RESUMEN
Dendritic cells (DCs) link innate and adaptive immunity and use a host of innate immune and inflammatory receptors to respond to pathogens and inflammatory stimuli. Although DC maturation via canonical NF-κB signaling is critical for many of these functions, the role of noncanonical NF-κB signaling via the serine/threonine kinase NIK (NF-κB-inducing kinase) remains unclear. Because NIK-deficient mice lack secondary lymphoid organs, we generated transgenic mice with targeted NIK deletion in CD11c(+) cells. Although these mice exhibited normal lymphoid organs, they were defective in cross-priming naive CD8(+) T cells following vaccination, even in the presence of anti-CD40 or polyinosinic:polycytidylic acid to induce DC maturation. This impairment reflected two intrinsic defects observed in splenic CD8(+) DCs in vitro, namely antigen cross-presentation to CD8(+) T cells and secretion of IL-12p40, a cytokine known to promote cross-priming in vivo. In contrast, antigen presentation to CD4(+) T cells was not affected. These findings reveal that NIK, and thus probably the noncanonical NF-κB pathway, is critical to allow DCs to acquire the capacity to cross-present antigen and prime CD8 T cells after exposure to licensing stimuli, such as an agonistic anti-CD40 antibody or Toll-like receptor 3 ligand.
Asunto(s)
Antígenos CD40/metabolismo , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Presentación de Antígeno/inmunología , Antígeno CD11c/metabolismo , Eliminación de Gen , Integrasas/metabolismo , Subunidad p40 de la Interleucina-12/metabolismo , Ratones Transgénicos , Bazo/citología , Quinasa de Factor Nuclear kappa BRESUMEN
Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, â¼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.
Asunto(s)
Exoma/genética , Fenómenos Inmunogenéticos/genética , Espectrometría de Masas , Mutación , Neoplasias/genética , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Inmunidad Celular/inmunología , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Neoplasias/inmunología , Péptidos/genética , Estructura Terciaria de ProteínaRESUMEN
Cancer metastases arise in part from disseminated tumor cells originating from the primary tumor and from residual disease persisting after therapy. The identification of biomarkers on micro-metastases, disseminated tumors, and residual disease may yield novel tools for early detection and treatment of these disease states prior to their development into metastases and recurrent tumors. Here we describe the molecular profiling of disseminated tumor cells in lungs, lung metastases, and residual tumor cells in the MMTV-PyMT breast cancer model. MMTV-PyMT mice were bred with actin-GFP mice, and focal hyperplastic lesions from pubertal MMTV-PyMT;actin-GFP mice were orthotopically transplanted into FVB/n mice to track single tumor foci. Tumor-bearing mice were treated with TAC chemotherapy (docetaxel, doxorubicin, cyclophosphamide), and residual and relapsed tumor cells were sorted and profiled by mRNA microarray analysis. Data analysis revealed enrichment of the Jak/Stat pathway, Notch pathway, and epigenetic regulators in residual tumors. Stat1 was significantly up-regulated in a DNA-damage-resistant population of residual tumor cells, and a pre-existing Stat1 sub-population was identified in untreated tumors. Tumor cells from adenomas, carcinomas, lung disseminated tumor cells, and lung metastases were also sorted from MMTV-PyMT transplant mice and profiled by mRNA microarray. Whereas disseminated tumors cells appeared similar to carcinoma cells at the mRNA level, lung metastases were genotypically very different from disseminated cells and primary tumors. Lung metastases were enriched for a number of chromatin-modifying genes and stem cell-associated genes. Histone analysis of H3K4 and H3K9 suggested that lung metastases had been reprogrammed during malignant progression. These data identify novel biomarkers of residual tumor cells and disseminated tumor cells and implicate pathways that may mediate metastasis formation and tumor relapse after therapy.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Virus del Tumor Mamario del Ratón , Células Neoplásicas Circulantes/metabolismo , Animales , Biomarcadores de Tumor/genética , Perfilación de la Expresión Génica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/terapia , Masculino , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Neoplasias Mamarias Experimentales/terapia , Ratones , Ratones Transgénicos , Metástasis de la Neoplasia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Neoplasia Residual , Células Neoplásicas Circulantes/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Factores de Transcripción STAT/metabolismoRESUMEN
Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.
