RESUMEN
Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.
Asunto(s)
Regeneración Nerviosa , Humanos , Neoplasias/tratamiento farmacológico , Regeneración Nerviosa/efectos de los fármacos , Isoformas de Proteínas/agonistas , Transducción de Señal/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase I/química , Fosfatidilinositol 3-Quinasa Clase I/efectos de los fármacos , Cardiotónicos/farmacología , Animales , Biocatálisis/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Neuritas/efectos de los fármacos , Daño por Reperfusión/prevención & control , Compresión Nerviosa , Proliferación Celular/efectos de los fármacosRESUMEN
A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.
Asunto(s)
Cromatina/metabolismo , ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Nucleosomas/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica/fisiología , Proteómica/métodos , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Factores de Elongación Transcripcional/metabolismoRESUMEN
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known for specifically killing cancer cells, whereas in resistant cancers, TRAIL/TRAIL-R can promote metastasis via Rac1 and PI3K. It remains unknown, however, whether and to what extent TRAIL/TRAIL-R signaling in cancer cells can affect the immune microenvironment. Here we show that TRAIL-triggered cytokine secretion from TRAIL-resistant cancer cells is FADD dependent and identify the TRAIL-induced secretome to drive monocyte polarization to myeloid-derived suppressor cells (MDSCs) and M2-like macrophages. TRAIL-R suppression in tumor cells impaired CCL2 production and diminished both lung MDSC presence and tumor growth. In accordance, the receptor of CCL2, CCR2, is required to facilitate increased MDSC presence and tumor growth. Finally, TRAIL and CCL2 are co-regulated with MDSC/M2 markers in lung adenocarcinoma patients. Collectively, endogenous TRAIL/TRAIL-R-mediated CCL2 secretion promotes accumulation of tumor-supportive immune cells in the cancer microenvironment, thereby revealing a tumor-supportive immune-modulatory role of the TRAIL/TRAIL-R system in cancer biology.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Citocinas/metabolismo , Neoplasias Pulmonares/metabolismo , Macrófagos/metabolismo , Receptores CCR2/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Microambiente Tumoral , Células A549 , Adenocarcinoma/genética , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Pulmón de Células no Pequeñas/patología , Caspasa 8/genética , Caspasa 8/metabolismo , Proliferación Celular , Quimiocina CCL2/metabolismo , Proteína de Dominio de Muerte Asociada a Fas/genética , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Femenino , Células HCT116 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Macrófagos/inmunología , Macrófagos/patología , Ratones Endogámicos C57BL , Ratones SCID , Fenotipo , Interferencia de ARN , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/genética , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Quinasas raf/química , Quinasas raf/metabolismo , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Línea Celular Tumoral , Edición Génica , Técnicas de Inactivación de Genes , Humanos , Fosforilación , Multimerización de Proteína , Proteínas ras/metabolismoRESUMEN
Protein biomarkers for epithelial ovarian cancer are critical for the early detection of the cancer to improve patient prognosis and for the clinical management of the disease to monitor treatment response and to detect recurrences. Unfortunately, the discovery of protein biomarkers is hampered by the limited availability of reliable and sensitive assays needed for the reproducible quantification of proteins in complex biological matrices such as blood plasma. In recent years, targeted mass spectrometry, exemplified by selected reaction monitoring (SRM) has emerged as a method, capable of overcoming this limitation. Here, we present a comprehensive SRM-based strategy for developing plasma-based protein biomarkers for epithelial ovarian cancer and illustrate how the SRM platform, when combined with rigorous experimental design and statistical analysis, can result in detection of predictive analytes.Our biomarker development strategy first involved a discovery-driven proteomic effort to derive potential N-glycoprotein biomarker candidates for plasma-based detection of human ovarian cancer from a genetically engineered mouse model of endometrioid ovarian cancer, which accurately recapitulates the human disease. Next, 65 candidate markers selected from proteins of different abundance in the discovery dataset were reproducibly quantified with SRM assays across a large cohort of over 200 plasma samples from ovarian cancer patients and healthy controls. Finally, these measurements were used to derive a 5-protein signature for distinguishing individuals with epithelial ovarian cancer from healthy controls. The sensitivity of the candidate biomarker signature in combination with CA125 ELISA-based measurements currently used in clinic, exceeded that of CA125 ELISA-based measurements alone. The SRM-based strategy in this study is broadly applicable. It can be used in any study that requires accurate and reproducible quantification of selected proteins in a high-throughput and multiplexed fashion.
