RESUMEN
Type I and II interferons (IFNs) stimulate pro-inflammatory programs that are critical for immune activation, but also induce immune-suppressive feedback circuits that impede control of cancer growth. Here, we sought to determine how these opposing programs are differentially induced. We demonstrated that the transcription factor interferon regulatory factor 2 (IRF2) was expressed by many immune cells in the tumor in response to sustained IFN signaling. CD8+ T cell-specific deletion of IRF2 prevented acquisition of the T cell exhaustion program within the tumor and instead enabled sustained effector functions that promoted long-term tumor control and increased responsiveness to immune checkpoint and adoptive cell therapies. The long-term tumor control by IRF2-deficient CD8+ T cells required continuous integration of both IFN-I and IFN-II signals. Thus, IRF2 is a foundational feedback molecule that redirects IFN signals to suppress T cell responses and represents a potential target to enhance cancer control.
Asunto(s)
Interferón Tipo I , Neoplasias , Humanos , Factor 2 Regulador del Interferón/genética , Linfocitos T CD8-positivos , Factores de Transcripción , Agotamiento de Células T , Neoplasias/patologíaRESUMEN
The molecular mechanisms whereby CD8+ T cells become "exhausted" in the tumor microenvironment remain unclear. Programmed death ligand-1 (PD-L1) is upregulated on tumor cells and PD-1-PD-L1 blockade has significant efficacy in human tumors; however, most patients do not respond, suggesting additional mechanisms underlying T cell exhaustion. B7 superfamily member 1 (B7S1), also called B7-H4, B7x, or VTCN1, negatively regulates T cell activation. Here we show increased B7S1 expression on myeloid cells from human hepatocellular carcinoma correlated with CD8+ T cell dysfunction. B7S1 inhibition suppressed development of murine tumors. Putative B7S1 receptor was co-expressed with PD-1 but not T cell immunoglobulin and mucin-domain containing-3 (Tim-3) at an activated state of early tumor-infiltrating CD8+ T cells, and B7S1 promoted T cell exhaustion, possibly through Eomes overexpression. Combinatorial blockade of B7S1 and PD-1 synergistically enhanced anti-tumor immune responses. Collectively, B7S1 initiates dysfunction of tumor-infiltrating CD8+ T cells and may be targeted for cancer immunotherapy.
Asunto(s)
Antígeno B7-H1/metabolismo , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/inmunología , Neoplasias Hepáticas/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Células Mieloides/inmunología , Inhibidor 1 de la Activación de Células T con Dominio V-Set/metabolismo , Animales , Carcinoma Hepatocelular/patología , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/patología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Dominio T Box/metabolismo , Inhibidor 1 de la Activación de Células T con Dominio V-Set/genéticaRESUMEN
Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
Asunto(s)
Envejecimiento/fisiología , Ácido Ascórbico/farmacología , Diabetes Mellitus Experimental/prevención & control , Proteína de Retinoblastoma/metabolismo , Animales , Senescencia Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Factor de Transcripción E2F1/metabolismo , Desarrollo Embrionario/genética , Femenino , Fibroblastos/efectos de los fármacos , Técnicas de Sustitución del Gen , Células Secretoras de Insulina/patología , Ratones , Fosforilación , Embarazo , Proteína de Retinoblastoma/genética , Telómero/genéticaRESUMEN
Activated T cells produce reactive oxygen species (ROS), which trigger the antioxidative glutathione (GSH) response necessary to buffer rising ROS and prevent cellular damage. We report that GSH is essential for T cell effector functions through its regulation of metabolic activity. Conditional gene targeting of the catalytic subunit of glutamate cysteine ligase (Gclc) blocked GSH production specifically in murine T cells. Gclc-deficient T cells initially underwent normal activation but could not meet their increased energy and biosynthetic requirements. GSH deficiency compromised the activation of mammalian target of rapamycin-1 (mTOR) and expression of NFAT and Myc transcription factors, abrogating the energy utilization and Myc-dependent metabolic reprogramming that allows activated T cells to switch to glycolysis and glutaminolysis. In vivo, T-cell-specific ablation of murine Gclc prevented autoimmune disease but blocked antiviral defense. The antioxidative GSH pathway thus plays an unexpected role in metabolic integration and reprogramming during inflammatory T cell responses.
