RESUMO
The inhibitor of apoptosis protein BIRC2 regulates fundamental cell death and survival signaling pathways. Here we show that BIRC2 accumulates in the nucleus via binding of its second and third BIR domains, BIRC2BIR2 and BIRC2BIR3, to the histone H3 tail and report the structure of the BIRC2BIR3-H3 complex. RNA-seq analysis reveals that the genes involved in interferon and defense response signaling and cell-cycle regulation are most affected by depletion of BIRC2. Overexpression of BIRC2 delays DNA damage repair and recovery of the cell-cycle progression. We describe the structural mechanism for targeting of BIRC2BIR3 by a potent but biochemically uncharacterized small molecule inhibitor LCL161 and demonstrate that LCL161 disrupts the association of endogenous BIRC2 with H3 and stimulates cell death in cancer cells. We further show that LCL161 mediates degradation of BIRC2 in human immunodeficiency virus type 1-infected human CD4+ T cells. Our findings provide mechanistic insights into the nuclear accumulation of and blocking BIRC2.
Assuntos
Proteínas Inibidoras de Apoptose , Tiazóis , Humanos , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Apoptose/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Pyroptosis is a lytic form of programmed cell death induced by the activation of gasdermins. The precise mechanism of gasdermin activation by upstream proteases remains incompletely understood. Here, we reconstituted human pyroptotic cell death in yeast by inducible expression of caspases and gasdermins. Functional interactions were reflected by the detection of cleaved gasdermin-D (GSDMD) and gasdermin-E (GSDME), plasma membrane permeabilization, and reduced growth and proliferative potential. Following overexpression of human caspases-1, -4, -5, and -8, GSDMD was cleaved. Similarly, active caspase-3 induced proteolytic cleavage of co-expressed GSDME. Caspase-mediated cleavage of GSDMD or GSDME liberated the ~ 30 kDa cytotoxic N-terminal fragments of these proteins, permeabilized the plasma membrane and compromised yeast growth and proliferation potential. Interestingly, the observation of yeast lethality mediated by co-expression of caspases-1 or -2 with GSDME signified functional cooperation between these proteins in yeast. The small molecule pan-caspase inhibitor Q-VD-OPh reduced caspase-mediated yeast toxicity, allowing us to expand the utility of this yeast model to investigate the activation of gasdermins by caspases that would otherwise be highly lethal to yeast. These yeast biological models provide handy platforms to study pyroptotic cell death and to screen for and characterize potential necroptotic inhibitors.
Assuntos
Piroptose , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/metabolismo , Gasderminas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Caspases/metabolismo , Caspase 1/metabolismo , Proteínas de Ligação a Fosfato , Inflamassomos/metabolismoRESUMO
The survival rate for metastatic osteosarcoma has not improved for several decades, since the introduction and refinement of chemotherapy as a treatment in addition to surgery. Over two thirds of metastatic osteosarcoma patients, many of whom are children or adolescents, fail to exhibit durable responses and succumb to their disease. Concerted efforts have been made to increase survival rates through identification of candidate therapies via animal studies and early phase trials of novel treatments, but unfortunately, this work has produced negligible improvements to the survival rate for metastatic osteosarcoma patients. This review summarizes data from clinical trials of metastatic osteosarcoma therapies as well as pre-clinical studies that report efficacy of novel drugs against metastatic osteosarcoma in vivo. Considerations regarding the design of animal studies and clinical trials to improve survival outcomes for metastatic osteosarcoma patients are also discussed.
Assuntos
Neoplasias Ósseas , Segunda Neoplasia Primária , Osteossarcoma , Adolescente , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Humanos , Estudos Longitudinais , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologiaRESUMO
BACKGROUND/AIM: Ewing sarcomas most commonly arise in the bones, but can also manifest as extraskeletal tumours in soft tissues. Metastases from extraskeletal Ewing sarcomas occur in more diverse anatomical sites than skeletal tumours, and have poorer survival rates. Few animal models replicate the extraskeletal form of Ewing sarcoma, and those that have been developed do not reflect the widespread metastatic spread of these cancers. MATERIALS AND METHODS: Luciferase-expressing Ewing sarcoma cells derived from a muscle tumour were intramuscularly or intravenously injected into nude mice. RESULTS: Both models achieved metastatic spread to numerous sites including the lungs, liver, kidneys, and brain. We characterized the cellular composition of primary and metastatic tumours, observing a greater level of immune cell infiltration in metastases compared to primary intramuscular tumours. CONCLUSION: These pre-clinical models will hopefully facilitate the evaluation of novel therapies and contribute to better understanding the disease progression of metastatic extraskeletal Ewing sarcoma.
