RESUMEN
The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included ß2-microglobulin, a known regulator of MR1 surface expression, and ATP13A1, a P5-type ATPase in the endoplasmic reticulum (ER) not previously known to be associated with MR1-mediated antigen presentation. CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.
Asunto(s)
Presentación de Antígeno , Antígenos de Histocompatibilidad Clase I , Complejo Mayor de Histocompatibilidad , Antígenos de Histocompatibilidad Menor , ATPasas Tipo P , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , ATPasas Tipo P/inmunologíaRESUMEN
In model organisms, classical genetic screening via random mutagenesis provides key insights into the molecular bases of genetic interactions, helping to define synthetic lethality, synthetic viability and drug-resistance mechanisms. The limited genetic tractability of diploid mammalian cells, however, precludes this approach. Here, we demonstrate the feasibility of classical genetic screening in mammalian systems by using haploid cells, chemical mutagenesis and next-generation sequencing, providing a new tool to explore mammalian genetic interactions.
Asunto(s)
Pruebas Genéticas , Genoma/efectos de los fármacos , Genoma/genética , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Mutagénesis/efectos de los fármacos , Animales , Línea Celular , RatonesRESUMEN
Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.
Asunto(s)
Neoplasias de la Mama/genética , Epigénesis Genética , Factor de Transcripción GATA3/genética , Antígenos de Histocompatibilidad/genética , N-Metiltransferasa de Histona-Lisina/genética , Neoplasias de la Mama/patología , Supervivencia sin Enfermedad , Resistencia a Antineoplásicos/genética , Estrógenos/genética , Estrógenos/metabolismo , Femenino , Mutación del Sistema de Lectura , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
The great majority of targeted anticancer drugs inhibit mutated oncogenes that display increased activity. Yet many tumors do not contain such actionable aberrations, such as those harboring loss-of-function mutations. The notion of targeting synthetic lethal vulnerabilities in cancer cells has provided an alternative approach to exploiting more of the genetic and epigenetic changes acquired during tumorigenesis. Here, we review synthetic lethality as a therapeutic concept that exploits the inherent differences between normal cells and cancer cells. Furthermore, we provide an overview of the screening approaches that can be used to identify synthetic lethal interactions in human cells and present several recently identified interactions that may be pharmacologically exploited. Finally, we indicate some of the challenges of translating synthetic lethal interactions into the clinic and how these may be overcome.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Diseño de Fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Terapia Genética/métodos , Terapia Molecular Dirigida/métodos , Neoplasias/terapia , Animales , Biomarcadores de Tumor/metabolismo , Muerte Celular/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal/efectos de los fármacos , Investigación Biomédica TraslacionalRESUMEN
Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.
Asunto(s)
Sistemas CRISPR-Cas/genética , Haploidia , Eliminación de Secuencia , Línea Celular , Perfilación de la Expresión Génica , Ingeniería Genética/métodos , Genómica , Humanos , CariotipoRESUMEN
MOTIVATION: Calling changes in DNA, e.g. as a result of somatic events in cancer, requires analysis of multiple matched sequenced samples. Events in low-mappability regions of the human genome are difficult to encode in variant call files and have been under-reported as a result. However, they can be described accurately through thesaurus annotation-a technique that links multiple genomic loci together to explicate a single variant. RESULTS: We here describe software and benchmarks for using thesaurus annotation to detect point changes in DNA from matched samples. In benchmarks on matched normal/tumor samples we show that the technique can recover between five and ten percent more true events than conventional approaches, while strictly limiting false discovery and being fully consistent with popular variant analysis workflows. We also demonstrate the utility of the approach for analysis of de novo mutations in parents/child families. AVAILABILITY AND IMPLEMENTATION: Software performing thesaurus annotation is implemented in java; available in source code on github at GeneticThesaurus (https://github.com/tkonopka/GeneticThesaurus) and as an executable on sourceforge at geneticthesaurus (https://sourceforge.net/projects/geneticthesaurus). Mutation calling is implemented in an R package available on github at RGeneticThesaurus (https://github.com/tkonopka/RGeneticThesaurus). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. CONTACT: tomasz.konopka@ludwig.ox.ac.uk.
