RESUMEN
Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/metabolismo , Proteoma/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Humanos , Espectrometría de Masas/métodos , Inestabilidad de Microsatélites , Mutación/genética , Proteómica/métodosRESUMEN
Elucidation of the mutational landscape of human cancer has progressed rapidly and been accompanied by the development of therapeutics targeting mutant oncogenes. However, a comprehensive mapping of cancer dependencies has lagged behind and the discovery of therapeutic targets for counteracting tumor suppressor gene loss is needed. To identify vulnerabilities relevant to specific cancer subtypes, we conducted a large-scale RNAi screen in which viability effects of mRNA knockdown were assessed for 7,837 genes using an average of 20 shRNAs per gene in 398 cancer cell lines. We describe findings of this screen, outlining the classes of cancer dependency genes and their relationships to genetic, expression, and lineage features. In addition, we describe robust gene-interaction networks recapitulating both protein complexes and functional cooperation among complexes and pathways. This dataset along with a web portal is provided to the community to assist in the discovery and translation of new therapeutic approaches for cancer.
Asunto(s)
Neoplasias/genética , Neoplasias/patología , Interferencia de ARN , Línea Celular Tumoral , Biblioteca de Genes , Redes Reguladoras de Genes , Humanos , Complejos Multiproteicos/metabolismo , Neoplasias/metabolismo , Oncogenes , ARN Interferente Pequeño , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.
Asunto(s)
Línea Celular Tumoral , Neoplasias/genética , Neoplasias/patología , Antineoplásicos/farmacología , Biomarcadores de Tumor , Metilación de ADN , Resistencia a Antineoplásicos , Etnicidad/genética , Edición Génica , Histonas/metabolismo , Humanos , MicroARNs/genética , Terapia Molecular Dirigida , Neoplasias/metabolismo , Análisis por Matrices de Proteínas , Empalme del ARNRESUMEN
Reliable methods to quantify dynamic signaling changes across diverse pathways are needed to better understand the effects of disease and drug treatment in cells and tissues but are presently lacking. Here, we present SigPath, a targeted mass spectrometry (MS) assay that measures 284 phosphosites in 200 phosphoproteins of biological interest. SigPath probes a broad swath of signaling biology with high throughput and quantitative precision. We applied the assay to investigate changes in phospho-signaling in drug-treated cancer cell lines, breast cancer preclinical models, and human medulloblastoma tumors. In addition to validating previous findings, SigPath detected and quantified a large number of differentially regulated phosphosites newly associated with disease models and human tumors at baseline or with drug perturbation. Our results highlight the potential of SigPath to monitor phosphoproteomic signaling events and to nominate mechanistic hypotheses regarding oncogenesis, response, and resistance to therapy.
Asunto(s)
Fosfoproteínas , Proteómica , Humanos , Espectrometría de Masas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Transducción de SeñalRESUMEN
Cell autonomous cancer dependencies are now routinely identified using CRISPR loss-of-function viability screens. However, a bias exists that makes it difficult to assess the true essentiality of genes located in amplicons, since the entire amplified region can exhibit lethal scores. These false-positive hits can either be discarded from further analysis, which in cancer models can represent a significant number of hits, or methods can be developed to rescue the true-positives within amplified regions. We propose two methods to rescue true positive hits in amplified regions by correcting for this copy number artefact. The Local Drop Out (LDO) method uses the relative lethality scores within genomic regions to assess true essentiality and does not require additional orthogonal data (e.g. copy number value). LDO is meant to be used in screens covering a dense region of the genome (e.g. a whole chromosome or the whole genome). The General Additive Model (GAM) method models the screening data as a function of the known copy number values and removes the systematic effect from the measured lethality. GAM does not require the same density as LDO, but does require prior knowledge of the copy number values. Both methods have been developed with single sample experiments in mind so that the correction can be applied even in smaller screens. Here we demonstrate the efficacy of both methods at removing the copy number effect and rescuing hits from some of the amplified regions. We estimate a 70-80% decrease of false positive hits with either method in regions of high copy number compared to no correction.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Variaciones en el Número de Copia de ADN/genética , Neoplasias/genética , Artefactos , Astrocitoma/genética , Astrocitoma/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular , Conjuntos de Datos como Asunto , Reacciones Falso Positivas , Genómica , Humanos , Modelos Teóricos , Neoplasias/patología , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologíaRESUMEN
The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2-Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2-Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.