Assuntos
Neoplasias Ósseas , Segunda Neoplasia Primária , Sarcoma de Ewing , Animais , Neoplasias Ósseas/genética , Modelos Animais de Doenças , Camundongos , Camundongos Nus , Sarcoma de Ewing/genéticaRESUMO
Osteosarcoma is the most common form of primary bone cancer and frequently metastasizes to the lungs. Current therapies fail to successfully treat over two thirds of patients with metastatic osteosarcoma, so there is an urgent imperative to develop therapies that effectively target established metastases. Smac mimetics are drugs that work by inhibiting the pro-survival activity of IAP proteins such as cIAP1 and cIAP2, which can be overexpressed in osteosarcomas. In vitro, osteosarcoma cells are sensitive to a range of Smac mimetics in combination with TNFα. This sensitivity has also been demonstrated in vivo using the Smac mimetic LCL161, which inhibited the growth of subcutaneous and intramuscular osteosarcomas. Here, we evaluated the efficacy of LCL161 using mice bearing osteosarcoma metastases without the presence of a primary tumor, modeling the scenario in which a patient's primary tumor had been surgically removed. We demonstrated the ability of LCL161 as a single agent and in combination with doxorubicin to inhibit the growth of, and in some cases eliminate, established pulmonary osteosarcoma metastases in vivo. Resected lung metastases from treated and untreated mice remained sensitive to LCL161 in combination with TNFα ex vivo. This suggested that there was little to no acquired resistance to LCL161 treatment in surviving osteosarcoma cells and implied that tumor microenvironmental factors underlie the observed variation in responses to LCL161.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Pulmonares/secundário , Osteossarcoma/secundário , Tiazóis/uso terapêutico , Animais , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Osteossarcoma/tratamento farmacológico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many human cancers exhibit defects in key DNA damage response elements that can render tumors insensitive to the cell death-promoting properties of DNA-damaging therapies. Using agents that directly induce apoptosis by targeting apoptotic components, rather than relying on DNA damage to indirectly stimulate apoptosis of cancer cells, may overcome classical blocks exploited by cancer cells to evade apoptotic cell death. However, there is increasing evidence that cells surviving sublethal exposure to classical apoptotic signaling may recover with newly acquired genomic changes which may have oncogenic potential, and so could theoretically spur the development of subsequent cancers in cured patients. Encouragingly, cells surviving sublethal necroptotic signaling did not acquire mutations, suggesting that necroptosis-inducing anti-cancer drugs may be less likely to trigger therapy-related cancers. We are yet to develop effective direct inducers of other cell death pathways, and as such, data regarding the consequences of cells surviving sublethal stimulation of those pathways are still emerging. This review details the currently known mutagenic consequences of cells surviving different cell death signaling pathways, with implications for potential oncogenic transformation. Understanding the mechanisms of mutagenesis associated (or not) with various cell death pathways will guide us in the development of future therapeutics to minimize therapy-related side effects associated with DNA damage.
Assuntos
Morte Celular , Dano ao DNA , Mutagênese , Mutação , Neoplasias/patologia , Animais , Humanos , Neoplasias/etiologiaRESUMO
The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.
