RESUMEN
Ubiquitination plays a crucial role in cellular homeostasis by regulating the degradation, localization, and activity of proteins, ensuring proper cell function and balance. Among E3 ubiquitin ligases, WW domain-containing protein 1 (WWP1) is implicated in cell proliferation, survival, and apoptosis. Notably WWP1 is frequently amplified in breast cancer and associated with poor prognosis. Here, we identify the protein cysteine and tyrosine-rich protein 1 (CYYR1) that had previously no assigned function, as a regulator of WWP1 activity and stability. We show that CYYR1 binds to the WW domains of the E3 ubiquitin ligase WWP1 through its PPxY motifs. This interaction triggers K63-linked autoubiquitination and subsequent degradation of WWP1. We furthermore demonstrate that CYYR1 localizes to late endosomal vesicles and directs polyubiquitinated WWP1 toward lysosomal degradation through binding to ANKyrin repeat domain-containing protein 13 A (ANKRD13A). Moreover, we found that CYYR1 expression attenuates breast cancer cell growth in anchorage-dependent and independent colony formation assays in a PPxY-dependent manner. Finally, we highlight that CYYR1 expression is significantly decreased in breast cancer and is associated with beneficial clinical outcome. Taken together our study suggests tumor suppressor properties for CYYR1 through regulation of WWP1 autoubiquitination and lysosomal degradation.
RESUMEN
BACKGROUND: E3 ubiquitin ligases play critical roles in regulating cellular signaling pathways by inducing ubiquitylation of key components. RNF111/Arkadia is a RING E3 ubiquitin ligase that activates TGF-ß signaling by inducing ubiquitylation and proteasomal degradation of the transcriptional repressor SKIL/SnoN. In this study, we have sought to identify novel regulators of the E3 ubiquitin ligase activity of RNF111 by searching for proteins that specifically interacts with its RING domain. RESULTS: We found that UBXN7, a member of the UBA-UBX family, directly interacts with the RING domain of RNF111 or its related E3 RNF165/ARK2C that shares high sequence homology with RNF111. We showed that UBXN7 docks on RNF111 or RNF165 RING domain through its UAS thioredoxin-like domain. Overexpression of UBXN7 or its UAS domain increases endogenous RNF111, while an UBXN7 mutant devoid of UAS domain has no effect. Conversely, depletion of UBXN7 decreases RNF111 protein level. As a consequence, we found that UBXN7 can modulate degradation of the RNF111 substrate SKIL in response to TGF-ß signaling. We further unveiled this mechanism of regulation by showing that docking of the UAS domain of UBXN7 inhibits RNF111 ubiquitylation by preventing interaction of the RING domain with the E2 conjugating enzymes. By analyzing the interactome of the UAS domain of UBXN7, we identified that it also interacts with the RING domain of the E3 TOPORS and similarly regulates its E3 ubiquitin ligase activity by impairing E2 binding. CONCLUSIONS: Taken together, our results demonstrate that UBXN7 acts as a direct regulator for the E3 ubiquitin ligases RNF111, RNF165, and TOPORS and reveal that a thioredoxin-like domain can dock on specific RING domains to regulate their E3 ubiquitin ligase activity.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitinas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Factor de Crecimiento Transformador beta/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-ß (TGF-ß) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-ß signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-ß signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-ß. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-ß pathway activation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Nucleares/genética , Proteoma/metabolismo , Proteínas Proto-Oncogénicas/genética , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The induction of Smad signalling by the extracellular ligand TGF-ß promotes tissue plasticity and cell migration in developmental and pathological contexts. Here, we show that vaccinia virus (VACV) stimulates the activity of Smad transcription factors and expression of TGF-ß/Smad-responsive genes at the transcript and protein levels. Accordingly, infected cells share characteristics to those undergoing TGF-ß/Smad-mediated epithelial-to-mesenchymal transition (EMT). Depletion of the Smad4 protein, a common mediator of TGF-ß signalling, results in an attenuation of viral cell-to-cell spread and reduced motility of infected cells. VACV induction of TGF-ß/Smad-responsive gene expression does not require the TGF-ß ligand or type I and type II TGF-ß receptors, suggesting a novel, non-canonical Smad signalling pathway. Additionally, the spread of ectromelia virus, a related orthopoxvirus that does not activate a TGF-ß/Smad response, is enhanced by the addition of exogenous TGF-ß. Together, our results indicate that VACV orchestrates a TGF-ß-like response via a unique activation mechanism to enhance cell migration and promote virus spread.
