RESUMEN
Importance: Clinical outcomes for glioblastoma remain poor. Treatment with immune checkpoint blockade has shown benefits in many cancer types. To our knowledge, data from a randomized phase 3 clinical trial evaluating a programmed death-1 (PD-1) inhibitor therapy for glioblastoma have not been reported. Objective: To determine whether single-agent PD-1 blockade with nivolumab improves survival in patients with recurrent glioblastoma compared with bevacizumab. Design, Setting, and Participants: In this open-label, randomized, phase 3 clinical trial, 439 patients with glioblastoma at first recurrence following standard radiation and temozolomide therapy were enrolled, and 369 were randomized. Patients were enrolled between September 2014 and May 2015. The median follow-up was 9.5 months at data cutoff of January 20, 2017. The study included 57 multicenter, multinational clinical sites. Interventions: Patients were randomized 1:1 to nivolumab 3 mg/kg or bevacizumab 10 mg/kg every 2 weeks until confirmed disease progression, unacceptable toxic effects, or death. Main Outcomes and Measures: The primary end point was overall survival (OS). Results: A total of 369 patients were randomized to nivolumab (n = 184) or bevacizumab (n = 185). The MGMT promoter was methylated in 23.4% (43/184; nivolumab) and 22.7% (42/185; bevacizumab), unmethylated in 32.1% (59/184; nivolumab) and 36.2% (67/185; bevacizumab), and not reported in remaining patients. At median follow-up of 9.5 months, median OS (mOS) was comparable between groups: nivolumab, 9.8 months (95% CI, 8.2-11.8); bevacizumab, 10.0 months (95% CI, 9.0-11.8); HR, 1.04 (95% CI, 0.83-1.30); P = .76. The 12-month OS was 42% in both groups. The objective response rate was higher with bevacizumab (23.1%; 95% CI, 16.7%-30.5%) vs nivolumab (7.8%; 95% CI, 4.1%-13.3%). Grade 3/4 treatment-related adverse events (TRAEs) were similar between groups (nivolumab, 33/182 [18.1%]; bevacizumab, 25/165 [15.2%]), with no unexpected neurological TRAEs or deaths due to TRAEs. Conclusions and Relevance: Although the primary end point was not met in this randomized clinical trial, mOS was comparable between nivolumab and bevacizumab in the overall patient population with recurrent glioblastoma. The safety profile of nivolumab in patients with glioblastoma was consistent with that in other tumor types. Trial Registration: ClinicalTrials.gov Identifier: NCT02017717.
Asunto(s)
Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Bevacizumab/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Nivolumab/uso terapéutico , Adulto , Anciano , Inhibidores de la Angiogénesis/efectos adversos , Antineoplásicos Inmunológicos/efectos adversos , Bevacizumab/efectos adversos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/radioterapia , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Femenino , Glioblastoma/genética , Glioblastoma/mortalidad , Glioblastoma/radioterapia , Humanos , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/mortalidad , Nivolumab/efectos adversos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Temozolomida/uso terapéutico , Resultado del Tratamiento , Proteínas Supresoras de Tumor/genética , Adulto JovenRESUMEN
Serum interleukin-8 (IL-8) levels and tumor neutrophil infiltration are associated with worse prognosis in advanced cancers. Here, using a large-scale retrospective analysis, we show that elevated baseline serum IL-8 levels are associated with poor outcome in patients (n = 1,344) with advanced cancers treated with nivolumab and/or ipilimumab, everolimus or docetaxel in phase 3 clinical trials, revealing the importance of assessing serum IL-8 levels in identifying unfavorable tumor immunobiology and as an independent biomarker in patients receiving immune-checkpoint inhibitors.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Biomarcadores Farmacológicos/sangre , Interleucina-8/sangre , Neoplasias/tratamiento farmacológico , Neutrófilos/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Biomarcadores de Tumor/sangre , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Estudios de Cohortes , Femenino , Humanos , Masculino , Neoplasias/sangre , Neoplasias/diagnóstico , Neoplasias/mortalidad , Infiltración Neutrófila/efectos de los fármacos , Pronóstico , Estudios Retrospectivos , Análisis de Supervivencia , Insuficiencia del Tratamiento , Microambiente Tumoral/inmunología , Regulación hacia ArribaRESUMEN
