RESUMEN

The cytokine macrophage migration inhibitory factor (MIF) possesses unique tautomerase enzymatic activity, which contributes to the biological functional activity of MIF. In this study, we investigated the effects of blocking the hydrophobic active site of the tautomerase activity of MIF in the pathogenesis of lung cancer. To address this, we initially established a Lewis lung carcinoma (LLC) murine model in Mif-KO and wild-type (WT) mice and compared tumor growth in a knock-in mouse model expressing a mutant MIF lacking enzymatic activity (Mif (P1G)). Primary tumor growth was significantly attenuated in both Mif-KO and Mif (P1G) mice compared with WT mice. We subsequently undertook a structure-based, virtual screen to identify putative small molecular weight inhibitors specific for the tautomerase enzymatic active site of MIF. From primary and secondary screens, the inhibitor SCD-19 was identified, which significantly attenuated the tautomerase enzymatic activity of MIF in vitro and in biological functional screens. In the LLC murine model, SCD-19, given intraperitoneally at the time of tumor inoculation, was found to significantly reduce primary tumor volume by 90% (p < 0.001) compared with the control treatment. To better replicate the human disease scenario, SCD-19 was given when the tumor was palpable (at d 7 after tumor inoculation) and, again, treatment was found to significantly reduce tumor volume by 81% (p < 0.001) compared with the control treatment. In this report, we identify a novel inhibitor that blocks the hydrophobic pocket of MIF, which houses its specific tautomerase enzymatic activity, and demonstrate that targeting this unique active site significantly attenuates lung cancer growth in in vitro and in vivo systems.

Asunto(s)

Antineoplásicos/uso terapéutico , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Isocumarinas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Línea Celular , Dinoprostona/metabolismo , Femenino , Oxidorreductasas Intramoleculares/genética , Oxidorreductasas Intramoleculares/metabolismo , Isocumarinas/farmacología , Lipopolisacáridos , Pulmón/efectos de los fármacos , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Factores Inhibidores de la Migración de Macrófagos/genética , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Carga Tumoral/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Active immunotherapy and cancer vaccines that promote host antitumor immune responses promise to be effective and less toxic alternatives to current cytotoxic drugs for the treatment of cancer. However, the success of tumor immunotherapeutics and vaccines is dependent on identifying approaches for circumventing the immunosuppressive effects of regulatory T (Treg) cells induced by the growing tumor and by immunotherapeutic molecules, including Toll-like receptor (TLR) agonists. Here, we show that tumors secrete high concentrations of active TGF-ß1, a cytokine that can convert naive T cells into Foxp3+ Treg cells. Silencing TGF-ß1 mRNA using small interfering RNA (siRNA) in tumor cells inhibited active TGF-ß1 production in vitro and restrained their growth in vivo. Prophylactic but not therapeutic administration of TGF-ß1 siRNA reduced the growth of CT26 tumors in vivo. Furthermore, suppressing TGF-ß1 expression at the site of a tumor, using siRNA before, during and after therapeutic administration of a TLR-activated antigen-pulsed dendritic cell vaccine significantly reduced the growth of B16 melanoma in mice. The protective effect of co-administering TGF-ß1 siRNA with the DC vaccine was associated with suppression of CD25+ Foxp3+ and CD25+ IL-10+ T cells and enhancement of tumor infiltrating CD4 and CD8 T cells. Our findings suggest that transient suppression of TGF-ß1 may be a promising approach for enhancing the efficacy of tumor vaccines in humans.

Asunto(s)

Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Neoplasias Experimentales/inmunología , Interferencia de ARN , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Terapia Combinada , Células Dendríticas/metabolismo , Citometría de Flujo , Humanos , Inmunoterapia Activa/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neoplasias Experimentales/genética , Neoplasias Experimentales/terapia , Linfocitos T Reguladores/patología , Factores de Tiempo , Receptor Toll-Like 4/inmunología , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Carga Tumoral/efectos de los fármacos , Carga Tumoral/inmunología