Asunto(s)
Transducción de Señal , Proteína Smad4/genética , Proteína Smad4/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Virus Vaccinia/fisiología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal , Células HT29 , Células HaCaT , Células HeLa , Humanos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Virus Vaccinia/efectos de los fármacosRESUMEN
Although E3 ubiquitin ligases are deemed to play key roles in normal cell function and homeostasis, whether their alterations contribute to cancer pathogenesis remains unclear. In this study, we sought to investigate potential mechanisms that govern WWP1/Tiul1 (WWP1) ubiquitin ligase activity, focusing on its ability to trigger degradation of TGFß type I receptor (TßRI) in conjunction with Smad7. Our data reveal that the WWP1 protein is very stable at steady states because its autopolyubiquitination activity is silenced due to an intra-interaction between the C2 and/or WW and Hect domains that favors WWP1 monoubiquitination at the expense of its polyubiquitination or polyubiquitination of TßRI. Upon binding of WWP1 to Smad7, this functional interplay is disabled, switching its monoubiquitination activity toward a polyubiquitination activity, thereby driving its own degradation and that of TßRI as well. Intriguingly, a WWP1 point mutation found in human prostate cancer disrupts this regulatory mechanism by relieving the inhibitory effects of C2 and WW on Hect and thereby causing WWP1 hyperactivation. That cancer-driven alteration of WWP1 culminates in excessive TßRI degradation and attenuated TGFß cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1.
Asunto(s)
Carcinogénesis/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación Puntual , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Carcinogénesis/genética , Carcinogénesis/patología , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal , Proteína smad7/genética , Proteína smad7/metabolismo , Factor de Crecimiento Transformador beta1/genética , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Arkadia is a RING-based ubiquitin ligase that positively regulates TGF-ß signaling by targeting several pathway components for ubiquitination and degradation. However, little is known about the mechanisms controlling Arkadia activity. Here we show that the LIM-only protein FHL2 binds and synergistically cooperates with Arkadia to activate Smad3/Smad4-dependent transcription. Knockdown of FHL2 by RNA interference decreases Arkadia level and restricts the amplitude of Arkadia-induced TGF-ß target gene responses. We found that Arkadia is ubiquitinated via K63- and K27-linked polyubiquitination. A single mutation at the RING domain that abolishes the E3 activity diminishes Arkadia ubiquitination, indicating that this modification partly involves autocatalytic process. Mutation of seven lysines at the C-terminal region of Arkadia severely impairs ubiquitination through the K27 but not the K63 linkage and slows down the turnover of Arkadia, suggesting that K27-linked polyubiquitination might promote proteolysis-dependent regulation of Arkadia. We show that FHL2 increases the half-life of Arkadia through inhibition of ubiquitin chain assembly on the protein, which provides a molecular basis for functional cooperation between Arkadia and FHL2 in enhancing TGF-ß signaling. Our study uncovers a novel regulatory mechanism of Arkadia by ubiquitination and identifies FHL2 as important regulator of Arkadia ubiquitination and TGF-ß signal transduction.
