RESUMEN
Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector ß-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/ß-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/ß-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/ß-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects.
Asunto(s)
Válvulas Cardíacas/embriología , Válvulas Cardíacas/metabolismo , Organogénesis , Vía de Señalización Wnt , Animales , Proteína Axina/metabolismo , Tipificación del Cuerpo/efectos de los fármacos , Tipificación del Cuerpo/genética , Proliferación Celular/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Cojinetes Endocárdicos/citología , Cojinetes Endocárdicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Transición Epitelial-Mesenquimal/genética , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Válvulas Cardíacas/efectos de los fármacos , Ratones Transgénicos , Válvula Mitral/efectos de los fármacos , Válvula Mitral/embriología , Válvula Mitral/metabolismo , Miocardio/metabolismo , Organogénesis/efectos de los fármacos , Organogénesis/genética , Vía de Señalización Wnt/efectos de los fármacosRESUMEN
Activating mutations in the KRAS oncogene occur in approximately 90% of pancreatic cancers, resulting in aberrant activation of the MAPK and the PI3K pathways, driving malignant progression. Significant efforts to develop targeted inhibitors of nodes within these pathways are underway and several are currently in clinical trials for patients with KRAS-mutant tumors, including patients with pancreatic cancer. To model MEK and PI3K inhibition in late-stage pancreatic cancer, we conducted preclinical trials with a mutant Kras-driven genetically engineered mouse model that faithfully recapitulates human pancreatic ductal adenocarcinoma development. Treatment of advanced disease with either a MEK (GDC-0973) or PI3K inhibitor (GDC-0941) alone showed modest tumor growth inhibition and did not significantly enhance overall survival. However, combination of the two agents resulted in a significant survival advantage as compared with control tumor-bearing mice. To model the clinical scenario, we also evaluated the combination of these targeted agents with gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer. The addition of MEK or PI3K inhibition to gemcitabine, or the triple combination regimen, incrementally enhanced overall survival as compared with gemcitabine alone. These results are reminiscent of the survival advantage conferred in this model and in patients by the combination of gemcitabine and erlotinib, an approved therapeutic regimen for advanced nonresectable pancreatic cancer. Taken together, these data indicate that inhibition of MEK and PI3K alone or in combination with chemotherapy do not confer a dramatic improvement as compared with currently available therapies for patients with pancreatic cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Azetidinas/administración & dosificación , Azetidinas/farmacología , Carcinoma Ductal Pancreático/genética , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Indazoles/administración & dosificación , Indazoles/farmacología , Ratones , Modelos Biológicos , Mutación , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Piperidinas/administración & dosificación , Piperidinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Nivel de Atención , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Transcriptional profiling is a powerful approach for understanding development and disease. Current cell type-specific RNA purification methods have limitations, including cell dissociation trauma or inability to identify all RNA species. Here, we describe "mouse thiouracil (TU) tagging," a genetic and chemical intersectional method for covalent labeling and purification of cell type-specific RNA in vivo. Cre-induced expression of uracil phosphoribosyltransferase (UPRT) provides spatial specificity; injection of 4-thiouracil (4TU) provides temporal specificity. Only UPRT(+) cells exposed to 4TU produce thio-RNA, which is then purified for RNA sequencing (RNA-seq). This method can purify transcripts from spatially complex and rare (<5%) cells, such as Tie2:Cre(+) brain endothelia/microglia (76% validated by expression pattern), or temporally dynamic transcripts, such as those acutely induced by lipopolysaccharide (LPS) injection. Moreover, generating chimeric mice via UPRT(+) bone marrow transplants identifies immune versus niche spleen RNA. TU tagging provides a novel method for identifying actively transcribed genes in specific cells at specific times within intact mice.
Asunto(s)
Biología Molecular/métodos , ARN/aislamiento & purificación , Coloración y Etiquetado/métodos , Tiouracilo/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Encéfalo/embriología , Encéfalo/metabolismo , Quimera , Perfilación de la Expresión Génica , Ratones , Transgenes/genéticaRESUMEN
The low rate of approval of novel anti-cancer agents underscores the need for better preclinical models of therapeutic response as neither xenografts nor early-generation genetically engineered mouse models (GEMMs) reliably predict human clinical outcomes. Whereas recent, sporadic GEMMs emulate many aspects of their human disease counterpart more closely, their ability to predict clinical therapeutic responses has never been tested systematically. We evaluated the utility of two state-of-the-art, mutant Kras-driven GEMMs--one of non-small-cell lung carcinoma and another of pancreatic adenocarcinoma--by assessing responses to existing standard-of-care chemotherapeutics, and subsequently in combination with EGFR and VEGF inhibitors. Standard clinical endpoints were modeled to evaluate efficacy, including overall survival and progression-free survival using noninvasive imaging modalities. Comparisons with corresponding clinical trials indicate that these GEMMs model human responses well, and lay the foundation for the use of validated GEMMs in predicting outcome and interrogating mechanisms of therapeutic response and resistance.
Asunto(s)
Modelos Animales de Enfermedad , Ingeniería Genética , Mutación/genética , Neoplasias/genética , Neoplasias/terapia , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapéutico , Clorhidrato de Erlotinib , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Ratones , Quinazolinas/uso terapéutico , Análisis de Supervivencia , Factor A de Crecimiento Endotelial Vascular/inmunología , GemcitabinaRESUMEN
[reaction: see text] 1-deoxy-D-xylulose-5-phosphate (DXP) reductoisomerase is a NADPH-dependent enzyme catalyzing the conversion of DXP to methyl-D-erythritol 4-phosphate (MEP). In this study, each of the hydroxyl groups in DXP and one of its C-1 hydrogen atoms, were separately replaced with a fluorine atom and the effect of the substitution on the catalytic turnover was examined. It was found that the 1-fluoro-DXP is a poor substrate, while both 3- and 4-fluoro-DXP behave as noncompetitive inhibitors.