Asunto(s)
Biomarcadores de Tumor/sangre , Carcinoma Epitelial de Ovario/sangre , Espectrometría de Masas/métodos , Neoplasias Ováricas/sangre , Proteómica/métodos , Animales , Antígenos de Neoplasias/sangre , Proteínas Sanguíneas/análisis , Antígeno Ca-125/sangre , Estudios de Casos y Controles , Estudios de Cohortes , Desmogleína 2/sangre , Femenino , Enfermedad de las Cadenas Pesadas/sangre , Humanos , Cadenas mu de Inmunoglobulina/sangre , Proteínas de la Membrana/sangre , Ratones Transgénicos , Molécula L1 de Adhesión de Célula Nerviosa/sangre , Sensibilidad y Especificidad , Trombospondina 1/sangreRESUMEN
Protein biomarkers have the potential to transform medicine as they are clinically used to diagnose diseases, stratify patients, and follow disease states. Even though a large number of potential biomarkers have been proposed over the past few years, almost none of them have been implemented so far in the clinic. One of the reasons for this limited success is the lack of technologies to validate proposed biomarker candidates in larger patient cohorts. This limitation could be alleviated by the use of antibody-independent validation methods such as selected reaction monitoring (SRM). Similar to measurements based on affinity reagents, SRM-based targeted mass spectrometry also requires the generation of definitive assays for each targeted analyte. Here, we present a library of SRM assays for 5568 N-glycosites enabling the multiplexed evaluation of clinically relevant N-glycoproteins as biomarker candidates. We demonstrate that this resource can be utilized to select SRM assay sets for cancer-associated N-glycoproteins for their subsequent multiplexed and consistent quantification in 120 human plasma samples. We show that N-glycoproteins spanning 5 orders of magnitude in abundance can be quantified and that previously reported abundance differences in various cancer types can be recapitulated. Together, the established N-glycoprotein SRMAtlas resource facilitates parallel, efficient, consistent, and sensitive evaluation of proposed biomarker candidates in large clinical sample cohorts.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/sangre , Glicoproteínas/sangre , Proteínas de Neoplasias/sangre , Neoplasias/sangre , Animales , Antígenos de Carbohidratos Asociados a Tumores/química , Estudios de Casos y Controles , Glicoproteínas/química , Humanos , Ratones , Anotación de Secuencia Molecular , Proteínas de Neoplasias/química , Biblioteca de Péptidos , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem/métodosRESUMEN
Primary cilia are antenna-like organelles which sense extracellular cues and act as signalling hubs. Cilia dysfunction causes a heterogeneous group of disorders known as ciliopathy syndromes affecting most organs. Cilia disassembly, the process by which cells lose their cilium, is poorly understood but frequently observed in disease and upon cell transformation. Here, we uncover a role for the PI3Kα signalling enzyme in cilia disassembly. Genetic PI3Kα-hyperactivation, as observed in PIK3CA-related overgrowth spectrum (PROS) and cancer, induced a ciliopathy-like phenotype during mouse development. Mechanistically, PI3Kα and PI3Kß produce the PIP3 lipid at the cilia transition zone upon disassembly stimulation. PI3Kα activation initiates cilia disassembly through a kinase signalling axis via the PDK1/PKCι kinases, the CEP170 centrosomal protein and the KIF2A microtubule-depolymerising kinesin. Our data suggest diseases caused by PI3Kα-activation may be considered 'Disorders with Ciliary Contributions', a recently-defined subset of ciliopathies in which some, but not all, of the clinical manifestations result from cilia dysfunction.
Asunto(s)
Cilios , Fosfatidilinositol 3-Quinasa Clase I , Transducción de Señal , Cilios/metabolismo , Animales , Ratones , Humanos , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/genética , Ciliopatías/metabolismo , Ciliopatías/genética , Ciliopatías/patología , Cinesinas/metabolismo , Cinesinas/genéticaRESUMEN
SWATH-MS is a data-independent acquisition method that generates, in a single measurement, a complete recording of the fragment ion spectra of all the analytes in a biological sample for which the precursor ions are within a predetermined m/z versus retention time window. To assess the performance and suitability of SWATH-MS-based protein quantification for clinical use, we compared SWATH-MS and SRM-MS-based quantification of N-linked glycoproteins in human plasma, a commonly used sample for biomarker discovery. Using dilution series of isotopically labeled heavy peptides representing biomarker candidates, the LOQ of SWATH-MS was determined to reach 0.0456 fmol at peptide level by targeted data analysis, which corresponds to a concentration of 5-10 ng protein/mL in plasma, while SRM reached a peptide LOQ of 0.0152 fmol. Moreover, the quantification of endogenous glycoproteins using SWATH-MS showed a high degree of reproducibility, with the mean CV of 14.90%, correlating well with SRM results (R(2) = 0.9784). Overall, SWATH-MS measurements showed a slightly lower sensitivity and a comparable reproducibility to state-of-the-art SRM measurements for targeted quantification of the N-glycosites in human blood. However, a significantly larger number of peptides can be quantified per analysis. We suggest that SWATH-MS analysis combined with N-glycoproteome enrichment in plasma samples is a promising integrative proteomic approach for biomarker discovery and verification.