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
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Trampas Extracelulares/metabolismo , Neoplasias Experimentales/terapia , Receptores de Quimiocina/agonistas , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Células HT29 , Humanos , Microscopía Intravital/métodos , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Receptores de Interleucina-8A/inmunología , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/inmunología , Receptores de Interleucina-8B/metabolismo , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Efforts to reduce immunosuppression in the solid tumor microenvironment by blocking the recruitment or polarization of tumor associated macrophages (TAM), or myeloid derived suppressor cells (MDSCs), have gained momentum in recent years. Expanding our knowledge of the immune cell types, cytokines, or recruitment factors that are associated with high-grade disease, both within the tumor and in circulation, is critical to identifying novel targets for immunotherapy. Furthermore, a better understanding of how therapeutic regimens, such as Dexamethasone (Dex), chemotherapy, and radiation, impact these factors will facilitate the design of therapies that can be targeted to the appropriate populations and retain efficacy when administered in combination with standard of care regimens. Here we perform quantitative analysis of tissue microarrays made of samples taken from grades I-III astrocytoma and glioblastoma (GBM, grade IV astrocytoma) to evaluate infiltration of myeloid markers CD163, CD68, CD33, and S100A9. Serum, flow cytometric, and Nanostring analysis allowed us to further elucidate the impact of Dex treatment on systemic biomarkers, circulating cells, and functional markers within tumor tissue. We found that common myeloid markers were elevated in Dex-treated grade I astrocytoma and GBM compared to non-neoplastic brain tissue and grade II-III astrocytomas. Cell frequencies in these samples differed significantly from those in Dex-naïve patients in a pattern that depended on tumor grade. In contrast, observed changes in serum chemokines or circulating monocytes were independent of disease state and were due to Dex treatment alone. Furthermore, these changes seen in blood were often not reflected within the tumor tissue. Conclusions: Our findings highlight the importance of considering perioperative treatment as well as disease grade when assessing novel therapeutic targets or biomarkers of disease.
RESUMEN
While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the in vivo setting and prioritization of combinations for clinical development remain low-throughput and resource intensive. Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies.
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Paclitaxel Unido a Albúmina/uso terapéutico , Compuestos de Anilina/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Paclitaxel Unido a Albúmina/administración & dosificación , Compuestos de Anilina/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis/efectos de los fármacos , Sinergismo Farmacológico , Ratones , Neoplasias Pancreáticas/patología , Sulfonamidas/administración & dosificaciónRESUMEN
A new species of Hylomyscus, H. heinrichorum, is described from mountains in western Angola. Based on morphological traits and cranial morphometry, the new species is assigned to the H. anselli species group and is hypothesized to be most closely related to H. anselli Bishop proper, a species named from Zambia. Members of both the H. anselli and H. denniae species groups occupy the Afromontane Biotic Zone, found in various mountain systems to the south and east of the Congo Basin. Evidence is reviewed that supports the independent radiation of these two species groups within montane forest from different Guineo-Congolian ancestral stocks.
