RESUMEN
RAS-MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1-3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF-MEK1-14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS-SHOC2-PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF-RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies-developmental syndromes caused by aberrant MAPK pathway activation6-14-and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)-RAS-driven tumours15-18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS-SHOC2-PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS-GTP molecules recruit RAF-14-3-3 and SHOC2-PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein-protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS-MAPK signalling, therefore providing the structural basis for therapeutic interventions.
Asunto(s)
Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular , Complejos Multiproteicos , Proteína Fosfatasa 1 , Proteínas ras , Proteínas 14-3-3 , Guanosina Trifosfato/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Complejos Multiproteicos/química , Mutación , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Proteína Fosfatasa 1/química , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Quinasas raf , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.
Asunto(s)
Genoma Humano , Proteoma/análisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Ciclopentanos/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Pirimidinas/farmacología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Defects in the structure or function of the primary cilium, an antennae-like structure whose functional integrity has been linked to the suppression of uncontrolled kidney epithelial cell proliferation, are a common feature of genetic disorders characterized by kidney cysts. However, the mechanisms by which primary cilia are maintained remain poorly defined. von Hippel-Lindau (VHL) disease is characterized by the development of premalignant renal cysts and arises because of functional inactivation of the VHL tumour suppressor gene product, pVHL. Here, we show that pVHL and glycogen synthase kinase (GSK)3beta are key components of an interlinked signalling pathway that maintains the primary cilium. Although inactivation of either pVHL or GSK3beta alone did not affect cilia maintenance, their combined inactivation leads to loss of cilia. In VHL patients, GSK3beta is subjected to inhibitory phosphorylation in renal cysts, but not in early VHL mutant lesions, and these cysts exhibit reduced frequencies of primary cilia. We propose that pVHL and GSK3beta function together in a ciliary-maintenance signalling network, disruption of which enhances the vulnerability of cells to lose their cilia, thereby promoting cyst formation.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Enfermedades Renales Quísticas/metabolismo , Túbulos Renales/metabolismo , Transducción de Señal , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Enfermedad de von Hippel-Lindau/metabolismo , Animales , Línea Celular , Cilios/metabolismo , Cilios/patología , Embrión de Mamíferos/citología , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Glucógeno Sintasa Quinasa 3/deficiencia , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Humanos , Enfermedades Renales Quísticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosforilación , Interferencia de ARN , Transfección , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/patologíaRESUMEN
Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.
Asunto(s)
Proteínas Portadoras , Quimera Dirigida a la Proteólisis , Ubiquitina-Proteína Ligasas , Proteínas Portadoras/metabolismo , Proteolisis , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
In this study, we describe the rapid identification of potent binders for the WD40 repeat domain (WDR) of DCAF1. This was achieved by two rounds of iterative focused screening of a small set of compounds selected on the basis of internal WDR domain knowledge followed by hit expansion. Subsequent structure-based design led to nanomolar potency binders with a clear exit vector enabling DCAF1-based bifunctional degrader exploration.
RESUMEN
In patients with von Hippel-Lindau (VHL) disease, renal cysts and clear cell renal cell carcinoma (ccRCC) arise from renal tubular epithelial cells containing biallelic inactivation of the VHL tumour suppressor gene. However, it is presumed that formation of renal cysts and their conversion to ccRCC involve additional genetic changes at other loci. Here, we show that cystic lesions in the kidneys of patients with VHL disease also demonstrate activation of the phosphatidylinositol-3-kinase (PI3K) pathway. Strikingly, combined conditional inactivation of Vhlh and the Pten tumour suppressor gene, which normally antagonises PI3K signalling, in the mouse kidney, elicits cyst formation after short latency, whereas inactivation of either tumour suppressor gene alone failed to produce such a phenotype. Interestingly, cells lining these cysts frequently lack a primary cilium, a microtubule-based cellular antenna important for suppression of uncontrolled kidney epithelial cell proliferation and cyst formation. Our results support a model in which the PTEN tumour suppressor protein cooperates with pVHL to suppress cyst development in the kidney.