Asunto(s)
Glicoproteínas/sangre , Espectrometría de Masas/métodos , Secuencia de Aminoácidos , Biomarcadores/sangre , Femenino , Glicoproteínas/química , Glicosilación , Humanos , Iones , Masculino , Espectrometría de Masas/normas , Datos de Secuencia Molecular , Péptidos/química , Proteómica/métodos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA, and R-loops, even in the absence of cohesin. Our results place SA1 on chromatin upstream of the cohesin ring and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.
Asunto(s)
Estructuras R-Loop , ARN , ARN/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina , Factor de Unión a CCCTC/metabolismo , CohesinasRESUMEN
The development of plasma biomarkers has proven to be more challenging than initially anticipated. Many studies have reported lists of candidate proteins rather than validated candidate markers with an assigned performance to a specific clinical objective. Biomarker research necessitates a clear rational framework with requirements on a multitude of levels. On the technological front, the platform needs to be effective to detect low abundant plasma proteins and be able to measure them in a high throughput manner over a large amount of samples reproducibly. At a conceptual level, the choice of the technological platform and available samples should be part of an overall clinical study design that depends on a joint effort between basic and clinical research. Solutions to these needs are likely to facilitate more feasible studies. Targeted proteomic workflows based on SRM mass spectrometry show the potential of fast verification of biomarker candidates in plasma and thereby closing the gap between discovery and validation in the biomarker development pipeline. Biological samples need to be carefully chosen based on well-established guidelines either for candidate discovery in the form of disease models with optimal fidelity to human disease or for candidate evaluation as well-designed and annotated clinical cohort groups. Most importantly, they should be representative of the target population and directly address the investigated clinical question. A conceptual structure of a biomarker study can be provided in the form of several sequential phases, each having clear objectives and predefined goals. Furthermore, guidelines for reporting the outcome of biomarker studies are critical to adequately assess the quality of the research, interpretation and generalization of the results. By being attentive to and applying these considerations, biomarker research should become more efficient and lead to directly translatable biomarker candidates into clinical evaluation.
Asunto(s)
Biomarcadores/sangre , Proteínas Sanguíneas/análisis , Técnicas y Procedimientos Diagnósticos , Proteómica , Animales , Humanos , Espectrometría de MasasRESUMEN
The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition.
Asunto(s)
Adhesión Celular/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Neoplasias/genética , Neurofibromina 2/genética , Proteínas Supresoras de Tumor/genética , Proliferación Celular/genética , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Inhibición de Contacto/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Neoplasias/patología , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Transducción de Señal/genéticaRESUMEN
Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos , Antineoplásicos , Endocitosis/efectos de los fármacos , Transporte de Proteínas , Membrana Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Receptores ErbB/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/etiología , Neoplasias de Cabeza y Cuello/metabolismo , Humanos , Lisosomas/metabolismo , Fosfoproteínas , Receptores de Superficie Celular , Factores de TranscripciónRESUMEN
The linear-ubiquitin chain assembly complex (LUBAC) modulates signalling via various immune receptors. In tumour necrosis factor (TNF) signalling, linear (also known as M1) ubiquitin enables full gene activation and prevents cell death. However, the mechanisms underlying cell death prevention remain ill-defined. Here, we show that LUBAC activity enables TBK1 and IKKε recruitment to and activation at the TNF receptor 1 signalling complex (TNFR1-SC). While exerting only limited effects on TNF-induced gene activation, TBK1 and IKKε are essential to prevent TNF-induced cell death. Mechanistically, TBK1 and IKKε phosphorylate the kinase RIPK1 in the TNFR1-SC, thereby preventing RIPK1-dependent cell death. This activity is essential in vivo, as it prevents TNF-induced lethal shock. Strikingly, NEMO (also known as IKKγ), which mostly, but not exclusively, binds the TNFR1-SC via M1 ubiquitin, mediates the recruitment of the adaptors TANK and NAP1 (also known as AZI2). TANK is constitutively associated with both TBK1 and IKKε, while NAP1 is associated with TBK1. We discovered a previously unrecognized cell death checkpoint that is mediated by TBK1 and IKKε, and uncovered an essential survival function for NEMO, whereby it enables the recruitment and activation of these non-canonical IKKs to prevent TNF-induced cell death.