Assuntos
Vesículas Extracelulares , Leite/citologia , Neoplasias Experimentais/patologia , Administração Oral , Animais , Disponibilidade Biológica , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Bovinos , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Vesículas Extracelulares/química , Vesículas Extracelulares/genética , Feminino , Humanos , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/terapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Most anticancer drugs provoke apoptotic signaling by damaging DNA or other means. Genotoxic therapies may enhance a patient's risk of developing "therapy-related cancers" due to the accumulation of oncogenic mutations that may occur in noncancerous cells. Mutations can also form upon apoptotic signaling due to sublethal caspase activity, implying that apoptosis activating drugs may also be oncogenic. Necroptosis is a different way of killing cancer cells: this version of caspase-independent cell death is characterized by receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase-like domain protein (MLKL) activation, leading to cell membrane rupture and controlled cell lysis. The mutagenic potential of sublethal necroptotic signaling has not yet been directly investigated. Smac mimetics drugs, which activate apoptotic or necroptotic cell death, do not induce mutations but the mechanistic basis for this lack of mutagenic activity has not been determined. In this study, we compared the mutagenic potential of these two cell death pathways by engineering cells to activate either apoptotic or necroptotic signaling by exposing them to Smac mimetics with or without TNFα, and/or enforcing or preventing expression of apoptotic or necroptotic regulators. We discovered that sublethal concentrations of Smac mimetics in contexts that activated apoptotic signaling provoked DNA damage and mutations in surviving cells. Mutagenesis was dependent on executioner caspase activation of the nuclease CAD. In contrast, RIPK3- and MLKL-dependent necroptotic signaling following Smac mimetic treatment was not mutagenic. Likewise, DNA damage was not provoked in cells expressing a lethal constitutively active MLKL mutant. These data reveal that cells surviving sublethal necroptotic signaling do not sustain genomic damage and provide hope for a reduced risk of therapy-related malignancies in patients treated with necroptosis-inducing drugs.
Assuntos
Dano ao DNA/genética , Hipoxantina Fosforribosiltransferase/genética , Mutação/genética , Necroptose/genética , Transdução de Sinais , Animais , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Camundongos , Mutagênese/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Serpinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tiazóis/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células U937 , Proteínas Virais/metabolismoRESUMO
Smac mimetics, or IAP antagonists, are a class of drugs currently being evaluated as anti-cancer therapeutics. These agents antagonize IAP proteins, including cIAP1/2 and XIAP, to induce cell death via apoptotic or, upon caspase-8 deficiency, necroptotic cell death pathways. Many cancer cells are unresponsive to Smac mimetic treatment as a single agent but can be sensitized to killing in the presence of the cytokine TNFα, provided either exogenously or via autocrine production. We found that high concentrations of a subset of Smac mimetics could provoke death in cells that did not produce TNFα, despite sensitization at lower concentrations by TNFα. The ability of these drugs to kill did not correlate with valency. These cells remained responsive to the lethal effects of Smac mimetics at high concentrations despite genetic or pharmacological impairments in apoptotic, necroptotic, pyroptotic, autophagic and ferroptotic cell death pathways. Analysis of dying cells revealed necrotic morphology, which was accompanied by the release of lactate dehydrogenase and cell membrane rupture without prior phosphatidylserine exposure implying cell lysis, which occurred over a several hours. Our study reveals that cells incapable of autocrine TNFα production are sensitive to some Smac mimetic compounds when used at high concentrations, and this exposure elicits a lytic cell death phenotype that occurs via a mechanism not requiring apoptotic caspases or necroptotic effectors RIPK3 or MLKL. These data reveal the possibility that non-canonical cell death pathways can be triggered by these drugs when applied at high concentrations.
Assuntos
Antineoplásicos/farmacologia , Azocinas/farmacologia , Compostos Benzidrílicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Oligopeptídeos/farmacologia , Triazóis/farmacologia , Acetilcisteína/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular Tumoral , Cicloexilaminas/farmacologia , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Humanos , Imidazóis/farmacologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mimetismo Molecular , Necroptose/efeitos dos fármacos , Necroptose/genética , Fenilenodiaminas/farmacologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Osteosarcoma is the most common form of primary bone cancer. Over 20% of osteosarcoma patients present with pulmonary metastases at diagnosis, and nearly 70% of these patients fail to respond to treatment. Previous work revealed that human and canine osteosarcoma cell lines are extremely sensitive to the therapeutic proteasome inhibitor bortezomib in vitro. However, bortezomib has proven disappointingly ineffective against solid tumors including sarcomas in animal experiments and clinical trials. Poor tumor penetration has been speculated to account for the inconsistency between in vitro and in vivo responses of solid tumors to bortezomib. Here we show that the second-generation proteasome inhibitor ixazomib, which reportedly has enhanced solid tumor penetration compared to bortezomib, is toxic to human and canine osteosarcoma cells in vitro. We used experimental osteosarcoma metastasis models to compare the efficacies of ixazomib and bortezomib against primary tumors and metastases derived from luciferase-expressing KRIB or 143B human osteosarcoma cell lines in athymic mice. Neither proteasome inhibitor reduced the growth of primary intramuscular KRIB tumors, however both drugs inhibited the growth of established pulmonary metastases created via intravenous inoculation with KRIB cells, which were significantly better vascularized than the primary tumors. Only ixazomib slowed metastases from KRIB primary tumors and inhibited the growth of 143B pulmonary and abdominal metastases, significantly enhancing the survival of mice intravenously injected with 143B cells. Taken together, these results suggest ixazomib exerts better single agent activity against osteosarcoma metastases than bortezomib. These data provide hope that incorporation of ixazomib, or other proteasome inhibitors that penetrate efficiently into solid tumors, into current regimens may improve outcomes for patients diagnosed with metastatic osteosarcoma.