Asunto(s)
Anafase/fisiología , Endopeptidasas/metabolismo , Ubiquitina/metabolismo , Anafase/efectos de los fármacos , Ciclosoma-Complejo Promotor de la Anafase , Proteínas de Unión al Calcio/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Endopeptidasas/deficiencia , Endopeptidasas/genética , Células HeLa , Humanos , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Proteínas Mad2 , Paclitaxel/farmacología , Proteínas Represoras/metabolismo , Reproducibilidad de los Resultados , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteasas Ubiquitina-EspecíficasRESUMEN
von Hippel-Lindau (VHL) gene inactivation occurs in von Hippel-Lindau (VHL) disease. The protein pVHL functions in a multi-subunit E3 ubiquitin ligase that targets the hypoxia-inducible transcription factor Hif1 alpha for proteasomal degradation during normoxia. We establish that pVHL binds to Tat-binding protein-1 (TBP-1), a component of the 19S regulatory complex of the proteasome. TBP-1 associates with the beta-domain of pVHL and complexes with pVHL and Hif1 alpha in vivo. Overexpression of TBP-1 promotes degradation of Hif1 alpha in a pVHL-dependent manner that requires the ATPase domain of TBP-1. Blockade of TBP-1 expression by small interfering RNA (siRNA) causes prolonged degradation kinetics of Hif1 alpha. Several distinct mutations in exon 2 of VHL disrupt binding of pVHL to TBP-1. A pVHL mutant containing a P154L substitution coimmunoprecipitates with Hif1 alpha, but not TBP-1, and does not promote degradation of Hif1 alpha. Thus, the ability of pVHL to degrade Hif1 alpha depends in part on its interaction with TBP-1 and suggests a new mechanism for Hif1 alpha stabilization in some pVHL-deficient tumors.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Supresoras de Tumor/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , ATPasas Asociadas con Actividades Celulares Diversas , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Humanos , Hidrólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Datos de Secuencia Molecular , Unión Proteica , ARN Interferente Pequeño , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-LindauRESUMEN
Various subunits of mammalian SWI/SNF chromatin remodeling complexes display loss-of-function mutations characteristic of tumor suppressors in different cancers, but an additional role for SWI/SNF supporting cell survival in distinct cancer contexts is emerging. In particular, genetic dependence on the catalytic subunit BRG1/SMARCA4 has been observed in acute myelogenous leukemia (AML), yet the feasibility of direct therapeutic targeting of SWI/SNF catalytic activity in leukemia remains unknown. Here, we evaluated the activity of dual BRG1/BRM ATPase inhibitors across a genetically diverse panel of cancer cell lines and observed that hematopoietic cancer cell lines were among the most sensitive compared with other lineages. This result was striking in comparison with data from pooled short hairpin RNA screens, which showed that only a subset of leukemia cell lines display sensitivity to BRG1 knockdown. We demonstrate that combined genetic knockdown of BRG1 and BRM is required to recapitulate the effects of dual inhibitors, suggesting that SWI/SNF dependency in human leukemia extends beyond a predominantly BRG1-driven mechanism. Through gene expression and chromatin accessibility studies, we show that the dual inhibitors act at genomic loci associated with oncogenic transcription factors, and observe a downregulation of leukemic pathway genes, including MYC, a well-established target of BRG1 activity in AML. Overall, small-molecule inhibition of BRG1/BRM induced common transcriptional responses across leukemia models resulting in a spectrum of cellular phenotypes. IMPLICATIONS: Our studies reveal the breadth of SWI/SNF dependency in leukemia and support targeting SWI/SNF catalytic function as a potential therapeutic strategy in AML.