Asunto(s)
Quistes/enzimología , Quistes/patología , Enfermedades Renales Quísticas/enzimología , Enfermedades Renales Quísticas/patología , Fosfohidrolasa PTEN/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Animales , Proliferación Celular , Cilios/enzimología , Cilios/patología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Túbulos Renales Distales/enzimología , Túbulos Renales Distales/patología , Sistema de Señalización de MAP Quinasas , Ratones , Mutación/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
While there are hundreds of predicted E3 ligases, characterizing their applications for targeted protein degradation has proved challenging. Here, we report a chemical biology approach to evaluate the ability of modified recombinant E3 ligase components to support neo-substrate degradation. Bypassing the need for specific E3 ligase binders, we use maleimide-thiol chemistry for covalent functionalization followed by E3 electroporation (COFFEE) in live cells. We demonstrate that electroporated recombinant von Hippel-Lindau (VHL) protein, covalently functionalized at its ligandable cysteine with JQ1 or dasatinib, induces degradation of BRD4 or tyrosine kinases, respectively. Furthermore, by applying COFFEE to SPSB2, a Cullin-RING ligase 5 receptor, as well as to SKP1, the adaptor protein for Cullin-RING ligase 1 F box (SCF) complexes, we validate this method as a powerful approach to define the activity of previously uncharacterized ubiquitin ligase components, and provide further evidence that not only E3 ligase receptors but also adaptors can be directly hijacked for neo-substrate degradation.
Asunto(s)
Ubiquitina-Proteína Ligasas/metabolismo , Línea Celular , Femenino , Humanos , Masculino , Proteínas Recombinantes/metabolismoRESUMEN
Bromodomain-containing protein 9 (BRD9), an essential component of the SWI/SNF chromatin remodeling complex termed ncBAF, has been established as a therapeutic target in a subset of sarcomas and leukemias. Here, we used novel small molecule inhibitors and degraders along with RNA interference to assess the dependency on BRD9 in the context of diverse hematological malignancies, including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM) model systems. Following depletion of BRD9 protein, AML cells undergo terminal differentiation, whereas apoptosis was more prominent in ALL and MM. RNA-seq analysis of acute leukemia and MM cells revealed both unique and common signaling pathways affected by BRD9 degradation, with common pathways including those associated with regulation of inflammation, cell adhesion, DNA repair and cell cycle progression. Degradation of BRD9 potentiated the effects of several chemotherapeutic agents and targeted therapies against AML, ALL, and MM. Our findings support further development of therapeutic targeting of BRD9, alone or combined with other agents, as a novel strategy for acute leukemias and MM.
Asunto(s)
Antineoplásicos , Leucemia Mieloide Aguda , Mieloma Múltiple , Factores de Transcripción , Antineoplásicos/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Interferencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
MYC oncoprotein is a multifunctional transcription factor that regulates the expression of a large number of genes involved in cellular growth, proliferation and metabolism. Altered MYC protein level lead to cellular transformation and tumorigenesis. MYC is deregulated in > 50% of human cancers, rendering it an attractive drug target. However, direct inhibition of this class of proteins using conventional small molecules is challenging due to their intrinsically disordered state. To discover novel posttranslational regulators of MYC protein stability and turnover, we established a genetic screen in mammalian cells by combining a fluorescent protein-based MYC abundance sensor, CRISPR/Cas9-based gene knockouts and next-generation sequencing. Our screen identifies UBR5, an E3 ligase of the HECT-type family, as a novel regulator of MYC degradation. Even in the presence of the well-described and functional MYC ligase, FBXW7, UBR5 depletion leads to accumulation of MYC in cells. We demonstrate interaction of UBR5 with MYC and reduced K48-linked ubiquitination of MYC upon loss of UBR5 in cells. Interestingly, in cancer cell lines with amplified MYC expression, depletion of UBR5 resulted in reduced cell survival, as a consequence of MYC stabilization. Finally, we show that MYC and UBR5 are co-amplified in more than 40% of cancer cells and that MYC copy number amplification correlates with enhanced transcriptional output of UBR5. This suggests that UBR5 acts as a buffer in MYC amplified settings and protects these cells from apoptosis.