RESUMEN

Prophylactic immunization of mice with autologous tumor-derived heat shock proteins (Hsp) generates effective anti-tumor immunity. However, this approach is ineffective when used therapeutically, partly due to the immunosuppressive effects of the growing tumor. Here we sought to overcome this problem by therapeutic vaccination with dendritic cells (DC) pulsed with Hsp70 and a COX-2 inhibitor. We found that Hsp70 induces IL-6 and IL-10 production and suppressed expression of CD40 on DC. Incubation of DC with tumor-conditioned medium attenuated Hsp70-induced expression of CD80 and induced expression of COX-2. Inhibition of COX-2 partially reversed the stimulatory effect of Hsp70 on DC IL-6 and IL-10 production and enhanced expression of CD80 and MHC classes I and II. Therapeutic administration of DC pulsed in vitro with Hsp70 in the presence of a COX-2 inhibitor significantly reduced progression of B16 tumors in mice and significantly enhanced survival. This was associated with a reduction in the frequency of IL-10-producing CD4(+) T cells and enhancement of IFN-gamma-producing CD8(+) T cells. Our findings provide a novel immunotherapeutic approach against cancer based on attenuation of COX-2-mediated immunosuppression using in vitro modulated DC.

Asunto(s)

Inhibidores de la Ciclooxigenasa 2/farmacología , Células Dendríticas/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Inmunoterapia Adoptiva/métodos , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Animales , Antígeno B7-1/biosíntesis , Antígenos CD40/antagonistas & inhibidores , Interleucina-10/biosíntesis , Interleucina-6/biosíntesis , Ratones , Análisis de Supervivencia

RESUMEN

TLR ligands are potent adjuvants and promote Th1 responses to coadministered Ags by inducing innate IL-12 production. We found that TLR ligands also promote the induction of IL-10-secreting regulatory T (Treg) cells through p38 MAPK-induced IL-10 production by dendritic cells (DC). Inhibition of p38 suppressed TLR-induced IL-10 and PGE(2) and enhanced IL-12 production in DC. Incubation of Ag-pulsed CpG-stimulated DC with a p38 inhibitor suppressed their ability to generate Treg cells, while enhancing induction of Th1 cells. In addition, inhibition of p38 enhanced the antitumor therapeutic efficacy of DC pulsed with Ag and CpG and this was associated with an enhanced frequency of IFN-gamma-secreting T cells and a reduction of Foxp3(+) Treg cells infiltrating the tumors. Furthermore, addition of a p38 inhibitor to a pertussis vaccine formulated with CpG enhanced its protective efficacy in a murine respiratory challenge model. These data demonstrate that the adjuvant activity of TLR agonists is compromised by coinduction of Treg cells, but this can be overcome by inhibiting p38 signaling in DC. Our findings suggest that p38 is an important therapeutic target and provides a mechanism to enhance the efficacy of TLR agonists as vaccine adjuvants and cancer immunotherapeutics.

Asunto(s)

Adyuvantes Inmunológicos/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/trasplante , Activación de Linfocitos/inmunología , Sistema de Señalización de MAP Quinasas/inmunología , Melanoma Experimental/terapia , Linfocitos T Reguladores/inmunología , Receptores Toll-Like/agonistas , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Adyuvantes Inmunológicos/administración & dosificación , Animales , Vacunas contra el Cáncer/administración & dosificación , Línea Celular Tumoral , Células Cultivadas , Células Dendríticas/enzimología , Células Dendríticas/metabolismo , Inmunoterapia Adoptiva , Ligandos , Melanoma Experimental/enzimología , Melanoma Experimental/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T Reguladores/metabolismo , Células TH1/inmunología , Receptores Toll-Like/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología

RESUMEN

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the repeat in toxin family of pore-forming toxins, which require posttranslational acylation to lyse eukaryotic cells. CyaA modulates dendritic cell (DC) and macrophage function upon stimulation with LPS. In this study, we examined the roles of acylation and enzymatic activity in the immunomodulatory and lytic effects of CyaA. The adenylate cyclase activity of CyaA was necessary for its modulatory effects on murine innate immune cells. In contrast, acylation was not essential for the immunomodulatory function of CyaA, but was required for maximal caspase-3 activation and cytotoxic activity. The wild-type acylated toxin (A-CyaA) and nonacylated CyaA (NA-CyaA), but not CyaA with an inactive adenylate cyclase domain (iAC-CyaA), enhanced TLR-ligand-induced IL-10 and inhibited IL-12, TNF-alpha, and CCL3 production by macrophages and DC. In addition, both A-CyaA and NA-CyaA, but not iAC-CyaA, enhanced surface expression of CD80 and decreased CpG-stimulated CD40 and ICAM-1 expression on immature DC. Furthermore, both A-CyaA and NA-CyaA promoted the induction of murine IgG1 Abs, Th2, and regulatory T cells against coadministered Ags in vivo, whereas iAC-CyaA had more limited adjuvant activity. In contrast, A-CyaA and iAC-CyaA induced caspase-3 activation and cell death in macrophages, but these effects were considerably reduced or absent with NA-CyaA. Our findings demonstrate that the enzymatic activity plays a critical role in the immunomodulatory effects of CyaA, whereas acylation facilitates the induction of apoptosis and cell lysis, and as such, NA-CyaA has considerable potential as a nontoxic therapeutic molecule with potent anti-inflammatory properties.

