RESUMO
IL-1ß is a cytokine of pivotal importance to the orchestration of inflammatory responses. Synthesized as an inactive pro-cytokine, IL-1ß requires proteolytic maturation to gain biological activity. Here, we identify intrinsic apoptosis as a non-canonical trigger of IL-1ß maturation. Guided by the discovery of the immunomodulatory activity of vioprolides, cyclic peptides isolated from myxobacteria, we observe IL-1ß maturation independent of canonical inflammasome pathways, yet dependent on intrinsic apoptosis. Mechanistically, vioprolides inhibit MCL-1 and BCL2, which in turn triggers BAX/BAK-dependent mitochondrial outer membrane permeabilization (MOMP). Induction of MOMP results in the release of pro-apoptotic factors initiating intrinsic apoptosis, as well as the depletion of IAPs (inhibitors of apoptosis proteins). IAP depletion, in turn, operates upstream of ripoptosome complex formation, subsequently resulting in caspase-8-dependent IL-1ß maturation. These results establish the ripoptosome/caspase-8 complex as a pro-inflammatory checkpoint that senses the perturbation of mitochondrial integrity.
Assuntos
Apoptose , Caspase 8/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Caspase 1/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Peptídeos Cíclicos/farmacologia , Permeabilidade , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.
Assuntos
Linfócitos B/metabolismo , Transformação Celular Neoplásica/metabolismo , Linfoma Difuso de Grandes Células B/metabolismo , Mutação de Sentido Incorreto , Fator 88 de Diferenciação Mieloide/biossíntese , Neoplasias Experimentais/metabolismo , Animais , Linfócitos B/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/patologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Inflamação/fisiopatologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Neovascularização Patológica , Receptores do Fator de Necrose Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The X-linked inhibitor of apoptosis protein (XIAP) is a potent caspase inhibitor, best known for its anti-apoptotic function in cancer. During apoptosis, XIAP is antagonized by SMAC, which is released from the mitochondria upon caspase-mediated activation of BID. Recent studies suggest that XIAP is involved in immune signaling. Here, we explore XIAP as an important mediator of an immune response against the enteroinvasive bacterium Shigella flexneri, both in vitro and in vivo. Our data demonstrate for the first time that Shigella evades the XIAP-mediated immune response by inducing the BID-dependent release of SMAC from the mitochondria. Unlike apoptotic stimuli, Shigella activates the calpain-dependent cleavage of BID to trigger the release of SMAC, which antagonizes the inflammatory action of XIAP without inducing apoptosis. Our results demonstrate how the cellular death machinery can be subverted by an invasive pathogen to ensure bacterial colonization.
Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/fisiologia , Proteínas de Transporte/metabolismo , Disenteria Bacilar/imunologia , Mitocôndrias/imunologia , Proteínas Mitocondriais/metabolismo , Shigella/imunologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologia , Animais , Apoptose , Proteínas Reguladoras de Apoptose , Western Blotting , Caspases/metabolismo , Proliferação de Células , Células Cultivadas , Disenteria Bacilar/microbiologia , Disenteria Bacilar/patologia , Feminino , Hepatócitos/imunologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Técnicas Imunoenzimáticas , Integrases/metabolismo , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas Mitocondriais/imunologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Shigella/patogenicidade , Transdução de Sinais , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
In the normal quiescent vasculature, only 0.01% of endothelial cells (ECs) are proliferating. However, this proportion increases dramatically following the angiogenic switch during tumor growth or wound healing. Recent evidence suggests that this angiogenic switch is accompanied by a metabolic switch. Here, we show that proliferating ECs increasingly depend on mitochondrial oxidative phosphorylation (OxPhos) for their increased energy demand. Under growth conditions, ECs consume three times more oxygen than quiescent ECs and work close to their respiratory limit. The increased utilization of the proton motif force leads to a reduced mitochondrial membrane potential in proliferating ECs and sensitizes to mitochondrial uncoupling. The benzoquinone embelin is a weak mitochondrial uncoupler that prevents neoangiogenesis during tumor growth and wound healing by exhausting the low respiratory reserve of proliferating ECs without adversely affecting quiescent ECs. We demonstrate that this can be exploited therapeutically by attenuating tumor growth in syngenic and xenograft mouse models. This novel metabolic targeting approach might be clinically valuable in controlling pathological neoangiogenesis while sparing normal vasculature and complementing cytostatic drugs in cancer treatment.
