RESUMO
Myc oncoproteins directly regulate transcription by binding to target genes, yet this only explains a fraction of the genes affected by Myc. mRNA turnover is controlled via AU-binding proteins (AUBPs) that recognize AU-rich elements (AREs) found within many transcripts. Analyses of precancerous and malignant Myc-expressing B cells revealed that Myc regulates hundreds of ARE-containing (ARED) genes and select AUBPs. Notably, Myc directly suppresses transcription of Tristetraprolin (TTP/ZFP36), an mRNA-destabilizing AUBP, and this circuit is also operational during B lymphopoiesis and IL7 signaling. Importantly, TTP suppression is a hallmark of cancers with MYC involvement, and restoring TTP impairs Myc-induced lymphomagenesis and abolishes maintenance of the malignant state. Further, there is a selection for TTP loss in malignancy; thus, TTP functions as a tumor suppressor. Finally, Myc/TTP-directed control of select cancer-associated ARED genes is disabled during lymphomagenesis. Thus, Myc targets AUBPs to regulate ARED genes that control tumorigenesis.
Assuntos
Genes Supressores de Tumor , Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tristetraprolina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estabilidade de RNA , RNA Mensageiro/químicaRESUMO
Macrophage activation is a critical step in host responses during bacterial infections. Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine metabolism, has been well studied in epithelial cells and is known to have essential roles in many different cellular functions. However, its role in regulating macrophage function during bacterial infections is not well characterized. We demonstrate that macrophage-derived ODC is a critical regulator of M1 macrophage activation during both Helicobacter pylori and Citrobacter rodentium infection. Myeloid-specific Odc deletion significantly increased gastric and colonic inflammation, respectively, and enhanced M1 activation. Add-back of putrescine, the product of ODC, reversed the increased macrophage activation, indicating that ODC and putrescine are regulators of macrophage function. Odc-deficient macrophages had increased histone 3, lysine 4 (H3K4) monomethylation, and H3K9 acetylation, accompanied by decreased H3K9 di/trimethylation both in vivo and ex vivo in primary macrophages. These alterations in chromatin structure directly resulted in up-regulated gene transcription, especially M1 gene expression. Thus, ODC in macrophages tempers antimicrobial, M1 macrophage responses during bacterial infections through histone modifications and altered euchromatin formation, leading to the persistence and pathogenesis of these organisms.
Assuntos
Infecções por Enterobacteriaceae/imunologia , Infecções por Helicobacter/imunologia , Histonas/metabolismo , Macrófagos/imunologia , Ornitina Descarboxilase/imunologia , Animais , Linhagem Celular , Citrobacter rodentium , Colite/imunologia , Colite/patologia , Colo/imunologia , Colo/patologia , Citocinas/imunologia , Infecções por Enterobacteriaceae/patologia , Mucosa Gástrica/imunologia , Mucosa Gástrica/patologia , Gastrite/imunologia , Gastrite/patologia , Infecções por Helicobacter/patologia , Helicobacter pylori , Humanos , Ativação de Macrófagos , Masculino , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Ornitina Descarboxilase/genética , Putrescina/metabolismoRESUMO
Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologs of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1, and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13-directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Autofagia/fisiologia , Proteínas de Ciclo Celular/fisiologia , Chaperoninas/fisiologia , Proteínas de Choque Térmico HSP90/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Linhagem Celular , Chaperoninas/metabolismo , Células Eritroides/citologia , Células Eritroides/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células K562 , Camundongos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
The source, specificity, and plasticity of the forkhead box transcription factor 3 (Foxp3)(+) regulatory T (Treg) and conventional T (Tconv) cell populations active at sites of autoimmune pathology are not well characterized. To evaluate this, we combined global repertoire analyses and functional assessments of isolated T cell receptors (TCR) from TCRalpha retrogenic mice with autoimmune encephalomyelitis. Treg and Tconv cell TCR repertoires were distinct, and autoantigen-specific Treg and Tconv cells were enriched in diseased tissue. Autoantigen sensitivity and fine specificity of these cells intersected, implying that differences in responsiveness were not responsible for lineage specification. Notably, autoreactive Treg and Tconv cells could be fully distinguished by an acidic versus aliphatic variation at a single TCR CDR3 residue. Our results imply that ontogenically distinct Treg and Tconv cell repertoires with convergent specificities for autoantigen respond during autoimmunity and argue against more than limited plasticity between Treg and Tconv cells during autoimmune inflammation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Encefalomielite Autoimune Experimental/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T Reguladores/imunologia , Animais , Autoantígenos/imunologia , Autoantígenos/metabolismo , Fatores de Transcrição Forkhead/imunologia , Glicoproteínas/imunologia , Glicoproteínas/farmacologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
