RESUMEN
c-Myc (MYC) is an oncogenic transcription factor that is commonly overexpressed in a wide variety of human tumors. In breast cancer, MYC has recently been linked to the triple-negative subtype, a subtype that lacks any targeted therapy. Previously, we demonstrated that MYC behaves as a potent repressor of YAP and TAZ, 2 transcriptional coactivators that function as downstream transducers of the Hippo pathway. In this previous study, MYC repressed YAP/TAZ not only in primary breast epithelial cells but also in mouse models of triple-negative tumors. Here, we extend our previous bioinformatic and experimental analyses and demonstrate that MYC deregulation in primary breast epithelial cells leads to a robust repression of TEAD transcription factor activity, the transcription factor family mainly responsible for YAP/TAZ recruitment. Surprisingly, we find that MYC and TEAD activity is able to stratify different breast cancer subtypes in large panels of breast cancer patients. Thus, a deep understanding of the MYC-YAP/TAZ circuitry might yield new insights into the establishment and maintenance of specific breast cancer subtypes.
Asunto(s)
Neoplasias de la Mama/clasificación , Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Epiteliales/metabolismo , Femenino , Humanos , Fosfoproteínas/metabolismo , Factores de Transcripción de Dominio TEA , Proteínas Señalizadoras YAPRESUMEN
MYC is an unstable protein, and its turnover is controlled by the ubiquitin system. Ubiquitination enhances MYC-dependent transactivation, but the underlying mechanism remains unresolved. Here we show that MYC proteasomal turnover is dispensable for loading of RNA polymerase II (RNAPII). In contrast, MYC turnover is essential for recruitment of TRRAP, histone acetylation, and binding of BRD4 and P-TEFb to target promoters, leading to phosphorylation of RNAPII and transcriptional elongation. In the absence of histone acetylation and P-TEFb recruitment, MYC associates with the PAF1 complex (PAF1C) through a conserved domain in the MYC amino terminus ("MYC box I"). Depletion of the PAF1C subunit CDC73 enhances expression of MYC target genes, suggesting that the MYC/PAF1C complex can inhibit transcription. Because several ubiquitin ligases bind to MYC via the same domain ("MYC box II") that interacts with TRRAP, we propose that degradation of MYC limits the accumulation of MYC/PAF1C complexes during transcriptional activation.
Asunto(s)
Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Elongación de la Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitinación , Acetilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Proliferación Celular , Ensamble y Desensamble de Cromatina , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Complejos Multiproteicos , Mutación , Proteínas Nucleares/genética , Fosfoproteínas/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Proteolisis , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , ARN Polimerasa II/metabolismo , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Proteínas Supresoras de Tumor/genéticaRESUMEN
In several developmental lineages, an increase in MYC expression drives the transition from quiescent stem cells to transit-amplifying cells. We show that MYC activates a stereotypic transcriptional program of genes involved in cell growth in mammary epithelial cells. This change in gene expression indirectly inhibits the YAP/TAZ co-activators, which maintain the clonogenic potential of these cells. We identify a phospholipase of the mitochondrial outer membrane, PLD6, as the mediator of MYC activity. MYC-dependent growth strains cellular energy resources and stimulates AMP-activated kinase (AMPK). PLD6 alters mitochondrial fusion and fission dynamics downstream of MYC. This change activates AMPK, which in turn inhibits YAP/TAZ. Mouse models and human pathological data show that MYC enhances AMPK and suppresses YAP/TAZ activity in mammary tumors.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Neoplasias de la Mama/metabolismo , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glándulas Mamarias Humanas/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular , Linaje de la Célula , Biología Computacional , Bases de Datos Genéticas , Activación Enzimática , Inducción Enzimática , Células Epiteliales/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Glándulas Mamarias Humanas/patología , Ratones Transgénicos , Mitocondrias/patología , Fenotipo , Fosfolipasa D/biosíntesis , Fosfolipasa D/genética , Fosfoproteínas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Transfección , Proteínas Señalizadoras YAPRESUMEN
Deregulated expression of MYC is a driver of colorectal carcinogenesis, necessitating novel strategies to inhibit MYC function. The ubiquitin ligase HUWE1 (HECTH9, ARF-BP1, MULE) associates with both MYC and the MYC-associated protein MIZ1. We show here that HUWE1 is required for growth of colorectal cancer cells in culture and in orthotopic xenograft models. Using high-throughput screening, we identify small molecule inhibitors of HUWE1, which inhibit MYC-dependent transactivation in colorectal cancer cells, but not in stem and normal colon epithelial cells. Inhibition of HUWE1 stabilizes MIZ1. MIZ1 globally accumulates on MYC target genes and contributes to repression of MYC-activated target genes upon HUWE1 inhibition. Our data show that transcriptional activation by MYC in colon cancer cells requires the continuous degradation of MIZ1 and identify a novel principle that allows for inhibition of MYC function in tumor cells.
