RESUMEN
In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3-/- mice, Stat3 is constitutively acetylated and naive CD4+ T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.
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Aminoácido Oxidorreductasas/metabolismo , Linfocitos T CD4-Positivos/enzimología , Colitis/enzimología , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT3/metabolismo , Acetilación , Aminoácido Oxidorreductasas/deficiencia , Aminoácido Oxidorreductasas/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Catálisis , Diferenciación Celular , Núcleo Celular/enzimología , Proliferación Celular , Colitis/genética , Colitis/inmunología , Modelos Animales de Enfermedad , Genotipo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Dominios Proteicos , Multimerización de Proteína , Interferencia de ARN , Factor de Transcripción STAT3/genética , Linfocitos T Reguladores/enzimología , Linfocitos T Reguladores/inmunología , Células Th17/enzimología , Células Th17/inmunología , Transcripción Genética , TransfecciónRESUMEN
Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.
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Anemia Hemolítica Congénita/genética , Hidropesía Fetal/genética , Canales Iónicos/genética , Adulto , Anemia Hemolítica Congénita/metabolismo , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Deshidratación/genética , Deshidratación/metabolismo , Eritrocitos/metabolismo , Familia , Femenino , Células HEK293 , Humanos , Hidropesía Fetal/metabolismo , Mutación INDEL , Recién Nacido , Canales Iónicos/metabolismo , Cinética , Masculino , Mutación Missense , Presión Osmótica/fisiologíaRESUMEN
Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Núcleo Celular/metabolismo , Histona Desacetilasas/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas Represoras/metabolismo , Acetilación , Transporte Activo de Núcleo Celular , Animales , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Citoplasma/metabolismo , Estrógenos/metabolismo , Histona Desacetilasa 6 , Histona Desacetilasas/química , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Espacio Intracelular/metabolismo , Carioferinas/metabolismo , Lisina , Ratones , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas Represoras/química , Survivin , Proteína Exportina 1RESUMEN
PURPOSE: In this first-in-human study (NCT03564691) in advanced solid tumors, we investigated a novel first-in-class human IgG4 monoclonal antibody targeting the immunoglobulin-like transcript 4 (ILT4) receptor, MK-4830, as monotherapy and in combination with pembrolizumab. PATIENTS AND METHODS: Patients with histologically/cytologically confirmed advanced solid tumors, measurable disease by RECIST v1.1, and evaluable baseline tumor sample received escalating doses of intravenous MK-4830 every 3 weeks as monotherapy (parts A and B) and in combination with pembrolizumab (part C). Safety and tolerability were the primary objectives. Pharmacokinetics, objective response rate per RECIST v1.1, and molecular biomarkers were also evaluated. RESULTS: Of 84 patients, 50 received monotherapy and 34 received combination therapy. No dose-limiting toxicities were observed; maximum tolerated dose was not reached. MK-4830 showed dose-related target engagement. Eleven of 34 patients in the dose-escalation phase who received combination therapy achieved objective responses; 5 previously had progressive disease on anti-PD-1/PD-L1 therapies. Exploratory evaluation of the association between response and pretreatment gene expression related to interferon-gamma signaling in tumors suggested higher sensitivity to T-cell inflammation with combination therapy than historically expected with pembrolizumab monotherapy, with greater response at more moderate levels of inflammation. CONCLUSIONS: This first-in-class MK-4830 antibody dosed as monotherapy and in combination with pembrolizumab was well tolerated with no unexpected toxicities, and demonstrated dose-related evidence of target engagement and antitumor activity. Inflammation intrinsic to the ILT4 mechanism may be facilitated by alleviating the myeloid-suppressive components of the tumor microenvironment, supporting the target of ILT4 as a potential novel immunotherapy in combination with an anti-PD-1/PD-L1 agent.