Asunto(s)
Proteínas con Homeodominio LIM/genética , Proteínas Musculares/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina/genética , Animales , Sitios de Unión , Línea Celular Tumoral , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Genes Reporteros , Semivida , Humanos , Proteínas con Homeodominio LIM/metabolismo , Luciferasas , Ratones , Proteínas Musculares/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transducción de Señal , Factores de Transcripción/metabolismo , Transfección , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
E3 ubiquitin ligases play important roles in regulating transforming growth factor beta (TGF-beta)/Smad signaling. Screening of an E3 ubiquitin ligase small interfering RNA library, using TGF-beta induction of a Smad3/Smad4-dependent luciferase reporter as a readout, revealed that Arkadia is an E3 ubiquitin ligase that is absolutely required for this TGF-beta response. Knockdown of Arkadia or overexpression of a dominant-negative mutant completely abolishes transcription from Smad3/Smad4-dependent reporters, but not from Smad1/Smad4-dependent reporters or from reporters driven by Smad2/Smad4/FoxH1 complexes. We show that Arkadia specifically activates transcription via Smad3/Smad4 binding sites by inducing degradation of the transcriptional repressor SnoN. Arkadia is essential for TGF-beta-induced SnoN degradation, but it has little effect on SnoN levels in the absence of signal. Arkadia interacts with SnoN and induces its ubiquitination irrespective of TGF-beta/Activin signaling, but SnoN is efficiently degraded only when it forms a complex with both Arkadia and phosphorylated Smad2 or Smad3. Finally, we describe an esophageal cancer cell line (SEG-1) that we show has lost Arkadia expression and is deficient for SnoN degradation. Reintroduction of wild-type Arkadia restores TGF-beta-induced Smad3/Smad4-dependent transcription and SnoN degradation in these cells, raising the possibility that loss of Arkadia function may be relevant in cancer.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína smad3/metabolismo , Proteína Smad4/metabolismo , Transcripción Genética , Adenocarcinoma , Animales , Esófago de Barrett , Línea Celular , Regulación de la Expresión Génica , Genes Reporteros , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiología , Proteína smad3/genética , Proteína Smad4/genética , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
Signaling by transforming growth factor-beta (TGF-beta) superfamily ligands to the nucleus is mediated by type I and type II receptors and the intracellular signal transducers, the Smads. Alteration of some of the components of these pathways has been observed in human tumors. These alterations can be deletions or mutations, or downregulation of components that act positively in the pathway, or alternatively, amplification or overexpression of inhibitors of the pathways. The selection of these alterations during tumor progression and their correlation with clinical outcomes, such as survival, risk of recurrence after tumor resection or tendency for metastatic spread, suggest that many are involved in tumor progression. Here, we review the genetic alterations and epigenetic modifications that occur in different components of the TGF-beta superfamily signaling pathways in human tumors and we discuss their correlation with clinical outcome. The evidence suggests that not all alterations of the TGF-beta superfamily signaling pathway components in human cancer have an equivalent effect on tumor progression and we discuss what implications this has for our understanding of the role of TGF-beta signaling in human cancer.
Asunto(s)
Familia de Multigenes/fisiología , Neoplasias/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/fisiología , Transducción de Señal/fisiología , Factor de Crecimiento Transformador beta/fisiología , Epigénesis Genética/fisiología , Humanos , Familia de Multigenes/genética , Neoplasias/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND: Recent progress in the treatment of sickle cell disease, in particular the use of hydroxyurea, has considerably modified the prognosis of this disease. Many more patients now reach reproductive age. The objective of this study was to assess the potential impact of hydroxyurea on the semen of patients. DESIGN AND METHODS: In this retrospective multicenter study, we evaluated the sperm parameters and fertility of 44 patients and analyzed the potential impact of hydroxyurea. RESULTS: We report data from the largest series so far of semen analyses in patients with sickle cell disease: 108 samples were analyzed, of which 76 were collected before treatment. We found that at least one sperm parameter was abnormal in 91% of the patients before treatment, in agreement with published literature. All sperm parameters seemed to be affected in semen samples collected during hydroxyurea treatment, and this impairment occurred in less than 6 months, later reaching a plateau. Furthermore, after hydroxyurea cessation, while global results in 30 patients were not statistically different before and after hydroxyurea treatment, in four individuals follow-up sperm parameters did not seem to recover quickly and the total number of spermatozoa per ejaculate fell below the normal range in about half the cases. CONCLUSIONS: The observed alterations of semen parameters due to sickle cell disease seem to be exacerbated by hydroxyurea treatment. Until prospective studies reveal reassuring findings, we suggest that a pre-treatment sperm analysis be performed and sperm cryopreservation be offered to patients before hydroxyurea treatment.