Asunto(s)
Glutamato-Cisteína Ligasa/deficiencia , Glutatión/metabolismo , Inflamación/metabolismo , Linfocitos T/metabolismo , Animales , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Metabolismo Energético/genética , Glutamato-Cisteína Ligasa/genética , Glutamina/metabolismo , Glucólisis , Immunoblotting , Inflamación/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción NFATC/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases, play key roles in cellular signaling. All Class I PTPs contain an essential active site cysteinyl residue, which executes a nucleophilic attack on substrate phosphotyrosyl residues. The high reactivity of the catalytic cysteine also predisposes PTPs to oxidation by reactive oxygen species, such as H(2)O(2). Reversible PTP oxidation is emerging as an important cellular regulatory mechanism and might contribute to diseases such as cancer. We exploited these unique features of PTP enzymology to develop proteomic methods, broadly applicable to cell and tissue samples, that enable the comprehensive identification and quantification of expressed classical PTPs (PTPome) and the oxidized subset of the PTPome (oxPTPome). We find that mouse and human cells and tissues, including cancer cells, display distinctive PTPomes and oxPTPomes, revealing additional levels of complexity in the regulation of protein-tyrosine phosphorylation in normal and malignant cells.
Asunto(s)
Proteínas Tirosina Fosfatasas/análisis , Proteómica/métodos , Animales , Línea Celular , Humanos , Ratones , Ratones Endogámicos C57BL , Neoplasias/metabolismo , Oxidación-Reducción , RatasRESUMEN
Understanding the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection. Mice infected with LCMV clone-13 have persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology. The IL-7-mediated effects were dependent on endogenous IL-6. These attributes of IL-7 have profound implications for its use as a therapeutic in the treatment of chronic viral diseases.
Asunto(s)
Interleucina-7/uso terapéutico , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Animales , Antígenos de Diferenciación/metabolismo , Regulación hacia Abajo , Factores de Transcripción Forkhead/metabolismo , Humanos , Interleucina-6/inmunología , Interleucina-7/inmunología , Ratones , Receptor de Muerte Celular Programada 1 , Proteínas Recombinantes/metabolismo , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Linfocitos T/inmunologíaRESUMEN
Mutations in IDH1, IDH2, and TET2 are recurrently observed in myeloid neoplasms. IDH1 and IDH2 encode isocitrate dehydrogenase isoforms, which normally catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). Oncogenic IDH1/2 mutations confer neomorphic activity, leading to the production of D-2-hydroxyglutarate (D-2-HG), a potent inhibitor of α-KG-dependent enzymes which include the TET methylcytosine dioxygenases. Given their mutual exclusivity in myeloid neoplasms, IDH1, IDH2, and TET2 mutations may converge on a common oncogenic mechanism. Contrary to this expectation, we observed that they have distinct, and even opposite, effects on hematopoietic stem and progenitor cells in genetically engineered mice. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172K and Tet2 loss-of-function have divergent consequences on the expression and activity of key hematopoietic and leukemogenic regulators. Notably, chromatin accessibility and transcriptional deregulation in Idh2R172K cells were partially disconnected from DNA methylation alterations. These results highlight unanticipated divergent effects of IDH1/2 and TET2 mutations, providing support for the optimization of genotype-specific therapies.