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Neoplasias , Receptor de Muerte Celular Programada 1 , Anticuerpos Monoclonales , Humanos , Dosis Máxima Tolerada , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Criterios de Evaluación de Respuesta en Tumores Sólidos , Microambiente TumoralRESUMEN
The multiple functions of the oncofetal protein survivin are dependent on its selective expression patterns within immunochemically distinct subcellular pools. The mechanism by which survivin localizes to these compartments, however, is only partly understood. Here we show that nuclear accumulation of survivin is promoted by CREB-binding protein (CBP)-dependent acetylation on lysine 129 (129K, Lys-129). We demonstrate a mechanism by which survivin acetylation at this position results in its homodimerization, while deacetylation promotes the formation of survivin monomers that heterodimerize with CRM1 and facilitate its nuclear export. Using proteomic analysis, we identified the oncogenic transcription factor STAT3 as a binding partner of nuclear survivin. We show that acetylated survivin binds to the N-terminal transcriptional activation domain of the STAT3 dimer and represses STAT3 transactivation of target gene promoters. Using multiplex PCR and DNA sequencing, we identified a single-nucleotide polymorphism (A â G) at Lys-129 that exists as a homozygous mutation in a neuroblastoma cell line and corresponds with a defect in survivin nuclear localization. Our results demonstrate that the dynamic equilibrium between survivin acetylation and deacetylation at amino acid 129 determines its interaction with CRM1, its subsequent subcellular localization, and its ability to inhibit STAT3 transactivation, providing a potential route for therapeutic intervention in STAT3-dependent tumors.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Factor de Transcripción STAT3/metabolismo , Acetilación , Transporte Activo de Núcleo Celular , Proteína de Unión a CREB/metabolismo , Línea Celular Tumoral , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Proteínas Inhibidoras de la Apoptosis , Carioferinas/metabolismo , Lisina/genética , Lisina/metabolismo , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Mutación , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patología , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Oncogénicas/química , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Polimorfismo de Nucleótido Simple , Unión Proteica , Multimerización de Proteína , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/genética , Survivin , Activación Transcripcional , Proteína Exportina 1RESUMEN
The E2A-HLF fusion transcription factor generated by t(17;19)(q22;p13) translocation is found in a small subset of pro-B cell acute lymphoblastic leukemias (ALLs) and promotes leukemogenesis by substituting for the antiapoptotic function of cytokines. Here we show that t(17;19)+ ALL cells express Survivin at high levels and that a dominant negative mutant of E2A-HLF suppresses Survivin expression. Forced expression of E2A-HLF in t(17;19)(-) leukemia cells up-regulated Survivin expression, suggesting that Survivin is a downstream target of E2A-HLF. Analysis using a counterflow centrifugal elutriator revealed that t(17;19)+ ALL cells express Survivin throughout the cell cycle. Reporter assays revealed that E2A-HLF induces survivin expression at the transcriptional level likely through indirect down-regulation of a cell cycle-dependent cis element in the promoter region. Down-regulation of Survivin function by a dominant negative mutant of Survivin or reduction of Survivin expression induced massive apoptosis throughout the cell cycle in t(17;19)+ cells mainly through caspase-independent pathways involving translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. AIF knockdown conferred resistance to apoptosis caused by down-regulation of Survivin function. These data indicated that reversal of AIF translocation by Survivin, which is induced by E2A-HLF throughout the cell cycle, is one of the key mechanisms in the protection of t(17;19)+ leukemia cells from apoptosis.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Proteínas Recombinantes de Fusión/metabolismo , Translocación Genética/genética , Regulación hacia Arriba , Animales , Apoptosis , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Caspasas/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 19/genética , Humanos , Proteínas Inhibidoras de la Apoptosis , Ratones , Datos de Secuencia Molecular , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Regiones Promotoras Genéticas/genética , Survivin , Activación TranscripcionalRESUMEN
OBJECTIVES: This first-in-human phase I study (NCT03179436) investigated anti-cytotoxic T-lymphocyte-associated protein 4 monoclonal antibody quavonlimab and anti-programmed death 1 monoclonal antibody pembrolizumab in patients with advanced solid tumors. The study was conducted in two parts: dose-escalation (part 1) and dose-confirmation (part 2). First-line treatment with quavonlimab + pembrolizumab conferred encouraging antitumor activity (objective response rate [ORR], 28%-40%) and was generally well tolerated (grade ≥ 3 treatment-related adverse events [TRAEs] were lowest with quavonlimab 25 mg every 6 weeks [Q6W] at 30% and highest with quavonlimab 75 mg Q3W at 57%) in non-small cell lung cancer. We present data from patients with extensive-stage small cell lung cancer (SCLC) receiving second-line or later therapy. MATERIALS AND