Asunto(s)
Adenosina Trifosfatasas , Leucemia Mieloide Aguda , Adenosina Trifosfatasas/genética , Animales , Carcinogénesis , Ensamble y Desensamble de Cromatina , ADN Helicasas/genética , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Mamíferos/genética , Mamíferos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Interferons (IFNs) are cytokines that play a critical role in limiting infectious and malignant diseases 1-4 . Emerging data suggest that the strength and duration of IFN signaling can differentially impact cancer therapies, including immune checkpoint blockade 5-7 . Here, we characterize the output of IFN signaling, specifically IFN-stimulated gene (ISG) signatures, in primary tumors from The Cancer Genome Atlas. While immune infiltration correlates with the ISG signature in some primary tumors, the existence of ISG signature-positive tumors without evident infiltration of IFN-producing immune cells suggests that cancer cells per se can be a source of IFN production. Consistent with this hypothesis, analysis of patient-derived tumor xenografts propagated in immune-deficient mice shows evidence of ISG-positive tumors that correlates with expression of human type I and III IFNs derived from the cancer cells. Mechanistic studies using cell line models from the Cancer Cell Line Encyclopedia that harbor ISG signatures demonstrate that this is a by-product of a STING-dependent pathway resulting in chronic tumor-derived IFN production. This imposes a transcriptional state on the tumor, poising it to respond to the aberrant accumulation of double-stranded RNA (dsRNA) due to increased sensor levels (MDA5, RIG-I and PKR). By interrogating our functional short-hairpin RNA screen dataset across 398 cancer cell lines, we show that this ISG transcriptional state creates a novel genetic vulnerability. ISG signature-positive cancer cells are sensitive to the loss of ADAR, a dsRNA-editing enzyme that is also an ISG. A genome-wide CRISPR genetic suppressor screen reveals that the entire type I IFN pathway and the dsRNA-activated kinase, PKR, are required for the lethality induced by ADAR depletion. Therefore, tumor-derived IFN resulting in chronic signaling creates a cellular state primed to respond to dsRNA accumulation, rendering ISG-positive tumors susceptible to ADAR loss.
Asunto(s)
Adenosina Desaminasa/metabolismo , Interferones/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Proteínas de la Membrana/metabolismo , Ratones Desnudos , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Supresión Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The caspase-associated ring proteins (CARP1 and CARP2) are distinguished from other caspase regulators by the presence of a FYVE-type zinc finger domain. FYVE-type domains are divided into two known classes: FYVE domains that specifically bind to phosphatidylinositol 3-phosphate in lipid bilayers and FYVE-related domains of undetermined function. Here, we report the crystal structure of the N-terminal region of CARP2 (44-139) including the FYVE-type domain and its associated helical bundle at 1.7 A resolution. The structure reveals a cramped phosphoinositide binding pocket and a blunted membrane insertion loop. These structural features indicate that the domain is not optimized to bind to phosphoinositides or insert into lipid bilayers. The CARP2 FYVE-like domain thus defines a third subfamily of FYVE-type domains that are functionally and structurally distinct. Structural analyses provide insights into the possible function of this unique subfamily of FYVE-type domains.
