RESUMO
The production of type I interferon is essential for viral clearance but is kept under tight control to avoid unnecessary tissue damage from hyperinflammatory responses. Here we found that OASL1 inhibited translation of IRF7, the master transcription factor for type I interferon, and thus negatively regulated the robust production of type I interferon during viral infection. OASL1 inhibited the translation of IRF7 mRNA by binding to the 5' untranslated region (UTR) of IRF7 and possibly by inhibiting scanning of the 43S preinitiation complex along the message. Oasl1-/- mice were resistant to viral infection because of the greater abundance of type I interferon, which suggests that OASL1 could be a potential therapeutic target for boosting the production of type I interferon during viral infection.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Regulação da Expressão Gênica , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Biossíntese de Proteínas , 2',5'-Oligoadenilato Sintetase/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Vírus da Encefalomiocardite/imunologia , Homozigoto , Humanos , Indutores de Interferon/administração & dosagem , Indutores de Interferon/farmacologia , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Poli I-C/administração & dosagem , Poli I-C/farmacologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismo , Viroses/genética , Viroses/imunologiaRESUMO
Interleukin-7 (IL-7) plays a vital role in the homeostasis of CD4+ and CD8+ T cells. Although IL-7 has been implicated in T helper (Th)1- and Th17-mediated autoinflammatory diseases, its role in Th2-type allergic disorders, such as atopic dermatitis (AD), remains unclear. Thus, to elucidate the effects of IL-7 deficiency on AD development, we generated IL-7-deficient AD-prone mice by backcrossing IL-7 knockout (KO) B6 mice onto the NC/Nga (NC) mouse strain, a model for human AD. As expected, IL-7 KO NC mice displayed defective development of conventional CD4+ and CD8+ T cells compared with wild type (WT) NC mice. However, IL-7 KO NC mice presented with enhanced AD clinical scores, IgE hyperproduction, and increased epidermal thickness compared with WT NC mice. Moreover, IL-7 deficiency decreased Th1, Th17, and IFN-γ-producing CD8+ T cells but increased Th2 cells in the spleen of NC mice, indicating that a reduced Th1/Th2 ratio correlates with severity of AD pathogenesis. Furthermore, significantly more basophils and mast cells infiltrated the skin lesions of IL-7 KO NC mice. Taken together, our findings suggest that IL-7 could be a useful therapeutic target for treating Th2-mediated skin inflammations, such as AD.
Assuntos
Dermatite Atópica , Dermatopatias , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/patologia , Citocinas , Dermatite Atópica/tratamento farmacológico , Modelos Animais de Doenças , Interleucina-7/genética , Interleucina-7/metabolismo , Pele/patologia , Dermatopatias/patologia , Células Th2RESUMO
The physiological functions of progesterone (P4) in female reproductive organs including the mammary glands are mediated via the progesterone receptor (PR), but not all P4 functions can be explained by PR-mediated signaling. Progesterone receptor membrane component 1 (PGRMC1), a potential mediator of P4 actions, plays an important role in the ovary and uterus in maintaining female fertility and pregnancy, but its function in mammary glands has not been elucidated. This study investigated the role of PGRMC1 in mouse mammary gland development. Unlike in the uterus, exogenous estrogen (E2) and/or P4 did not alter PGRMC1 expression in the mammary gland, and Pgrmc1-knockout (KO) mice displayed reduced ductal elongation and side branching in response to hormone treatment. During pregnancy, PGRMC1 was expressed within both the luminal and basal epithelium and gradually increased with gestation and decreased rapidly after parturition. Moreover, although lactogenic capacity was normal after parturition, Pgrmc1 KO resulted in defective mammary gland development from puberty until midpregnancy, while the expression of PR and its target genes was not significantly different between wild-type and Pgrmc1-KO mammary gland. These data suggest that PGRMC1 is essential for mammary gland development during puberty and pregnancy in a PR-independent manner.