Asunto(s)
Murinae/clasificación , Angola , Distribución Animal , Estructuras Animales/anatomía & histología , Estructuras Animales/crecimiento & desarrollo , Animales , Tamaño Corporal , Ecosistema , Femenino , Masculino , Murinae/anatomía & histología , Murinae/genética , Murinae/crecimiento & desarrollo , Tamaño de los Órganos , FilogeniaRESUMEN
PURPOSE: nab-paclitaxel demonstrates improved clinical efficacy compared with conventional Cremophor EL (CrEL)-paclitaxel in multiple tumor types. This study explored the distinctions in drug distribution between nab-paclitaxel and CrEL-paclitaxel and the underlying mechanisms. METHODS: Uptake and transcytosis of paclitaxel were analyzed by vascular permeability assay across human endothelial cell monolayers. The tissue penetration of paclitaxel within tumors was evaluated by local injections into tumor xenografts and quantitative image analysis. The distribution profile of paclitaxel in solid-tumor patients was assessed using pharmacokinetic modeling and simulation. RESULTS: Live imaging demonstrated that albumin and paclitaxel were present in punctae in endothelial cells and could be observed in very close proximity, suggesting cotransport. Uptake and transport of albumin, nab-paclitaxel and paclitaxel were inhibited by clinically relevant CrEL concentrations. Further, nab-paclitaxel causes greater mitotic arrest in wider area within xenografted tumors than CrEL- or dimethyl sulfoxide-paclitaxel following local microinjection, demonstrating enhanced paclitaxel penetration and uptake by albumin within tumors. Modeling of paclitaxel distribution in patients with solid tumors indicated that nab-paclitaxel is more dependent upon transporter-mediated pathways for drug distribution into tissues than CrEL-paclitaxel. The percent dose delivered to tissue via transporter-mediated pathways is predicted to be constant with nab-paclitaxel but decrease with increasing CrEL-paclitaxel dose. CONCLUSIONS: Compared with CrEL-paclitaxel, nab-paclitaxel demonstrated more efficient transport across endothelial cells, greater penetration and cytotoxic induction in xenograft tumors, and enhanced extravascular distribution in patients that are attributed to carrier-mediated transport. These observations are consistent with the distinct clinical efficacy and toxicity profile of nab-paclitaxel.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Endotelio Vascular/metabolismo , Nanopartículas/química , Paclitaxel/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Albúmina Sérica/química , Animales , Antineoplásicos Fitogénicos/metabolismo , Antineoplásicos Fitogénicos/farmacocinética , Antineoplásicos Fitogénicos/uso terapéutico , Transporte Biológico/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Carcinoma/tratamiento farmacológico , Carcinoma/metabolismo , Carcinoma/patología , Línea Celular Tumoral , Células Cultivadas , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/patología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Infusiones Intravenosas , Ratones Desnudos , Microinyecciones , Paclitaxel/metabolismo , Paclitaxel/farmacocinética , Paclitaxel/uso terapéutico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Albúmina Sérica/metabolismo , Albúmina Sérica Humana , Distribución Tisular , Moduladores de Tubulina/administración & dosificación , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacocinética , Moduladores de Tubulina/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.
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Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales/métodos , Linfoma/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Animales , Biomarcadores , Línea Celular Tumoral , Ciclofosfamida/análogos & derivados , Ciclofosfamida/química , Perros , Doxorrubicina/química , Sistemas de Liberación de Medicamentos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Trasplante de Neoplasias , Prednisolona/química , Serina-Treonina Quinasas TOR/metabolismo , Vincristina/químicaRESUMEN
Gradations in extracellular regulated kinase (ERK) signaling have been implicated in essentially every developmental checkpoint or differentiation process encountered by lymphocytes. Yet, despite intensive effort, the molecular basis by which differences in ERK activation specify alternative cell fates remains poorly understood. We report here that differential ERK signaling controls lymphoid-fate specification through an alternative mode of action. While ERK phosphorylates most substrates, such as RSK, by targeting them through its D-domain, this well-studied mode of ERK action was dispensable for development of γδ T cells. Instead, development of γδ T cells was dependent upon an alternative mode of action mediated by the DEF-binding pocket (DBP) of ERK. This domain enabled ERK to bind a distinct and select set of proteins required for specification of the γδ fate. These data provide the first in vivo demonstration for the role of DBP-mediated interactions in orchestrating alternate ERK-dependent developmental outcomes.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Linfocitos T/inmunología , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Activación Enzimática/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estabilidad Proteica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Transducción de Señal/genética , Especificidad por Sustrato/genéticaRESUMEN
We describe how pathway engineering can be used to convert a single intermediate derived from lipid biosynthesis, fatty aldehydes, into a variety of biofuel precursors including alkanes, free fatty acids and wax esters. In cyanobacteria, long-chain acyl-ACPs can be reduced to fatty aldehydes, and then decarbonylated to alkanes. We discovered a cyanobacteria class-3 aldehyde-dehydrogenase, AldE, that was necessary and sufficient to instead oxidize fatty aldehyde precursors into fatty acids. Overexpression of enzymes in this pathway resulted in production of 50 to 100 fold more fatty acids than alkanes, and the fatty acids were secreted from the cell. Co-expression of acyl-ACP reductase, an alcohol-dehydrogenase and a wax-ester-synthase resulted in a third fate for fatty aldehydes: conversion to wax esters, which accumulated as intracellular lipid bodies. Conversion of acyl-ACP to fatty acids using endogenous cyanobacterial enzymes may allow biofuel production without transgenesis.