METHODS: Patients with stage III/IV SCLC received quavonlimab 75 mg Q6W plus pembrolizumab 200 mg Q3W for ≤ 2 years. Primary endpoints were safety and tolerability; ORRs as assessed by blinded independent central review per Response Evaluation Criteria In Solid Tumorsv1.1 was a secondary endpoint. Progression-free survival (PFS), overall survival (OS), and the correlation of response with PD-L1 expression were exploratory endpoints. RESULTS: Forty patients with extensive-stage SCLC received treatment; median follow-up was 13 months. Dose-limiting toxicity occurred in 4 patients (10%). TRAEs occurred in 80% of patients; grade 3 events occurred in 33% of patients and no grade 4/5 events were reported. Confirmed ORRs (95% CI) were 18% (7-33) among all patients, 7% (<1-34) for PD-L1-positive tumors (n = 14), and 19% (5-42) for PD-L1-negative tumors (n = 21). Response duration ranged from 2.9 to 19.1+ months. Median PFS was 2.0 months; 6-month PFS rate was 26%. Median OS was 11.0 months; 6-month OS rate was 66%. CONCLUSIONS: Encouraging antitumor activity was observed with quavonlimab + pembrolizumab in patients with extensive-stage SCLC; responses were observed in PD-L1-positive and PD-L1-negative tumors. The combination was tolerable with manageable toxicities.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Anticuerpos Monoclonales Humanizados , Antígeno B7-H1 , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológicoRESUMEN
BACKGROUND: Postnatal expansion of the pancreatic ß-cell mass is required to maintain glucose homeostasis immediately after birth. This ß-cell expansion is regulated by multiple growth factors, including glucose, insulin, insulin-like growth factor (IGF-1) and epidermal growth factor (EGF). These mitogens signal through several downstream pathways (AKT, ERK, STAT3, and JNK) to regulate the survival and proliferation of ß-cells. Survivin, an oncofetal protein with both pro-proliferative and anti-apoptotic properties, is a known transcriptional target of both IGF-1 and EGF in cancer cells. Here, we analyzed the effects of the ß-cell mitogens IGF-1 and EGF on survivin regulation in the established pancreatic ß-cell model cell lines, MIN6 and INS-1 and in primary mouse islets. RESULTS: In pancreatic ß-cells, treatment with glucose, insulin, or EGF increased survivin protein levels at early time points. By contrast, no significant effects on survivin were observed following IGF-1 treatment. EGF-stimulated increases in survivin protein were abrogated in the presence of downstream inhibitors of the Raf-1/MEK/ERK pathway. EGF had no significant effect on survivin transcription however it prolonged the half-life of the survivin protein and stabilized survivin protein levels by inhibiting surviving ubiquitination. CONCLUSIONS: This study defines a novel mechanism of survivin regulation by EGF through the Raf-1/MEK/ERK pathway in pancreatic ß-cells, via prolongation of survivin protein half-life and inhibition of the ubiquitin-mediated proteasomal degradation pathway. This mechanism may be important for regulating ß-cell expansion after birth.
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Factor de Crecimiento Epidérmico/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/fisiología , Animales , Línea Celular , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/genética , Glucosa/metabolismo , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Insulina/metabolismo , Células Secretoras de Insulina/citología , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-raf/genética , Ratas , Proteínas Represoras/genética , Survivin , UbiquitinaciónAsunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Antineoplásicos/farmacología , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Femenino , Humanos , Transporte de Proteínas/efectos de los fármacos , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , SurvivinRESUMEN
Survivin is an antiapoptotic protein highly expressed in malignant cells that confers resistance to cytotoxic therapy. Granzyme B is a potent cytotoxic protein that is released from mammalian natural killer cells and CTLs following noxious stimuli, including foreign invaders. Here, we took advantage of the properties of these two functionally divergent molecules to create a molecular agent that specifically activates Granzyme B within tumor cells. We designed Survivin and Granzyme B-induced apoptosis (SAGA), which consists of a fusion of the Survivin gene promoter to the coding sequence of active Granzyme B. In cultured human tumor cells transfected with SAGA DNA, Granzyme B is rapidly expressed and results in significant tumor cell death. In vivo, mice harboring human ovarian tumors had statistically significant clinical responses to SAGA treatment that were magnified following combination therapy with SAGA and paclitaxel. At the completion of a 3-week therapeutic trial, 3 of 15 animals were free of disease in the SAGA-treated group, and an additional eight animals had tumors that were nonpalpable and only detected on surgical resection. In contrast, 15 of 15 animals in the control and paclitaxel-only-treated groups had tumors at end of therapy. Treatment with SAGA with or without paclitaxel also prevented disease dissemination in 19 of 20 animals. These results strongly suggest that SAGA has the potential to be a potent agent for the treatment of primary and recurrent human ovarian carcinoma. Moreover, we predict that SAGA will be useful therapeutically in any human cancer that expresses Survivin.