Assuntos
Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Receptores de Progesterona/metabolismo , Animais , Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Lactação , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovariectomia , Gravidez , Progesterona/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Progesterona/genética , Maturidade Sexual/efeitos dos fármacos , Maturidade Sexual/genética , Maturidade Sexual/fisiologiaRESUMO
Protein-peptide interactions are involved in a wide range of biological processes and are attractive targets for therapeutic purposes because of their small interfaces. Therefore, effective protein-peptide docking techniques can provide the basis for potential therapeutic applications by enabling an atomic-level understanding of protein interactions. With the increasing number of protein-peptide structures deposited in the protein data bank, the prediction accuracy of protein-peptide docking can be enhanced by utilizing the information provided by the database. The GalaxyPepDock web server, which is freely accessible at http://galaxy.seoklab.org/pepdock, performs similarity-based docking by finding templates from the database of experimentally determined structures and building models using energy-based optimization that allows for structural flexibility. The server can therefore effectively model the structural differences between the template and target protein-peptide complexes. The performance of GalaxyPepDock is superior to those of the other currently available web servers when tested on the PeptiDB set and on recently released complex structures. When tested on the CAPRI target 67, GalaxyPepDock generates models that are more accurate than the best server models submitted during the CAPRI blind prediction experiment.
Assuntos
Simulação de Acoplamento Molecular/métodos , Peptídeos/química , Conformação Proteica , Software , Sítios de Ligação , Internet , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismoRESUMO
Type I interferon (IFN-I) plays a critical role in antiviral and antitumor defense. In our previous studies, we showed that IFN-I-inducible 2'-5' oligoadenylate synthetase-like 1 (OASL1) negatively regulates IFN-I production upon viral infection by specifically inhibiting translation of the IFN-I-regulating master transcription factor, interferon regulatory factor 7 (IRF7). In this study, we investigated whether OASL1 plays a negative role in the anti-tumor immune response by using OASL1-deficient (Oasl1 (-/-)) mice and transplantable syngeneic tumor cell models. We found that Oasl1 (-/-) mice demonstrate enhanced resistance to lung metastatic tumors and subcutaneously implanted tumors compared to wild-type (WT) mice. Additionally, we found that cytotoxic effector cells such as CD8(+) T cells (including tumor antigen-specific CD8(+) T cells) and NK cells as well as CD8α(+) DCs (the major antigen cross-presenting cells) were much more frequent (>fivefold) in the Oasl1 (-/-) mouse tumors. Furthermore, the cytotoxic effector cells in Oasl1 (-/-) mouse tumors seemed to be more functionally active. However, the proportion of immunosuppressive myeloid-derived suppressor cells within hematopoietic cells and of regulatory T cells within CD4(+) T cells in Oasl1 (-/-) mouse tumors did not differ significantly from that of WT mice. Tumor-challenged Oasl1 (-/-) mice expressed increased levels of IFN-I and IRF7 protein in the growing tumor, indicating that the enhanced antitumor immune response observed in Oasl1 (-/-) mice was caused by higher IFN-I production in Oasl1 (-/-) mice. Collectively, these results show that OASL1 deficiency promotes the antitumor immune response, and thus, OASL1 could be a good therapeutic target for treating tumors.
Assuntos
2',5'-Oligoadenilato Sintetase/deficiência , Imunidade/genética , Interferon Tipo I/biossíntese , Neoplasias/etiologia , Neoplasias/metabolismo , 2',5'-Oligoadenilato Sintetase/genética , Animais , Testes Imunológicos de Citotoxicidade , Modelos Animais de Doenças , Imunofenotipagem , Fator Regulador 7 de Interferon/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Contagem de Linfócitos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental , Camundongos , Camundongos Knockout , Neoplasias/mortalidade , Neoplasias/patologiaRESUMO
Knowledge of ligand-binding sites of proteins provides invaluable information for functional studies, drug design and protein design. Recent progress in ligand-binding-site prediction methods has demonstrated that using information from similar proteins of known structures can improve predictions. The GalaxySite web server, freely accessible at http://galaxy.seoklab.org/site, combines such information with molecular docking for more precise binding-site prediction for non-metal ligands. According to the recent critical assessments of structure prediction methods held in 2010 and 2012, this server was found to be superior or comparable to other state-of-the-art programs in the category of ligand-binding-site prediction. A strong merit of the GalaxySite program is that it provides additional predictions on binding ligands and their binding poses in terms of the optimized 3D coordinates of the protein-ligand complexes, whereas other methods predict only identities of binding-site residues or copy binding geometry from similar proteins. The additional information on the specific binding geometry would be very useful for applications in functional studies and computer-aided drug discovery.