Asunto(s)
Proteínas de Unión al ADN , Dioxigenasas , Isocitrato Deshidrogenasa , Células Madre , Animales , Ratones , Dioxigenasas/genética , Proteínas de Unión al ADN/genética , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mutación , Neoplasias , Células Madre/metabolismoRESUMEN
Asbestos is the main cause of malignant mesothelioma. Previous studies have linked asbestos-induced mesothelioma to the release of HMGB1 from the nucleus to the cytoplasm, and from the cytoplasm to the extracellular space. In the cytoplasm, HMGB1 induces autophagy impairing asbestos-induced cell death. Extracellularly, HMGB1 stimulates the secretion of TNFα. Jointly, these two cytokines kick-start a chronic inflammatory process that over time promotes mesothelioma development. Whether the main source of extracellular HMGB1 were the mesothelial cells, the inflammatory cells, or both was unsolved. This information is critical to identify the targets and design preventive/therapeutic strategies to interfere with asbestos-induced mesothelioma. To address this issue, we developed the conditional mesothelial HMGB1-knockout (Hmgb1ΔpMeso) and the conditional myelomonocytic-lineage HMGB1-knockout (Hmgb1ΔMylc) mouse models. We establish here that HMGB1 is mainly produced and released by the mesothelial cells during the early phases of inflammation following asbestos exposure. The release of HMGB1 from mesothelial cells leads to atypical mesothelial hyperplasia, and in some animals, this evolves over the years into mesothelioma. We found that Hmgb1ΔpMeso, whose mesothelial cells cannot produce HMGB1, show a greatly reduced inflammatory response to asbestos, and their mesothelial cells express and secrete significantly reduced levels of TNFα. Moreover, the tissue microenvironment in areas of asbestos deposits displays an increased fraction of M1-polarized macrophages compared to M2 macrophages. Supporting the biological significance of these findings, Hmgb1ΔpMeso mice showed a delayed and reduced incidence of mesothelioma and an increased mesothelioma-specific survival. Altogether, our study provides a biological explanation for HMGB1 as a driver of asbestos-induced mesothelioma.
Asunto(s)
Amianto , Proteína HMGB1 , Mesotelioma Maligno , Mesotelioma , Animales , Ratones , Factor de Necrosis Tumoral alfa/genética , Proteína HMGB1/genética , Mesotelioma/inducido químicamente , Mesotelioma/genética , Amianto/toxicidad , Inflamación , Microambiente TumoralRESUMEN
The E3 ligase ARIH2 has an unusual structure and mechanism of elongating ubiquitin chains. To understand its physiological role, we generated gene-targeted mice deficient in ARIH2. ARIH2 deficiency resulted in the embryonic death of C57BL/6 mice. On a mixed genetic background, the lethality was attenuated, with some mice surviving beyond weaning and then succumbing to an aggressive multiorgan inflammatory response. We found that in dendritic cells (DCs), ARIH2 caused degradation of the inhibitor IκBß in the nucleus, which abrogated its ability to sequester, protect and transcriptionally coactivate the transcription factor subunit p65 in the nucleus. Loss of ARIH2 caused dysregulated activation of the transcription factor NF-κB in DCs, which led to lethal activation of the immune system in ARIH2-sufficent mice reconstituted with ARIH2-deficient hematopoietic stem cells. Our data have therapeutic implications for targeting ARIH2 function.
Asunto(s)
Células Dendríticas/inmunología , Desarrollo Embrionario/inmunología , Insuficiencia Multiorgánica/inmunología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Desarrollo Embrionario/genética , Hematopoyesis/genética , Humanos , Sistema Inmunológico/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terapia Molecular Dirigida , Insuficiencia Multiorgánica/genética , FN-kappa B/metabolismo , Activación Transcripcional/inmunología , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Ubiquitinación/inmunologíaRESUMEN
PI3K and PTEN lipid phosphatase control the level of cellular phosphatidylinositol (3,4,5)-trisphosphate, an activator of AKT kinases that promotes cell growth and survival. Mutations activating AKT are commonly observed in human cancers. We report here that ENTPD5, an endoplasmic reticulum (ER) enzyme, is upregulated in cell lines and primary human tumor samples with active AKT. ENTPD5 hydrolyzes UDP to UMP to promote protein N-glycosylation and folding in ER. Knockdown of ENTPD5 in PTEN null cells causes ER stress and loss of growth factor receptors. ENTPD5, together with cytidine monophosphate kinase-1 and adenylate kinase-1, constitute an ATP hydrolysis cycle that converts ATP to AMP, resulting in a compensatory increase in aerobic glycolysis known as the Warburg effect. The growth of PTEN null cells is inhibited both in vitro and in mouse xenograft tumor models. ENTPD5 is therefore an integral part of the PI3K/PTEN regulatory loop and a potential target for anticancer therapy.