Asunto(s)
Neoplasias Colorrectales/enzimología , Proteína Oncogénica p55(v-myc)/antagonistas & inhibidores , Proteína Oncogénica p55(v-myc)/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/fisiopatología , Regulación Neoplásica de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones SCID , Proteína Oncogénica p55(v-myc)/genética , Unión Proteica , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Activación Transcripcional , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Colorectal cancer is the third most common cancer worldwide. Although the transcription factor c-MYC is misregulated in the majority of colorectal tumors, it is difficult to target directly. The deubiquitinase USP28 stabilizes oncogenic factors, including c-MYC; however, the contribution of USP28 in tumorigenesis, particularly in the intestine, is unknown. Here, using murine genetic models, we determined that USP28 antagonizes the ubiquitin-dependent degradation of c-MYC, a known USP28 substrate, as well as 2 additional oncogenic factors, c-JUN and NOTCH1, in the intestine. Mice lacking Usp28 had no apparent adverse phenotypes, but exhibited reduced intestinal proliferation and impaired differentiation of secretory lineage cells. In a murine model of colorectal cancer, Usp28 deletion resulted in fewer intestinal tumors, and importantly, in established tumors, Usp28 deletion reduced tumor size and dramatically increased lifespan. Moreover, we identified Usp28 as a c-MYC target gene highly expressed in murine and human intestinal cancers, which indicates that USP28 and c-MYC form a positive feedback loop that maintains high c-MYC protein levels in tumors. Usp28 deficiency promoted tumor cell differentiation accompanied by decreased proliferation, which suggests that USP28 acts similarly in intestinal homeostasis and colorectal cancer models. Hence, inhibition of the enzymatic activity of USP28 may be a potential target for cancer therapy.
Asunto(s)
Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/etiología , Ubiquitina Tiolesterasa/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Neoplasias Colorrectales/genética , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica , Genes myc , Células HCT116 , Células HEK293 , Células HeLa , Homeostasis , Humanos , Intestinos/enzimología , Intestinos/patología , Ratones , Ratones Noqueados , Mutación , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptor Notch1/metabolismo , Ubiquitina Tiolesterasa/deficiencia , Ubiquitina Tiolesterasa/genéticaRESUMEN
The HRD ubiquitin ligase recognizes and ubiquitylates proteins of the endoplasmic reticulum that display structural defects. Here, we apply quantitative proteomics to characterize the substrate spectrum of the HRD complex. Among the identified substrates is Erg3p, a glycoprotein involved in sterol synthesis. We characterize Erg3p and demonstrate that the elimination of Erg3p requires Htm1p and Yos9p, two proteins that take part in the glycan-dependent turnover of aberrant proteins. We further show that the HRD ligase also mediates the breakdown of Erg3p and CPY* engineered to lack N-glycans. The degradation of these nonglycosylated substrates is enhanced by a mutant variant of Yos9p that has lost its affinity for oligosaccharides, indicating that Yos9p has a previously unrecognized role in the quality control of nonglycosylated proteins.
Asunto(s)
Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Carboxipeptidasas/metabolismo , Técnicas de Inactivación de Genes , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
The SCFFbw7 ubiquitin ligase mediates growth-factor-regulated turnover of the Myc oncoprotein. Here we show that SCFß-TrCP binds to Myc by means of a characteristic phosphodegron and ubiquitylates Myc; this results in enhanced Myc stability. SCFFbw7 and SCFß-TrCP can exert these differential effects through polyubiquitylation of the amino terminus of Myc. Whereas SCFFbw7 with the Cdc34 ubiquitin-conjugating enzyme specifically requires lysine 48 (K48) of ubiquitin, SCFß-TrCP uses the UbcH5 ubiquitin-conjugating enzyme to form heterotypic polyubiquitin chains on Myc. Ubiquitylation of Myc by SCFß-TrCP is required for Myc-dependent acceleration of cell cycle progression after release from an arrest in S phase. Therefore, alternative ubiquitylation events at the N terminus can lead to the ubiquitylation-dependent stabilization of Myc.