RESUMEN
The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Caspasa 8/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Neoplasias Ováricas , Sulfonamidas/farmacologíaRESUMEN
We investigated the role of the ubiquitously expressed calpain 2 isoform in breast tumor cell growth, migration, signaling, and tumorigenesis. RNAi-mediated knockdown of the capn2 transcript was used to manipulate expression of the catalytic subunit of calpain 2 in the AC2M2 mouse mammary carcinoma cell line. Stable knockdown of capn2 correlated with reduced in vitro proliferation rates, soft agar colony formation efficiency, and migration rates, indicating roles for calpain 2 in mitogenesis, survival, and motogenesis. Biochemical analysis showed increased levels of protein phosphatase 2A and reduced levels of activated Akt in calpain 2-deficient cells, and this correlated with increased levels of the FoxO3a target gene product p27(Kip1), a key regulator of cell proliferation. Calpain 2 deficiency in the AC2M2 cells correlated with enhanced nuclear localization of FoxO3a, consistent with it being in a derepressed state capable of regulating transcriptional targets. Orthotopically engrafted calpain 2 knockdown AC2M2 cells generated tumors with reduced growth rates and enhanced in vivo expression of p27(Kip1). In summary, calpain 2 deficiency correlated with reduced Akt activity, increased protein phosphatase 2A levels, derepression of FoxO3a, and enhanced expression of the p27(Kip1) tumor suppressor. These observations argue that calpain 2 promotes tumor cell growth both in vitro and in vivo through the PI3K-Akt-FoxO-p27(Kip1) signaling cascade. Inhibition of calpain 2 might therefore provide therapeutic benefits in the treatment of cancer.
Asunto(s)
Calpaína/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción Forkhead/metabolismo , Neoplasias Mamarias Animales/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Calpaína/genética , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Núcleo Celular/metabolismo , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Citoplasma/metabolismo , Femenino , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Desnudos , Microscopía Fluorescente , Proteínas Proto-Oncogénicas c-akt/genética , Interferencia de ARN , Transducción de Señal/genética , Carga Tumoral/genéticaRESUMEN
Injury-induced expression of the p75 neurotrophin receptor (p75NTR) in the CNS facilitates neuronal apoptosis and prevents neuronal regrowth, but the mechanisms regulating p75NTR expression are poorly characterized. In this study, we showed that hypo-osmolarity induces p75NTR expression in primary neurons, and, using a comparative genomics approach, we identified conserved elements in the 25 kb upstream sequences of the rat, mouse, and human p75NTR genes. We found that only one of these, a proximal region rich in Sp1 sites, responds to changes in hypo-osmolarity. We then showed that Sp1 DNA binding activity is increased in cells exposed to hypo-osmolarity, established that hypo-osmolarity enhanced Sp1 binding to the endogenous p75NTR promoter, and showed that Sp1 is required for p75NTR expression induced by hypo-osmolarity. We examined how Sp1 is regulated to effect these changes and established that Sp1 turnover is strongly inhibited by hypo-osmolarity. We propose that stress-induced Sp1 accumulation that results from reductions in Sp1 turnover rate contributes to injury-induced gene expression.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Soluciones Hipotónicas/farmacología , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Receptores de Factor de Crecimiento Nervioso/genética , Factor de Transcripción Sp1/fisiología , Animales , Sitios de Unión , Línea Celular , Corteza Cerebral/citología , Secuencia de Consenso , Cicloheximida/farmacología , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Genes Dominantes , Humanos , Riñón , Ratones , Mutación , Proteínas del Tejido Nervioso/biosíntesis , Neuronas/metabolismo , Presión Osmótica , Fosfatidilinositol 3-Quinasas/fisiología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , ARN Interferente Pequeño/farmacología , Ratas , Receptores de Factores de Crecimiento , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Proteínas Recombinantes de Fusión/fisiología , Homología de Secuencia de Ácido Nucleico , Factor de Transcripción Sp1/química , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3/metabolismo , Especificidad de la Especie , Transcripción Genética/genética , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/fisiologíaRESUMEN
We describe a group of small-molecule inhibitors of Jun kinase (JNK)-dependent apoptosis. AEG3482, the parental compound, was identified in a screening effort designed to detect compounds that reduce apoptosis of neonatal sympathetic neurons after NGF withdrawal. We show that AEG3482 blocks apoptosis induced by the p75 neurotrophin receptor (p75NTR) or its cytosolic interactor, NRAGE, and demonstrate that AEG3482 blocks proapoptotic JNK activity. We show that AEG3482 induces production of heat shock protein 70 (HSP70), an endogenous inhibitor of JNK, and establish that HSP70 accumulation is required for the AEG3482-induced JNK blockade. We show that AEG3482 binds HSP90 and induces HSF1-dependent HSP70 mRNA expression and find that AEG3482 facilitates HSP70 production while retaining HSP90 chaperone activity. These studies establish that AEG3482 inhibits JNK activation and apoptosis by a mechanism involving induced expression of HSP proteins.
Asunto(s)
Apoptosis/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Sulfonamidas/farmacología , Tiadiazoles/farmacología , Animales , Antígenos de Neoplasias/fisiología , Benzoquinonas , Activación Enzimática , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lactamas Macrocíclicas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Células PC12 , Fosforilación , Quinonas/farmacología , Ratas , Receptor de Factor de Crecimiento Nervioso/antagonistas & inhibidores , Receptor de Factor de Crecimiento Nervioso/fisiologíaRESUMEN
The primary goal of chemotherapy is to cause cancer cell death. However, a side effect of many commonly used chemotherapeutic drugs is the activation of nuclear factor-kappaB (NF-kappaB), a potent inducer of antiapoptotic genes, which may blunt the therapeutic efficacy of these compounds. We have assessed the effect of doxorubicin, an anthracycline in widespread clinical use, on NF-kappaB activation and expression of antiapoptotic genes in breast cancer cells. We show that doxorubicin treatment activates NF-kappaB signaling and produces NF-kappaB complexes that are competent for NF-kappaB binding in vitro. Surprisingly, these NF-kappaB complexes suppress, rather than activate, constitutive- and cytokine-induced NF-kappaB-dependent transcription. We show that doxorubicin treatment produces RelA, which is deficient in phosphorylation and acetylation and which blocks NF-kappaB signaling in a histone deacetylase-independent manner, and we show that NF-kappaB activated by doxorubicin does not remain stably bound to kappaB elements in vivo. Together these data show that NF-kappaB signaling induced by doxorubicin reduces expression of NF-kappaB-dependent genes in cancer cells.