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Fusión Artificial Génica , Carcinoma/terapia , Terapia Genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Neoplasias Ováricas/terapia , Serina Endopeptidasas/genética , Animales , Apoptosis , Carcinoma/tratamiento farmacológico , Carcinoma/secundario , Proliferación Celular , Terapia Combinada , Femenino , Granzimas , Humanos , Proteínas Inhibidoras de la Apoptosis , Ratones , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Paclitaxel/uso terapéutico , Regiones Promotoras Genéticas , Survivin , TransfecciónRESUMEN
Survivin is an inhibitor of apoptosis protein that also plays critical roles in regulating the cell cycle and mitosis. Its prominent expression in essentially all human malignancies, and low or absent expression in most normal tissues, suggests that it would be an ideal target for cancer-directed therapy. Impeding development of safe and effective survivin antagonists for clinical use is a lack of understanding of the molecular mechanisms by which survivin differentially affects apoptosis and cell division, in normal and malignant cells. We show that the diverse functional roles of survivin can be explained, in part, by its heterodimerization with survivin splice variants in tumor cells. Survivin and survivin-DeltaEx3 interact within the mitochondria where they may inhibit mitochondrial-dependent apoptosis. If the expression of all survivin forms is eliminated by siRNA transfections, cells undergo both apoptosis and defective cell division. Overall, we provide new insights suggesting that targeting specific survivin isoforms, rather than survivin alone, may selectively and effectively destroy tumor cells. These findings are likely to have a significant impact in the design of biologic agents for clinical therapy.
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Empalme Alternativo , Muerte Celular/genética , División Celular/genética , Proteínas Asociadas a Microtúbulos/genética , Apoptosis/genética , Neoplasias Encefálicas , Línea Celular Tumoral , Neoplasias Cerebelosas , Variación Genética , Células HeLa , Humanos , Proteínas Inhibidoras de la Apoptosis , Meduloblastoma , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de Neoplasias , Reacción en Cadena de la Polimerasa , Survivin , TransfecciónRESUMEN
Apoptosis is an essential process during normal neuronal development. Approximately one-half of the neurons produced during neurogenesis die before completion of CNS maturation. To characterize the role of the inhibitor of apoptosis gene, survivin, during neurogenesis, we used the Cre-loxP-system to generate mice lacking survivin in neuronal precursor cells. Conditional deletion of survivin starting at embryonic day 10.5 leads to massive apoptosis of neuronal precursor cells in the CNS. Conditional mutants were born at the expected Mendelian ratios; however, these died shortly after birth from respiratory insufficiency, without primary cardiopulmonary pathology. Newborn conditional mutants showed a marked reduction in the size of the brain associated with severe, mutifocal apoptosis in the cerebrum, cerebellum, brainstem, spinal cord, and retina. Caspase-3 and caspase-9 activities in the mutant brains were significantly elevated, whereas bax expression was unchanged from controls. These results show that survivin is critically required for the survival of developing CNS neurons, and may impact on our understanding of neural repair, neural development, and neurodegenerative diseases. Our study is the first to solidify a role for survivin as an antiapoptotic protein during normal neuronal development in vivo.