Assuntos
Benzoquinonas/farmacologia , Respiração Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Desacopladores/farmacologia , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Camundongos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Desacopladores/uso terapêutico , Cicatrização/efeitos dos fármacosRESUMO
Mast cells modulate autoimmune diseases such as psoriasis and multiple sclerosis. Fumaric acid esters (FAEs) are widely used for the treatment of psoriasis, and dimethylfumarate (DMF) has recently been approved for multiple sclerosis. In this study, we analysed the cytotoxic effect of FAEs on human mast cells. Specifically, cell death was analysed in the human mast cell line HMC-1 and in primary cord blood-derived mast cells (CBMCs) after incubation with fumaric acid (FA), monomethylfumarate (MMF), DMF and calcium bis(monomethylfumarate) (Ca-MF). Our data show that only DMF potently induces apoptotic cell death in HMC-1 cells and CBMCs. DMF-mediated apoptosis was associated with increased expression of Bax and Bak and activation of caspase-9 and caspase-6. Interestingly, DMF also enhanced the sensitivity of CBMCs towards TRAIL- and dexamethasone-induced apoptosis. These findings demonstrate for the first time that DMF induces apoptosis of human mast cells, primarily via the mitochondrial apoptotic pathway. Our study contributes to the understanding of the beneficial effects of FAEs in autoimmune diseases and provides a rationale for exploiting FAEs for other diseases associated with mast cells.
Assuntos
Apoptose/efeitos dos fármacos , Fármacos Dermatológicos/química , Fumaratos/química , Mastócitos/efeitos dos fármacos , Psoríase/patologia , Cálcio/química , Caspase 6/metabolismo , Caspase 9/metabolismo , Morte Celular , Linhagem Celular , Dexametasona/química , Fumarato de Dimetilo , Etoposídeo/química , Humanos , Interleucina-8/metabolismo , Maleatos/química , Metotrexato/química , Ligante Indutor de Apoptose Relacionado a TNF/química , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Obesity increases the incidence of hepatocellular carcinoma (HCC) development in part through the activation of obesity-associated proinflammatory signaling. Here, we show that in lean mice, abrogation of IL-6Rα signaling protects against diethylnitrosamine (DEN)-induced HCC development. HCC protection occurs via Mcl-1 destabilization, thus promoting hepatocyte apoptosis. IL-6 regulates Mcl-1 stability via the inhibition of PP-1α expression, promoting GSK-3ß inactivation. In addition, IL-6 suppresses expression of the Mcl-1 E3 ligase (Mule). Consequently, IL-6Rα deficiency activates PP-1α and Mule expression, resulting in increased Mcl-1 turnover and protection against HCC development. In contrast, in obesity, inhibition of PP-1α and Mule expression, leading to Mcl-1 stabilization, occurs independently of IL-6 signaling. Collectively, this study provides evidence that obesity inhibits hepatocyte apoptosis through Mcl-1 stabilization independent of IL-6 signaling, thus promoting liver carcinogenesis.
Assuntos
Carcinogênese/metabolismo , Neoplasias Hepáticas Experimentais/etiologia , Neoplasias Hepáticas Experimentais/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Obesidade/complicações , Receptores de Interleucina-6/metabolismo , Animais , Apoptose , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células Hep G2 , Hepatócitos/metabolismo , Humanos , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Knockout , Obesidade/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Estabilidade Proteica , Receptores de Interleucina-6/genética , Transdução de Sinais , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Basophilia is associated with allergic and parasitic diseases and advanced chronic myeloid leukemia. In the present study, we characterized the expression and function of the death receptors Fas/CD95 and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors in basophils from healthy donors compared to neoplastic basophils. Peripheral blood basophils obtained from healthy donors (HD-PBB) and from patients with chronic myeloid leukemia (CML-PBB) were found to express high levels of Fas/CD95 and low levels of TRAIL-R2, whereas the basophil-like chronic myeloid leukemia cell line KU-812 expressed significant levels of TRAIL-R1 and TRAIL-R2. HD-PBB underwent apoptosis in response to anti-Fas/CD95, but showed resistance to TRAIL, unless they were co-treated with actinomycin D. Interestingly, CML-PBB and KU-812 cells exhibited the opposite response pattern with resistance to anti-Fas/CD95, but significant susceptibility to TRAIL-induced apoptosis. Our data show that anti-Fas/CD95 and TRAIL differentially regulate apoptosis of normal and neoplastic human basophils, which may direct the development of novel therapeutic strategies.
Assuntos
Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Basófilos/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Receptor fas/antagonistas & inibidores , Basófilos/metabolismo , Basófilos/patologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cicloexanos , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/metabolismoRESUMO
Clinical resistance to epidermal growth factor receptor (EGFR) inhibition in lung cancer has been linked to the emergence of the EGFR T790M resistance mutation or amplification of MET. Additional mechanisms contributing to EGFR inhibitor resistance remain elusive. By applying combined analyses of gene expression, copy number, and biochemical analyses of EGFR inhibitor responsiveness, we identified homozygous loss of PTEN to segregate EGFR-dependent and EGFR-independent cells. We show that in EGFR-dependent cells, PTEN loss partially uncouples mutant EGFR from downstream signaling and activates EGFR, thereby contributing to erlotinib resistance. The clinical relevance of our findings is supported by the observation of PTEN loss in 1 out of 24 primary EGFR-mutant non-small cell lung cancer (NSCLC) tumors. These results suggest a novel resistance mechanism in EGFR-mutant NSCLC involving PTEN loss.