RESUMO
Two thirds of metastatic osteosarcoma patients die within 5 years of diagnosis. Improved experimental models of osteosarcoma metastasis will facilitate the development of more effective therapies. Intravenous cancer cell injection can produce lung metastases in nude mice, but this "experimental metastasis" technique has been predominantly applied to a single osteosarcoma cell line (143B) and required injection of 1-2 million cells. Using two human osteosarcoma cell lines, we discovered that transient Natural Killer cell depletion dramatically enhanced the efficiency of experimental pulmonary osteosarcoma metastasis. This technique for modeling osteosarcoma metastasis may enable the identification of better treatments for this aggressive cancer.
Assuntos
Células Matadoras Naturais/metabolismo , Neoplasias Pulmonares/secundário , Osteossarcoma/terapia , Administração Intravenosa , Animais , Feminino , Camundongos , Camundongos NusRESUMO
High throughput cell viability screening assays often capitalize on the ability of active enzymes or molecules within viable cells to catalyze a quantifiable chemical reaction. The tetrazolium reduction (MTT) assay relies on oxidoreductases to reduce tetrazolium into purple formazan crystals that are solubilized so absorbance reflects viability, while other assays use cellular ATP to catalyze a luminescence-emitting reaction. It is therefore important to know how accurately these assays report cellular responses, as cytotoxic anti-cancer agents promote cell death via a variety of signaling pathways, some of which may alter how these assays work. In this study, we compared the magnitude of cytotoxicity to different cell types provoked by currently used anti-cancer agents, using three different cell viability assays. We found the three assays were consistent in reporting the viability of cells treated with chemotherapy drugs or the BH3 mimetic navitoclax, but the MTT assay underreported the killing capacity of proteasome inhibitors. Additionally, the MTT assay failed to confirm the induction of caspase-mediated cell death by bortezomib at physiologically relevant concentrations, thereby mischaracterizing the mode of cell death. While the cell viability assays used allow for the rapid identification of novel cytotoxic compounds, our study emphasizes the importance for these screening assays to be complemented with a direct measure of cell death or another independent measure of cell viability. We caution researchers against using MTT assays for monitoring cytotoxicity induced by proteasome inhibitors.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , NADH Tetrazólio Redutase/metabolismo , Sais de Tetrazólio/metabolismo , Antineoplásicos/farmacologia , Bioensaio , Caspases/metabolismo , Catálise , Morte Celular/efeitos dos fármacos , Formazans/química , Formazans/farmacologia , Humanos , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Sais de Tetrazólio/química , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologiaRESUMO
Billions of cells undergo apoptosis daily and often fragment into small, membrane-bound extracellular vesicles termed apoptotic bodies (ApoBDs). We demonstrate that apoptotic monocytes undergo a highly coordinated disassembly process and form long, beaded protrusions (coined as beaded apoptopodia), which fragment to release ApoBDs. Here, we find that the protein plexin B2 (PlexB2), a transmembrane receptor that regulates axonal guidance in neurons, is enriched in the ApoBDs of THP1 monocytes and is a caspase 3/7 substrate. To determine whether PlexB2 is involved in the disassembly of apoptotic monocytes, we generate PlexB2-deficient THP1 monocytes and demonstrate that lack of PlexB2 impairs the formation of beaded apoptopodia and ApoBDs. Consequently, the loss of PlexB2 in apoptotic THP1 monocytes impairs their uptake by both professional and non-professional phagocytes. Altogether, these data identify PlexB2 as a positive regulator of apoptotic monocyte disassembly and demonstrate the importance of this process in apoptotic cell clearance.