Asunto(s)
Proliferación Celular , Retículo Endoplásmico/metabolismo , Glicosilación , Proteínas Oncogénicas/metabolismo , Adenosina Trifosfato/metabolismo , Aerobiosis , Animales , Línea Celular Tumoral , Glucólisis , Guanosina Monofosfato/metabolismo , Humanos , Ratones , Trasplante de Neoplasias , Proteína Oncogénica v-akt/metabolismo , Proteínas Oncogénicas/genética , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Pirofosfatasas , Trasplante Heterólogo , Uridina Monofosfato/metabolismoRESUMEN
A hallmark of cancer, including pancreatic ductal adenocarcinoma (PDA), is a massive stromal and inflammatory reaction. Many efforts have been made to identify the anti- or protumoral role of cytokines and immune subpopulations within the stroma. Here, we investigated the role of interleukin-17A (IL17A) and its effect on tumor fibroblasts and the tumor microenvironment. We used a spontaneous PDA mouse model (KPC) crossed to IL17A knockout mice to show an extensive desmoplastic reaction, without impaired immune infiltration. Macrophages, especially CD80+ and T cells, were more abundant at the earlier time point. In T cells, a decrease in FoxP3+ cells and an increase in CD8+ T cells were observed in KPC/IL17A-/- mice. Fibroblasts isolated from IL17A+/+ and IL17A-/- KPC mice revealed very different messenger RNA (mRNA) and protein profiles. IL17A-/- fibroblasts displayed the ability to restrain tumor cell invasion by producing factors involved in extracellular matrix remodeling, increasing T cell recruitment, and producing higher levels of cytokines and chemokines favoring T helper 1 cell recruitment and activation and lower levels of those recruiting myeloid/granulocytic immune cells. Single-cell quantitative PCR on isolated fibroblasts confirmed a very divergent profile of IL17A-proficient and -deficient cells. All these features can be ascribed to increased levels of IL17F observed in the sera of IL17A-/- mice, and to the higher expression of its cognate receptor (IL17RC) specifically in IL17A-/- cancer-associated fibroblasts (CAFs). In addition to the known effects on neoplastic cell transformation, the IL17 cytokine family uniquely affects fibroblasts, representing a suitable candidate target for combinatorial immune-based therapies in PDA.
Asunto(s)
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Interleucina-17/genética , Receptores de Interleucina/genética , Adenocarcinoma/patología , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinogénesis/genética , Carcinoma Ductal Pancreático/patología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/genética , Humanos , Ratones , Ratones Noqueados , Microambiente Tumoral/genéticaRESUMEN
Isocitrate dehydrogenase (IDH) mutations are common genetic alterations in myeloid disorders, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Epigenetic changes, including abnormal histone and DNA methylation, have been implicated in the pathogenic build-up of hematopoietic progenitors, but it is still unclear whether and how IDH mutations themselves affect hematopoiesis. Here, we show that IDH1-mutant mice develop myeloid dysplasia in that these animals exhibit anemia, ineffective erythropoiesis, and increased immature progenitors and erythroblasts. In erythroid cells of these mice, D-2-hydroxyglutarate, an aberrant metabolite produced by the mutant IDH1 enzyme, inhibits oxoglutarate dehydrogenase activity and diminishes succinyl-coenzyme A (CoA) production. This succinyl-CoA deficiency attenuates heme biosynthesis in IDH1-mutant hematopoietic cells, thus blocking erythroid differentiation at the late erythroblast stage and the erythroid commitment of hematopoietic stem cells, while the exogenous succinyl-CoA or 5-ALA rescues erythropoiesis in IDH1-mutant erythroid cells. Heme deficiency also impairs heme oxygenase-1 expression, which reduces levels of important heme catabolites such as biliverdin and bilirubin. These deficits result in accumulation of excessive reactive oxygen species that induce the cell death of IDH1-mutant erythroid cells. Our results clearly show the essential role of IDH1 in normal erythropoiesis and describe how its mutation leads to myeloid disorders. These data thus have important implications for the devising of new treatments for IDH-mutant tumors.