Asunto(s)
Variación Genética , Genoma Humano , Genómica , Humanos , Programas Informáticos , Vocabulario ControladoRESUMEN
The upswing in US Food and Drug Administration and European Medicines Agency drug approvals in 2014 may have marked an end to the dry spell that has troubled the pharmaceutical industry over the past decade. Regardless, the attrition rate of drugs in late clinical phases remains high, and a lack of target validation has been highlighted as an explanation. This has led to a resurgence in appreciation of phenotypic drug screens, as these may be more likely to yield compounds with relevant modes of action. However, cell-based screening approaches do not directly reveal cellular targets, and hence target deconvolution and a detailed understanding of drug action are needed for efficient lead optimization and biomarker development. Here, recently developed functional genomics technologies that address this need are reviewed. The approaches pioneered in model organisms, particularly in yeast, and more recently adapted to mammalian systems are discussed. Finally, areas of particular interest and directions for future tool development are highlighted.
Asunto(s)
Genómica , Preparaciones Farmacéuticas , Animales , HumanosRESUMEN
High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that avoids the need for chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small-molecule screen against human protein kinases, including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes.
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Ensayos Analíticos de Alto Rendimiento , Luz , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Células HEK293 , Humanos , Inhibidores de Proteínas Quinasas/química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Detecting genetic variation is one of the main applications of high-throughput sequencing, but is still challenging wherever aligning short reads poses ambiguities. Current state-of-the-art variant calling approaches avoid such regions, arguing that it is necessary to sacrifice detection sensitivity to limit false discovery. We developed a method that links candidate variant positions within repetitive genomic regions into clusters. The technique relies on a resource, a thesaurus of genetic variation, that enumerates genomic regions with similar sequence. The resource is computationally intensive to generate, but once compiled can be applied efficiently to annotate and prioritize variants in repetitive regions. We show that thesaurus annotation can reduce the rate of false variant calls due to mappability by up to three orders of magnitude. We apply the technique to whole genome datasets and establish that called variants in low mappability regions annotated using the thesaurus can be experimentally validated. We then extend the analysis to a large panel of exomes to show that the annotation technique opens possibilities to study variation in hereto hidden and under-studied parts of the genome.
Asunto(s)
Variación Genética , Genoma Humano , Genómica/métodos , Vocabulario Controlado , Línea Celular Tumoral , Exoma , Humanos , Anotación de Secuencia Molecular , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Histone lysine demethylases (KDMs) are of critical importance in the epigenetic regulation of gene expression, yet there are few selective, cell-permeable inhibitors or suitable tool compounds for these enzymes. We describe the discovery of a new class of inhibitor that is highly potent towards the histone lysine demethylases KDM2A/7A. A modular synthetic approach was used to explore the chemical space and accelerate the investigation of key structure-activity relationships, leading to the development of a small molecule with around 75-fold selectivity towards KDM2A/7A versus other KDMs, as well as cellular activity at low micromolar concentrations.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Proteínas F-Box/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Proteínas F-Box/metabolismo , Células HeLa , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-ß, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.
Asunto(s)
Biblioteca de Genes , Haploidia , Mutagénesis Insercional/métodos , Genética Inversa/métodos , Línea Celular Tumoral , Genoma Humano , Humanos , Datos de Secuencia MolecularRESUMEN
Some mutations in cancer cells can be exploited for therapeutic intervention. However, for many cancer subtypes, including triple-negative breast cancer (TNBC), no frequently recurring aberrations could be identified to make such an approach clinically feasible. Characterized by a highly heterogeneous mutational landscape with few common features, many TNBCs cluster together based on their 'basal-like' transcriptional profiles. We therefore hypothesized that targeting TNBC cells on a systems level by exploiting the transcriptional cell state might be a viable strategy to find novel therapies for this highly aggressive disease. We performed a large-scale chemical genetic screen and identified a group of compounds related to the drug PKC412 (midostaurin). PKC412 induced apoptosis in a subset of TNBC cells enriched for the basal-like subtype and inhibited tumor growth in vivo. We employed a multi-omics approach and computational modeling to address the mechanism of action and identified spleen tyrosine kinase (SYK) as a novel and unexpected target in TNBC. Quantitative phosphoproteomics revealed that SYK inhibition abrogates signaling to STAT3, explaining the selectivity for basal-like breast cancer cells. This non-oncogene addiction suggests that chemical SYK inhibition may be beneficial for a specific subset of TNBC patients and demonstrates that targeting cell states could be a viable strategy to discover novel treatment strategies.
