RESUMO
Post-mitotic, differentiated cells exhibit a variety of characteristics that contrast with those of actively growing neoplastic cells, such as the expression of cell-cycle inhibitors and differentiation factors. We hypothesized that the gene expression profiles of these differentiated cells could reveal the identities of genes that may function as tumour suppressors. Here we show, using in vitro and in vivo studies in mice and humans, that the mitochondrial protein LACTB potently inhibits the proliferation of breast cancer cells. Its mechanism of action involves alteration of mitochondrial lipid metabolism and differentiation of breast cancer cells. This is achieved, at least in part, through reduction of the levels of mitochondrial phosphatidylserine decarboxylase, which is involved in the synthesis of mitochondrial phosphatidylethanolamine. These observations uncover a novel mitochondrial tumour suppressor and demonstrate a connection between mitochondrial lipid metabolism and the differentiation program of breast cancer cells, thereby revealing a previously undescribed mechanism of tumour suppression.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Supressoras de Tumor/metabolismo , beta-Lactamases/metabolismo , Animais , Neoplasias da Mama/genética , Carboxiliases/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metabolismo dos Lipídeos/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Fosfatidiletanolaminas/metabolismo , Proteínas Supressoras de Tumor/genética , beta-Lactamases/genéticaRESUMO
Val-boroPro (Talabostat, PT-100), a nonselective inhibitor of post-proline cleaving serine proteases, stimulates mammalian immune systems through an unknown mechanism of action. Despite this lack of mechanistic understanding, Val-boroPro has attracted substantial interest as a potential anticancer agent, reaching phase 3 trials in humans. Here we show that Val-boroPro stimulates the immune system by triggering a proinflammatory form of cell death in monocytes and macrophages known as pyroptosis. We demonstrate that the inhibition of two serine proteases, DPP8 and DPP9, activates the pro-protein form of caspase-1 independent of the inflammasome adaptor ASC. Activated pro-caspase-1 does not efficiently process itself or IL-1ß but does cleave and activate gasdermin D to induce pyroptosis. Mice lacking caspase-1 do not show immune stimulation after treatment with Val-boroPro. Our data identify what is to our knowledge the first small molecule that induces pyroptosis and reveals a new checkpoint that controls the activation of the innate immune system.
Assuntos
Ácidos Borônicos/farmacologia , Caspase 1/metabolismo , Dipeptidases/antagonistas & inibidores , Dipeptídeos/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Leucócitos Mononucleares/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Animais , Ácidos Borônicos/química , Caspase 1/deficiência , Linhagem Celular , Dipeptidases/metabolismo , Dipeptídeos/química , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Relação Dose-Resposta a Droga , Humanos , Leucócitos Mononucleares/enzimologia , Leucócitos Mononucleares/patologia , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos , Conformação Molecular , Inibidores de Serina Proteinase/química , Relação Estrutura-AtividadeRESUMO
Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4+ and CD8+ T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ciclo Celular , Dipeptidases/antagonistas & inibidores , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Inflamassomos/metabolismo , Linfócitos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Dipeptidases/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Camundongos , Proteínas NLR , Inibidores de Proteases/farmacologia , Piroptose/efeitos dos fármacos , RatosRESUMO
Intracellular pathogenic structures or activities stimulate the formation of inflammasomes, which recruit and activate caspase-1 and trigger an inflammatory form of cell death called pyroptosis. The well-characterized mammalian inflammasome sensor proteins all detect one specific type of signal, for example double-stranded DNA or bacterial flagellin. Remarkably, NLRP1 was the first protein discovered to form an inflammasome, but the pathogenic signal that NLRP1 detects has not yet been identified. NLRP1 is highly polymorphic, even among inbred rodent strains, and it has been suggested that these diverse NLRP1 alleles may have evolved to detect entirely different stimuli. Intriguingly, inhibitors of the serine proteases DPP8 and DPP9 (DPP8/9) were recently shown to activate human NLRP1, its homolog CARD8, and several mouse NLRP1 alleles. Here, we show now that DPP8/9 inhibitors activate all functional rodent NLRP1 alleles, indicating that DPP8/9 inhibition induces a signal detected by all NLRP1 proteins. Moreover, we discovered that the NLRP1 allele sensitivities to DPP8/9 inhibitor-induced and Toxoplasma gondii-induced pyroptosis are strikingly similar, suggesting that DPP8/9 inhibition phenocopies a key activity of T. gondii. Overall, this work indicates that the highly polymorphic NLRP1 inflammasome indeed senses a specific signal like the other mammalian inflammasomes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Alelos , Proteínas Reguladoras de Apoptose/genética , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos de Bactérias/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/farmacologia , Ácidos Borônicos/farmacologia , Dipeptídeos/farmacologia , Feminino , Células HEK293 , Humanos , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Proteínas do Tecido Nervoso/metabolismo , Piroptose/efeitos dos fármacos , Células RAW 264.7 , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Inibidores de Serina Proteinase/farmacologia , Toxoplasma/imunologia , TransfecçãoRESUMO
Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptosis. The anthrax lethal factor metalloprotease and small-molecule DPP8/9 inhibitors both activate the NLRP1B inflammasome, but the molecular mechanism of NLRP1B activation is unknown. In this study, we used genome-wide CRISPR-Cas9 knockout screens to identify genes required for NLRP1B-mediated pyroptosis. We discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves NLRP1B, inducing the N-end rule-mediated degradation of the NLRP1B N terminus and freeing the NLRP1B C terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the NLRP1B N terminus but not via the N-end rule pathway. Thus, N-terminal degradation is the common activation mechanism of this innate immune sensor.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Toxinas Bacterianas/metabolismo , Inflamassomos/metabolismo , Proteólise , Piroptose/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Sistemas CRISPR-Cas , Caspase 1/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Técnicas de Inativação de Genes , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Piroptose/genética , Células RAW 264.7 , Inibidores de Serina Proteinase/farmacologia , Células THP-1 , Ubiquitina-Proteína Ligases/genéticaRESUMO
Val-boroPro (PT-100, Talabostat) induces powerful anti-tumor immune responses in syngeneic cancer models, but its mechanism of action has not yet been established. Val-boroPro is a non-selective inhibitor of post-proline-cleaving serine proteases, and the inhibition of the highly related cytosolic serine proteases Dpp8 and Dpp9 (Dpp8/9) by Val-boroPro was recently demonstrated to trigger an immunostimulatory form of programmed cell death known as pyroptosis selectively in monocytes and macrophages. Here we show that Dpp8/9 inhibition activates the inflammasome sensor protein Nlrp1b, which in turn activates pro-caspase-1 to mediate pyroptosis. This work reveals a previously unrecognized mechanism for activating an innate immune pattern recognition receptor and suggests that Dpp8/9 serve as an intracellular checkpoint to restrain Nlrp1b and the innate immune system.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Dipeptidases/metabolismo , Inflamassomos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/química , Ácidos Borônicos/química , Ácidos Borônicos/metabolismo , Ácidos Borônicos/farmacologia , Caspase 1/metabolismo , Dipeptidases/antagonistas & inibidores , Dipeptídeos/química , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Feminino , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Piroptose/efeitos dos fármacos , Células RAW 264.7RESUMO
Small-molecule inhibitors of the serine dipeptidases DPP8 and DPP9 (DPP8/9) induce a lytic form of cell death called pyroptosis in mouse and human monocytes and macrophages1,2. In mouse myeloid cells, Dpp8/9 inhibition activates the inflammasome sensor Nlrp1b, which in turn activates pro-caspase-1 to mediate cell death3, but the mechanism of DPP8/9 inhibitor-induced pyroptosis in human myeloid cells is not yet known. Here we show that the CARD-containing protein CARD8 mediates DPP8/9 inhibitor-induced pro-caspase-1-dependent pyroptosis in human myeloid cells. We further show that DPP8/9 inhibitors induce pyroptosis in the majority of human acute myeloid leukemia (AML) cell lines and primary AML samples, but not in cells from many other lineages, and that these inhibitors inhibit human AML progression in mouse models. Overall, this work identifies an activator of CARD8 in human cells and indicates that its activation by small-molecule DPP8/9 inhibitors represents a new potential therapeutic strategy for AML.
Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Inibidores de Proteases/uso terapêutico , Piroptose/efeitos dos fármacos , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Linhagem Celular Tumoral , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Progressão da Doença , Células HEK293 , Humanos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologiaRESUMO
Pyroptosis is a lytic form of programmed cell death mediated by the inflammatory caspase-1, -4, and -5. We recently discovered that small-molecule inhibitors of the serine peptidases DPP8 and DPP9 (DPP8/9) induce pro-caspase-1-dependent pyroptosis in monocytes and macrophages. Notably, DPP8/9 inhibitors, unlike microbial agents, absolutely require caspase-1 to induce cell death. Therefore, DPP8/9 inhibitors are useful probes to study caspase-1 in cells. Here, we show that, in the absence of the pyroptosis-mediating substrate gasdermin D (GSDMD), caspase-1 activates caspase-3 and -7 and induces apoptosis, demonstrating that GSDMD is the only caspase-1 substrate that induces pyroptosis. Conversely, we found that, during apoptosis, caspase-3/-7 specifically block pyroptosis by cleaving GSDMD at a distinct site from the inflammatory caspases that inactivates the protein. Overall, this work reveals bidirectional crosstalk between apoptosis and pyroptosis in monocytes and macrophages, further illuminating the complex interplay between cell death pathways in the innate immune system.