RESUMEN
NOTCH1 gain-of-function mutations are recurrent in B-cell chronic lymphocytic leukemia (B-CLL), where they are associated with accelerated disease progression and refractoriness to chemotherapy. The specific role of NOTCH1 in the development and progression of this malignancy is unclear. Here, we assess the impact of loss of Notch signaling and pathway hyperactivation in an in vivo mouse model of CLL (IgH.TEµ) that faithfully replicates many features of the human pathology. Ablation of canonical Notch signaling using conditional gene inactivation of RBP-J in immature hematopoietic or B-cell progenitors delayed CLL induction and reduced incidence of mice developing disease. In contrast, forced expression of a dominant active form of Notch resulted in more animals developing CLL with early disease onset. Comparative analysis of gene expression and epigenetic features of Notch gain-of-function and control CLL cells revealed direct and indirect regulation of cell cycle-associated genes, which led to increased proliferation of Notch gain-of-function CLL cells in vivo. These results demonstrate that Notch signaling facilitates disease initiation and promotes CLL cell proliferation and disease progression.
Asunto(s)
Regulación Leucémica de la Expresión Génica , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Animales , Leucemia Linfocítica Crónica de Células B/genética , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Receptor Notch1/genéticaRESUMEN
Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.
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Receptores Notch/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Activación Transcripcional/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Drosophila , Resistencia a Antineoplásicos/efectos de los fármacos , Células HeLa , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/química , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Intestino Delgado/efectos de los fármacos , Intestino Delgado/metabolismo , Ratones , Mutación , Fenotipo , Multimerización de Proteína , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéuticoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1003161.].
RESUMEN
UNLABELLED: Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE: Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.
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Antirretrovirales/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Adulto , Estudios de Cohortes , ADN Viral/análisis , ADN Viral/genética , Femenino , Infecciones por VIH/inmunología , Humanos , Masculino , Persona de Mediana Edad , Factores de Tiempo , Resultado del TratamientoRESUMEN
HIV-1 infects CD4+ T cells and completes its replication cycle in approximately 24 hours. We employed repeated measurements in a standardized cell system and rigorous mathematical modeling to characterize the emergence of the viral replication intermediates and their impact on the cellular transcriptional response with high temporal resolution. We observed 7,991 (73%) of the 10,958 expressed genes to be modulated in concordance with key steps of viral replication. Fifty-two percent of the overall variability in the host transcriptome was explained by linear regression on the viral life cycle. This profound perturbation of cellular physiology was investigated in the light of several regulatory mechanisms, including transcription factors, miRNAs, host-pathogen interaction, and proviral integration. Key features were validated in primary CD4+ T cells, and with viral constructs using alternative entry strategies. We propose a model of early massive cellular shutdown and progressive upregulation of the cellular machinery to complete the viral life cycle.
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Linfocitos T CD4-Positivos/fisiología , Regulación Viral de la Expresión Génica , VIH-1/fisiología , Replicación Viral/genética , Linfocitos T CD4-Positivos/virología , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Modelos Estadísticos , Factores de Tiempo , Transcriptoma , Regulación hacia ArribaRESUMEN
Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.
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Células Dendríticas/metabolismo , Células Dendríticas/virología , Gangliósidos/metabolismo , Infecciones por VIH/inmunología , VIH-1/fisiología , Membrana Dobles de Lípidos/metabolismo , Lectina 1 Similar a Ig de Unión al Ácido Siálico/metabolismo , Células Dendríticas/efectos de los fármacos , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Silenciador del Gen/efectos de los fármacos , Células HEK293 , Infecciones por VIH/patología , Infecciones por VIH/virología , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Lipopolisacáridos/farmacología , Liposomas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Virión/efectos de los fármacos , Virión/metabolismoRESUMEN
UNLABELLED: The liver is characterized by sexually dimorphic gene expression translating into sex-specific differences in lipid, drug, steroid hormone, and xenobiotic metabolism, with distinct responses of males and females to environmental challenges. Here, we investigated the role of the Krüppel-associated box (KRAB)-associated protein 1 (KAP1) epigenetic regulator in this process. Liver-specific KAP1 knockout (KO) led to strikingly sexually dimorphic phenotypic disturbances, including male-predominant steatosis and hepatic tumors with up-regulation of protein kinase B and extracellular signal-related kinases 1/2 mitogen-activated protein kinase signaling. This correlated with the sex-specific transcriptional dysregulation of a wide range of metabolic genes, notably those involved in retinol and sex hormone processing as well as in detoxification. Furthermore, chromatin immunoprecipitation followed by deep sequencing indicated that a number of dysregulated genes are direct targets of the KRAB/KAP1 repression system. Those genes include sexually dimorphic cytochrome P 450 Cyp2d9, glutathione S-transferase π, Cyp2a, Cyp2b, and Cyp3a gene clusters. Additionally, we identified a male-restricted KAP1-binding site in the fat-specific protein 27 gene, correlating with its male-predominant up-regulation upon Kap1 deletion, suggesting that the latter might be an important trigger in the development of male-specific hepatosteatosis and secondary tumorigenesis. CONCLUSION: This work reveals KRAB/KAP1-mediated transcriptional regulation as a central event in metabolic control hormones, drugs, and xenobiotics in the liver and further links disturbances in these processes with hepatic carcinogenesis.