Asunto(s)
Benzocicloheptenos/farmacología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma/mortalidad , Carcinoma/patología , Proteínas Oncogénicas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Triazoles/farmacología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzocicloheptenos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma/tratamiento farmacológico , Femenino , Células HeLa , Humanos , Células K562 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas , Análisis de Supervivencia , Triazoles/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
PURPOSE: Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. METHODS: Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. RESULTS: R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. CONCLUSIONS: R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Microscopía Fluorescente/métodos , Norbornanos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Aurora Quinasas , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Citometría de Flujo , Células HL-60 , Células HeLa , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones Desnudos , Ratones SCID , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A focus of contemporary cancer therapeutic development is the targeting of both the transformed cell and the supporting cellular microenvironment. Cell migration is a fundamental cellular behavior required for the complex interplay between multiple cell types necessary for tumor development. We therefore developed a novel retroviral-based screening technology in primary human endothelial cells to discover genes that control cell migration. We identified the receptor tyrosine kinase Axl as a novel regulator of endothelial cell haptotactic migration towards the matrix factor vitronectin. Using small interfering RNA-mediated silencing and overexpression of wild-type or mutated receptor proteins, we show that Axl is a key regulator of multiple angiogenic behaviors including endothelial cell migration, proliferation, and tube formation in vitro. Moreover, using sustained, retrovirally delivered short hairpin RNA (shRNA) Axl knockdown, we show that Axl is necessary for in vivo angiogenesis in a mouse model. Furthermore, we show that Axl is also required for human breast carcinoma cells to form a tumor in vivo. These findings indicate that Axl regulates processes vital for both neovascularization and tumorigenesis. Disruption of Axl signaling using a small-molecule inhibitor will hence simultaneously affect both the tumor and stromal cell compartments and thus represents a unique approach for cancer therapeutic development.
Asunto(s)
Neoplasias de la Mama/enzimología , Transformación Celular Neoplásica/metabolismo , Proteínas Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/fisiología , Animales , Neoplasias de la Mama/irrigación sanguínea , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/genética , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Técnicas de Cocultivo , Células Endoteliales/citología , Células Endoteliales/enzimología , Humanos , Ratones , Ratones SCID , Neovascularización Patológica/enzimología , Neovascularización Patológica/genética , Neovascularización Fisiológica/genética , Neovascularización Fisiológica/fisiología , Proteínas Oncogénicas/antagonistas & inhibidores , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/genética , Proteínas Proto-Oncogénicas , ARN Interferente Pequeño/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/genética , Transducción de Señal , Transfección , Trasplante Heterólogo , Vitronectina/farmacología , Tirosina Quinasa del Receptor AxlRESUMEN
Early cellular events associated with tumorigenesis often include loss of cell cycle checkpoints or alteration in growth signaling pathways. Identification of novel genes involved in cellular proliferation may lead to new classes of cancer therapeutics. By screening a tetracycline-inducible cDNA library in A549 cells for genes that interfere with proliferation, we have identified a fragment of UHRF1 (ubiquitin-like protein containing PHD and RING domains 1), a nuclear RING finger protein, that acts as a dominant negative effector of cell growth. Reduction of UHRF1 levels using an UHRF1-specific shRNA decreased growth rates in several tumor cell lines. In addition, treatment of A549 cells with agents that activated different cell cycle checkpoints resulted in down-regulation of UHRF1. The primary sequence of UHRF1 contains a PHD and a RING motif, both of which are structural hallmarks of ubiquitin E3 ligases. We have confirmed using an in vitro autoubiquitination assay that UHRF1 displays RING-dependent E3 ligase activity. Overexpression of a GFP-fused UHRF1 RING mutant that lacks ligase activity sensitizes cells to treatment with various chemotherapeutics. Taken together, our results suggest a general requirement for UHRF1 in tumor cell proliferation and implicate the RING domain of UHRF1 as a functional determinant of growth regulation.