Assuntos
Apoptose , Monócitos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células A549 , Animais , Células HeLa , Humanos , Camundongos , Monócitos/citologia , Proteínas do Tecido Nervoso/genética , Células THP-1RESUMO
BACKGROUND: Current therapies fail to cure over a third of osteosarcoma patients and around three quarters of those with metastatic disease. "Smac mimetics" (also known as "IAP antagonists") are a new class of anti-cancer agents. Previous work revealed that cells from murine osteosarcomas were efficiently sensitized by physiologically achievable concentrations of some Smac mimetics (including GDC-0152 and LCL161) to killing by the inflammatory cytokine TNFα in vitro, but survived exposure to Smac mimetics as sole agents. METHODS: Nude mice were subcutaneously or intramuscularly implanted with luciferase-expressing murine 1029H or human KRIB osteosarcoma cells. The impacts of treatment with GDC-0152, LCL161 and/or doxorubicin were assessed by caliper measurements, bioluminescence, 18FDG-PET and MRI imaging, and by weighing resected tumors at the experimental endpoint. Metastatic burden was examined by quantitative PCR, through amplification of a region of the luciferase gene from lung DNA. ATP levels in treated and untreated osteosarcoma cells were compared to assess in vitro sensitivity. Immunophenotyping of cells within treated and untreated tumors was performed by flow cytometry, and TNFα levels in blood and tumors were measured using cytokine bead arrays. RESULTS: Treatment with GDC-0152 or LCL161 suppressed the growth of subcutaneously or intramuscularly implanted osteosarcomas. In both models, co-treatment with doxorubicin and Smac mimetics impeded average osteosarcoma growth to a greater extent than either drug alone, although these differences were not statistically significant. Co-treatments were also more toxic. Co-treatment with LCL161 and doxorubicin was particularly effective in the KRIB intramuscular model, impeding primary tumor growth and delaying or preventing metastasis. Although the Smac mimetics were effective in vivo, in vitro they only efficiently killed osteosarcoma cells when TNFα was supplied. Implanted tumors contained high levels of TNFα, produced by infiltrating immune cells. Spontaneous osteosarcomas that arose in genetically-engineered immunocompetent mice also contained abundant TNFα. CONCLUSIONS: These data imply that Smac mimetics can cooperate with TNFα secreted by tumor-associated immune cells to kill osteosarcoma cells in vivo. Smac mimetics may therefore benefit osteosarcoma patients whose tumors contain Smac mimetic-responsive cancer cells and TNFα-producing infiltrating cells.
Assuntos
Antineoplásicos/farmacologia , Cicloexanos/farmacologia , Pirróis/farmacologia , Tiazóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Poxviruses encode many proteins that enable them to evade host anti-viral defense mechanisms. Spi-2 proteins, including Cowpox virus CrmA, suppress anti-viral immune responses and contribute to poxviral pathogenesis and lethality. These proteins are 'serpin' protease inhibitors, which function via a pseudosubstrate mechanism involving initial interactions between the protease and a cleavage site within the serpin. A conformational change within the serpin interrupts the cleavage reaction, deforming the protease active site and preventing dissociation. Spi-2 proteins like CrmA potently inhibit caspases-1, -4 and -5, which produce proinflammatory cytokines, and caspase-8, which facilitates cytotoxic lymphocyte-mediated target cell death. It is not clear whether both of these functions are equally perilous for the virus, or whether only one must be suppressed for poxviral infectivity and spread but the other is coincidently inhibited merely because these caspases are biochemically similar. We compared the caspase specificity of CrmA to three orthologs from orthopoxviruses and four from more distant chordopoxviruses. All potently blocked caspases-1, -4, -5 and -8 activity but exhibited negligible inhibition of caspases-2, -3 and -6. The orthologs differed markedly in their propensity to inhibit non-mammalian caspases. We determined the specificity of CrmA mutants bearing various residues in positions P4, P3 and P2 of the cleavage site. Almost all variants retained the ability to inhibit caspase-1, but many lacked caspase-8 inhibitory activity. The retention of Spi-2 proteins' caspase-8 specificity during chordopoxvirus evolution, despite this function being readily lost through cleavage site mutagenesis, suggests that caspase-8 inhibition is crucial for poxviral pathogenesis and spread.
