and beta-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and beta-catenin-induced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on beta-catenin/TCF-mediated transcription since it can block transactivation of a consensus beta-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage beta-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores beta-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that beta-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to beta-catenin, which could explain its negative effect on beta-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as beta-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Factor de Unión 1 al Potenciador Linfoide/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Fusión Oncogénica/fisiología , Factor 1 de Transcripción de Linfocitos T/antagonistas & inhibidores , Factores de Transcripción/fisiología , beta Catenina/antagonistas & inhibidores , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Línea Celular Tumoral/metabolismo , Transformación Celular Neoplásica/genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HeLa/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Familia de Multigenes , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Mapeo de Interacción de Proteínas , Proteína Proto-Oncogénica c-fli-1 , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sarcoma de Ewing/patología , Factor de Transcripción 4 , Factores de Transcripción/genética , Transcripción Genética , Transgenes , Proteínas Wnt/fisiologíaRESUMEN
Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR-/-) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR-/- mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/ß-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR-/- liver. Consistently, PI3K, ERK, and Wnt/ß-catenin inhibition partially rescued polyploidy in AhR-/- mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.
RESUMEN
Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.
Asunto(s)
Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas de Pez Cebra , Proteínas Adaptadoras Transductoras de Señales , Proteínas del Citoesqueleto/metabolismo , Proteínas Dishevelled , Péptidos y Proteínas de Señalización Intercelular , Proteínas Relacionadas con Receptor de LDL , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Fosforilación , Proteínas/metabolismo , Receptores de LDL/metabolismo , Transactivadores/metabolismo , Proteínas Wnt , Proteína Wnt-5a , Proteína Wnt1 , beta CateninaRESUMEN
Secreted signaling proteins of the Wnt family are known to regulate a diverse range of developmental processes, and their signaling pathway through beta-catenin is frequently activated in cancer. The identification of both Frizzled and LRP5/6 (LRP: low-density lipoprotein receptor-related protein) proteins as components of cell-surface receptors for Wnt proteins has raised questions about their individual functions. We have investigated this issue through a structure-function analysis of Frizzled and LRP proteins that have been implicated in Wnt1 signaling. Consistent with other reports, we find that LRP6/Arrow proteins deleted for their extracellular domain are able to activate the Wnt/beta-catenin signaling pathway. Importantly, our results demonstrate that this signaling from LRP6/Arrow derivatives can occur in a Frizzled- and ligand-independent manner. Furthermore, we show that the PPSP motifs within the intracellular domain of LRP6 are required for signaling. In contrast to results with LRP6, overexpression of Frizzled proteins did not activate the pathway. Based on evidence of ligand binding to both Frizzled and LRP6, current models suggest that both proteins are components of a Wnt receptor complex that signals to beta-catenin. In light of these models, our data imply that LRP5/6/Arrow proteins constitute the distal signal-initiating component of these receptors. The results also support the notion that LRP5/6 are candidate oncogenes.
Asunto(s)
Proteínas del Citoesqueleto/fisiología , Proteínas/fisiología , Receptores de LDL/fisiología , Transactivadores/fisiología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Drosophila melanogaster , Receptores Frizzled , Genes Reporteros , Humanos , Proteínas Relacionadas con Receptor de LDL , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Luciferasas/análisis , Oncogenes , Receptores de LDL/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Transducción de Señal/fisiología , Transfección , beta CateninaRESUMEN
The relation between thyroid status and diseases and cancer is unclear. No detailed analysis of thyroid hormone receptor (TR) expression in human breast cancer has been reported. We have analysed the expression and mutational status of the TRalpha1, encoded by the c-erbA proto-oncogene, TRbeta1 and TRbeta2 isoforms in 70 sporadic breast cancers. Alterations in the RNA level of TRbeta1, TRalpha1, or both were found in a number of patients. No expression of TRbeta2 RNA was detected. Western blotting analysis confirmed the differences in expression at the protein level in those cases where sufficient tumor sample was available. Additionally, tumor-specific truncated TRbeta1 RNA was found in six patients. Strikingly, three transcripts shared the same breakpoint. Only one tumor carried the corresponding deletion at the genomic DNA level, suggesting that the remaining abnormal TRbeta1 transcripts are aberrant splicing products. Though no significant correlation was found between TRbeta1 alteration and any clinical parameter, it showed a tendency to associate with early age of onset (<50 years). Our results reveal specific alterations in the expression of TRbeta and TRalpha genes in a subset of breast cancer patients, suggesting that deregulation of thyroid hormone target genes may be involved in the generation of this neoplasia.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , Genes erbA , Proteínas de Neoplasias/biosíntesis , Receptores de Hormona Tiroidea/biosíntesis , Adulto , Alelos , Animales , Neoplasias de la Mama/metabolismo , Carcinoma/metabolismo , Transformación Celular Neoplásica/genética , Análisis Mutacional de ADN , Femenino , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Proto-Oncogenes Mas , Empalme del ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Eliminación de Secuencia , Hormonas Tiroideas/fisiologíaRESUMEN
Thyroid hormone (triiodothyronine, T3) is a pleiotropic regulator of growth, differentiation and tissue homeostasis in higher organisms that acts through the control of target gene expression. Most, if not all, major T3 actions are mediated by specific high affinity nuclear receptors (TR) which are encoded by two genes, THRA and THRB. Several TRalpha and TRbeta receptor isoforms are expressed. Abundant and contradictory literature exists on the relationship between circulating thyroid hormone levels, thyroid diseases and human cancer. In 1986, a connection between TR and cancer became evident when the chicken TRalpha1 was characterized as the c-erbA proto-oncogene, the cellular counterpart of the retroviral v-erbA oncogene. V-erbA causes erythroleukemias and sarcomas in birds, and hepatocellular carcinomas in transgenic mice. In recent years, many studies have analyzed the presence of quantitative (abnormal levels) or qualitative (mutations) alterations in the expression of THR genes in different types of human neoplasias. While their role in tumor generation or progression is currently unclear, both gross chromosomal and minor mutations (deletions, aberrant splicing, point mutations) and changes in the level of expression of THRA and THRB genes have been found. Together with other in vitro data indicating connections between TR and p53, Rb, cyclin D and other cell cycle regulators and oncogenes, these results suggest that THRA and THRB may be involved in human cancer.