RESUMEN
Toll-like receptors (TLRs) are key players in the innate immune system. Recent studies have suggested that they may affect the growth of pancreatic cancer, a disease with no cure. Among them, TLR7 shows promise for therapy but may also promotes tumor growth. Thus, we aimed to clarify the therapeutic potential of TLR7 ligands in experimental pancreatic cancer models, to open the door for clinical applications. In vitro, we found that TLR7 ligands strongly inhibit the proliferation of both human and murine pancreatic cancer cells, compared with TLR2 agonists. Hence, TLR7 treatment alters cancer cells' cell cycle and induces cell death by apoptosis. In vivo, TLR7 agonist therapy significantly delays the growth of murine pancreatic tumors engrafted in immunodeficient mice. Remarkably, TLR7 ligands administration instead increases tumor growth and accelerates animal death when tumors are engrafted in immunocompetent models. Further investigations revealed that TLR7 agonists modulate the intratumoral content and phenotype of macrophages and that depleting such tumor-associated macrophages strongly hampers TLR7 agonist-induced tumor growth. Collectively, our findings shine a light on the duality of action of TLR7 agonists in experimental cancer models and call into question their use for pancreatic cancer therapy.
Asunto(s)
Neoplasias Pancreáticas , Receptor Toll-Like 7 , Animales , Humanos , Ligandos , Macrófagos/metabolismo , Glicoproteínas de Membrana , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Inherited retinal degenerations are blinding diseases characterized by the loss of photoreceptors. Their extreme genetic heterogeneity complicates treatment by gene therapy. This has motivated broader strategies for transplantation of healthy retinal pigmented epithelium to protect photoreceptors independently of the gene causing the disease. The limited clinical benefit for visual function reported up to now is mainly due to dedifferentiation of the transplanted cells that undergo an epithelial-mesenchymal transition. We have studied this mechanism in vitro and revealed the role of the homeogene OTX2 in preventing dedifferentiation through the regulation of target genes. We have overexpressed OTX2 in retinal pigmented epithelial cells before their transplantation in the eye of a model of retinitis pigmentosa carrying a mutation in Mertk, a gene specifically expressed by retinal pigmented epithelial cells. OTX2 increases significantly the protection of photoreceptors as seen by histological and functional analyses. We observed that the beneficial effect of OTX2 is non-cell autonomous, and it is at least partly mediated by unidentified trophic factors. Transplantation of OTX2-genetically modified cells may be medically effective for other retinal diseases involving the retinal pigmented epithelium as age-related macular degeneration.
Asunto(s)
Células Epiteliales/metabolismo , Factores de Transcripción Otx/genética , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/trasplante , Epitelio Pigmentado de la Retina/citología , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Animales , Biomarcadores , Pollos , Transición Epitelial-Mesenquimal , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Ratas , Elementos de Respuesta , PorcinosRESUMEN
Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.
Asunto(s)
Transducción de Señal , Proteína de Unión al GTP rhoB , Proteína de Unión al GTP rhoB/genética , Proteína de Unión al GTP rhoB/química , Proteína de Unión al GTP rhoB/metabolismo , GTP Fosfohidrolasas/metabolismo , Membrana Celular/metabolismo , Guanosina Trifosfato/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) patients frequently suffer from undetected micro-metastatic disease. This clinical situation would greatly benefit from additional investigation. Therefore, we set out to identify key signalling events that drive metastatic evolution from the pancreas. We searched for a gene signature that discriminate localised PDAC from confirmed metastatic PDAC and devised a preclinical protocol using circulating cell-free DNA (cfDNA) as an early biomarker of micro-metastatic disease to validate the identification of key signalling events. An unbiased approach identified, amongst actionable markers of disease progression, the PI3K pathway and a distinctive PI3Kα activation signature as predictive of PDAC aggressiveness and prognosis. Pharmacological or tumour-restricted genetic PI3Kα-selective inhibition prevented macro-metastatic evolution by hindering tumoural cell migratory behaviour independently of genetic alterations. We found that PI3Kα inhibition altered the quantity and the species composition of the produced lipid second messenger PIP3 , with a selective decrease of C36:2 PI-3,4,5-P3 . Tumoural PI3Kα inactivation prevented the accumulation of pro-tumoural CD206-positive macrophages in the tumour-adjacent tissue. Tumour cell-intrinsic PI3Kα promotes pro-metastatic features that could be pharmacologically targeted to delay macro-metastatic evolution.