Asunto(s)
Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/tratamiento farmacológico , Hidroxiurea/uso terapéutico , Infertilidad Masculina/complicaciones , Semen/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Adolescente , Adulto , Fertilidad , Heterocigoto , Homocigoto , Humanos , Infertilidad Masculina/etiología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios RetrospectivosRESUMEN
In response to transforming growth factor beta (TGF-beta), Smad4 forms complexes with activated Smad2 and Smad3, which accumulate in the nucleus, where they both positively and negatively regulate TGF-beta target genes. Mutation or deletion of Smad4 is found in about 50% of pancreatic tumors and in about 15% of colorectal tumors. As Smad4 is a central component of the TGF-beta/Smad pathway, we have determined whether Smad4 is absolutely required for all TGF-beta responses, to evaluate the effect of its loss during human tumor development. We have generated cell lines from the immortalized human keratinocyte cell line HaCaT or the pancreatic tumor cell line Colo-357, which stably express a tetracyline-inducible small interfering RNA targeted against Smad4. In response to tetracycline, Smad4 expression is effectively silenced. Large-scale microarray analysis identifies two populations of TGF-beta target genes that are distinguished by their dependency on Smad4. Some genes absolutely require Smad4 for their regulation, while others do not. Functional analysis also indicates a differential Smad4 requirement for TGF-beta-induced functions; TGF-beta-induced cell cycle arrest and migration, but not epithelial-mesenchymal transition, are abolished after silencing of Smad4. Altogether our results suggest that loss of Smad4 might promote TGF-beta-mediated tumorigenesis by abolishing tumor-suppressive functions of TGF-beta while maintaining some tumor-promoting TGF-beta responses.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Pancreáticas/genética , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Ciclo Celular/genética , Diferenciación Celular , Línea Celular Transformada , Movimiento Celular , Proteínas de Unión al ADN/genética , Células Epiteliales/fisiología , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Queratinocitos/fisiología , Mesodermo/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/metabolismo , Regiones Promotoras Genéticas/efectos de los fármacos , ARN Interferente Pequeño/genética , Proteína Smad4 , Tetraciclina/farmacología , Transactivadores/genética , Células Tumorales CultivadasRESUMEN
Lysine acetylation modulates the activities of nonhistone regulatory proteins and plays a critical role in the regulation of cellular gene transcription. In this study, we showed that the transcriptional coactivator p300 acetylated beta-catenin at lysine 345, located in arm repeat 6, in vitro and in vivo. Acetylation of this residue increased the affinity of beta-catenin for Tcf4, and the cellular Tcf4-bound pool of beta-catenin was significantly enriched in acetylated form. We demonstrated that the acetyltransferase activity of p300 was required for efficient activation of transcription mediated by beta-catenin/Tcf4 and that the cooperation between p300 and beta-catenin was severely reduced by the K345R mutation, implying that acetylation of beta-catenin plays a part in the coactivation of beta-catenin by p300. Interestingly, acetylation of beta-catenin had opposite, negative effects on the binding of beta-catenin to the androgen receptor. Our data suggest that acetylation of beta-catenin in the arm 6 domain regulates beta-catenin transcriptional activity by differentially modulating its affinity for Tcf4 and the androgen receptor. Thus, our results describe a new mechanism by which p300 might regulate beta-catenin transcriptional activity.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Transactivadores/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra , Acetilación , Secuencia de Aminoácidos , Línea Celular , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Genes Reporteros , Histona Acetiltransferasas , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Péptidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción TCF , Transactivadores/química , Transactivadores/genética , Proteína 2 Similar al Factor de Transcripción 7 , Transcripción Genética , Proteínas Wnt , beta Catenina , Factores de Transcripción p300-CBPRESUMEN