Asunto(s)
Proteínas Portadoras/química , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Membrana Celular/metabolismo , Cristalización , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Fosfatidilinositoles/metabolismo , Estructura Terciaria de ProteínaRESUMEN
UNLABELLED: CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. We compared the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies across multiple cancer cell lines. CRISPR dropout screens consistently identified more lethal genes than RNAi, implying that the identification of many cellular dependencies may require full gene inactivation. However, in two aneuploid cancer models, we found that all genes within highly amplified regions, including nonexpressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits. In addition, using a CRISPR tiling screen, we found that sgRNAs targeting essential domains generate the strongest lethality phenotypes and thus provide a strategy to rapidly define the protein domains required for cancer dependence. Collectively, these findings not only demonstrate the utility of CRISPR screens in the identification of cancer-essential genes, but also reveal the need to carefully control for false-positive results in chromosomally unstable cancer lines. SIGNIFICANCE: We show in this study that CRISPR-based screens have a significantly lower false-negative rate compared with RNAi-based screens, but have specific liabilities particularly in the interrogation of regions of genome amplification. Therefore, this study provides critical insights for applying CRISPR-based screens toward the systematic identification of new cancer targets. Cancer Discov; 6(8); 900-13. ©2016 AACR.See related commentary by Sheel and Xue, p. 824See related article by Aguirre et al., p. 914This article is highlighted in the In This Issue feature, p. 803.
Asunto(s)
Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Amplificación de Genes , Genoma Humano , Genómica , Neoplasias/genética , Línea Celular Tumoral , Estudios de Asociación Genética , Genómica/métodos , Genómica/normas , Ensayos Analíticos de Alto Rendimiento , Humanos , Fenotipo , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , ARN Guía de Kinetoplastida/genética , ARN Interferente Pequeño/genética , Reproducibilidad de los ResultadosRESUMEN
PURPOSE: To explore the genetic landscape of tumors from patients enrolled on the BOLERO-2 trial to identify potential correlations between genetic alterations and efficacy of everolimus treatment. The BOLERO-2 trial has previously demonstrated that the addition of everolimus to exemestane prolonged progression-free survival by more than twofold in patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative, advanced breast cancer previously treated with nonsteroidal aromatase inhibitors. PATIENTS AND METHODS: Next-generation sequencing was used to analyze genetic status of cancer-related genes in 302 archival tumor specimens from patients representative of the BOLERO-2 study population. Correlations between the most common somatic alterations and degree of chromosomal instability, and treatment effect of everolimus were investigated. RESULTS: Progression-free survival benefit with everolimus was maintained regardless of alteration status of PIK3CA, FGFR1, and CCND1 or the pathways of which they are components. However, quantitative differences in everolimus benefit were observed between patient subgroups defined by the exon-specific mutations in PIK3CA (exon 20 v 9) or by different degrees of chromosomal instability in the tumor tissues. CONCLUSION: The data from this exploratory analysis suggest that the efficacy of everolimus was largely independent of the most commonly altered genes or pathways in hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. The potential impact of chromosomal instabilities and low-frequency genetic alterations on everolimus efficacy warrants further investigation.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Everolimus/uso terapéutico , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Anciano , Inhibidores de la Aromatasa/uso terapéutico , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Técnicas para Inmunoenzimas , Persona de Mediana Edad , Mutación/genética , Invasividad Neoplásica , Metástasis de la Neoplasia , Estadificación de Neoplasias , Posmenopausia , Pronóstico , Tasa de SupervivenciaRESUMEN