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Apoptosis/fisiología , Encéfalo/embriología , Proteínas Asociadas a Microtúbulos/fisiología , Neuronas/citología , Retina/embriología , Animales , Encéfalo/citología , Encéfalo/fisiología , Femenino , Eliminación de Gen , Proteínas Inhibidoras de la Apoptosis , Integrasas/genética , Proteínas de Filamentos Intermediarios/genética , Masculino , Ratones , Ratones Mutantes , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Nestina , Neuronas/fisiología , Fenotipo , Embarazo , Proteínas Represoras , Retina/citología , Retina/fisiología , SurvivinRESUMEN
Targeted next-generation sequencing (NGS) identified a novel loss of function mutation in GARS, a gene linked to Charcot-Marie-Tooth disease (CMT), in a paediatric acute lymphoblastic leukaemia patient with severe chemotherapy-induced peripheral neuropathy (CIPN) due to vincristine. The patient was clinically asymptomatic, and lacked a family history of neuropathy. The effect of the mutation was modelled in a zebrafish knockdown system that recapitulated the symptoms of the patient both prior to and after treatment with vincristine. Confocal microscopy of pre- and post-synaptic markers revealed that the GARS knockdown results in changes to peripheral motor neurons, acetylcholine receptors and their co-localisation in neuromuscular junctions (NMJs), whereas a sensitive and reproducible stimulus-response assay demonstrated that the changes correlating with the GARS mutation in themselves fail to produce peripheral neuropathy symptoms. However, with vincristine treatment the GARS knockdown exacerbates decreased stimulus response and NMJ lesions. We propose that there is substantial benefit in the use of a targeted NGS screen of cancer patients who are to be treated with microtubule targeting agents for deleterious mutations in CMT linked genes, and for the screening in zebrafish of reagents that might inhibit CIPN.
RESUMEN
BACKGROUND: Survivin and its alternative splice forms are involved in critical cellular processes, including cell division and programmed cell death. Survivin is expressed in the majority of human cancers, but minimally in differentiated normal tissues. Expression levels correlate with tumor aggressiveness and resistance to therapy. RESULTS: In the present study, we identify and characterize a novel survivin isoform that we designate survivin 2alpha. Structurally, the transcript consists of 2 exons: exon 1 and exon 2, as well as a 3' 197 bp region of intron 2. Acquisition of a new in-frame stop codon within intron 2 results in an open reading frame of 225 nucleotides, predicting a truncated 74 amino acid protein. Survivin 2alpha is expressed at high levels in several malignant cell lines and primary tumors. Functional assays show that survivin 2alpha attenuates the anti-apoptotic activity of survivin. Subcellular localization and immunoprecipitation of survivin 2alpha suggests a physical interaction with survivin. CONCLUSION: We characterized a novel survivin splice variant that we designated survivin 2alpha. We hypothesize that survivin 2alpha can alter the anti-apoptotic functions of survivin in malignant cells. Thus survivin 2alpha may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy.
Asunto(s)
Empalme Alternativo/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Apoptosis , Secuencia de Bases , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/ultraestructura , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Homología Estructural de Proteína , SurvivinRESUMEN
Inhibition of XPO1 (CRM1)-mediated nuclear export of multiple tumor suppressor proteins has been proposed as a novel cancer therapeutic strategy to turn off oncogenic signals and enhance tumor suppression. Survivin is a multifunctional protein with oncogenic properties when expressed in the cytoplasm that requires the XPO1-RanGTP complex for its nuclear export. We investigated the antitumor mechanisms of the drug-like selective inhibitors of nuclear export (SINE) XPO1 antagonists KPT-185, KPT-251 KPT-276, and KPT-330 in estrogen receptor-positive and triple-negative breast cancer (TNBC) cell lines and xenograft models of human breast tumors. KPT compounds significantly inhibited breast cancer cell growth and induced tumor cell death, both in vitro and in vivo. These drugs initially promoted survivin accumulation within tumor cell nuclei. However, their major in vitro effect was to decrease survivin cytoplasmic protein levels, correlating with the onset of apoptosis. XPO1 inhibition repressed Survivin transcription by inhibiting CREB-binding protein-mediated STAT3 acetylation, and blocking STAT3 binding to the Survivin promoter. In addition, caspase-3 was activated to cleave survivin, rendering it unavailable to bind X-linked inhibitor of apoptosis protein and block the caspase cascade. Collectively, these data demonstrate that XPO1 inhibition by SINE compounds represses STAT3 transactivation to block the selective oncogenic properties of survivin and supports their clinical use in TNBC.