Assuntos
Simulação de Acoplamento Molecular/métodos , Proteínas/química , Proteínas/metabolismo , Software , Sítios de Ligação , Internet , Ligantes , Conformação ProteicaRESUMO
The forced reduction of global DNA methylation suppresses tumor development in several cancer models in vivo. Nevertheless, the mechanisms underlying these suppressive effects remain unclear. In this report, we describe our findings showing that a genome-wide reduction in the DNA methylation levels induces cellular differentiation in association with decreased cell proliferation in Apc (Min/+) mouse colon tumor cells in vivo. Colon tumor-specific DNA methylation at Cdx1 is reduced in the DNA-hypomethylated tumors accompanied by Cdx1 derepression and an increased expression of intestinal differentiation-related genes. Furthermore, a histological analysis revealed that Cdx1 derepression in the DNA-hypomethylated tumors is correlated with the differentiation of colon tumor cells. Similarly, the treatment of human colon cancer cell lines with a hypomethylating agent induces differentiation-related genes, including CDX1. We herein propose that DNA demethylation exerts a tumor suppressive effect in the colon by inducing tumor cell differentiation.
Assuntos
Diferenciação Celular/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Metilação de DNA , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Fator de Transcrição CDX2 , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Regiões Promotoras Genéticas , Análise Serial de Tecidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The type I interferons (IFN-Is) are critical not only in early viral control but also in prolonged T-cell immune responses. However, chronic viral infections such as those of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in humans and lymphocytic choriomeningitis virus (LCMV) in mice overcome this early IFN-I barrier and induce viral persistence and exhaustion of T-cell function. Although various T-cell-intrinsic and -extrinsic factors are known to contribute to induction of chronic conditions, the roles of IFN-I negative regulators in chronic viral infections have been largely unexplored. Herein, we explored whether 2'-5' oligoadenylate synthetase-like 1 (OASL1), a recently defined IFN-I negative regulator, plays a key role in the virus-specific T-cell response and viral defense against chronic LCMV. To this end, we infected Oasl1 knockout and wild-type mice with LCMV CL-13 (a chronic virus) and monitored T-cell responses, serum cytokine levels, and viral titers. LCMV CL-13-infected Oasl1 KO mice displayed a sustained level of serum IFN-I, which was primarily produced by splenic plasmacytoid dendritic cells, during the very early phase of infection (2-3 days post-infection). Oasl1 deficiency also led to the accelerated elimination of viremia and induction of a functional antiviral CD8 T-cell response, which critically depended on IFN-I receptor signaling. Together, these results demonstrate that OASL1-mediated negative regulation of IFN-I production at an early phase of infection permits viral persistence and suppresses T-cell function, suggesting that IFN-I negative regulators, including OASL1, could be exciting new targets for preventing chronic viral infection.
Assuntos
2',5'-Oligoadenilato Sintetase/metabolismo , Linfócitos T CD8-Positivos/imunologia , Regulação para Baixo , Interferon Tipo I/metabolismo , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptor de Interferon alfa e beta/metabolismo , 2',5'-Oligoadenilato Sintetase/biossíntese , 2',5'-Oligoadenilato Sintetase/genética , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Resistência à Doença , Feminino , Imunidade Inata , Fator Regulador 7 de Interferon/biossíntese , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/sangue , Interferon Tipo I/genética , Coriomeningite Linfocítica/sangue , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/genética , Transdução de Sinais , Viremia/sangue , Viremia/imunologia , Viremia/metabolismo , Viremia/virologiaRESUMO
Trehalose 6,6'-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincleâ»/â» mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.