5-Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine salvage pathway. The MTAP gene is frequently deleted in human cancers because of its chromosomal proximity to the tumor suppressor gene CDKN2A. By interrogating data from a large-scale short hairpin RNA-mediated screen across 390 cancer cell line models, we found that the viability of MTAP-deficient cancer cells is impaired by depletion of the protein arginine methyltransferase PRMT5. MTAP-deleted cells accumulate the metabolite methylthioadenosine (MTA), which we found to inhibit PRMT5 methyltransferase activity. Deletion of MTAP in MTAP-proficient cells rendered them sensitive to PRMT5 depletion. Conversely, reconstitution of MTAP in an MTAP-deficient cell line rescued PRMT5 dependence. Thus, MTA accumulation in MTAP-deleted cancers creates a hypomorphic PRMT5 state that is selectively sensitized toward further PRMT5 inhibition. Inhibitors of PRMT5 that leverage this dysregulated metabolic state merit further investigation as a potential therapy for MTAP/CDKN2A-deleted tumors.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Metionina/metabolismo , Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Desoxiadenosinas/metabolismo , Eliminación de Gen , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína-Arginina N-Metiltransferasas/genética , Purina-Nucleósido Fosforilasa/genética , ARN Interferente Pequeño/genética , Tionucleósidos/metabolismoRESUMEN
The DNA damage response (DDR) is brought about by a protein kinase cascade that orchestrates DNA repair through transcriptional and posttranslational mechanisms. Cell cycle arrest is a hallmark of the DDR. We screened for cells that lacked damage-induced cell cycle arrest and uncovered a critical role for Fanconi anemia and homologous recombination proteins in ATR (ataxia telangiectasia and Rad3-related) signaling. Three DDR candidates, the RNA processing protein INTS7, the circadian transcription factor CLOCK, and a previously uncharacterized protein RHINO, were recruited to sites of DNA damage. RHINO independently bound the Rad9-Rad1-Hus1 complex (9-1-1) and the ATR activator TopBP1. RHINO was recruited to sites of DNA damage by the 9-1-1 complex to promote Chk1 activation. We suggest that RHINO functions together with the 9-1-1 complex and TopBP1 to fully activate ATR.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/metabolismo , Quimiocinas/fisiología , Reparación del ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Quimiocinas/genética , Quimiocinas CXC , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Exonucleasas/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismoAsunto(s)
Genes Supresores de Tumor , Técnicas Genéticas , Técnicas del Sistema de Dos Híbridos , Animales , Citoplasma/metabolismo , ADN Complementario/metabolismo , Biblioteca de Genes , Vectores Genéticos , Humanos , Litio/farmacología , Modelos Genéticos , Plásmidos/metabolismo , Transcripción Genética , beta-Galactosidasa/metabolismoRESUMEN
Caspase 8/10-associated RING proteins (CARPs) are a recently described family of protein ubiquitin ligases that interact with and negatively regulate death receptor-mediated apoptosis. Because CARPs are overexpressed in cancer and their silencing reduces cell viability and sensitizes tumor cells to chemotherapeutic agents, we investigated their relationship to p53 tumor suppressor signaling. p53 is a major determinant of chemosensitivity, and its levels are increased following DNA damage through N-terminal phosphorylation and inhibition of degradation. Although p53 is well known to be negatively regulated by several ubiquitin ligases including MDM2, none are known to target phosphorylated p53 for degradation. CARPs physically interact with and ubiquitinate p53, targeting it for degradation in the absence of MDM2. Serine 20-phosphorylated p53 is also ubiquitinated by CARPs. CARP silencing stimulates p53 expression and promotes downstream effects, including transcriptional activation and tumor suppression.
Asunto(s)
Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Serina , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Apoptosis , Línea Celular , Supervivencia Celular , Daño del ADN , Vectores Genéticos , Humanos , Ratones , Proteínas Recombinantes/metabolismo , Proteína p53 Supresora de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Fanconi anemia (FA) is a developmental and cancer-predisposition syndrome caused by mutations in genes controlling DNA interstrand crosslink repair. Several FA proteins form a ubiquitin ligase that controls monoubiquitination of the FANCD2 protein in an ATR-dependent manner. Here we describe the FA protein FANCI, identified as an ATM/ATR kinase substrate required for resistance to mitomycin C. FANCI shares sequence similarity with FANCD2, likely evolving from a common ancestral gene. The FANCI protein associates with FANCD2 and, together, as the FANCI-FANCD2 (ID) complex, localize to chromatin in response to DNA damage. Like FANCD2, FANCI is monoubiquitinated and unexpectedly, ubiquitination of each protein is important for the maintenance of ubiquitin on the other, indicating the existence of a dual ubiquitin-locking mechanism required for ID complex function. Mutation in FANCI is responsible for loss of a functional FA pathway in a patient with Fanconi anemia complementation group I.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Ciclo Celular , Línea Celular , Daño del ADN , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Humanos , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación , Fase S , Strongylocentrotus purpuratusRESUMEN