Asunto(s)
Antineoplásicos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Terapia Molecular Dirigida , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT3/metabolismo , Estaurosporina/análogos & derivados , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Simulación del Acoplamiento Molecular , Dominios y Motivos de Interacción de Proteínas , Proteómica/métodos , Análisis de Secuencia de ARN , Transducción de Señal , Estaurosporina/farmacología , Quinasa Syk , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Measuring the impact of combinations of genetic or chemical perturbations on cellular fitness, sometimes referred to as synthetic lethal screening, is a powerful method for obtaining novel insights into gene function and drug action. Especially when performed at large scales, gene-gene or gene-drug interaction screens can reveal complex genetic interactions or drug mechanism of action or even identify novel therapeutics for the treatment of diseases.The result of such large-scale screen results can be represented as a matrix with a numeric score indicating the cellular fitness (e.g. viability or doubling time) for each double perturbation. In a typical screen, the majority of combinations do not impact the cellular fitness. Thus, it is critical to first discern true "hits" from noise. Subsequent data exploration and visualization methods can assist to extract meaningful biological information from the data. However, despite the increasing interest in combination perturbation screens, no user friendly open-source program exists that combines statistical analysis, data exploration tools and visualization. RESULTS: We developed TOPS (Tool for Combination Perturbation Screen Analysis), a Java and R-based software tool with a simple graphical user interface that allows the user to import, analyze, filter and plot data from double perturbation screens as well as other compatible data. TOPS was designed in a modular fashion to allow the user to add alternative importers for data formats or custom analysis scripts not covered by the original release.We demonstrate the utility of TOPS on two datasets derived from functional genetic screens using different methods. Dataset 1 is a gene-drug interaction screen and is based on Luminex xMAP technology. Dataset 2 is a gene-gene short hairpin (sh)RNAi screen exploring the interactions between deubiquitinating enzymes and a number of prominent oncogenes using massive parallel sequencing (MPS). CONCLUSIONS: TOPS provides the benchtop scientist with a free toolset to analyze, filter and visualize data from functional genomic gene-gene and gene-drug interaction screens with a flexible interface to accommodate different technologies and analysis algorithms in addition to those already provided here. TOPS is freely available for academic and non-academic users and is released as open source.
Asunto(s)
Evaluación Preclínica de Medicamentos , Genes , Programas Informáticos , Algoritmos , Neoplasias de la Mama/genética , Línea Celular Tumoral , Gráficos por Computador , Interpretación Estadística de Datos , Femenino , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Lineales , Interferencia de ARNRESUMEN
Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identify new therapeutic applications. However, there has been no systematic approach for analyzing gene-drug interactions in human cells. Here we establish a multiplexed assay to study the cellular fitness of a panel of engineered isogenic cancer cells in response to a collection of drugs, enabling the systematic analysis of thousands of gene-drug interactions. Applying this approach to breast cancer revealed various synthetic-lethal interactions and drug-resistance mechanisms, some of which were known, thereby validating the method. NOTCH pathway activation, which occurs frequently in breast cancer, unexpectedly conferred resistance to phosphoinositide 3-kinase (PI3K) inhibitors, which are currently undergoing clinical trials in breast cancer patients. NOTCH1 and downstream induction of c-MYC over-rode the dependency of cells on the PI3K-mTOR pathway for proliferation. These data reveal a new mechanism of resistance to PI3K inhibitors with direct clinical implications.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Inhibidores de las Quinasa Fosfoinosítidos-3 , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Mutación , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Monoubiquitination is a reversible post-translational protein modification that has an important regulatory function in many biological processes, including DNA repair. Deubiquitinating enzymes (DUBs) are proteases that are negative regulators of monoubiquitination, but little is known about their regulation and contribution to the control of conjugated-substrate levels. Here, we show that the DUB ubiquitin specific protease 1 (USP1) deubiquitinates the DNA replication processivity factor, PCNA, as a safeguard against error-prone translesion synthesis (TLS) of DNA. Ultraviolet (UV) irradiation inactivates USP1 through an autocleavage event, thus enabling monoubiquitinated PCNA to accumulate and to activate TLS. Significantly, the site of USP1 cleavage is immediately after a conserved internal ubiquitin-like diglycine (Gly-Gly) motif. This mechanism is reminiscent of the processing of precursors of ubiquitin and ubiquitin-like modifiers by DUBs. Our results define a regulatory mechanism for protein ubiquitination that involves the signal-induced degradation of an inhibitory DUB.