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Adenoma/genética , Transformación Celular Neoplásica/genética , Hígado Graso/genética , Predisposición Genética a la Enfermedad , Neoplasias Hepáticas/genética , Proteínas Nucleares/metabolismo , Proteínas Represoras/metabolismo , Adenoma/patología , Animales , Biopsia con Aguja , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Hígado Graso/patología , Femenino , Regulación de la Expresión Génica , Inmunohistoquímica , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución Aleatoria , Sensibilidad y Especificidad , Factores Sexuales , Proteína 28 que Contiene Motivos Tripartito , Dedos de Zinc/genéticaRESUMEN
Krüppel-associated box domain-zinc finger proteins (KRAB-ZFPs) are tetrapod-specific transcriptional repressors encoded in the hundreds by the human genome. In order to explore their as yet ill-defined impact on gene expression, we developed an ectopic repressor assay, allowing the study of KRAB-mediated transcriptional regulation at hundreds of different transcriptional units. By targeting a drug-controllable KRAB-containing repressor to gene-trapping lentiviral vectors, we demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases (kb) away from their DNA binding sites, with an efficiency which is generally higher for promoters located within 15 kb or less. Silenced promoters exhibit a loss of histone H3-acetylation, an increase in H3 lysine 9 trimethylation (H3K9me3), and a drop in RNA Pol II recruitment, consistent with a block of transcriptional initiation following the establishment of silencing marks. Furthermore, we reveal that KRAB-mediated repression is established by the long-range spreading of H3K9me3 and heterochromatin protein 1 beta (HP1beta) between the repressor binding site and the promoter. We confirm the biological relevance of this phenomenon by documenting KAP1-dependent transcriptional repression at an endogenous KRAB-ZFP gene cluster, where KAP1 binds to the 3' end of genes and mediates propagation of H3K9me3 and HP1beta towards their 5' end. Together, our data support a model in which KRAB/KAP1 recruitment induces long-range repression through the spread of heterochromatin. This finding not only suggests auto-regulatory mechanisms in the control of KRAB-ZFP gene clusters, but also provides important cues for interpreting future genome-wide DNA binding data of KRAB-ZFPs and KAP1.
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Heterocromatina/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Dedos de Zinc , Acetilación , Emparejamiento Base , Sitios de Unión , Línea Celular , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Silenciador del Gen , Histonas/metabolismo , Humanos , Metilación , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , Proteína 28 que Contiene Motivos TripartitoRESUMEN
BACKGROUND: KRAB-ZFPs (Krüppel-associated box domain-zinc finger proteins) are vertebrate-restricted transcriptional repressors encoded in the hundreds by the mouse and human genomes. They act via an essential cofactor, KAP1, which recruits effectors responsible for the formation of facultative heterochromatin. We have recently shown that KRAB/KAP1 can mediate long-range transcriptional repression through heterochromatin spreading, but also demonstrated that this process is at times countered by endogenous influences. METHOD: To investigate this issue further we used an ectopic KRAB-based repressor. This system allowed us to tether KRAB/KAP1 to hundreds of euchromatic sites within genes, and to record its impact on gene expression. We then correlated this KRAB/KAP1-mediated transcriptional effect to pre-existing genomic and chromatin structures to identify specific characteristics making a gene susceptible to repression. RESULTS: We found that genes that were susceptible to KRAB/KAP1-mediated silencing carried higher levels of repressive histone marks both at the promoter and over the transcribed region than genes that were insensitive. In parallel, we found a high enrichment in euchromatic marks within both the close and more distant environment of these genes. CONCLUSION: Together, these data indicate that high levels of gene activity in the genomic environment and the pre-deposition of repressive histone marks within a gene increase its susceptibility to KRAB/KAP1-mediated repression.