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Proteínas Inhibidoras de la Apoptosis/genética , Carioferinas/genética , Receptores Citoplasmáticos y Nucleares/genética , Factor de Transcripción STAT3/genética , Neoplasias de la Mama Triple Negativas/genética , Transporte Activo de Núcleo Celular/genética , Apoptosis/efectos de los fármacos , Caspasa 3 , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Femenino , Terapia Genética , Humanos , Carioferinas/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Survivin , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/terapia , Proteína Exportina 1RESUMEN
Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.
Asunto(s)
Neuroblastoma/metabolismo , Fosfoproteínas/metabolismo , Proteómica , Proteínas Proto-Oncogénicas c-ret/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Transducción de Señal , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas , Anotación de Secuencia Molecular , Células-Madre Neurales/metabolismo , Neuroblastoma/genética , Fosfatidilinositol 3-Quinasas , Fosfoproteínas/genética , Mapas de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Proteoma , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Receptor IGF Tipo 1/antagonistas & inhibidores , Receptor de Insulina/antagonistas & inhibidores , Quinasas raf/metabolismoRESUMEN
CRM1 (Chromosomal Maintenance 1, also known as Exportin 1) is the major mammalian export protein that facilitates the transport of large macromolecules including RNA and protein across the nuclear membrane to the cytoplasm. The gene encoding CRM1 was originally identified in yeast as required to maintain higher order chromosome structure. In mammalian cells, CRM1 was found to bind several nuclear pore proteins hence its role in nuclear-cytosolic transport was discovered. In addition to nuclear-cytosolic transport, CRM1 also plays a role in centrosome duplication and spindle assembly, especially in response to DNA damage. The crystal structure of CRM1 suggests a complex protein that binds the Ran protein bound to GTP, allowing for a conformational change that facilitates binding to different cargo proteins through a nuclear export signal (NES). Included in the cadre of cargo are multiple tumor suppressor and oncoproteins as p53, BRCA1, Survivin, NPM, and APC, which function in the nucleus to regulate transcription or aid in chromosomal assembly and movement. An imbalance in the cytosolic level of these proteins has been observed in cancer cells, resulting in either inactivation (tumor suppressor) or an excess of anti-apoptotic activity (oncoprotein). Thus, the concept of inhibiting CRM1 has been explored as a potential therapeutic intervention. Indeed, inhibition of CRM1 by a variety of small molecules that interfere with cargo-NES binding results in cancer cell death. Whether all of these proteins together are responsible for this phenotype or whether specific proteins are required for this effect is unclear at this time.
RESUMEN
Neuroblastoma is the most common solid tumor of infancy, accounting for 15% of all cancer cell deaths in children. Expression of the anti-apoptotic protein survivin in these tumors correlates with poor prognostic features and resistance to therapy. The mammalian target of rapamycin (mTOR) protein is being explored as a potential therapeutic target in patients with this disease. The objective of this study was to test the hypothesis that rapamycin regulates survivin expression and function in neuroblastoma cells. To explore this hypothesis, we treated two different neuroblastoma lines (NB7, NB8) and a well-characterized control lung cancer cell line, A549, with varying doses of rapamycin (0.1-10µM) for serial time points (2-48 hours). RNA and protein expression levels were then evaluated by quantitative RT-PCR and western blotting, respectively. Cell proliferation and apoptosis were assayed by WST-1 and Annexin V. The results showed a rapamycin-dependent increase in survivin mRNA and protein levels in the neuroblastoma cell lines in a dose- and time-dependent fashion, while a decrease in these levels was observed in control cells. Rapamycin inhibited cell proliferation in both A549 and neuroblastoma cells however neuroblastoma cells had less apoptosis than A549 cells (9% vs. 20%). In summary, our results indicate that rapamycin induces expression of the anti-apoptotic protein survivin in neuroblastoma cells which may protect these cells from programmed cell death. Induction of survivin by rapamycin could therefore be a potential mechanism of neuroblastoma tumor cell resistance and rapamycin may not be an effective therapeutic agent for these tumors.