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias/patología , Proteolisis , Proteínas Proto-Oncogénicas c-myc/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Apoptosis , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Renal cysts and clear cell renal cell carcinoma are common clinical manifestations of people with germ-line mutations of the von Hippel-Lindau tumor suppressor gene, VHL. Recent cell biological evidence suggests that the VHL gene product, pVHL, functions to maintain the primary cilium, a microtubule-based antenna-like structure whose functional integrity is believed to have an important role in cell-cycle control. As VHL mutations are common in sporadic clear cell renal cell carcinoma, but not papillary renal cell carcinoma, we asked whether there is an association between VHL status and primary cilia in vivo. VHL status was assessed in 20 cases of clear cell renal cell carcinoma and 9 cases of papillary renal cell carcinoma by DNA sequencing and by immunohistochemical staining for the hypoxia-inducible factor-alpha target gene products CA9 and GLUT-1. Of 20, 18 clear cell renal cell carcinomas, but only 1 of 9 papillary renal cell carcinomas, displayed evidence of VHL inactivation. In clear cell renal cell carcinoma the frequency of ciliated tumor cells ranged from 0 to 22% (median value 7.8+/-6.0%), whereas cilia frequency was significantly higher (P<0.0001) in papillary renal cell carcinoma (range 12-83%, median value 43.3+/-21.3%). There was no correlation between Ki-67 staining and cilia frequency, suggesting that the observed differences between the tumor types in cilia frequency are not accounted for by differences in cellular proliferation rates and that primary cilia degeneration in sporadic clear cell renal cell carcinoma depends on VHL inactivation. We propose that the different ciliation status of clear cell and papillary renal cell carcinoma may contribute, at least in part, to the different biological behaviors of these tumor types.
Asunto(s)
Carcinoma de Células Renales/patología , Cilios/patología , Neoplasias Renales/patología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Carcinoma de Células Renales/genética , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Neoplasias Renales/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is linked to the development of tumors of the eyes, kidneys, and central nervous system. VHL encodes two gene products, pVHL30 and pVHL19, of which one, pVHL30, associates functionally with microtubules (MTs) to regulate their stability. Here we report that pVHL30 is a novel substrate of glycogen synthase kinase 3 (GSK3) in vitro and in vivo. Phosphorylation of pVHL on serine 68 (S68) by GSK3 requires a priming phosphorylation event at serine 72 (S72) mediated in vitro by casein kinase I. Functional analysis of pVHL species carrying nonphosphorylatable or phosphomimicking mutations at S68 and/or S72 reveals a central role for these phosphorylation events in the regulation of pVHL's MT stabilization (but not binding) activity. Taken together, our results identify pVHL as a novel priming-dependent substrate of GSK3 and suggest a dual-kinase mechanism in the control of pVHL's MT stabilization function. Since GSK3 is a component of multiple signaling pathways that are altered in human cancer, our results further imply that normal operation of the GSK3-pVHL axis may be a critical aspect of pVHL's tumor suppressor mechanism through the regulation of MT dynamics.
Asunto(s)
Glucógeno Sintasa Quinasa 3/metabolismo , Fragmentos de Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Glucógeno Sintasa Quinasa 3/genética , Humanos , Ratones , Microtúbulos/metabolismo , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/genética , Fosforilación , Unión Proteica , Isoformas de Proteínas/genética , Alineación de Secuencia , Serina/metabolismo , Transducción de Señal/fisiología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Chromosomal instability enables tumor development, enabled in part by aberrant expression of the mitotic checkpoint protein Mad2. Here we identify a novel regulatory mechanism for Mad2 expression involving miR-28-5p-mediated inhibition of Mad2 translation, and we demonstrate that this mechanism is triggered by inactivation of the tumor suppressor VHL, the most common event in clear cell renal cell carcinoma (ccRCC). In VHL-positive cancer cells, enhanced expression of miR-28-5p diminished Mad2 levels and promoted checkpoint weakness and chromosomal instability. Conversely, in checkpoint-deficient VHL-negative renal carcinoma cells, inhibition of miR-28-5p function restored Mad2 levels, mitotic checkpoint proficiency, and chromosomal stability. Notably, chromosome missegregation errors and aneuploidy that were produced in a mouse model of acute renal injury (as a result of kidney-specific ablation of pVHL function) were reverted in vivo also by genetic inhibition of miR-28-5p. Finally, bioinformatic analyses in human ccRCC associated loss of VHL with increased miR-28-5p expression and chromosomal instability. Together, our results defined miR-28-5p as a critical regulator of Mad2 translation and mitotic checkpoint function. By identifying a potential mediator of chromosomal instability in VHL-associated cancers, our work also suggests a novel microRNA-based therapeutic strategy to target aneuploid cells in VHL-associated cancers.