Asunto(s)
Daño del ADN/efectos de la radiación , Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis , Replicación del ADN , Endopeptidasas/química , Endopeptidasas/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación G de la Anemia de Fanconi/fisiología , Humanos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Proteasas Ubiquitina-Específicas , Rayos UltravioletaRESUMEN
Systemic mastocytosis is a neoplastic disease of mast cells harboring the activating KIT mutation D816V. In most patients, mast cell infiltration in the bone marrow is accompanied by marked microenvironment alterations, including increased angiogenesis, osteosclerosis, and sometimes fibrosis. Little is known about the mast cell-derived molecules contributing to these bone marrow alterations. We show here that neoplastic mast cells in patients with systemic mastocytosis express oncostatin M (OSM), a profibrogenic and angiogenic modulator. To study the regulation of OSM expression, KIT D816V was inducibly expressed in Ba/F3 cells and was found to up-regulate OSM mRNA and protein levels, suggesting that OSM is a KIT D816V-dependent mediator. Correspondingly, KIT D816V(+) HMC-1.2 cells expressed significantly higher amounts of OSM than the KIT D816V(-) HMC-1.1 subclone. RNA interference-induced knockdown of STAT5, a key transcription factor in KIT D816V(+) mast cells, inhibited OSM expression in HMC-1 cells, whereas a constitutively activated STAT5 mutant induced OSM expression. Finally, OSM secreted from KIT D816V(+) mast cells stimulated growth of endothelial cells, fibroblasts, and osteoblasts, suggesting that mast cell-derived OSM may serve as a key modulator of the marrow microenvironment and thus contribute to the pathology of systemic mastocytosis.
Asunto(s)
Médula Ósea/patología , Mastocitosis Sistémica/metabolismo , Mastocitosis Sistémica/patología , Oncostatina M/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Western Blotting , Médula Ósea/metabolismo , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , Immunoblotting , Inmunohistoquímica , Mastocitos/metabolismo , Mastocitos/patología , Mastocitosis Sistémica/genética , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT5/metabolismoRESUMEN
UNLABELLED: Signal transducer and activator of transcription 3 (Stat3) is activated in a variety of malignancies, including hepatocellular carcinoma (HCC). Activation of Ras occurs frequently at advanced stages of HCC by aberrant signaling through growth factor receptors or inactivation of effectors negatively regulating Ras signaling. Here, we addressed the role of Stat3 in Ras-dependent HCC progression in the presence and absence of p19(ARF) /p14(ARF) . We show that constitutive active (ca) Stat3 is tumor suppressive in Ras-transformed p19(ARF-/-) hepatocytes, whereas the expression of Stat3 lacking Tyr(705) phosphorylation (U-Stat3) enhances tumor formation. Accordingly, Ras-transformed Stat3(Δhc) /p19(ARF-/-) hepatocytes (lacking Stat3 and p19(ARF) ) showed increased tumor growth, compared to those expressing Stat3, demonstrating a tumor-suppressor activity of Stat3 in cells lacking p19(ARF) . Notably, endogenous expression of p19(ARF) in Ras-transformed hepatocytes conveyed oncogenic Stat3 functions, resulting in augmented or reduced HCC progression after the expression of caStat3 or U-Stat3, respectively. In accord with these data, the knockdown of p14(ARF) (the human homolog of p19(ARF) ) in Hep3B cells was associated with reduced pY-Stat3 levels during tumor growth to circumvent the tumor-suppressive effect of Stat3. Inhibition of Janus kinases (Jaks) revealed that Jak causes pY-Stat3 activation independently of p14(ARF) levels, indicating that p14(ARF) controls the oncogenic function of pY-Stat3 downstream of Jak. CONCLUSION: These data show evidence that p19(ARF) /p14(ARF) determines the pro- or anti-oncogenic activity of U-Stat3 and pY-Stat3 in Ras-dependent HCC progression.