Assuntos
Adjuvantes Imunológicos/efeitos adversos , Fatores Corda/efeitos adversos , Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Animais , Antígeno CD11b/genética , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígenos CD18/genética , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Fatores Corda/química , Fatores Corda/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Granuloma do Sistema Respiratório/induzido quimicamente , Granuloma do Sistema Respiratório/genética , Granuloma do Sistema Respiratório/imunologia , Granuloma do Sistema Respiratório/metabolismo , Granuloma do Sistema Respiratório/patologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Neutrófilos/patologia , Pneumonia/genética , Pneumonia/imunologia , Pneumonia/patologia , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Proteínas Quinases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/patologiaRESUMO
IL-7 is a required factor for T-cell homeostasis. Because of low expression levels and poor reagent availability, the cellular sources of IL-7 have proven challenging to characterize. In this study, we describe a reporter mouse in which enhanced GFP is expressed from the endogenous Il7 locus. We show that IL-7 is produced by lymphatic endothelial cells (LECs) distributed throughout the systemic lymphatic vasculature as well as by fibroblastic reticular cells, and that phosphorylation of STAT5 in lymphocytes is higher in lymphatics than in blood. Furthermore, in nodes depleted of lymphocytes, Il7 transcription is increased in stromal but not in myeloid subsets. These data support recent findings that lymphocyte homeostasis is influenced by access to secondary lymphoid organs and point to LECs as an important in vivo source of IL-7, bathing trafficking immune cells under both resting and lymphopenic conditions.
Assuntos
Células Endoteliais/imunologia , Interleucina-7/biossíntese , Sistema Linfático/citologia , Linfopenia/imunologia , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Proteínas de Fluorescência Verde/metabolismo , Interleucina-7/imunologia , Interleucina-7/metabolismo , Linfopenia/metabolismo , Camundongos , Camundongos Transgênicos , Fosforilação , Fator de Transcrição STAT5/metabolismoRESUMO
Certain bacterial zinc-containing anti-sigma (ZAS) factors respond sensitively to thiol-induced oxidative stress by undergoing conformational changes, which in turn reduce binding affinities for their cognate sigma factors. This redox sensitivity provides a mechanism for coping with oxidative stress by activating the transcription of antioxidant genes. Not all ZAS proteins are redox-sensitive, but the mechanism of redox sensitivity is not fully understood. Here we propose that alternative zinc-binding sites determine redox sensitivity. To support this proposal, we performed protein modeling and zinc docking on redox-sensitive and redox-insensitive ZAS proteins complexed with their cognate sigma factors. At least one strong alternative zinc-binding pocket was detected for all known redox-sensitive ZAS factors in actinomycetes, while no strong alternative zinc-binding pocket was identified in redox-insensitive ZAS factors, except for one controversial case. This hypothesis of alternative zinc-binding sites can also explain residue-specific contributions to the redox sensitivity of RsrA, a redox-sensing ZAS protein from Streptomyces coelicolor, for which alanine mutagenesis experiments are available. Our results suggest a mechanistic model for redox sensitivity as follows: zinc ion can probabilistically occupy multiple sites in redox-sensitive ZAS proteins, increasing the susceptibility of zinc-coordinating cysteine residues to oxidation. This picture of probabilistic zinc occupation agrees with a previous structure and energy analysis on zinc finger proteins, and thus it may be more widely applicable to other classes of reactive zinc-binding proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Fator sigma/antagonistas & inibidores , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Zinco/química , Zinco/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Sensibilidade e EspecificidadeRESUMO
Self-assembled peptide nanostructures with actively folded secondary structures have potential to mimic the function of proteins. We here show that α-helix-stabilized self-assembled peptide nanostructures (αSSPNs), whose sizes are comparable to those of proteins, have potential to be developed as protein-protein interaction (PPI) inhibitors along with several unprecedented properties. Using p53-MDM2 PPI as a model system, the molecular recognition and modulation of PPIs by αSSPN grafted with a p53 α-helix (p53 αSSPN) were investigated. The competition assay showed that the p53 αSSPN can inhibit the p53-MDM2 interaction. Interestingly, the p53 αSSPN was far more resistant to degradation by the protease chymotrypsin than the monomeric p53 peptide and had high thermal stability. These results suggest that the αSSPN scaffold holds great potential to be developed as a novel class of PPI inhibitors.