Inappropriate activation of the Wnt/beta-catenin signaling has been implicated in the development of hepatocellular carcinoma (HCC), but exactly how beta-catenin works remains to be elucidated. To identify, in vivo, the target genes of beta-catenin in the liver, we have used the suppression subtractive hybridization technique and transgenic mice expressing an activated beta-catenin in the liver that developed hepatomegaly. We identified three genes involved in glutamine metabolism, encoding glutamine synthetase (GS), ornithine aminotransferase (OAT) and the glutamate transporter GLT-1. By Northern blot and immunohistochemical analysis we demonstrated that these three genes were specifically induced by activation of the beta-catenin pathway in the liver. In different mouse models bearing an activated beta-catenin signaling in the liver known to be associated with hepatocellular proliferation we observed a marked up-regulation of these three genes. The cellular distribution of GS and GLT-1 parallels beta-catenin activity. By contrast no up-regulation of these three genes was observed in the liver in which hepatocyte proliferation was induced by a signal-independent of beta-catenin. In addition, the GS promoter was activated in the liver of GS(+/LacZ) mice by adenovirus vector-mediated beta-catenin overexpression. Strikingly, the overexpression of the GS gene in human HCC samples was strongly correlated with beta-catenin activation. Together, our results indicate that GS is a target of the Wnt/beta-catenin pathway in the liver. Because a linkage of the glutamine pathway to hepatocarcinogenesis has already been demonstrated, we propose that regulation of these three genes of glutamine metabolism by beta-catenin is a contributing factor to liver carcinogenesis.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Glutamina/metabolismo , Hígado/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Cartilla de ADN , Transportador 2 de Aminoácidos Excitadores/genética , Glutamato-Amoníaco Ligasa/genética , Inmunohistoquímica , Hígado/enzimología , Ratones , Ratones Transgénicos , Ornitina-Oxo-Ácido Transaminasa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , beta CateninaRESUMEN
PHRF1 functions as an essential component of the TGF-ß tumor suppressor pathway by triggering degradation of the homeodomain repressor factor TGIF. This leads to redistribution of cPML into the cytoplasm, where it coordinates phosphorylation and activation of Smad2 by the TGF-ß receptor. In acute promyelocytic leukemia (APL), acquisition of PML-RARα is known to impede critical aspects of TGF-ß signaling, including myeloid differentiation. Although these defects are thought to rely on suppression of cPML activity, the mechanisms underlying this phenomenon remain enigmatic. Here, we find that an abnormal function of PML-RARα is to interfere with TGIF breakdown, presumably by competing with PHRF1 for binding to TGIF, culminating in cPML sequestration and inactivation. Enforcing PHRF1 activity is sufficient to restore TGF-ß cytostatic signaling in human blasts and suppress APL formation in a mouse model of APL, providing proof-of-concept data that suppression of PHRF1 activity by PML-RARα represents a critical determinant in APL pathogenesis.
RESUMEN
Many types of human cancers having hyperactivated Wnt signaling display no causative alterations in known effectors of this pathway. Here, we report a function of TGIF in Wnt signaling. TGIF associates with and diverts Axin1 and Axin2 from the ß-catenin destruction complex, therefore allowing ß-catenin accrual. Intriguingly, activation of Wnt signaling induces the expression of TGIF, which unveils a feed-forward loop that ensures effective integration of Wnt signaling. In triple-negative breast cancers (TNBC), elevated levels of TGIF correlate with high Wnt signaling and poor survival of patients. Moreover, genetic experiments revealed that Tgif1 ablation impeded mammary tumor development in MMTV-Wnt1 mice, further underscoring a requirement of TGIF for oncogenic Wnt signaling.
Asunto(s)
Proteínas de Homeodominio/fisiología , Neoplasias Mamarias Experimentales/metabolismo , Proteínas Represoras/fisiología , Vía de Señalización Wnt , Transporte Activo de Núcleo Celular , Animales , Proteína Axina/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Modelos Biológicos , Proteínas Represoras/metabolismo , beta Catenina/metabolismoRESUMEN
Hepatocellular carcinoma (HCC) is a major type of primary liver cancer and one of the rare human neoplasms etiologically linked to viral factors. Chronic infections with the hepatitis B virus (HBV) and the hepatitis C virus (HCV) have been implicated in about 80% of cases worldwide, and other known environmental risk factors, including alcohol abuse and dietary intake of aflatoxin B1, might synergize with viral infections. Recent insight into the molecular mechanisms leading to HCC development has been provided by the identification of major genetic abnormalities revealed by genomewide allelotype studies and molecular cytogenetic analysis. Moreover, several oncogenic pathways have been implicated in malignant transformation of liver cells. Inactivation of the p53 tumor suppressor gene by mutations and allelic deletions in about 30% of HCC cases has been associated predominantly with exposure to aflatoxin B1 and HBV infection. By contrast, a mutation in the beta-catenin gene in around 22% of HCCs is more rare in HBV-associated tumors. Activation of cyclin D1 and disruption of the Rb pathway are also commonly involved in liver tumorigenesis. New major challenges include the identification of candidate genes located in frequently altered chromosomal regions and that of oncogenic pathways driven by different risk factors. This search might shed some light on the tumorigenic role of HBV and HCV. It might also permit accurate evaluation of major targets for prognostic and therapeutic intervention.