Asunto(s)
Carcinoma Hepatocelular/fisiopatología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/fisiología , Neoplasias Hepáticas/fisiopatología , Factor de Transcripción STAT3/fisiología , Animales , Carcinoma Hepatocelular/patología , Proliferación Celular , Células Cultivadas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/deficiencia , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Hepatocitos/patología , Quinasas Janus/fisiología , Neoplasias Hepáticas/patología , Ratones , Ratones Noqueados , Transducción de Señal/fisiologíaRESUMEN
Protein modification by the conjugation of ubiquitin moieties--ubiquitination--plays a major part in many biological processes, including cell cycle and apoptosis. The enzymes that mediate ubiquitin-conjugation have been well-studied, but much less is known about the ubiquitin-specific proteases that mediate de-ubiquitination of cellular substrates. To study this gene family, we designed a collection of RNA interference vectors to suppress 50 human de-ubiquitinating enzymes, and used these vectors to identify de-ubiquitinating enzymes in cancer-relevant pathways. We report here that inhibition of one of these enzymes, the familial cylindromatosis tumour suppressor gene (CYLD), having no known function, enhances activation of the transcription factor NF-kappaB. We show that CYLD binds to the NEMO (also known as IKKgamma) component of the IkappaB kinase (IKK) complex, and appears to regulate its activity through de-ubiquitination of TRAF2, as TRAF2 ubiquitination can be modulated by CYLD. Inhibition of CYLD increases resistance to apoptosis, suggesting a mechanism through which loss of CYLD contributes to oncogenesis. We show that this effect can be relieved by aspirin derivatives that inhibit NF-kappaB activity, which suggests a therapeutic intervention strategy to restore growth control in patients suffering from familial cylindromatosis.
Asunto(s)
Apoptosis , FN-kappa B/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Apoptosis/efectos de los fármacos , Aspirina/análogos & derivados , Aspirina/farmacología , Línea Celular , Enzima Desubiquitinante CYLD , Humanos , Quinasa I-kappa B , FN-kappa B/antagonistas & inhibidores , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Interferencia de ARN , Factor 2 Asociado a Receptor de TNF , Transfección , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina/metabolismoRESUMEN
Intratumor heterogeneity is a key hallmark of cancer that contributes to progression and therapeutic resistance. Phenotypic heterogeneity is in part caused by Darwinian selection of subclones that arise by random (epi)genetic aberrations. In addition, cancer cells are endowed with increased cellular plasticity compared with their normal counterparts, further adding to their heterogeneous behavior. However, the molecular mechanisms underpinning cancer cell plasticity are incompletely understood. Here, I outline the hypothesis that cancer-associated perturbations collectively disrupt normal gene regulatory networks (GRNs) by increasing their entropy. Importantly, in this model both somatic driver and passenger alterations contribute to 'perturbation-driven entropy', thereby increasing phenotypic heterogeneity and evolvability. This additional layer of heterogeneity may contribute to our understanding of cancer evolution and therapeutic resistance.
Asunto(s)
Evolución Molecular , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Heterogeneidad Genética , Neoplasias/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Plasticidad de la Célula/genética , Resistencia a Antineoplásicos/genética , Entropía , Epigénesis Genética , Humanos , Modelos Genéticos , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Células Madre Neoplásicas/patologíaRESUMEN
Forward genetic screens in mammalian cell lines, such as RNAi and CRISPR-Cas9 screens, have made major contributions to the elucidation of diverse signaling pathways. Here, we exploited human haploid cells as a robust comparative screening platform and report a set of quantitative forward genetic screens for identifying regulatory mechanisms of mTORC1 signaling, a key growth control pathway that senses diverse metabolic states. Selected chemical and genetic perturbations in this screening platform, including rapamycin treatment and genetic ablation of the Ragulator subunit LAMTOR4, revealed the known core mTORC1 regulatory signaling complexes and the intimate interplay of the mTORC1 pathway with lysosomal function, validating the approach. In addition, we identified a differential requirement for LAMTOR4 and LAMTOR5 in regulating the mTORC1 pathway under fed and starved conditions. Furthermore, we uncovered a previously unknown "synthetic-sick" interaction between the tumor suppressor folliculin and LAMTOR4, which may have therapeutic implications in cancer treatment. Together, our study demonstrates the use of iterative "perturb and observe" genetic screens to uncover regulatory mechanisms driving complex mammalian signaling networks.