RESUMEN
Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.
Asunto(s)
Enterovirus/patogenicidad , Interacciones Huésped-Patógeno/inmunología , Proteínas de la Membrana/metabolismo , Replicación Viral/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Resfriado Común/inmunología , Resfriado Común/virología , Enterovirus/genética , Enterovirus/inmunología , Enterovirus/metabolismo , Células HeLa , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Lipoilación , Proteínas de la Membrana/agonistas , Mutación , Dominios Proteicos/genética , Transducción de Señal , Especificidad de la Especie , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses.
Asunto(s)
Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Evasión Inmune/inmunología , L-Lactato Deshidrogenasa/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Línea Celular Tumoral , Proteínas Ligadas a GPI/inmunología , Glioma/inmunología , Células HEK293 , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunidad Innata/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Isoenzimas/inmunología , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Lactato Deshidrogenasa 5 , Monocitos/citología , Monocitos/inmunología , Células Mieloides/citología , Células Mieloides/inmunologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest solid cancers and thus identifying more effective therapies is a major unmet need. In this study we characterized the super enhancer (SE) landscape of human PDAC to identify novel, potentially targetable, drivers of the disease. Our analysis revealed that MICAL2 is a super enhancer-associated gene in human PDAC. MICAL2 is a flavin monooxygenase that induces actin depolymerization and indirectly promotes SRF transcription by modulating the availability of serum response factor coactivators myocardin related transcription factors (MRTF-A and MRTF-B). We found that MICAL2 is overexpressed in PDAC and correlates with poor patient prognosis. Transcriptional analysis revealed that MICAL2 upregulates KRAS and EMT signaling pathways, contributing to tumor growth and metastasis. In loss and gain of function experiments in human and mouse PDAC cells, we observed that MICAL2 promotes both ERK1/2 and AKT activation. Consistent with its role in actin depolymerization and KRAS signaling, loss of MICAL2 expression also inhibited macropinocytosis. Through in vitro phenotypic analyses, we show that MICAL2, MRTF-A and MRTF-B influence PDAC cell proliferation, migration and promote cell cycle progression. Importantly, we demonstrate that MICAL2 is essential for in vivo tumor growth and metastasis. Interestingly, we find that MRTF-B, but not MRTF-A, phenocopies MICAL2-driven phenotypes in vivo . This study highlights the multiple ways in which MICAL2 impacts PDAC biology and suggests that its inhibition may impede PDAC progression. Our results provide a foundation for future investigations into the role of MICAL2 in PDAC and its potential as a target for therapeutic intervention.
RESUMEN
The development of a mouse model for Kaposi's sarcoma-associated herpesvirus (KSHV) infection has been impeded by the limited host range of the virus. Here, we have examined the molecular basis of this host range restriction. KSHV efficiently enters murine cells and establishes latency. However, ectopic expression of the lytic switch protein RTA (replication and transcription activator) in these cells induces little viral gene expression and no virus production. Upon treatment with histone deacetylase inhibitors, KSHV-infected murine cells display more extensive but aberrant viral transcription and do not support either viral DNA synthesis or the production of infectious virions. These aberrantly infected cells also display markedly enhanced apoptosis. Genetic ablation of the mitochondrial apoptotic pathway in these cells prolongs their survival and permits viral DNA replication but does not rescue the generation of virions. We conclude that multiple defects, both prior to and following DNA synthesis, restrict lytic KSHV infection in murine cells.
Asunto(s)
Apoptosis , Herpesvirus Humano 8/fisiología , Replicación Viral , Animales , Línea Celular , Genes Virales , Herpesvirus Humano 8/genética , Humanos , RatonesRESUMEN
We characterized the enhancer landscape of 66 patients with acute myeloid leukemia (AML), identifying 6 novel subgroups and their associated regulatory loci. These subgroups are defined by their superenhancer (SE) maps, orthogonal to somatic mutations, and are associated with distinct leukemic cell states. Examination of transcriptional drivers for these epigenomic subtypes uncovers a subset of patients with a particularly strong SE at the retinoic acid receptor alpha (RARA) gene locus. The presence of a RARA SE and concomitant high levels of RARA mRNA predisposes cell lines and ex vivo models to exquisite sensitivity to a selective agonist of RARα, SY-1425 (tamibarotene). Furthermore, only AML patient-derived xenograft (PDX) models with high RARA mRNA were found to respond to SY-1425. Mechanistically, we show that the response to SY-1425 in RARA-high AML cells is similar to that of acute promyelocytic leukemia treated with retinoids, characterized by the induction of known retinoic acid response genes, increased differentiation, and loss of proliferation.Significance: We use the SE landscape of primary human AML to elucidate transcriptional circuitry and identify novel cancer vulnerabilities. A subset of patients were found to have an SE at RARA, which is predictive for response to SY-1425, a potent and selective RARα agonist, in preclinical models, forming the rationale for its clinical investigation in biomarker-selected patients. Cancer Discov; 7(10); 1136-53. ©2017 AACR.See related commentary by Wang and Aifantis, p. 1065.This article is highlighted in the In This Issue feature, p. 1047.