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Silenciador del Gen , Genómica , Proteínas Represoras/metabolismo , Transcripción Genética/genética , Cromatina/genética , Células HeLa , Histonas/genética , Humanos , Proteína 28 que Contiene Motivos TripartitoRESUMEN
Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and antiangiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras G12D/+;p53 -/- NSCLC, including a mismatch repair-deficient variant (Kras G12D/+;p53 -/-;Msh2 -/-) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, antiangiogenic therapy, and chemotherapy. Antiangiogenic blockade of vascular endothelial growth factor A and angiopoietin-2 markedly slowed progression of autochthonous lung tumors, but contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that antiangiogenic treatment facilitated tumor infiltration by PD-1+ regulatory T cells (Tregs), which were more efficiently targeted by the PD-1 antibody than CD8+ T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R) dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-ß-rich tumor microenvironment that supported PD-1+ Tregs Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated Tregs, redirected the PD-1 antibody to CD8+ T cells, and improved the efficacy of antiangiogenic immunotherapy, achieving regression of most tumors.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Antígeno B7-H1 , Linfocitos T CD8-positivos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral , Factor A de Crecimiento Endotelial VascularRESUMEN
Neutrophils are the most abundant circulating leucocytes and are essential for innate immunity. In cancer, pro- or antitumor properties have been attributed to tumor-associated neutrophils (TAN). Here, focusing on TAN accumulation within lung tumors, we identify GLUT1 as an essential glucose transporter for their tumor supportive behavior. Compared with normal neutrophils, GLUT1 and glucose metabolism increased in TANs from a mouse model of lung adenocarcinoma. To elucidate the impact of glucose uptake on TANs, we used a strategy with two recombinases, dissociating tumor initiation from neutrophil-specific Glut1 deletion. Loss of GLUT1 accelerated neutrophil turnover in tumors and reduced a subset of TANs expressing SiglecF. In the absence of GLUT1 expression by TANs, tumor growth was diminished and the efficacy of radiotherapy was augmented. Our results demonstrate the importance of GLUT1 in TANs, which may affect their pro- versus antitumor behavior. These results also suggest targeting metabolic vulnerabilities to favor antitumor neutrophils. SIGNIFICANCE: Lung tumor support and radiotherapy resistance depend on GLUT1-mediated glucose uptake in tumor-associated neutrophils, indicating that metabolic vulnerabilities should be considered to target both tumor cells as well as innate immune cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2345/F1.large.jpg.
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Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/radioterapia , Proliferación Celular/genética , Transportador de Glucosa de Tipo 1/deficiencia , Transportador de Glucosa de Tipo 1/metabolismo , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/radioterapia , Neutrófilos/inmunología , Insuficiencia del Tratamiento , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Estudios de Casos y Controles , Línea Celular Tumoral , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Transportador de Glucosa de Tipo 1/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
High T-cell infiltration in colorectal cancer (CRC) correlates with a favorable disease outcome and immunotherapy response. This, however, is only observed in a small subset of CRC patients. A better understanding of the factors influencing tumor T-cell responses in CRC could inspire novel therapeutic approaches to achieve broader immunotherapy responsiveness. Here, we investigated T cell-suppressive properties of different myeloid cell types in an inducible colon tumor mouse model. The most potent inhibitors of T-cell activity were tumor-infiltrating neutrophils. Gene expression analysis and combined in vitro and in vivo tests indicated that T-cell suppression is mediated by neutrophil-secreted metalloproteinase activation of latent TGFß. CRC patient neutrophils similarly suppressed T cells via TGFß in vitro, and public gene expression datasets suggested that T-cell activity is lowest in CRCs with combined neutrophil infiltration and TGFß activation. Thus, the interaction of neutrophils with a TGFß-rich tumor microenvironment may represent a conserved immunosuppressive mechanism in CRC.