Ataxia-telangiectasia mutated (ATM) plays a central role in DNA damage responses, and its loss leads to development of T-cell malignancies. Here, we show that ATM loss also leads to intrinsic mitochondrial abnormalities in thymocytes, including elevated reactive oxygen species, increased aberrant mitochondria, high cellular respiratory capacity, and decreased mitophagy. A fraction of ATM protein is localized in mitochondria, and it is rapidly activated by mitochondrial dysfunction. Unexpectedly, allelic loss of the autophagy regulator Beclin-1 significantly delayed tumor development in ATM-null mice. This effect was not associated with rescue of DNA damage signaling but rather with a significant reversal of the mitochondrial abnormalities. These data support a model in which ATM plays direct roles in modulating mitochondrial homeostasis and suggest that mitochondrial dysfunction and associated increases in mitochondrial reactive oxygen species contribute to the cancer-prone phenotype observed in organisms lacking ATM. Thus, ataxia-telangiectasia should be considered, at least in part, as a mitochondrial disease.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Ataxia Telangiectasia/fisiopatologia , Proteínas Mutadas de Ataxia Telangiectasia , Autofagia , Proteína Beclina-1 , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Immunoblotting , Estimativa de Kaplan-Meier , Linfoma de Células T/genética , Linfoma de Células T/metabolismo , Potencial da Membrana Mitocondrial , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias/genética , Mitocôndrias/fisiologia , Consumo de Oxigênio , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timócitos/metabolismo , Timócitos/ultraestrutura , Proteínas Supressoras de Tumor/genéticaRESUMO
IL-10 is a critical anti-inflammatory cytokine, the deficiency of which leads to spontaneous autoimmunity. However, therapeutically administered or ectopically expressed IL-10 can either suppress or promote disease. Distinct lineage-specific activities may explain the contradictory effects of IL-10. To dissect the T cell-specific response to IL-10 during organ-specific autoimmunity, we generated mice with a selective deletion of IL-10Rα in T cells and analyzed its effects in an autoimmune model, experimental allergic encephalomyelitis (EAE). Surprisingly, the T cell response to IL-10 increased EAE severity. This did not result from altered T cell functional potential; T cell cytokine profile was preserved. IL-10 also diminished the proliferation of T cells in situ within the target organ, an effect that would be expected to restrain disease. However, IL-10 acted cell autonomously to sustain the autoreactive T cells essential for immunopathogenesis, promoting their accumulation and distorting the regulatory and effector T cell balance. Indeed, in chimeric mice and after adoptive transfer, wild type T cells showed a competitive advantage over cells deficient in IL-10Rα. Therefore, T cell specific actions of IL-10 can support autoimmune inflammation, and this appears to result from an overall increase in the long term fitness of pathologic T cells. Lineage-restricted, disease-promoting activities of IL-10 should be considered in the therapeutic manipulation of the IL-10 pathway.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Interleucina-10/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Técnicas de Cocultura , Encefalomielite Autoimune Experimental/genética , Epitopos de Linfócito T/imunologia , Mediadores da Inflamação/administração & dosagem , Mediadores da Inflamação/fisiologia , Interleucina-10/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Receptores de Interleucina-10/deficiência , Receptores de Interleucina-10/genética , Receptores de Interleucina-10/fisiologia , Índice de Gravidade de Doença , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Harnessing the immune system to kill tumors has been revolutionary and, as a result, has had an enormous benefit for patients in extending life and resulting in effective cures in some. However, activation of the immune system can come at the cost of undesirable adverse events such as cytokine release syndrome, immune-related adverse events, on-target/off-tumor toxicity, neurotoxicity and tumor lysis syndrome, which are safety risks that can be challenging to assess non-clinically. This article provides a review of the biology and mechanisms that can result in immune-mediated adverse effects and describes industry approaches using in vitro and in vivo models to aid in the nonclinical safety risk assessments for immune-oncology modalities. Challenges and limitations of knowledge and models are also discussed.
Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias , Humanos , Neoplasias/tratamento farmacológico , Medição de RiscoRESUMO
Woodchuck (Marmota monax) infected with woodchuck hepatitis virus (WHV) is the most pathogenically compatible naturally occurring model of human hepatitis B virus (HBV) infection, chronic hepatitis B, and HBV-induced hepatocellular carcinoma. This system plays a crucial role in discovery and preclinical evaluation of anti-HBV therapies. Its utilization remains tempered by the relatively narrow range of validated immunologic and molecular tools. We evaluated commercial antibodies against immune cell phenotypic markers and T cell molecules for cross-reactivity with woodchuck antigenic equivalents. The confirmed antibodies against programed cell death protein-1 (PD-1) and its ligand (PD-L1) were examined for ex vivo ability to activate WHV-specific, global and bystander cytotoxic T cells (CTLs) in chronic hepatitis and asymptomatic infection persisting after self-resolved acute hepatitis. Examination of 65 antibodies led to identification or confirmation of 23 recognizing woodchuck T, regulatory T, B and natural killer cells, T cell-associated PD-1, PD-L1, CTLA-4 and TIM-3 molecules, CD25 and CD69 markers of T cell activation, and interferon gamma (IFNγ). Antibodies against woodchuck PD-1 and PD-L1 triggered in vitro highly individualized WHV-specific and global activation of CTLs in both chronic hepatitis and persistent occult infection. WHV-specific CTLs were more robustly augmented by anti-PD-1 than by anti-PD-L1 in chronic hepatitis, while global IFNγ-positive CTL response was significantly suppressed in chronic hepatitis compared to persistent occult infection. Anti-PD-1 and anti-PD-L1 also occasionally activated CTLs to specificities other than those tested suggesting their potency to trigger side effects. This was particularly apparent when T cells from chronic hepatitis were treated with anti-PD-L1. The current findings indicate that inhibition of the PD-1/PD-L1 pathway could reactivate virus-specific and global T cell responses in both chronic hepatitis and asymptomatic persistent infection. They suggest a mechanism of potential reactivation of clinically silent infection during anti-PD-1/PD-L1 treatment and indicate that this therapy may also subdue occult HBV infection.
RESUMO
MYC family oncoproteins are regulators of metabolic reprogramming that sustains cancer cell anabolism. Normal cells adapt to nutrient-limiting conditions by activating autophagy, which is required for amino acid (AA) homeostasis. Here we report that the autophagy pathway is suppressed by Myc in normal B cells, in premalignant and neoplastic B cells of Eµ-Myc transgenic mice, and in human MYC-driven Burkitt lymphoma. Myc suppresses autophagy by antagonizing the expression and function of transcription factor EB (TFEB), a master regulator of autophagy. Mechanisms that sustained AA pools in MYC-expressing B cells include coordinated induction of the proteasome and increases in AA transport. Reactivation of the autophagy-lysosomal pathway by TFEB disabled the malignant state by disrupting mitochondrial functions, proteasome activity, AA transport, and AA and nucleotide metabolism, leading to metabolic anergy, growth arrest, and apoptosis. This phenotype provides therapeutic opportunities to disable MYC-driven malignancies, including AA restriction and treatment with proteasome inhibitors. SIGNIFICANCE: MYC suppresses TFEB and autophagy and controls amino acid homeostasis by upregulating amino acid transport and the proteasome, and reactivation of TFEB disables the metabolism of MYC-driven tumors.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Lisossomos , Proteínas Proto-Oncogênicas c-myc , Aminoácidos/metabolismo , Animais , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Homeostase , Humanos , Lisossomos/metabolismo , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
After stimulation, T cells enter a transient refractory period, promoted by IL-2, during which they are resistant to re-stimulation. We previously demonstrated that these IL-2- and Ag-stimulated refractory T cells are able to suppress the Ag-induced proliferation of naive T cells in vitro. We show here that, after adoptive transfer, these T cells are also able to suppress naive T cell proliferation in vivo. More interestingly, potently suppressive T cells can be generated directly in vivo by stimulation with Ag and supplemental IL-2. The activity of the suppressive cells is dose dependent, and the suppressor and suppressed T cells need not be restricted to the same MHC or Ag. Similar to its role in promoting T cell-mediated suppression in vitro, IL-2 is critical for the induction of suppressive activity in activated T cells in vivo. Supplemental IL-2, however, cannot overcome the suppressive activity in target T cells, indicating that suppression is not mediated by competition for this cytokine. Although the activated T cells block naive T cell proliferation, the naive cells do engage Ag and up-regulate the CD25 and CD69 activation markers after stimulation. Therefore, activated T cells stimulated in the presence of IL-2 develop MHC- and Ag-unrestricted suppressive activity. These results provide a new mechanism for competition among CD4(+) T lymphocytes, in which initial waves of responding T cells may inhibit subsequently recruited naive T cells. They further suggest a novel negative feedback loop limiting the expansion of T cell responses that may be present during vigorous immune responses or after IL-2 immunotherapy.