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Aldehídos/metabolismo , Biocombustibles , Cianobacterias/metabolismo , Ácidos Grasos/metabolismo , Aldehído Deshidrogenasa/metabolismo , Cianobacterias/genética , Enoil-ACP Reductasa (NADPH Específica B)/genética , Enoil-ACP Reductasa (NADPH Específica B)/metabolismo , Ácidos Grasos/biosíntesis , Expresión Génica , Cinética , Ceras/metabolismoRESUMEN
Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).
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Ensayos Analíticos de Alto Rendimiento , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Apoptosis/efectos de los fármacos , Apoptosis/genética , División Celular/efectos de los fármacos , División Celular/genética , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Reparación del ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Interferencia de ARN/efectos de los fármacos , Reproducibilidad de los Resultados , Transducción de Señal/efectos de los fármacosRESUMEN
Tumor suppressor genes BRCA1 and BRCA2 function in a complex gene network that regulates homologous recombination and DNA double-strand break repair. Disruption of the BRCA-network through gene mutation, deletion, or RNAi-mediated silencing can sensitize cells to small molecule inhibitors of poly (ADP-ribose) polymerase (PARPi). Here, we demonstrate that BRCA-network disruption in the presence of PARPi leads to the selective induction and enhancement of interferon pathway and apoptotic gene expression in cultured tumor cells. In addition, we report PARPi cytotoxicity in BRCA1-deficient tumor cells is enhanced >10-fold when combined with interferon-γ. These findings establish a link between synthetic lethality of PARPi in BRCA-network disrupted cells and interferon pathway activation triggered by genetic instability.
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Proteína BRCA1/genética , Redes Reguladoras de Genes/genética , Interferón gamma/metabolismo , Interferón gamma/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteína BRCA1/metabolismo , Ciclo Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Células HT29 , Células HeLa , Humanos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal/genéticaRESUMEN
αß and γδ lineage T cells are thought to arise from a common CD4(-)CD8(-) progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We demonstrated recently that a ubiquitously expressed ribosomal protein, Rpl22, is selectively required for the development of αß lineage T cells. Germline ablation of Rpl22 impairs development of αß lineage, but not γδ lineage, T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors used by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22(-/-) thymocytes, including miR-34a, PUMA, p21(waf), Bax, and Noxa. Notably, the proapoptotic factor Bim, while not a direct p53 target, was also strongly induced in Rpl22(-/-) T cells. Gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in Rpl22-deficient T cells; however, only a few p53 targets alleviated developmental arrest when individually ablated by gene targeting or knockdown. Co-elimination of PUMA and Bim resulted in a nearly complete restoration of development of Rpl22(-/-) thymocytes, indicating that p53-mediated arrest is enforced principally through effects on cell survival. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21(waf)) and apoptosis (PUMA) actually abrogated the partial rescue caused by loss of PUMA alone, suggesting that the G1 checkpoint protein p21(waf) facilitates thymocyte development in some contexts.