Asunto(s)
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/genética , Humanos , Macrófagos , Neoplasias Pancreáticas/genética , Fosfatidilinositol 3-Quinasas/genéticaRESUMEN
Blue light is an identified risk factor for age-related macular degeneration (AMD). The production of vascular endothelial growth factor (VEGF), leading to neovascularization, is a major complication of the wet form of this disease. We investigated how blue light affects VEGF expression and secretion using A2E-loaded retinal pigment epithelium (RPE) cells, a cell model of AMD. Incubation of RPE cells with A2E resulted in a significant increase in VEGF mRNA and, intracellular and secreted VEGF protein levels, but not mRNA levels of VEGFR1 or VEGFR2. Blue light exposure of A2E-loaded RPE cells resulted in a decrease in VEGF mRNA and protein levels, but an increase in VEGFR1 levels. The toxicity of 440 nm light on A2E-loaded RPE cells was enhanced by VEGF supplementation. Our results suggest that age-related A2E accumulation may result in VEGF synthesis and release. This synthesis of VEGF, which enhances blue light toxicity for the RPE cells, is itself suppressed by blue light. Anti-VEGF therapy may therefore improve RPE survival in AMD.
Asunto(s)
Proteínas del Ojo/metabolismo , Luz , Degeneración Macular/metabolismo , Modelos Biológicos , Epitelio Pigmentado de la Retina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Apoptosis , Supervivencia Celular , Células Cultivadas , Técnicas In Vitro , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/efectos de la radiación , PorcinosRESUMEN
AIMS: Blue light is an identified risk factor for age-related macular degeneration (AMD). We investigated oxidative stress markers and mitochondrial changes in A2E-loaded retinal pigment epithelium cells under the blue-green part of the solar spectrum that reaches the retina to better understand the mechanisms underlying light-elicited toxicity. RESULTS: Primary retinal pigment epithelium cells were loaded with a retinal photosensitizer, AE2, to mimic aging. Using a custom-made illumination device that delivers 10 nm-wide light bands, we demonstrated that A2E-loaded RPE cells generated high levels of both hydrogen peroxide (H2O2) and superoxide anion (O2â¢-) when exposed to blue-violet light. In addition, they exhibited perinuclear clustering of mitochondria with a decrease of both their mitochondrial membrane potential and their respiratory activities. The increase of oxidative stress resulted in increased levels of the oxidized form of glutathione and decreased superoxide dismutase (SOD) and catalase activities. Furthermore, mRNA expression levels of the main antioxidant enzymes (SOD2, catalase, and GPX1) also decreased. CONCLUSIONS: Using an innovative illumination device, we measured the precise action spectrum of the oxidative stress mechanisms on A2E-loaded retinal pigment epithelium cells. We defined 415-455 nm blue-violet light, within the solar spectrum reaching the retina, to be the spectral band that generates the highest amount of reactive oxygen species and produces the highest level of mitochondrial dysfunction, explaining its toxic effect. This study further highlights the need to filter these wavelengths from the eyes of AMD patients.
Asunto(s)
Luz/efectos adversos , Mitocondrias/efectos de la radiación , Estrés Oxidativo/efectos de los fármacos , Fármacos Fotosensibilizantes/efectos adversos , Epitelio Pigmentado de la Retina/efectos de la radiación , Espectro de Acción , Animales , Catalasa/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Humanos , Degeneración Macular/genética , Degeneración Macular/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Retina/metabolismo , Retina/efectos de la radiación , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Superóxido Dismutasa/metabolismo , PorcinosRESUMEN
To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.