RESUMEN
Tumorigenesis depends on intricate interactions between genetically altered tumor cells and their surrounding microenvironment. While oncogenic drivers in lung squamous carcinoma (LUSC) have been described, the role of stroma in modulating tissue architecture, particularly cell polarity, remains unclear. Here, we report the establishment of a 3D coculture system of LUSC epithelial cells with cancer-associated fibroblasts (CAFs) and extracellular matrix that together capture key components of the tumor microenvironment (TME). Single LUSC epithelial cells develop into acinar-like structures with 0.02% efficiency, and addition of CAFs provides proper tumor-stromal interactions within an appropriate 3D architectural context. Using this model, we recapitulate key pathological changes during tumorigenesis, from hyperplasia to dysplasia and eventually invasion, in malignant LUSC spheroids that undergo phenotypic switching in response to cell intrinsic and extrinsic changes. Overexpression of SOX2 is sufficient to mediate the transition from hyperplasia to dysplasia in LUSC spheroids, while the presence of CAFs makes them invasive. Unexpectedly, CAFs suppress the activity of high SOX2 levels, restore hyperplasia, and enhance the formation of acinar-like structures. Taken together, these observations suggest that stromal factors can override cell intrinsic oncogenic changes in determining the disease phenotype, thus providing fundamental evidence for the existence of dynamic reciprocity between the nucleus and the TME of LUSC.
Asunto(s)
Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Factores de Transcripción SOXB1/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Escamosas/genética , Polaridad Celular , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Humanos , Hiperplasia , Neoplasias Pulmonares/genética , Modelos Biológicos , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Factores de Transcripción SOXB1/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Microambiente Tumoral/genética , Regulación hacia ArribaRESUMEN
The purinergic receptor P2Y(13) has been shown to play a role in the uptake of holo-HDL particles in in vitro hepatocyte experiments. In order to determine the role of P2Y(13) in lipoprotein metabolism in vivo, we ablated the expression of this gene in mice. Here we show that P2Y(13) knockout mice have lower fecal concentrations of neutral sterols (-27%±2.1% in males) as well as small decreases in plasma HDL (-13.1%±3.2% in males; -17.5%±4.0% in females) levels. In addition, significant decreases were detected in serum levels of fatty acids and glycerol in female P2Y(13) knockout mice. Hepatic mRNA profiling analyses showed increased expression of SREBP-regulated cholesterol and fatty acid biosynthesis genes, while fatty acid ß-oxidation genes were significantly decreased. Liver gene signatures also identified changes in PPARα-regulated transcript levels. With the exception of a small increase in bone area, P2Y(13) knockout mice do not show any additional major abnormalities, and display normal body weight, fat mass and lean body mass. No changes in insulin sensitivity and oral glucose tolerance could be detected. Taken together, our experiments assess a role for the purinergic receptor P2Y(13) in the regulation of lipoprotein metabolism and demonstrate that modulating its activity could be of benefit to the treatment of dyslipidemia in people.
Asunto(s)
Lipoproteínas/metabolismo , Receptores Purinérgicos P2/fisiología , Animales , Femenino , Perfilación de la Expresión Génica , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , ARN Mensajero/genética , Receptores Purinérgicos P2/genéticaRESUMEN
Leukotrienes (LTs) are signaling molecules derived from arachidonic acid that initiate and amplify innate and adaptive immunity. In turn, how their synthesis is organized on the nuclear envelope of myeloid cells in response to extracellular signals is not understood. We define the supramolecular architecture of LT synthesis by identifying the activation-dependent assembly of novel multiprotein complexes on the outer and inner nuclear membranes of mast cells. These complexes are centered on the integral membrane protein 5-Lipoxygenase-Activating Protein, which we identify as a scaffold protein for 5-Lipoxygenase, the initial enzyme of LT synthesis. We also identify these complexes in mouse neutrophils isolated from inflamed joints. Our studies reveal the macromolecular organization of LT synthesis.
Asunto(s)
Araquidonato 5-Lipooxigenasa/metabolismo , Proteínas Portadoras/metabolismo , Leucotrienos/biosíntesis , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/análisis , Membrana Nuclear/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Animales , Artritis/enzimología , Artritis/metabolismo , Proteínas de la Membrana/análisis , Ratones , Células Mieloides/química , Células Mieloides/metabolismo , Neutrófilos/química , Neutrófilos/metabolismo , Membrana Nuclear/químicaRESUMEN
Aberrant NOTCH3 signaling and overexpression is oncogenic, associated with cancer stem cells and drug resistance, yet therapeutic targeting remains elusive. Here, we develop NOTCH3-targeted antibody drug conjugates (NOTCH3-ADCs) by bioconjugation of an auristatin microtubule inhibitor through a protease cleavable linker to two antibodies with differential abilities to inhibit signaling. The signaling inhibitory antibody rapidly induces ligand-independent receptor clustering and internalization through both caveolin and clathrin-mediated pathways. The non-inhibitory antibody also efficiently endocytoses via clathrin without inducing receptor clustering but with slower lysosomal co-localization kinetics. In addition, DLL4 ligand binding to the NOTCH3 receptor mediates transendocytosis of NOTCH3-ADCs into ligand-expressing cells. NOTCH3-ADCs internalize into receptor and ligand cells independent of signaling and induce cell death in both cell types representing an atypical mechanism of ADC cytotoxicity. Treatment of xenografts with NOTCH3-ADCs leads to sustained tumor regressions, outperforms standard-of-care chemotherapy, and allows targeting of tumors that overexpress NOTCH3 independent of signaling inhibition.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Inmunoconjugados/farmacología , Receptor Notch3/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Humanos , Inmunoconjugados/metabolismo , Oncogenes/efectos de los fármacos , Receptor Notch3/inmunología , Receptores Notch/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The nuclear membrane protein 5-lipoxygenase-activating protein (FLAP) plays an essential role in leukotriene synthesis. Recombinant full-length human FLAP with a C-terminal hexahistidine tag has been expressed and purified from the cytoplasmic membrane of Escherichia coli. Diffraction-quality crystals of FLAP in complex with leukotriene-synthesis inhibitor MK-591 and with an iodinated analogue of MK-591 have been grown using the sitting-drop vapor-diffusion method. The crystals exhibit tetragonal symmetry (P42(1)2) and diffracted to a resolution limit of 4 A.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Expresión Génica , Leucotrienos/biosíntesis , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Activadoras de la 5-Lipooxigenasa , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Estructura MolecularRESUMEN
Leukotrienes are proinflammatory products of arachidonic acid oxidation by 5-lipoxygenase that have been shown to be involved in respiratory and cardiovascular diseases. The integral membrane protein FLAP is essential for leukotriene biosynthesis. We describe the x-ray crystal structures of human FLAP in complex with two leukotriene biosynthesis inhibitors at 4.0 and 4.2 angstrom resolution, respectively. The structures show that inhibitors bind in membrane-embedded pockets of FLAP, which suggests how these inhibitors prevent arachidonic acid from binding to FLAP and subsequently being transferred to 5-lipoxygenase, thereby preventing leukotriene biosynthesis. This structural information provides a platform for the development of therapeutics for respiratory and cardiovascular diseases.