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Diferenciación Celular/inmunología , Marcación de Gen , Inhibidores de Crecimiento/inmunología , Proteínas Ribosómicas/deficiencia , Subgrupos de Linfocitos T/inmunología , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/genética , Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteína 11 Similar a Bcl2 , Diferenciación Celular/genética , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/inmunología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Marcación de Gen/métodos , Inhibidores de Crecimiento/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/deficiencia , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/genética , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Timo/inmunología , Timo/metabolismo , Timo/patología , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/deficiencia , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/deficiencia , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
The hypoxia-inducible factor (HIF) pathway is essential for cell survival under low oxygen and plays an important role in tumor cell homeostasis. We investigated the function of miR-210, the most prominent microRNA upregulated by hypoxia and a direct transcriptional target of HIFs. miR-210 expression was elevated in multiple cancer types and correlated with metastasis of breast and melanoma tumors. miR-210 overexpression in cancer cell lines bypassed hypoxia-induced cell cycle arrest and partially reversed the hypoxic gene expression signature. We identified MNT, a known MYC antagonist, as a miR-210 target. MNT mRNA contains multiple miR-210 binding sites in the 3' UTR and its knockdown phenocopied miR-210 overexpression. Furthermore, loss of MYC abolished miR-210-mediated override of hypoxia-induced cell cycle arrest. Comparison of miR-210 and MYC overexpression with MNT knockdown signatures also indicated that miR-210 triggered a "MYC-like" transcriptional response. Thus, miR-210 influences the hypoxia response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.
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Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Ciclo Celular , Hipoxia de la Célula , Línea Celular Tumoral , Análisis por Conglomerados , Técnicas de Silenciamiento del Gen , Humanos , Factor 1 Inducible por Hipoxia/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genética , Regulación hacia ArribaRESUMEN
Cell cycle arrest in response to DNA damage is an important antitumorigenic mechanism. MicroRNAs (miRNAs) were recently shown to play key regulatory roles in cell cycle progression. For example, miR-34a is induced in response to p53 activation and mediates G(1) arrest by down-regulating multiple cell cycle-related transcripts. Here we show that genotoxic stress promotes the p53-dependent up-regulation of the homologous miRNAs miR-192 and miR-215. Like miR-34a, activation of miR-192/215 induces cell cycle arrest, suggesting that multiple miRNA families operate in the p53 network. Furthermore, we define a downstream gene expression signature for miR-192/215 expression, which includes a number of transcripts that regulate G(1) and G(2) checkpoints. Of these transcripts, 18 transcripts are direct targets of miR-192/215, and the observed cell cycle arrest likely results from a cooperative effect among the modulations of these genes by the miRNAs. Our results showing a role for miR-192/215 in cell proliferation combined with recent observations that these miRNAs are underexpressed in primary cancers support the idea that miR-192 and miR-215 function as tumor suppressors.
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Ciclo Celular/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias/genética , Neoplasias/patología , Proteína p53 Supresora de Tumor/genética , División Celular/genética , Daño del ADN , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/genética , Fase G1/genética , Fase G2/genética , Perfilación de la Expresión Génica , Silenciador del Gen , Genes p53 , Células HCT116 , Humanos , MicroARNs/biosíntesis , Neoplasias/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transfección , Proteína p53 Supresora de Tumor/biosíntesis , Regulación hacia ArribaRESUMEN
Notch1 signalling is essential for the commitment of multipotent lymphocyte precursors towards the alphabeta T-cell lineage and plays an important role in regulating beta-selection in CD4(-)CD8(-) double-negative (DN) thymocytes. However, the role played by Notch in promoting the development of CD4(+)CD8(+) double-positive (DP) thymocytes is poorly characterized. Here, we demonstrate that the introduction of a constitutively active Notch1 (ICN1) construct into RAG(-/-) lymphocyte precursors resulted in the generation of DP thymocytes in in vitro T-cell culture systems. Notably, developmental rescue was dependent not only on the presence of an intact Notch1 RAM domain but also on Delta-like signals, as ICN1-induced DP development in RAG(-/-) thymocytes occurred within an intact thymus or in OP9-DL1 co-cultures, but not in OP9-control co-cultures. Interestingly, ICN1 expression in SLP-76(-/-) precursors resulted in only a minimal developmental rescue to the immature CD8(+) single-positive stage, suggesting that Notch is utilizing the same signalling pathway as the pre-TCR complex. In support of this, ICN1 introduction resulted in the activation of the ERK-MAPK-signalling cascade in RAG(-/-) thymocytes. Taken together, these studies demonstrate that constitutive Notch signalling can bypass beta-selection during early T-cell development by inducing pre-TCR-like signals within a T-cell-promoting environment.