Assuntos
Nanoestruturas/química , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteína Supressora de Tumor p53/química , Sequência de Aminoácidos , Quimotripsina/química , Dicroísmo Circular , Meia-Vida , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteólise , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Adoptive cell therapy (ACT) with antigen-specific T cells is a promising treatment approach for solid cancers. Interleukin-2 (IL-2) has been utilized in boosting the efficacy of ACT. However, the clinical applications of IL-2 in combination with ACT is greatly limited by short exposure and high toxicities. Herein, a complex coacervate was designed to intratumorally deliver IL-2 in a sustained manner and protect against proteolysis. The complex coacervate consisted of fucoidan, a specific IL-2 binding glycosaminoglycan, and poly-l-lysine, a cationic counterpart (FPC2). IL-2-laden FPC2 exhibited a preferential bioactivity in ex vivo expansion of CD8+T cells over Treg cells. Additionally, FPC2 was embedded in pH modulating injectable gel (FPC2-IG) to endure the acidic tumor microenvironment. A single intratumoral administration of FPC2-IG-IL-2 increased expansion of tumor-infiltrating cytotoxic lymphocytes and reduced frequencies of myeloid populations. Notably, the activation and persistency of tumor-reactive T cells were observed only in the tumor site, not in the spleen, confirming a localized effect of FPC2-IG-IL-2. The immune-favorable tumor microenvironment induced by FPC2-IG-IL-2 enabled adoptively transferred TCR-engineered T cells to effectively eradicate tumors. FPC2-IG delivery system is a promising strategy for T-cell-based immunotherapies.
RESUMO
Aberrant activation of cytokine and growth factor signal transduction pathways confers enhanced survival and proliferation properties to acute myeloid leukemia (AML) cells. However, the mechanisms underlying the deregulation of signaling pathways in leukemia cells are unclear. To identify genes capable of independently supporting cytokine-independent growth, we employed a genome-wide CRISPR/Cas9-mediated loss-of-function screen in GM-CSF-dependent human AML TF-1 cells. More than 182 genes (p < 0.01) were found to suppress the cytokine-independent growth of TF-1 cells. Among the top hits, genes encoding key factors involved in sialylation biosynthesis were identified; these included CMAS, SLC35A1, NANS, and GNE. Knockout of either CMAS or SLC35A1 enabled cytokine-independent proliferation and survival of AML cells. Furthermore, NSG (NOD/SCID/IL2Rγ-/-) mice injected with CMAS or SLC35A1-knockout TF-1 cells exhibited a shorter survival than mice injected with wild-type cells. Mechanistically, abrogation of sialylation biosynthesis in TF-1 cells induced a strong activation of ERK signaling, which sensitized cells to MEK inhibitors but conferred resistance to JAK inhibitors. Further, the surface level of α2,3-linked sialic acids was negatively correlated with the sensitivity of AML cell lines to MEK/ERK inhibitors. We also found that sialylation modulated the expression and stability of the CSF2 receptor. Together, these results demonstrate a novel role of sialylation in regulating oncogenic transformation and drug resistance development in leukemia. We propose that altered sialylation could serve as a biomarker for targeted anti-leukemic therapy.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Leucemia Mieloide Aguda/genética , Animais , Carcinogênese , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Transdução de SinaisRESUMO
Pgrmc1 is a non-canonical progesterone receptor related to the lethality of various types of cancer. PGRMC1 has been reported to exist in co-precipitated protein complexes with epidermal growth factor receptor (EGFR), which is considered a useful therapeutic target in hepatocellular carcinoma (HCC). Here, we investigated whether Pgrmc1 is involved in HCC progression. In clinical datasets, PGRMC1 transcription level was positively correlated with EGFR levels; importantly, PGRMC1 level was inversely correlated with the survival duration of HCC patients. In a diethylnitrosamine (DEN)-induced murine model of HCC, the global ablation of Pgrmc1 suppressed the development of HCC and prolonged the survival of HCC-bearing mice. We further found that increases in hepatocyte death and suppression of compensatory proliferation in the livers of DEN-injured Pgrmc1-null mice were concomitant with decreases in nuclear factor κB (NF-κB)-dependent production of interleukin-6 (IL-6). Indeed, silencing of Pgrmc1 in murine macrophages led to reductions in NF-κB activity and IL-6 production. We found that the anti-proinflammatory effect of Pgrmc1 loss was mediated by reductions in EGFR level and its effect was not observed after exposure of the EGFR inhibitor erlotinib. This study reveals a novel cooperative role of Pgrmc1 in supporting the EGFR-mediated development of hepatocellular carcinoma, implying that pharmacological suppression of Pgrmc1 may be a useful strategy in HCC treatment.