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Neoplasias del Colon , Linfocitos Infiltrantes de Tumor/inmunología , Metaloproteinasas de la Matriz/metabolismo , Neutrófilos , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Neoplasias del Colon/inmunología , Humanos , Ratones , Neutrófilos/inmunología , Microambiente TumoralRESUMEN
Cancers induced by human papillomaviruses (HPV) should be responsive to immunotherapy by virtue of expressing the immunogenic oncoproteins E6/E7. However, advanced forms of cervical cancer, driven by HPV, are poorly responsive to immune response-enhancing treatments involving therapeutic vaccination against these viral neoantigens. Leveraging a transgenic mouse model of HPV-derived cancers, K14HPV16/H2b, we demonstrated that a potent nanoparticle-based E7 vaccine, but not a conventional "liquid" vaccine, induced E7 tumor antigen-specific CD8+ T cells in cervical tumor-bearing mice. Vaccination alone or in combination with anti-PD-1/anti-CTLA4 did not elicit tumor regression nor increase CD8+ T cells in the tumor microenvironment (TME), suggesting the presence of immune-suppressive barriers. Patients with cervical cancer have poor dendritic cell functions, have weak cytotoxic lymphocyte responses, and demonstrate an accumulation of myeloid cells in the periphery. Here, we illustrated that myeloid cells in K14HPV16/H2b mice possess potent immunosuppressive activity toward antigen-presenting cells and CD8+ T cells, dampening antitumor immunity. These immune-inhibitory effects inhibited synergistic effects of combining our oncoprotein vaccine with immune checkpoint-blocking antibodies. Our data highlighted a link between HPV-induced cancers, systemic amplification of myeloid cells, and the detrimental effects of myeloid cells on CD8+ T-cell activation and recruitment into the TME. These results established immunosuppressive myeloid cells in lymphoid organs as an HPV+ cancer-induced means of circumventing tumor immunity that will require targeted abrogation to enable the induction of efficacious antitumor immune responses.
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Antineoplásicos Inmunológicos/farmacología , Linfocitos T CD8-positivos/inmunología , Células Mieloides/inmunología , Papillomaviridae/inmunología , Infecciones por Papillomavirus/inmunología , Vacunas contra Papillomavirus/inmunología , Neoplasias del Cuello Uterino/inmunología , Animales , Antígeno CTLA-4/antagonistas & inhibidores , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Terapia de Inmunosupresión , Inmunoterapia/métodos , Ratones , Células Mieloides/efectos de los fármacos , Nanopartículas/administración & dosificación , Proteínas E7 de Papillomavirus/inmunología , Proteínas E7 de Papillomavirus/metabolismo , Infecciones por Papillomavirus/tratamiento farmacológico , Infecciones por Papillomavirus/virología , Vacunas contra Papillomavirus/administración & dosificación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Microambiente Tumoral , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virologíaRESUMEN
Glucose utilization increases in tumors, a metabolic process that is observed clinically by 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). However, is increased glucose uptake important for tumor cells, and which transporters are implicated in vivo? In a genetically-engineered mouse model of lung adenocarcinoma, we show that the deletion of only one highly expressed glucose transporter, Glut1 or Glut3, in cancer cells does not impair tumor growth, whereas their combined loss diminishes tumor development. 18F-FDG-PET analyses of tumors demonstrate that Glut1 and Glut3 loss decreases glucose uptake, which is mainly dependent on Glut1. Using 13C-glucose tracing with correlated nanoscale secondary ion mass spectrometry (NanoSIMS) and electron microscopy, we also report the presence of lamellar body-like organelles in tumor cells accumulating glucose-derived biomass, depending partially on Glut1. Our results demonstrate the requirement for two glucose transporters in lung adenocarcinoma, the dual blockade of which could reach therapeutic responses not achieved by individual targeting.
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Adenocarcinoma del Pulmón/fisiopatología , Eliminación de Gen , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 2/genética , Glucosa/metabolismo , Animales , Línea Celular Tumoral , Femenino , Fluorodesoxiglucosa F18/química , Transportador de Glucosa de Tipo 1/metabolismo , Transportador de Glucosa de Tipo 2/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Tomografía de Emisión de Positrones , Espectrometría de Masa de Ion SecundarioRESUMEN
Herein, we report that the TGFß superfamily receptor ALK7 is a suppressor of tumorigenesis and metastasis, as revealed by functional studies in mouse models of pancreatic neuroendocrine and luminal breast cancer, complemented by experimental metastasis assays. Activation in neoplastic cells of the ALK7 signaling pathway by its principal ligand activin B induces apoptosis. During tumorigenesis, cancer cells use two different approaches to evade this barrier, either downregulating activin B and/or downregulating ALK7. Suppressing ALK7 expression additionally contributes to the capability for metastatic seeding. ALK7 is associated with shorter relapse-free survival of various human cancers and distant-metastasis-free survival of breast cancer patients. This study introduces mechanistic insights into primary and metastatic tumor development, in the form of a protective barrier that triggers apoptosis in cells that are not "authorized" to proliferate within a particular tissue, by virtue of those cells expressing ALK7 in a tissue microenvironment bathed in its ligand.