Asunto(s)
Proteínas Portadoras/química , Indoles/química , Proteínas de la Membrana/química , Quinolinas/química , Proteínas Activadoras de la 5-Lipooxigenasa , Araquidonato 5-Lipooxigenasa/metabolismo , Ácido Araquidónico/metabolismo , Sitios de Unión , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Citosol/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Indoles/metabolismo , Indoles/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Mutagénesis , Membrana Nuclear/química , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Quinolinas/metabolismo , Quinolinas/farmacologíaRESUMEN
Chemistry was developed to synthesize the title series of compounds. The ability of these novel ligands to bind to the glucocorticoid receptor was investigated. These compounds were also tested in a series of functional assays and some were found to display the profile of a dissociated glucocorticoid. The SAR of the 6,5-bicyclic series differed markedly from the previously reported 6,6-series. Molecular modeling studies were employed to understand the conformational differences between the two series of compounds, which may explain their divergent activity. Two compounds were profiled in vivo and shown to reduce inflammation in a mouse model. An active metabolite is suspected in one case.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Bicíclicos con Puentes/química , Glucocorticoides/química , Pirazoles/química , Receptores de Glucocorticoides/efectos de los fármacos , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Humanos , Ligandos , Ratones , Modelos Químicos , Modelos Moleculares , Receptores de Glucocorticoides/metabolismo , Relación Estructura-ActividadRESUMEN
Current immunoassays for the measurement of leukotriene B(4) (LTB(4)) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB(4) antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB(4) ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647-LTB(4), the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z=0.8; S/N=250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<0.10 dollars/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB(4) giving 50% inhibition (IC(50)) was ca. 55pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB(4) and 12-epi LTB(4). Measurements of LTB(4) determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.
Asunto(s)
Fluorometría , Inmunoensayo , Leucotrieno B4/análisis , Humanos , Inmunoensayo/métodos , Leucotrieno B4/sangre , Neutrófilos/metabolismoRESUMEN
A series of novel ligands for the glucocorticoid receptor containing two heterocycles were synthesized. These compounds were investigated for a dissociative profile using transrepression and transactivation assays. Several compounds were tested in vivo and showed the ability to reduce inflammation in a mouse.
Asunto(s)
Glucocorticoides/química , Compuestos Heterocíclicos/química , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/química , Antiinflamatorios/farmacología , Línea Celular , Glucocorticoides/administración & dosificación , Glucocorticoides/farmacología , Compuestos Heterocíclicos/administración & dosificación , Compuestos Heterocíclicos/farmacología , Humanos , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratas , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
A novel series of selective ligands for the human glucocorticoid receptor is described. Structure-activity studies focused on variation of B-ring size, ketal ring size, and ketal substitution. These analogs were found to be potent and selective ligands for GR and have partial agonist profiles in functional assays for transactivation (TAT, GS) and transrepression (IL-6). Of these compounds, 27, 28, and 35 were evaluated further in a mouse LPS-induced TNF-alpha secretion model. Compound 28 had an ED(50) of 14.1 mg/kg compared with 0.5 mg/kg for prednisolone in the same assay.
Asunto(s)
Receptores de Glucocorticoides/metabolismo , Animales , Células Cultivadas , Humanos , Técnicas In Vitro , Ligandos , RatonesRESUMEN
Cell signaling leading to the formation of leukotriene (LT)C(4) requires the localization of the four key biosynthetic enzymes on the outer nuclear membrane and endoplasmic reticulum. Whether any macromolecular organization of these proteins exists is unknown. By using fluorescence lifetime imaging microscopy and biochemical analysis, we demonstrate the presence of two distinct multimeric complexes that regulate the formation of LTs in RBL-2H3 cells. One complex consists of multimers of LTC(4) synthase and the 5-lipoxygenase activating protein (FLAP). The second complex consists of multimers of FLAP. Surprisingly, all LTC(4) synthase was found to be in association with FLAP. The results indicate that the formation of LTC(4) and LTB(4) may be determined by the compartmentalization of biosynthetic enzymes in discrete molecular complexes.