RESUMO
Type I interferon (IFN-I) plays a critical role in the antitumor immune response. In our previous study, we showed that IFN-I-inducible 2'-5' oligoadenylate synthetase-like 1 (OASL1) negatively regulated IFN-I production upon tumor challenge similar to that of viral infection. Thus, OASL1-deficient (Oasl1 -/-) mice were more resistant to implanted tumor growth than wild-type (WT) mice. In this study, we investigated whether targeting or suppressing OASL1 could show synergistic effects on tumor clearance with conventional cancer therapies (such as chemotherapy and radiotherapy) using Oasl1 -/- mice and a transplantable lung metastatic tumor cell model. Upon treatment with the anticancer drug cisplatin, we found that Oasl1 -/- mice showed enhanced resistance to injected tumors compared to untreated Oasl1 -/- mice. Similarly, irradiated Oasl1 -/- mice showed better resistance to tumor challenge than untreated Oasl1 -/- mice. Additionally, we found that Oasl1 -/- mice applied with both types of the cancer therapies contained more cytotoxic effector cells, such as CD8+ T cells and NK cells, and produced more cytotoxic effector cytokine IFN-γ as well as IFN-I in their tumor-containing lungs compared to untreated Oasl1 -/- mice. Collectively, these results show that targeting OASL1 together with conventional cancer therapies could be an effective strategy to enhance treatment efficacy.
Assuntos
2',5'-Oligoadenilato Sintetase/antagonistas & inibidores , Cisplatino/uso terapêutico , Interferon Tipo I/imunologia , Neoplasias Pulmonares/imunologia , 2',5'-Oligoadenilato Sintetase/genética , Animais , Antineoplásicos , Interferon gama/imunologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Metástase NeoplásicaRESUMO
BACKGROUND: Very early-onset inflammatory bowel disease (VEO-IBD) is often associated with monogenetic disorders. IL-10RA deficiency is one of the major causal mutations in VEO-IBD. Here, we aimed to identify the causal mutation associated with severe IBD in a 1-year-old patient, validate the pathogenicity of the mutation, and characterize the mutant protein. METHODS: To identify the causal mutation, targeted exome sequencing (ES) was performed using the genomic DNA from the patient. To validate the pathogenicity, IL-10RA functional tests were performed using the patient's peripheral blood mononuclear cells (PBMCs). Additionally, flow cytometry analysis, confocal microscopy on overexpressed green fluorescent protein-fused mutants, and computational analysis on the structures of IL-10RA proteins were performed. RESULTS: We identified a novel compound heterozygote mutation p.[Tyr91Cys];[Pro146Alafs*40] in the IL10RA gene of the patient. The missense variant p.Tyr91Cys was previously identified but not functionally tested, and a frameshift variant, p.Pro146Alafs*40, is novel and nonfunctional. PBMCs from the patient showed defective signal transducer and activator of transcription 3 activation. The p.Tyr91Cys mutant protein failed to properly localize on the plasma membrane. The p.Tyr91Cys mutation seems to disrupt the hydrophobic core structure surrounding the tyrosine 91 residue, causing structural instability. CONCLUSIONS: Targeted ES and linkage analysis identified novel compound heterozygous mutations p.[Tyr91Cys];[Pro146Alafs*40] in the IL10RA gene of a child with severe VEO-IBD. p.Tyr91Cys proteins were functionally defective in IL-10RA signaling and failed to properly localize on the plasma membrane, probably due to its structural instability.