Assuntos
Retroalimentação Fisiológica , Tolerância Imunológica/efeitos dos fármacos , Interleucina-2/farmacologia , Ativação Linfocitária , Subpopulações de Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos , Antígenos/imunologia , Linfócitos T CD4-Positivos , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos TransgênicosAssuntos
Anticorpos Antinucleares/imunologia , DNA/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antinucleares/genética , Afinidade de Anticorpos , Especificidade de Anticorpos , Autoantígenos/imunologia , Doenças Autoimunes/imunologia , Subpopulações de Linfócitos B/imunologia , Cruzamentos Genéticos , Modelos Animais de Doenças , Genes de Imunoglobulinas , Tolerância Imunológica , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Dados de Sequência Molecular , Hipermutação Somática de ImunoglobulinaRESUMO
Autophagy is crucial for maintaining cellular homeostasis, coping with metabolic stress, and limiting oxidative damage. Several autophagy-deficient or knockout models show increased tumor incidence, implicating autophagy as a tumor suppressor. Autophagy is involved in multiple processes that may curb transformation, including the control of oncogene-induced senescence (OIS), which can limit progression to full malignancy, and efficient antigen presentation, which is crucial for immune cell recognition and elimination of nascent cancer cells. Activation of the autophagy pathway may therefore hold promise as a chemoprevention strategy. Caloric restriction, bioactive dietary compounds, or specific pharmacological activators of the autophagy pathway are all possible avenues to explore in harnessing the autophagy pathway in cancer prevention.
Assuntos
Autofagia , Quimioprevenção , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Transdução de Sinais , Animais , Transformação Celular Neoplásica/patologia , Humanos , Modelos BiológicosRESUMO
Autophagy is an ancient cell survival pathway that is induced by metabolic stress and that helps prevent bioenergetic failure. This pathway has emerged as a promising new target in cancer treatment, where agents that inhibit autophagic degradation have efficacy in preventing cancer and in treating resistant disease when combined with conventional chemotherapeutics, which generally activate the pathway. However, agents that specifically target the autophagy pathway are currently lacking, and monitoring the effects of therapeutics on the autophagy pathway raises several challenges. Here we review the potential roles of the autophagy pathway in tumor progression and in maintenance of the malignant state, and introduce novel methods that we have developed that allow one to monitor autophagic activity ex vivo and in vivo.
Assuntos
Autofagia/fisiologia , Neoplasias/metabolismo , Animais , Autofagia/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Linfoma/genética , Linfoma/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Lupus-prone (NZB x NZW)F(1) (BWF(1)) mice were made transgenic (Tg) for an anti-DNA Ab inherited either as a conventional V(H)3H9- micro H chain Tg (3H9- micro ) with or without a conventional V(kappa)8-kappa Tg, or a V(H)3H9 V(H) knock-in Tg allele (3H9R) with or without a V(kappa)4 V(kappa) knock-in Tg allele (V(kappa)4R). V(H)3H9 yields an anti-DNA Ab with most L chains including an anti-ssDNA with the V(kappa)8 Tg and an anti-dsDNA with the V(kappa)4 Tg. BWF(1) mice that inherited the conventional 3H9- micro had normal serum IgM, little to none of which was encoded by 3H9- micro, and only a small percentage of those mice had serum anti-DNA, none of which was transgene encoded. B cells expressing the conventional 3H9- micro Tg were anergic. BWF(1) mice that inherited the knock-in 3H9R Tg allele also had normal serum IgM, one-half of which was encoded by 3H9R, and produced anti-DNA encoded by the Tg allele. Most B cells expressing the knock-in 3H9R Tg also had an anergic phenotype. The results indicate that autoimmune-prone BWF(1) mice initially develop effective B cell tolerance to DNA through anergy, and anergy was sustained in 3H9- micro Tg peripheral B cells but not in 3H9R Tg B cells. B cells expressing the 3H9R knock-in Tg allele were able to achieve an activation threshold that B cells expressing the 3H9- micro conventional Tg could not. The maintenance of B cell tolerance to DNA in autoimmune-prone BWF(1) mice appears to differ from both normal mice and autoimmune-prone MRL(lpr/lpr) mice.