Asunto(s)
Inestabilidad Cromosómica , Proteínas Mad2/genética , MicroARNs/genética , Iniciación de la Cadena Peptídica Traduccional , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Regiones no Traducidas 3' , Aneuploidia , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Puntos de Control del Ciclo Celular , Segregación Cromosómica , Femenino , Células HCT116 , Células HeLa , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Proteínas Mad2/metabolismo , Ratones , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Descubrimiento de Drogas/métodos , Neoplasias , Esferoides Celulares/fisiología , Microambiente Tumoral/fisiología , Animales , Antineoplásicos/administración & dosificación , Técnicas de Cultivo de Célula/tendencias , Descubrimiento de Drogas/tendencias , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Cancer-associated mutations in oncogene products and tumor suppressors contributing to tumor progression manifest themselves, at least in part, by deregulating microtubule-dependent cellular processes that play important roles in many cell biological pathways, including intracellular transport, cell architecture, and primary cilium and mitotic spindle organization. An essential characteristic of microtubules in the performance of these varied cell processes is their ability to continuously remodel, a phenomenon known as dynamic instability. It is therefore conceivable that part of the normal function of certain cancer-causing genes is to regulate microtubule dynamic instability. Here, we report the results of a high-resolution live-cell image-based RNA interference screen targeting a collection of 70 human tumor suppressor genes to uncover cancer genes affecting microtubule dynamic instability. Extraction and computational analysis of microtubule dynamics from EB3-GFP time-lapse image sequences identified the products of the tumor suppressor genes NF1 and NF2 as potent microtubule-stabilizing proteins. Further in-depth characterization of NF2 revealed that it binds to and stabilizes microtubules through attenuation of tubulin turnover by lowering both rates of microtubule polymerization and depolymerization as well as by reducing the frequency of microtubule catastrophes. The latter function appears to be mediated, in part, by inhibition of hydrolysis of tubulin-bound GTP on the growing microtubule plus end.
Asunto(s)
Microtúbulos/metabolismo , Neoplasias/metabolismo , Neurofibromina 2/metabolismo , Genes Supresores de Tumor , Humanos , Microtúbulos/genética , Microtúbulos/fisiología , Neoplasias/genética , Neurofibromina 2/genética , Neurofibromina 2/fisiología , Transducción de SeñalRESUMEN
The von Hippel-Lindau tumour suppressor protein (pVHL) controls distinct cellular responses ranging from targeting hypoxia inducible factor α (HIFα) subunits for degradation and promotion of chromosomal stability to the regulation of microtubule dynamics. pVHL is produced in mammalian cells as a long and a short isoform, derived from alternate translational initiation sites in a single Vhl mRNA. However, it is unclear whether these isoforms have different cell biological activities that may represent different tumour suppressor activities of pVHL. Through a knock-in strategy to mutate the first translational initiation site from methionine to leucine (M1L) we have genetically deleted the pVHL long protein isoform in mice, allowing dissection of isoform-specific functions of pVHL. Vhl(M1L/M1L) mice exhibit no obvious phenotypic abnormalities. While numerous pVHL-mediated activities, including degradation of HIFα transcription factors, are unaffected, microtubule dynamics are altered in primary cells derived from Vhl(M1L/M1L) mice to an extent similar to that seen following complete loss of pVHL function. We conclude that the microtubule-regulating function and the HIFα-regulating function of pVHL are separable activities mediated by different protein isoforms.