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Receptores de Activinas Tipo I/metabolismo , Activinas/metabolismo , Neoplasias/metabolismo , Animales , Apoptosis/fisiología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinogénesis , Línea Celular Tumoral , Transformación Celular Neoplásica/metabolismo , Femenino , Xenoinjertos , Homeostasis , Humanos , Masculino , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Ratones SCID , Metástasis de la Neoplasia , Neoplasias/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Transducción de Señal , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Microambiente TumoralRESUMEN
Calcineurin/NFAT signaling is active in endothelial cells and is proposed to be an essential component of the tumor angiogenic response. Here, we investigated the role of endothelial calcineurin signaling in vivo in physiological and pathological angiogenesis and tumor metastasis. We show that this pathway is dispensable for retinal and tumor angiogenesis, but it is implicated in vessel stabilization. While ablation of endothelial calcineurin does not affect the progression of primary tumors or tumor cell extravasation, it does potentiate the outgrowth of lung metastases. We identify Bmp2 as a downstream target of the calcineurin/NFAT pathway in lung endothelium, potently inhibiting cancer cell growth by stimulating differentiation. We reveal a dual role of calcineurin/NFAT signaling in vascular regression or stabilization and in the tissue-specific production of an angiocrine factor restraining cancer cell outgrowth. Our results suggest that, besides targeting the immune system, post-transplantation immunosuppressive therapy with calcineurin inhibitors directly targets the endothelium, contributing to aggressive cancer progression.
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Proteína Morfogenética Ósea 2/metabolismo , Calcineurina/metabolismo , Células Endoteliales/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Pulmonares/secundario , Melanoma/patología , Ratones , Metástasis de la Neoplasia , Micrometástasis de Neoplasia , Neovascularización Patológica/metabolismo , Vasos Retinianos/metabolismoRESUMEN
The epithelial-mesenchymal transition-inducing transcription factor Snail contributes to tumor progression in different malignancies. In the present study, we used a transcriptomics approach to elucidate the mechanism of Snail-mediated tumor growth promotion in a KrasLSL-G12D/+;p53fl/fl mouse model of lung adenocarcinoma. We discovered that Snail mediated the downregulation of the imprinted Dlk1-Dio3 locus, a complex genomic region containing protein-coding genes and non-coding RNAs that has been linked to tumor malignancy in lung cancer patients. The Dlk1-Dio3 locus repression mediated by Snail was found to occur specifically in several populations of tumor-infiltrating immune cells. It could be reproduced in primary splenocytes upon ex vivo culture with conditioned medium from Snail-expressing cancer cell lines, which suggests that a Snail-induced soluble factor secreted by the cancer cells mediates the Dlk1-Dio3 locus repression in immune cells, particularly in lymphocytes. Our findings furthermore point towards the contribution of Snail to an inflammatory tumor microenvironment, which is in line with our previous report of the Snail-mediated recruitment of pro-tumorigenic neutrophils to the lung tumors. This underlines an important role for Snail in influencing the immune compartment of lung tumors and thus contributing to disease progression.
RESUMEN
INTRODUCTION: NSCLC is the leading cause of cancer mortality. Recent retrospective clinical analyses suggest that blocking the receptor activator of NF-κB (RANK) signaling pathway inhibits the growth of NSCLC and might represent a new treatment strategy. METHODS: Receptor activator of NF-κB gene (RANK) and receptor activator of NF-κB ligand gene (RANKL) expression in human lung adenocarcinoma was interrogated from publicly available gene expression data sets. Several genetically engineered mouse models were used to evaluate treatment efficacy of RANK-Fc to block RANKL, with primary tumor growth measured longitudinally using microcomputed tomography. A combination of RANKL blockade with cisplatin was tested to mirror an ongoing clinical trial. RESULTS: In human lung adenocarcinoma data sets, RANKL expression was associated with decreased survival and KRAS mutation, with the highest levels in tumors with co-occurring KRAS and liver kinase B1 gene (LKB1) mutations. In KrasLSL-G12D/WT, KrasLSL-G12D/WT; Lkb1Flox/Flox and KrasLSL-G12D/WT; p53Flox/Flox mouse models of lung adenocarcinoma, we monitored an impaired progression of tumors upon RANKL blockade. Despite elevated expression of RANKL and RANK in immune cells, treatment response was not associated with major changes in the tumor immune microenvironment. Combined RANK-Fc with cisplatin revealed increased efficacy compared with that of single agents in p53- but not in Lkb1-deficient tumors. CONCLUSIONS: RANKL blocking agents impair the growth of primary lung tumors in several mouse models of lung adenocarcinoma and suggest that patients with KRAS-mutant lung tumors will benefit from such treatments.