Asunto(s)
Quinasa I-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células A549 , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Células HeLa , Humanos , Ratones Noqueados , Fosforilación/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquitinación/efectos de los fármacosRESUMEN
Cancer is mostly incurable when diagnosed at a metastatic stage, making its early detection via blood proteins of immense clinical interest. Proteomic changes in tumor tissue may lead to changes detectable in the protein composition of circulating blood plasma. Using a proteomic workflow combining N-glycosite enrichment and SWATH mass spectrometry, we generate a data resource of 284 blood samples derived from patients with different types of localized-stage carcinomas and from matched controls. We observe whether the changes in the patient's plasma are specific to a particular carcinoma or represent a generic signature of proteins modified uniformly in a common, systemic response to many cancers. A quantitative comparison of the resulting N-glycosite profiles discovers that proteins related to blood platelets are common to several cancers (e.g., THBS1), whereas others are highly cancer-type specific. Available proteomics data, including a SWATH library to study N-glycoproteins, will facilitate follow-up biomarker research into early cancer detection.
Asunto(s)
Carcinoma/sangre , Carcinoma/patología , Glicoproteínas/sangre , Espectrometría de Masas/métodos , Algoritmos , Plaquetas/metabolismo , Carcinoma/genética , Estudios de Cohortes , Humanos , Estadificación de Neoplasias , Oncogenes , Proteoma/metabolismo , Curva ROCRESUMEN
To identify novel effectors and processes regulated by PI3K pathway activation, we performed an unbiased phosphoproteomic screen comparing two common events of PI3K deregulation in cancer: oncogenic Pik3ca mutation (Pik3caH1047R) and deletion of Pten. Using mouse embryonic fibroblast (MEF) models that generate inducible, low-level pathway activation as observed in cancer, we quantified 7566 unique phosphopeptides from 3279 proteins. A number of proteins were found to be differentially-regulated by Pik3caH1047R and Pten loss, suggesting unique roles for these two events in processes such as vesicular trafficking, DNA damage repair and RNA splicing. We also identified novel PI3K effectors that were commonly-regulated, including putative AKT substrates. Validation of one of these hits, confirmed NT5C (5',3'-Nucleotidase, Cytosolic) as a novel AKT substrate, with an unexpected role in actin cytoskeleton regulation via an interaction with the ARP2/3 complex. This study has produced a comprehensive data resource and identified a new link between PI3K pathway activation and actin regulation.
Asunto(s)
5'-Nucleotidasa/metabolismo , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfoproteínas/análisis , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Células Cultivadas , Fosfatidilinositol 3-Quinasa Clase I , Eliminación de Gen , Regulación de la Expresión Génica , Ratones , Células Madre Embrionarias de Ratones , Mutación , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
Recruitment of the deubiquitinase CYLD to signaling complexes is mediated by its interaction with HOIP, the catalytically active component of the linear ubiquitin chain assembly complex (LUBAC). Here, we identify SPATA2 as a constitutive direct binding partner of HOIP that bridges the interaction between CYLD and HOIP. SPATA2 recruitment to TNFR1- and NOD2-signaling complexes is dependent on HOIP, and loss of SPATA2 abolishes CYLD recruitment. Deficiency in SPATA2 exerts limited effects on gene activation pathways but diminishes necroptosis induced by tumor necrosis factor (TNF), resembling loss of CYLD. In summary, we describe SPATA2 as a previously unrecognized factor in LUBAC-dependent signaling pathways that serves as an adaptor between HOIP and CYLD, thereby enabling recruitment of CYLD to signaling complexes.