Asunto(s)
Fibroblastos/metabolismo , Microtúbulos/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Embrión de Mamíferos/citología , Femenino , Fibroblastos/citología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Fluorescente , Datos de Secuencia Molecular , FN-kappa B/genética , FN-kappa B/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Cancer cells in vivo are coordinately influenced by an interactive 3D microenvironment. However, identification of drug targets and initial target validations are usually performed in 2D cell culture systems. The opportunity to design 3D co-culture models that reflect, at least in part, these heterotypic interactions, when coupled with RNA interference, would enable investigations on the phenotypic impact of gene function in a model that more closely resembles tumor growth in vivo. Here we describe a high-throughput-compatible method to discover cancer gene functions in a co-culture 3D tumor microtissue model system composed of human DLD1 colon cancer cells together with murine fibroblasts. Strikingly, DLD1 cells in this model failed to expand upon siRNA-mediated depletion of Kif11/Eg5, a member of the mitotic kinesin-like motor protein family. In contrast, these cancer cells proved to be more resistant to Kif11/Eg5 depletion when grown as a 2D monolayer. These results suggest that growth of certain cancer cells in 3D versus 2D can unveil differential dependencies on specific genes for their survival. Moreover, they denote that the high-throughput-compatible, hanging drop technology-based 3D co-culture model will enable the discovery, characterization, and validation of gene functions in key biological and pathological processes.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Interferencia de ARN , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Técnicas de Cocultivo , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Ratones , Células 3T3 NIH , ARN Interferente Pequeño/genéticaRESUMEN
The identification of cell surface accessible biomarkers enabling diagnosis, disease monitoring, and treatment of renal cell carcinoma (RCC) is as challenging as the biology and progression of RCC is unpredictable. A hallmark of most RCC is the loss-of-function of the von Hippel-Lindau (pVHL) protein by mutation of its gene (VHL). Using the cell surface capturing (CSC) technology, we screened and identified cell surface N-glycoproteins in pVHL-negative and positive 786-O cells. One hundred six cell surface N-glycoproteins were identified. Stable isotope labeling with amino acids in cell culture-based quantification of the CSC screen revealed 23 N-glycoproteins whose abundance seemed to change in a pVHL-dependent manner. Targeted validation experiments using transcriptional profiling of primary RCC samples revealed that nine glycoproteins, including CD10 and AXL, could be directly linked to pVHL-mediated transcriptional regulation. Subsequent human tumor tissue analysis of these cell surface candidate markers showed a correlation between epithelial AXL expression and aggressive tumor phenotype, indicating that pVHL-dependent regulation of glycoproteins may influence the biologic behavior of RCC. Functional characterization of the metalloprotease CD10 in cell invasion assays demonstrated a diminished penetrating behavior of pVHL-negative 786-O cells on treatment with the CD10-specific inhibitor thiorphan. Our proteomic surfaceome screening approach in combination with transcriptional profiling and functional validation suggests pVHL-dependent cell surface glycoproteins as potential diagnostic markers for therapeutic targeting and RCC patient monitoring.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Neoplasias Renales/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/genética , Línea Celular , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Neoplasias Renales/genética , Proteínas de la Membrana/genética , Neprilisina/sangre , Neprilisina/genética , Neprilisina/metabolismo , Proteómica , Reproducibilidad de los ResultadosRESUMEN
Aneuploidy, as a result of numerical changes in chromosome number, was observed in tumours almost a century ago. The molecular mechanisms underlying this phenomenon and their impact on tumour development are still poorly understood. A series of recent observations provide direct linkages between the normal function of tumour suppressor proteins and the suppression of aneuploidy. The prospects that these findings offer for understanding the role of aneuploidy in cancer are discussed in this review.
Asunto(s)
Aneuploidia , Neoplasias/genética , Proteínas Supresoras de Tumor/genética , Proteína de la Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Inestabilidad Cromosómica/genética , Genes cdc , Humanos , Neoplasias/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell-level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.