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Células Progenitoras Linfoides/citología , Células Progenitoras Linfoides/metabolismo , Receptor Notch1/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/inmunología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Activación de Linfocitos , Células Progenitoras Linfoides/inmunología , Ratones , Ratones Mutantes , Transducción de Señal , Linfocitos T/citología , Timo/citología , Timo/metabolismoRESUMEN
MicroRNAs (microRNAs) are abundant, approximately 21-25 nucleotide (nt) non-coding RNAs that mediate sequence-specific, post-transcriptional repression of mRNA targets. Emerging evidence suggests that several microRNAs target transcripts that encode proteins directly or indirectly involved in cell cycle progression and cellular proliferation. Moreover, alteration of microRNA levels can contribute to pathological conditions, including tumorigenesis, that are associated with loss of cell cycle control. In this review we highlight recent data linking microRNAs to mammalian cell cycle regulation. We describe how specific miRNAs function within pathways that control cell cycle checkpoints. We discuss emerging evidence that support the idea that some microRNA activity may be cell cycle dependent, and we outline how the coordinate regulation of microRNA targets may influence cell cycle progression.
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Ciclo Celular/genética , MicroARNs/metabolismo , Animales , Apoptosis , Diferenciación Celular , Humanos , MicroARNs/genética , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
microRNAs (miRNAs) are abundant, approximately 21-nucleotide, noncoding regulatory RNAs. Each miRNA may regulate hundreds of mRNA targets, but the identities of these targets and the processes they regulate are poorly understood. Here we have explored the use of microarray profiling and functional screening to identify targets and biological processes triggered by the transfection of human cells with miRNAs. We demonstrate that a family of miRNAs sharing sequence identity with miRNA-16 (miR-16) negatively regulates cellular growth and cell cycle progression. miR-16-down-regulated transcripts were enriched with genes whose silencing by small interfering RNAs causes an accumulation of cells in G(0)/G(1). Simultaneous silencing of these genes was more effective at blocking cell cycle progression than disruption of the individual genes. Thus, miR-16 coordinately regulates targets that may act in concert to control cell cycle progression.
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Ciclo Celular/genética , Ciclo Celular/fisiología , MicroARNs/clasificación , MicroARNs/genética , Familia de Multigenes/genética , Transcripción Genética , Línea Celular , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , FenotipoRESUMEN
RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/-) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity approximately 4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.
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Antineoplásicos/toxicidad , Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA2/antagonistas & inhibidores , Cisplatino/toxicidad , Neoplasias/tratamiento farmacológico , Neoplasias/patología , ARN Interferente Pequeño/fisiología , Proteína p53 Supresora de Tumor/deficiencia , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Células HeLa , Humanos , Neoplasias/genética , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
KIF14 is a microtubule motor protein whose elevated expression is associated with poor-prognosis breast cancer. Here we demonstrate KIF14 accumulation in mitotic cells, where it associated with developing spindle poles and spindle microtubules. Cells at later stages of mitosis were characterized by the concentration of KIF14 at the midbody. Time-lapse microscopy revealed that strong RNA interference (RNAi)-mediated silencing of KIF14 induced cytokinesis failure, causing several rounds of endoreduplication and resulting in multinucleated cells. Additionally, less efficacious KIF14-specific short interfering RNAs (siRNAs) induced multiple phenotypes, all of which resulted in acute apoptosis. Our data demonstrate the ability of siRNA-mediated silencing to generate epiallelic hypomorphs associated with KIF14 depletion. Furthermore, the link we observed between siRNA efficacy and phenotypic outcome indicates that distinct stages during cell cycle progression are disrupted by the differential modulation of KIF14 expression.