Asunto(s)
Eritropoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Hemo/biosíntesis , Isocitrato Deshidrogenasa/genética , Mutación Missense , Mutación Puntual , Preleucemia/genética , Acilcoenzima A/biosíntesis , Acilcoenzima A/deficiencia , Anemia/genética , Animales , Médula Ósea/patología , Eritroblastos/metabolismo , Técnicas de Sustitución del Gen , Glutaratos/metabolismo , Hemo/deficiencia , Hemo-Oxigenasa 1/metabolismo , Isocitrato Deshidrogenasa/fisiología , Complejo Cetoglutarato Deshidrogenasa/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/patología , Mielopoyesis/genética , Preleucemia/metabolismo , Preleucemia/patología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/metabolismo , Esplenomegalia/etiología , Trombocitopenia/genéticaRESUMEN
Despite development of new antiviral drugs, viral infections are still a major health problem. The most potent antiviral defense mechanism is the innate production of type I interferon (IFN-I), which not only limits virus replication but also promotes antiviral T cell immunity through mechanisms, which remain insufficiently studied. Using the murine lymphocytic choriomeningitis virus model system, we show here that IFN-I signaling on T cells prevented their rapid elimination in vivo. Microarray analyses uncovered that IFN-I triggered the expression of selected inhibitory NK-cell-receptor ligands. Consequently, T cell immunity of IFN-I receptor (IFNAR)-deficient T cells could be restored by NK cell depletion or in NK-cell-deficient hosts (Nfil3(-/-)). The elimination of Ifnar1(-/-) T cells was dependent on NK-cell-mediated perforin expression. In summary, we identified IFN-I as a key player regulating the protection of T cells against regulatory NK cell function.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citotoxicidad Inmunológica , Interferón Tipo I/inmunología , Células Asesinas Naturales/inmunología , Coriomeningitis Linfocítica/inmunología , Receptor de Interferón alfa y beta/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células Cultivadas , Inmunidad Innata , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Perforina/biosíntesis , Receptor de Interferón alfa y beta/genética , Transducción de Señal/inmunología , Replicación Viral/inmunologíaRESUMEN
The p53 family of proteins consists of p53, p63 and p73, which are transcription factors that affect both cancer and development. It is now emerging that these proteins also regulate maternal reproduction. Whereas p63 is important for maturation of the egg, p73 ensures normal mitosis in the developing blastocyst. p53 subsequently regulates implantation of the embryo through transcriptional control of leukaemia inhibitory factor. Elucidating the cell biological basis of how these factors regulate female fertility may lead to new approaches to the control of human maternal reproduction.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Proteína p53 Supresora de Tumor/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Blastocisto/citología , Blastocisto/fisiología , Proteínas de Unión al ADN/genética , Femenino , Fertilidad/genética , Fertilidad/fisiología , Humanos , Masculino , Ratones , Ratones Noqueados , Modelos Biológicos , Proteínas Nucleares/genética , Oocitos/citología , Oocitos/fisiología , Fosfoproteínas/genética , Fosfoproteínas/fisiología , Reproducción/genética , Reproducción/fisiología , Transactivadores/genética , Factores de Transcripción , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
The cell cycle is an evolutionarily conserved process necessary for mammalian cell growth and development. Because cell-cycle aberrations are a hallmark of cancer, this process has been the target of anti-cancer therapeutics for decades. However, despite numerous clinical trials, cell-cycle-targeting agents have generally failed in the clinic. This review briefly examines past cell-cycle-targeted therapeutics and outlines how experience with these agents has provided valuable insight to refine and improve anti-mitotic strategies. An overview of emerging anti-mitotic approaches with promising pre-clinical results is provided, and the concept of exploiting the genomic instability of tumor cells through therapeutic inhibition of mitotic checkpoints is discussed. We believe this strategy has a high likelihood of success given its potential to enhance therapeutic index by targeting tumor-specific vulnerabilities. This reasoning stimulated our development of novel inhibitors targeting the critical regulators of genomic stability and the mitotic checkpoint: AURKA, PLK4, and Mps1/TTK.