Assuntos
Heterozigoto , Doenças Inflamatórias Intestinais/etiologia , Subunidade alfa de Receptor de Interleucina-10/genética , Mutação , Idade de Início , Humanos , Lactente , Doenças Inflamatórias Intestinais/patologia , Masculino , Prognóstico , Sequenciamento do ExomaRESUMO
The expression of major histocompatibility complex class I (MHC I) in tumor cells is regulated by interferon signaling, and it is an important factor in the efficacy of cytotoxic T cell-dependent immunotherapy. To determine the impact of immune cells in MHC I expression on tumor cells, we compared the expression of MHC I in tumor cells derived from primary breast cancers and patient-derived xenograft (PDX) models. MHC I and myxovirus resistance gene A (MxA) expression were analyzed using immunohistochemistry in 23 cases of tumor tissue and corresponding primary and secondary PDXs. The median H score of MHC I was 210 (0-300) in patient tumor tissues, 197.5 (0-300) in primary PDX tumors, and 157.5 (5-300) in secondary PDX tumors. Cases were divided into four groups based on the difference in MHC I expression between the patient tumor tissues and secondary PDXs. Eleven cases constituted the high MHC I group, four constituted the low MHC I group, six comprised the decreased MHC I group, and two comprised the increased MHC I group. MHC I and MxA expressions in each tumor were weakly correlated within patients' tumors, while strongly correlated within PDX models. Retained or altered expression of MHC I in breast cancer PDXs reveals the presence of intrinsic and extrinsic interferon signaling pathways in tumor cells. Thus, considering MHC I expression in PDX is important when using PDX models to evaluate the efficacy of cancer immunotherapy in a preclinical setting.
Assuntos
Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Antígenos de Histocompatibilidade Classe I/imunologia , Animais , Modelos Animais de Doenças , Feminino , Antígenos HLA/imunologia , Humanos , Imuno-Histoquímica/métodos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Invading pathogens are recognized by diverse germline-encoded pattern-recognition receptors (PRRs) which are distributed in three different cellular compartments: extracellular, membrane, and cytoplasmic. In mammals, the major extracellular PRRs such as complements may first encounter the invading pathogens and opsonize them for clearance by phagocytosis which is mediated by membrane-associated phagocytic receptors including complement receptors. The major membrane-associated PRRs, Toll-like receptors, recognize diverse pathogens and generate inflammatory signals to coordinate innate immune responses and shape adaptive immune responses. Furthemore, certain membrane-associated PRRs such as Dectin-1 can mediate phagocytosis and also induce inflammatory response. When these more forefront detection systems are avoided by the pathogens, cytoplasmic PRRs may play major roles. Cytoplasmic caspase-recruiting domain (CARD) helicases such as retinoic acid-inducible protein I (RIG-I)melanoma differentiation-associated gene 5 (MDA5), mediate antiviral immunity by inducing the production of type I interferons. Certain members of nucleotide-binding oligomerization domain (NOD)-like receptors such as NALP3 present in the cytosol form inflammasomes to induce inflammatory responses upon ligand recognition. Thus, diverse families of PRRs coordinately mediate immune responses against diverse types of pathogens.
Assuntos
Membrana Celular/metabolismo , Citoplasma/metabolismo , Espaço Extracelular/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Transdução de Sinais , Animais , Humanos , FagocitoseRESUMO
Adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs) has been successful in treating a considerable proportion of patients with metastatic melanoma. In addition, some patients with several other solid tumors were recently reported to have benefited clinically from such ACT. However, it remains unclear whether ACT using TILs is broadly applicable in breast cancer, the most common cancer in women. In this study, the utility of TILs as an ACT source in breast cancers was explored by deriving TILs from a large number of breast cancer samples and assessing their biological potentials. We successfully expanded TILs ex vivo under a standard TIL culture condition from over 100 breast cancer samples, including all breast cancer subtypes. We also found that the information about the percentage of TIL and presence of tertiary lymphoid structure in the tumor tissues could be useful for estimating the number of obtainable TILs after ex vivo culture. The ex vivo expanded TILs contained a considerable level of central memory phenotype T cells (about 20%), and a large proportion of TIL samples were reactive to autologous tumor cells in vitro. Furthermore, the in vitro tumor-reactive autologous TILs could also function in vivo in a xenograft mouse model implanted with the primary tumor tissue. Collectively, these results strongly indicate that ACT using ex vivo expanded autologous TILs is a feasible option in treating patients with breast cancer.