Asunto(s)
Carcinoma Hepatocelular/genética , Virus de la Hepatitis B/genética , Neoplasias Hepáticas/genética , Transactivadores , Carcinoma Hepatocelular/virología , Aberraciones Cromosómicas , Proteínas del Citoesqueleto/genética , Regulación de la Expresión Génica , Virus de la Hepatitis B/fisiología , Humanos , Neoplasias Hepáticas/virología , Pérdida de Heterocigocidad , Virus Oncogénicos/fisiología , Proteínas Supresoras de Tumor/genética , beta CateninaRESUMEN
Arkadia is a RING domain E3 ubiquitin ligase that activates the transforming growth factor ß (TGF-ß) pathway by inducing degradation of the inhibitor SnoN/Ski. Here we show that Arkadia contains three successive SUMO-interacting motifs (SIMs) that mediate noncovalent interaction with poly-SUMO2. We identify the third SIM (VVDL) of Arkadia to be the most relevant one in this interaction. Furthermore, we provide evidence that Arkadia can function as a SUMO-targeted ubiquitin ligase (STUBL) by ubiquitinating SUMO chains. While the SIMs of Arkadia are not essential for SnoN/Ski degradation in response to TGF-ß, we show that they are necessary for the interaction of Arkadia with polysumoylated PML in response to arsenic and its concomitant accumulation into PML nuclear bodies. Moreover, Arkadia depletion leads to accumulation of polysumoylated PML in response to arsenic, highlighting a requirement of Arkadia for arsenic-induced degradation of polysumoylated PML. Interestingly, Arkadia homodimerizes but does not heterodimerize with RNF4, the other STUBL involved in PML degradation, suggesting that these two E3 ligases do not act synergistically but most probably act independently during this process. Altogether, these results identify Arkadia to be a novel STUBL that can trigger degradation of signal-induced polysumoylated proteins.
Asunto(s)
Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arsénico/farmacología , Línea Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
TGF-ß can act as a tumor suppressor at early stages of cancer progression and as a tumor promoter at later stages. The E3 ubiquitin ligase Arkadia (RNF111) is a critical component of the TGF-ß signaling pathway, being required for a subset of responses, those mediated by Smad3-Smad4 complexes. It acts by mediating ligand-induced degradation of Ski and SnoN (SKIL), which are 2 potent transcriptional repressors. Here, we investigate the role of Arkadia in cancer using model systems to address both potential tumor-suppressive and tumor-promoting roles. Stable reexpression of Arkadia in lung carcinoma NCI-H460 cells, which we show contain a hemizygous nonsense mutation in the Arkadia/RNF111 gene, efficiently restored TGF-ß-induced Smad3-dependent transcription, and substantially decreased the ability of these cells to grow in soft agar in vitro. However, it had no effect on tumor growth in vivo in mouse models. Moreover, loss of Arkadia in cancer cell lines and human tumors is rare, arguing against a prominent tumor-suppressive role. In contrast, we have uncovered a potent tumor-promoting function for Arkadia. Using 3 different cancer cell lines whose tumorigenic properties are driven by TGF-ß signaling, we show that loss of Arkadia function, either by overexpression of dominant negative Arkadia or by siRNA-induced knockdown, substantially inhibited lung colonization in tail vein injection experiments in immunodeficient mice. Our findings indicate that Arkadia is not critical for regulating tumor growth per se, but is required for the early stages of cancer cell colonization at the sites of metastasis.
Asunto(s)
Metástasis de la Neoplasia/prevención & control , Proteínas Nucleares/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Biocatálisis , Western Blotting , Línea Celular Tumoral , Humanos , Ratones , Mutación , Proteínas Nucleares/genética , Proteína smad3/fisiología , Transcripción Genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The homeodomain protein TGIF (TG-interacting factor) restricts TGF-ß/Smad cytostatic signaling by interfering with the nucleocytoplasmic transit of the tumor suppressor cPML. Here, we identify PHRF1 as a ubiquitin ligase that enforces TGIF decay by driving its ubiquitination at lysine 130. In so doing, PHRF1 ensures redistribution of cPML into the cytoplasm, where it associates with SARA and coordinates activation of Smad2 by the TGF-ß receptor. The PHRF1 gene resides within the tumor suppressor locus 11p15.5, which displays frequent loss in a wide variety of malignancies, including breast cancer. Remarkably, we found that the PHRF1 gene is deleted or silenced in a high proportion of human breast cancer samples and cancer cell lines. Reconstitution of PHRF1 into deficient cells impeded their propensity to form tumors in vivo, most likely because of the reemergence of TGF-ß responsiveness. These findings unveil a paradigm behind inactivation of the cPML tumor suppressor network in human malignancies.