Asunto(s)
Antineoplásicos/farmacología , Mitosis/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/uso terapéutico , Aurora Quinasa A/antagonistas & inhibidores , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inestabilidad Genómica/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
Asbestos causes malignant transformation of primary human mesothelial cells (HM), leading to mesothelioma. The mechanisms of asbestos carcinogenesis remain enigmatic, as exposure to asbestos induces HM death. However, some asbestos-exposed HM escape cell death, accumulate DNA damage, and may become transformed. We previously demonstrated that, upon asbestos exposure, HM and reactive macrophages releases the high mobility group box 1 (HMGB1) protein that becomes detectable in the tissues near asbestos deposits where HMGB1 triggers chronic inflammation. HMGB1 is also detectable in the sera of asbestos-exposed individuals and mice. Searching for additional biomarkers, we found higher levels of the autophagy marker ATG5 in sera from asbestos-exposed individuals compared to unexposed controls. As we investigated the mechanisms underlying this finding, we discovered that the release of HMGB1 upon asbestos exposure promoted autophagy, allowing a higher fraction of HM to survive asbestos exposure. HMGB1 silencing inhibited autophagy and increased asbestos-induced HM death, thereby decreasing asbestos-induced HM transformation. We demonstrate that autophagy was induced by the cytoplasmic and extracellular fractions of HMGB1 via the engagement of the RAGE receptor and Beclin 1 pathway, while nuclear HMGB1 did not participate in this process. We validated our findings in a novel unique mesothelial conditional HMGB1-knockout (HMGB1-cKO) mouse model. Compared to HMGB1 wild-type mice, mesothelial cells from HMGB1-cKO mice showed significantly reduced autophagy and increased cell death. Autophagy inhibitors chloroquine and desmethylclomipramine increased cell death and reduced asbestos-driven foci formation. In summary, HMGB1 released upon asbestos exposure induces autophagy, promoting HM survival and malignant transformation.
Asunto(s)
Amianto/efectos adversos , Autofagia/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Proteína HMGB1/metabolismo , Mesotelioma/metabolismo , Adulto , Anciano , Animales , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Exposición ProfesionalRESUMEN
Little is known about how mammalian cells maintain cell size homeostasis. We conducted a novel genetic screen to identify cell-size-controlling genes and isolated Largen, the product of a gene (PRR16) that increased cell size upon overexpression in human cells. In vitro evidence indicated that Largen preferentially stimulates the translation of specific subsets of mRNAs, including those encoding proteins affecting mitochondrial functions. The involvement of Largen in mitochondrial respiration was consistent with the increased mitochondrial mass and greater ATP production in Largen-overexpressing cells. Furthermore, Largen overexpression led to increased cell size in vivo, as revealed by analyses of conditional Largen transgenic mice. Our results establish Largen as an important link between mRNA translation, mitochondrial functions, and the control of mammalian cell size.
Asunto(s)
Tamaño de la Célula/efectos de los fármacos , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Proteínas/genética , ARN Mensajero/genética , Animales , Línea Celular Tumoral , Escherichia coli/genética , Escherichia coli/metabolismo , Vectores Genéticos , Ensayos Analíticos de Alto Rendimiento , Humanos , Células Jurkat , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacologíaRESUMEN
The mitotic protein polo-like kinase 4 (PLK4) plays a critical role in centrosome duplication for cell division. By using immunofluorescence, we confirm that PLK4 is localized to centrosomes. In addition, we find that phospho-PLK4 (pPLK4) is cleaved and distributed to kinetochores (metaphase and anaphase), spindle midzone/cleavage furrow (anaphase and telophase), and midbody (cytokinesis) during cell division in immortalized epithelial cells as well as breast, ovarian, and colorectal cancer cells. The distribution of pPLK4 midzone/cleavage furrow and midbody positions pPLK4 to play a functional role in cytokinesis. Indeed, we found that inhibition of PLK4 kinase activity with a small-molecule inhibitor, CFI-400945, prevents translocation to the spindle midzone/cleavage furrow and prevents cellular abscission, leading to the generation of cells with polyploidy, increased numbers of duplicated centrosomes, and vulnerability to anaphase or mitotic catastrophe. The regulatory role of PLK4 in cytokinesis makes it a potential target for therapeutic intervention in appropriately selected cancers.
Asunto(s)
Citocinesis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Anafase/fisiología , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Centrosoma/metabolismo , Células HCT116 , Células HT29 , Humanos , Cinetocoros/metabolismo , Células MCF-7 , Mitosis/fisiología , Huso Acromático/metabolismoRESUMEN
The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti-PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.
Asunto(s)
Antígeno B7-H1/metabolismo , Inmunosupresores/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Animales , Antígeno B7-H1/genética , Neoplasias de la Mama/metabolismo , Butionina Sulfoximina/farmacología , Línea Celular Tumoral , Quimiocinas , Quimioterapia , Femenino , Glutatión/metabolismo , Humanos , Ratones , Paclitaxel/farmacología , Fenotipo , ARN Mensajero/metabolismo , Neoplasias de la Mama Triple Negativas , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.