Asunto(s)

Toxina de Adenilato Ciclasa/química , Toxina de Adenilato Ciclasa/fisiología , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/fisiología , Bordetella pertussis/enzimología , Bordetella pertussis/inmunología , Inmunidad Activa , Inmunidad Innata , Acilación , Toxina de Adenilato Ciclasa/antagonistas & inhibidores , Toxina de Adenilato Ciclasa/aislamiento & purificación , Adyuvantes Inmunológicos/antagonistas & inhibidores , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Antígeno CD11b/fisiología , Caspasa 3 , Caspasas/metabolismo , Muerte Celular/inmunología , Diferenciación Celular/inmunología , Línea Celular , Células Cultivadas , AMP Cíclico/química , Citocinas/metabolismo , Pruebas Inmunológicas de Citotoxicidad , Proteínas de Unión al ADN/fisiología , Células Dendríticas/citología , Células Dendríticas/inmunología , Activación Enzimática/inmunología , Femenino , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Oligodesoxirribonucleótidos/química , Receptores de Superficie Celular/fisiología , Transducción de Señal/inmunología , Receptor Toll-Like 9

RESUMEN

Members of the caspase family of cysteine proteases have been firmly established to play key roles in signal transduction cascades that culminate in apoptosis (programmed cell death). Caspases are normally expressed as inactive precursor enzymes (zymogens) that become activated during apoptosis and proceed to dismantle the cell from within. To date, three major apoptosis-associated pathways to caspase activation have been elucidated. Certain caspases, such as caspase-1, also occupy important positions in signaling pathways associated with immune responses to microbial pathogens. In this situation, caspase activation is associated with the maturation of pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and IL-18, and not apoptosis per se. Here, we discuss the current understanding of how caspases are activated during apoptosis and inflammation and the roles these proteases play in either context.

Asunto(s)

Apoptosis/inmunología , Caspasas/inmunología , Activación Enzimática/inmunología , Transducción de Señal/inmunología , Animales , Humanos

RESUMEN

The transmembrane proto-oncogene receptor tyrosine kinase (RTK) ROS is an orphan receptor that is aberrantly expressed in neoplasms of the central nervous system. Here, we report the fusion of its carboxy-terminal kinase domain to the amino-terminal portion of a protein called FIG (Fused in Glioblastoma) in a human glioblastoma multiforme (GBM). By characterizing both FIG and ROS genes in normal and in U118MG GBM cells, we determined that an intra-chromosomal homozygous deletion of 240 kilobases on 6q21 is responsible for the formation of the FIG-ROS locus. The FIG-ROS transcript is encoded by 7 FIG exons and 9 ROS-derived exons. We also demonstrate that the FIG-ROS locus encodes for an in-frame fusion protein with a constitutively active kinase activity, suggesting that FIG-ROS may act as an oncogene. This is the first example of a fusion RTK protein that results from an intra-chromosomal deletion, and it represents the first fusion RTK protein isolated from a human astrocytoma.

Asunto(s)

Proteínas Portadoras/genética , Deleción Cromosómica , Cromosomas Humanos Par 6/genética , Glioblastoma/enzimología , Glioblastoma/genética , Proteínas de la Membrana/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Secuencia de Bases/genética , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Dominio Catalítico/genética , Chlorocebus aethiops , Mapeo Cromosómico , Proteínas de la Matriz de Golgi , Humanos , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Fusión Oncogénica/química , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/química , Sistemas de Lectura/genética , Proteínas Tirosina Quinasas Receptoras/química , Células Tumorales Cultivadas