Assuntos
Caspase 1 , Caspase 8 , Vírus da Varíola Bovina , Proteólise , Serpinas , Proteínas Virais , Caspase 1/química , Caspase 1/genética , Caspase 1/metabolismo , Caspase 8/química , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , Vírus da Varíola Bovina/química , Vírus da Varíola Bovina/genética , Vírus da Varíola Bovina/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Serpinas/química , Serpinas/genética , Serpinas/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Genotoxic anti-cancer therapies such as chemotherapy and radiotherapy can contribute to an increase in second malignancies in cancer survivors due to their oncogenic effects on non-cancerous cells. Inhibition of histone deacetylase (HDAC) proteins or the proteasome differ from chemotherapy in that they eliminate cancer cells by regulating gene expression or cellular protein equilibrium, respectively. As members of these drug classes have been approved for clinical use in recent times, we investigated whether these two drug classes exhibit similar mutagenic capabilities as chemotherapy. The HDAC inhibitors vorinostat/SAHA and romidepsin/FK288 were found to induce DNA damage, and mis-repair of this damage manifested into mutations in clonogenically viable surviving cells. DNA damage and mutations were also detected in cells treated with the proteasome inhibitor bortezomib. Exposure to both drug classes stimulated caspase activation consistent with apoptotic cell death. Inhibition of caspases protected cells from bortezomib-induced acute (but not clonogenic) death and mutagenesis, implying caspases were required for the mutagenic action of bortezomib. This was also observed for second generation proteasome inhibitors. Cells deficient in caspase-activated DNase (CAD) also failed to acquire DNA damage or mutations following treatment with bortezomib. Surprisingly, vorinostat and romidepsin maintained an equivalent level of killing and mutagenic ability regardless of caspase or CAD activity. Our findings indicate that both drug classes harbour mutagenic potential in vitro. If recapitulated in vivo, the mutagenicity of these agents may influence the treatment of cancer patients who are more susceptible to oncogenic mutations due to dysfunctional DNA repair pathways.
Assuntos
Caspases/metabolismo , Desoxirribonucleases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Mutação/efeitos dos fármacos , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Inibidores de Proteassoma/farmacologia , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Inibidores de Caspase/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Desoxirribonucleases/deficiência , Depsipeptídeos/farmacologia , Humanos , Hipoxantina Fosforribosiltransferase/genética , Hipoxantina Fosforribosiltransferase/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/microbiologia , Mutagênese , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , Transdução de Sinais/efeitos dos fármacos , Vorinostat/farmacologiaRESUMO
DNA damaging therapies can spur the formation of therapy-related cancers, due to mis-repair of lesions they create in non-cancerous cells. This risk may be amplified in patients with impaired DNA damage responses. We disabled key DNA damage response pathways using genetic and pharmacological approaches, and assessed the impact of these deficiencies on the mutagenicity of chemotherapy drugs or the "Smac mimetic" GDC-0152, which kills tumor cells by targeting XIAP, cIAP1 and 2. Doxorubicin and cisplatin provoked mutations in more surviving cells deficient in ATM, p53 or the homologous recombination effector RAD51 than in wild type cells, but suppressing non-homologous end joining (NHEJ) by disabling DNA-PKcs prevented chemotherapy-induced mutagenesis. Vincristine-induced mutagenesis required p53 and DNA-PKcs but was not affected by ATM status, consistent with it provoking ATM-independent p53-mediated activation of caspases and CAD, which creates DNA lesions in surviving cells that could be mis-repaired by NHEJ. Encouragingly, GDC-0152 failed to stimulate mutations in cells with proficient or defective DNA damage response pathways. This study highlights the elevated oncogenic risk associated with treating DNA repair-deficient patients with genotoxic anti-cancer therapies, and suggests a potential advantage for Smac mimetic drugs over traditional therapies: a reduced risk of therapy-related cancers.