Cellular responses to DNA damage are mediated by a number of protein kinases, including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related). The outlines of the signal transduction portion of this pathway are known, but little is known about the physiological scope of the DNA damage response (DDR). We performed a large-scale proteomic analysis of proteins phosphorylated in response to DNA damage on consensus sites recognized by ATM and ATR and identified more than 900 regulated phosphorylation sites encompassing over 700 proteins. Functional analysis of a subset of this data set indicated that this list is highly enriched for proteins involved in the DDR. This set of proteins is highly interconnected, and we identified a large number of protein modules and networks not previously linked to the DDR. This database paints a much broader landscape for the DDR than was previously appreciated and opens new avenues of investigation into the responses to DNA damage in mammals.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Sitios de Unión , Ciclo Celular/fisiología , Línea Celular , Biología Computacional , Secuencia de Consenso , Replicación del ADN/fisiología , Humanos , Inmunoprecipitación , Marcaje Isotópico , Ratones , Células 3T3 NIH , Fosforilación , Proteoma/aislamiento & purificación , Proteoma/fisiología , ARN Interferente Pequeño , Transducción de Señal , Especificidad por SustratoRESUMEN
The destruction of cellular targets during apoptosis is carried out by caspases, which are negatively regulated by the inhibitor of apoptosis proteins (IAP); however, death effector domain (DED) caspases of the extrinsic pathway are refractory to the IAP family. We have isolated a family of apoptotic inhibitors [caspases-8- and -10-associated RING proteins (CARPs)] that bind to and negatively regulate DED caspases. When overexpressed, CARPs, via an IAP-like RING domain, can contribute to the ubiquitin-mediated proteolysis of DED caspases. Furthermore, CARPs are rapidly cleaved during apoptosis. However, in tumors and tumor cell lines, they are overexpressed, and their silencing leads to restoration of efficient apoptosis via enhanced activation of DED caspases. Long-term inhibition of CARP expression results in suppression of cancer cell growth, highlighting their importance in tumor cell survival.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , División Celular , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Línea Celular Tumoral , Clonación Molecular , Humanos , Hidrólisis , Ligandos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Fosfolípidos/metabolismo , ARN Interferente Pequeño/fisiología , Homología de Secuencia de AminoácidoRESUMEN
Many tumor cell types are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Incubation of TRAIL-sensitive cells with TRAIL invariably leads to resistant survivors even when high doses of TRAIL are used. Because the emergence of resistance to apoptosis is a major concern in successful treatment of cancer, and TRAIL survivors may contribute to therapeutic failure, we investigated potential resistance mechanisms. We selected TRAIL-resistant SW480 human colon adenocarcinoma cells by repeatedly treating them with high and/or low doses of TRAIL. The resulting TRAIL-resistant clones were not cross-resistant to Fas or paclitaxel. Expression of modulators of apoptosis was not changed in the resistant cells, including TRAIL receptors, cFLIP, Bax, Bid, or IAP proteins. Surprisingly, we found that DISC formation was deficient in multiple selected TRAIL-resistant clones. DR4 was not recruited to the DISC upon TRAIL treatment, and caspase-8 was not activated at the DISC. Although total cellular DR4 mRNA and protein were virtually identical in TRAIL-sensitive parental and TRAIL-resistant clones, DR4 protein expression on the cell surface was essentially undetectable in the TRAIL-resistant clones. Moreover, exogenous DR4 and KILLER/DR5 were not properly transported to the cell surface in the TRAIL-resistant cells. Interestingly, TRAIL-resistant cells were resensitized to TRAIL by tunicamycin pretreatment, which increased cell surface expression of DR4 and KILLER/DR5. Our data suggest that tumor cells may become resistant to TRAIL through regulation of the death receptor cell surface transport and that resistance to TRAIL may be overcome by the glycosylation inhibitor/endoplasmic reticulum stress-inducing agent tunicamycin.