Asunto(s)
Benzoatos/administración & dosificación , Elementos de Facilitación Genéticos , Epigenómica/métodos , Leucemia Mieloide Aguda/tratamiento farmacológico , Receptor alfa de Ácido Retinoico/genética , Tetrahidronaftalenos/administración & dosificación , Anciano , Animales , Benzoatos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Receptor alfa de Ácido Retinoico/agonistas , Tetrahidronaftalenos/farmacología , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Excessive demands on the protein-folding capacity of the endoplasmic reticulum (ER) cause irremediable ER stress and contribute to cell loss in a number of cell degenerative diseases, including type 2 diabetes and neurodegeneration. The signals communicating catastrophic ER damage to the mitochondrial apoptotic machinery remain poorly understood. We used a biochemical approach to purify a cytosolic activity induced by ER stress that causes release of cytochrome c from isolated mitochondria. We discovered that the principal component of the purified pro-apoptotic activity is the proto-oncoprotein CRK (CT10-regulated kinase), an adaptor protein with no known catalytic activity. Crk(-/-) cells are strongly resistant to ER-stress-induced apoptosis. Moreover, CRK is cleaved in response to ER stress to generate an amino-terminal M(r)~14K fragment with greatly enhanced cytotoxic potential. We identified a putative BH3 (BCL2 homology 3) domain within this N-terminal CRK fragment, which sensitizes isolated mitochondria to cytochrome c release and when mutated significantly reduces the apoptotic activity of CRK in vivo. Together these results identify CRK as a pro-apoptotic protein that signals irremediable ER stress to the mitochondrial execution machinery.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Mitocondrias/metabolismo , Proteína Oncogénica v-crk/metabolismo , Animales , Apoptosis/fisiología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Caspasas/metabolismo , Línea Celular Transformada , Línea Celular Tumoral , Células Cultivadas , Citocromos c/metabolismo , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/fisiología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Células Jurkat , Ratones , Mutación , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Apoptosis of motor neurons is a well-documented feature in amyotrophic lateral sclerosis (ALS) and related motor neuron diseases (MNDs). However, the role of apoptosis in the pathogenesis of these diseases remains unresolved. One possibility is that the affected motor neurons only succumb to apoptosis once they have exhausted functional capacity. If true, blocking apoptosis should confer no therapeutic benefit. To directly investigate this idea, we tested whether tissue-specific deletion in the mouse CNS of BCL2-associated X protein (BAX) and BCL2-homologous antagonist/killer (BAK), 2 proapoptotic BCL-2 family proteins that together represent an essential gateway to the mitochondrial apoptotic pathway, would protect against motor neuron degeneration. We found that neuronal deletion of Bax and Bak in a mouse model of familial ALS not only halted neuronal loss, but prevented axonal degeneration, symptom onset, weight loss, and paralysis and extended survival. These results show that motor neurons damaged in ALS activate the mitochondrial apoptotic pathway early in the disease process and that apoptotic signaling directly contributes to neuromuscular degeneration and neuronal dysfunction. Hence, inhibiting apoptosis upstream of mitochondrial permeabilization represents a possible therapeutic strategy for preserving functional motor neurons in ALS and other MNDs.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Apoptosis , Mitocondrias/fisiología , Neuronas Motoras/fisiología , Proteína Destructora del Antagonista Homólogo bcl-2/fisiología , Proteína X Asociada a bcl-2/fisiología , Esclerosis Amiotrófica Lateral/terapia , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Superóxido Dismutasa/fisiología , Superóxido Dismutasa-1 , Proteína Destructora del Antagonista Homólogo bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/antagonistas & inhibidoresRESUMEN
The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax-/- Bak-/- mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.