Asunto(s)
Macrófagos/metabolismo , Proteínas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Sitios de Unión , Clonación Molecular , Enzima Desubiquitinante CYLD , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Células HeLa , Humanos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Proteína Adaptadora de Señalización NOD2/genética , Proteína Adaptadora de Señalización NOD2/metabolismo , Plásmidos , Cultivo Primario de Células , Unión Proteica , Proteínas/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Supresoras de Tumor/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The current management of colorectal cancer (CRC) would greatly benefit from non-invasive prognostic biomarkers indicative of clinicopathological tumor characteristics. Here, we employed targeted proteomic profiling of 80 glycoprotein biomarker candidates across plasma samples of a well-annotated patient cohort with comprehensive CRC characteristics. Clinical data included 8-year overall survival, tumor staging, histological grading, regional localization, and molecular tumor characteristics. The acquired quantitative proteomic dataset was subjected to the development of biomarker signatures predicting prognostic clinical endpoints. Protein candidates were selected into the signatures based on significance testing and a stepwise protein selection, each within 10-fold cross-validation. A six-protein biomarker signature of patient outcome could predict survival beyond clinical stage and was able to stratify patients into groups of better and worse prognosis. We further evaluated the performance of the signature on the mRNA level and assessed its prognostic value in the context of previously published transcriptional signatures. Additional signatures predicting regional tumor localization and disease dissemination were also identified. The integration of rich clinical data, quantitative proteomic technologies, and tailored computational modeling facilitated the characterization of these signatures in patient circulation. These findings highlight the value of a simultaneous assessment of important prognostic disease characteristics within a single measurement.
Asunto(s)
Biomarcadores de Tumor/sangre , Técnicas de Laboratorio Clínico/métodos , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/patología , Plasma/química , Proteómica/métodos , Humanos , PronósticoRESUMEN
Ubiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes. LUBAC-generated linear (M1) ubiquitin is important for signaling via various immune receptors. We show here that the deubiquitinases CYLD and A20, but not OTULIN, are recruited to the TNFR1- and NOD2-associated signaling complexes (TNF-RSC and NOD2-SC), at which they cooperate to limit gene activation. Whereas CYLD recruitment depends on its interaction with LUBAC, but not on LUBAC's M1-chain-forming capacity, A20 recruitment requires this activity. Intriguingly, CYLD and A20 exert opposing effects on M1 chain stability in the TNF-RSC and NOD2-SC. While CYLD cleaves M1 chains, and thereby sensitizes cells to TNF-induced death, A20 binding to them prevents their removal and, consequently, inhibits cell death. Thus, CYLD and A20 cooperatively restrict gene activation and regulate cell death via their respective activities on M1 chains. Hence, the interplay between LUBAC, M1-ubiquitin, CYLD, and A20 is central for physiological signaling through innate immune receptors.
Asunto(s)
Muerte Celular/fisiología , Activación Transcripcional/fisiología , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Línea Celular , Proteínas de Unión al ADN/metabolismo , Enzima Desubiquitinante CYLD , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Transducción de Señal/fisiología , Transducción Genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Non-invasive detection of colorectal cancer with blood-based markers is a critical clinical need. Here we describe a phased mass spectrometry-based approach for the discovery, screening, and validation of circulating protein biomarkers with diagnostic value. Initially, we profiled human primary tumor tissue epithelia and characterized about 300 secreted and cell surface candidate glycoproteins. These candidates were then screened in patient systemic circulation to identify detectable candidates in blood plasma. An 88-plex targeting method was established to systematically monitor these proteins in two large and independent cohorts of plasma samples, which generated quantitative clinical datasets at an unprecedented scale. The data were deployed to develop and evaluate a five-protein biomarker signature for colorectal cancer detection.
Asunto(s)
Biomarcadores de Tumor/sangre , Técnicas de Laboratorio Clínico/métodos , Neoplasias Colorrectales/diagnóstico , Espectrometría de Masas/métodos , Plasma/química , HumanosRESUMEN
Targeted proteomics based on selected reaction monitoring (SRM) mass spectrometry is commonly used for accurate and reproducible quantification of protein analytes in complex biological mixtures. Strictly hypothesis-driven, SRM assays quantify each targeted protein by collecting measurements on its peptide fragment ions, called transitions. To achieve sensitive and accurate quantitative results, experimental design and data analysis must consistently account for the variability of the quantified transitions. This consistency is especially important in large experiments, which increasingly require profiling up to hundreds of proteins over hundreds of samples. Here we describe a robust and automated workflow for the analysis of large quantitative SRM data sets that integrates data processing, statistical protein identification and quantification, and dissemination of the results. The integrated workflow combines three software tools: mProphet for peptide identification via probabilistic scoring; SRMstats for protein significance analysis with linear mixed-effect models; and PASSEL, a public repository for storage, retrieval and query of SRM data. The input requirements for the protocol are files with SRM traces in mzXML format, and a file with a list of transitions in a text tab-separated format. The protocol is especially suited for data with heavy isotope-labeled peptide internal standards. We demonstrate the protocol on a clinical data set in which the abundances of 35 biomarker candidates were profiled in 83 blood plasma samples of subjects with ovarian cancer or benign ovarian tumors. The time frame to realize the protocol is 1-2 weeks, depending on the number of replicates used in the experiment.