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Adenocarcinoma del Pulmón/genética , Ingeniería Genética/métodos , Neoplasias Pulmonares/genética , Ligando RANK/genética , Adenocarcinoma del Pulmón/patología , Animales , Modelos Animales de Enfermedad , Neoplasias Pulmonares/patología , Ratones , Estudios Retrospectivos , Transducción de SeñalRESUMEN
The metabolic health benefits of fermented milks have already been investigated using clinical biomarkers but the development of transcriptomic analytics in blood offers an alternative approach that may help to sensitively characterise such effects. We aimed to assess the effects of probiotic yoghurt intake, compared to non-fermented, acidified milk intake, on clinical biomarkers and gene expression in peripheral blood. To this end, a randomised, crossover study was conducted in fourteen healthy, young men to test the two dairy products. For a subset of seven subjects, RNA sequencing was used to measure gene expression in blood collected during postprandial tests and after two weeks daily intake. We found that the postprandial response in insulin was different for probiotic yoghurt as compared to that of acidified milk. Moreover changes in several clinical biomarkers were associated with changes in the expression of genes representing six metabolic genesets. Assessment of the postprandial effects of each dairy product on gene expression by geneset enrichment analysis revealed significant, similar modulation of inflammatory and glycolytic genes after both probiotic yoghurt and acidified milk intake, although distinct kinetic characteristics of the modulation differentiated the dairy products. The aryl hydrocarbon receptor was a major contributor to the down-regulation of the inflammatory genesets and was also positively associated with changes in circulating insulin at 2h after yoghurt intake (p = 0.05). Daily intake of the dairy products showed little effect on the fasting blood transcriptome. Probiotic yoghurt and acidified milk appear to affect similar gene pathways during the postprandial phase but differences in the timing and the extent of this modulation may lead to different physiological consequences. The functional relevance of these differences in gene expression is supported by their associations with circulating biomarkers.
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Leche , Probióticos , Transcriptoma/genética , Yogur , Adulto , Animales , Apetito , Biomarcadores/sangre , Estudios Cruzados , Productos Lácteos Cultivados , Método Doble Ciego , Perfilación de la Expresión Génica , Marcadores Genéticos , Humanos , Masculino , Periodo Posprandial/genética , ARN/sangre , ARN/genética , Adulto JovenRESUMEN
Loss of epithelial differentiation and extracellular matrix (ECM) remodeling are known to facilitate cancer progression and are associated with poor prognosis in patients with lung cancer. We have identified Receptor-interacting serine/threonine protein kinase 4 (RIP4) as a regulator of tumor differentiation in lung adenocarcinoma (AC). Bioinformatics analyses of human lung AC samples showed that poorly differentiated tumors express low levels of RIP4, whereas high levels are associated with better overall survival. In vitro, lung tumor cells expressing reduced RIP4 levels showed enhanced activation of STAT3 signaling and had a greater ability to invade through collagen. In contrast, overexpression of RIP4 inhibited STAT3 activation, which abrogated interleukin-6-dependent induction of lysyl oxidase, a collagen cross-linking enzyme. In an autochthonous mouse model of lung AC initiated by Kras(G12D) expression with loss of p53, Rip4 knockdown tumors progressed to a poorly differentiated state marked by an increase in Hmga2, reduced Ttf1, and enrichment of genes regulating extracellular remodeling and Jak-Stat signaling. Tail vein injections of cells overexpressing Rip4 showed a reduced potential to invade and form tumors, which was restored by co-expression of Stat3. Altogether, our work has identified that loss of RIP4 enhances STAT3 signaling in lung cancer cells, promoting the expression of ECM remodeling genes and cancer dedifferentiation.