Assuntos
Materiais Biomiméticos/farmacologia , Cicloexanos/farmacologia , Dano ao DNA , Sistemas de Liberação de Medicamentos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Mitocondriais , Proteínas de Neoplasias , Neoplasias , Pirróis/farmacologia , Proteínas Reguladoras de Apoptose , Linhagem Celular Tumoral , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Células HEK293 , Humanos , Mutagênese/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
Chemotherapy drugs interfere with cellular processes to generate genotoxic lesions that activate cell death pathways. Sustained DNA damage induced by these drugs can provoke mutations in surviving non-cancerous cells, potentially increasing the risk of therapy-related cancers. Ligation of death receptors by ligands such as TRAIL, and subsequent activation of extrinsic apoptotic pathways, also provokes mutations. In this study, we show that executioner caspase activation of the apoptotic nuclease CAD/DFF40 is essential for TRAIL-induced mutations in surviving cells. As exposure to chemotherapy drugs also activates apoptotic caspases and presumably CAD, we hypothesized that these pathways may also contribute to the mutagenesis induced by conventional chemotherapy drugs, perhaps augmenting the mutations that arise from direct DNA damage provoked by these agents. Interestingly, vincristine-mediated mutations were caspase and CAD dependent. Executioner caspases accounted for some of the mutations caused by the topoisomerase poisons doxorubicin and SN38, but were dispensable for mutagenesis following treatment with cisplatin or temozolomide. These data highlight a non-apoptotic role of caspases in mutagenesis mediated by death receptor agonists, microtubule poisons and topoisomerase inhibitors, and provide further evidence for a potential carcinogenic consequence of sublethal apoptotic signaling stimulated by anticancer therapies.
Assuntos
Aspartato Carbamoiltransferase/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Caspases/genética , Di-Hidro-Orotase/genética , Neoplasias/genética , Ligante Indutor de Apoptose Relacionado a TNF/genética , Apoptose/efeitos dos fármacos , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/análogos & derivados , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Dano ao DNA/efeitos dos fármacos , Dacarbazina/administração & dosagem , Dacarbazina/efeitos adversos , Dacarbazina/análogos & derivados , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Ativação Enzimática/efeitos dos fármacos , Humanos , Irinotecano , Mutagênese/efeitos dos fármacos , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Transdução de Sinais/efeitos dos fármacos , Temozolomida , Vincristina/administração & dosagem , Vincristina/efeitos adversosRESUMO
Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1-procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions.IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as downstream procaspase-3 cleavage in several cell types and under multiple conditions. M1 is the first poxviral protein reported to associate with and prevent the function of the apoptosome, giving a more detailed picture of the threats VACV encounters during infection. Dysregulation of apoptosis is associated with several human diseases. One potential treatment of apoptosis-related diseases is through the use of designed ANK repeat proteins (DARPins), similar to M1, as caspase inhibitors. Thus, the study of the novel antiapoptosis effects of M1 via apoptosome association will be helpful for understanding how to control apoptosis using either natural or synthetic molecules.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Apoptossomas/metabolismo , Vaccinia virus/genética , Animais , Repetição de Anquirina , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 9/genética , Caspase 9/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Saccharomyces cerevisiae/genética , Estaurosporina/farmacologia , Vaccinia virus/efeitos dos fármacos , Vaccinia virus/metabolismoRESUMO
Subversion of host cell apoptotic responses is a prominent feature of viral immune evasion strategies to prevent premature clearance of infected cells. Numerous poxviruses encode structural and functional homologs of the Bcl-2 family of proteins, and vaccinia virus harbors antiapoptotic F1L that potently inhibits the mitochondrial apoptotic checkpoint. Recently F1L has been assigned a caspase-9 inhibitory function attributed to an N-terminal α helical region of F1L spanning residues 1-15 (1) preceding the domain-swapped Bcl-2-like domains. Using a reconstituted caspase inhibition assay in yeast we found that unlike AcP35, a well characterized caspase-9 inhibitor from the insect virus Autographa californica multiple nucleopolyhedrovirus, F1L does not prevent caspase-9-mediated yeast cell death. Furthermore, we found that deletion of the F1L N-terminal region does not impede F1L antiapoptotic activity in the context of a viral infection. Solution analysis of the F1L N-terminal regions using small angle x-ray scattering indicates that the region of F1L spanning residues 1-50 located N-terminally from the Bcl-2 fold is an intrinsically unstructured region. We conclude that the N terminus of F1L is not involved in apoptosis inhibition and may act as a regulatory element in other signaling pathways in a manner reminiscent of other unstructured regulatory elements commonly found in mammalian prosurvival Bcl-2 members including Bcl-xL and Mcl-1.