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1.
Development ; 143(6): 1041-54, 2016 Mar 15.
Article in English | MEDLINE | ID: mdl-26893350

ABSTRACT

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.


Subject(s)
Heart Valves/embryology , Heart Valves/metabolism , Organogenesis , Wnt Signaling Pathway , Animals , Axin Protein/metabolism , Body Patterning/drug effects , Body Patterning/genetics , Cell Proliferation/drug effects , Embryonic Development/drug effects , Endocardial Cushions/cytology , Endocardial Cushions/drug effects , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation, Developmental/drug effects , Heart Valves/drug effects , Mice, Transgenic , Mitral Valve/drug effects , Mitral Valve/embryology , Mitral Valve/metabolism , Myocardium/metabolism , Organogenesis/drug effects , Organogenesis/genetics , Wnt Signaling Pathway/drug effects
2.
Mol Cancer Ther ; 14(1): 40-7, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25376606

ABSTRACT

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.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Carcinoma, Pancreatic Ductal/drug therapy , Deoxycytidine/analogs & derivatives , MAP Kinase Kinase 1/antagonists & inhibitors , Pancreatic Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/administration & dosage , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Azetidines/administration & dosage , Azetidines/pharmacology , Carcinoma, Pancreatic Ductal/genetics , Cell Line, Tumor , Deoxycytidine/administration & dosage , Deoxycytidine/pharmacology , Dose-Response Relationship, Drug , Humans , Indazoles/administration & dosage , Indazoles/pharmacology , Mice , Models, Biological , Mutation , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Piperidines/administration & dosage , Piperidines/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Standard of Care , Sulfonamides/administration & dosage , Sulfonamides/pharmacology , Xenograft Model Antitumor Assays , Gemcitabine
3.
Genes Dev ; 27(1): 98-115, 2013 Jan 01.
Article in English | MEDLINE | ID: mdl-23307870

ABSTRACT

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.


Subject(s)
Molecular Biology/methods , RNA/isolation & purification , Staining and Labeling/methods , Thiouracil/metabolism , Animals , Bone Marrow Cells/metabolism , Bone Marrow Transplantation , Brain/embryology , Brain/metabolism , Chimera , Gene Expression Profiling , Mice , Transgenes/genetics
4.
Nat Biotechnol ; 28(6): 585-93, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20495549

ABSTRACT

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.


Subject(s)
Disease Models, Animal , Genetic Engineering , Mutation/genetics , Neoplasms/genetics , Neoplasms/therapy , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/pathology , Deoxycytidine/analogs & derivatives , Deoxycytidine/therapeutic use , Erlotinib Hydrochloride , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Mice , Quinazolines/therapeutic use , Survival Analysis , Vascular Endothelial Growth Factor A/immunology , Gemcitabine
5.
Org Lett ; 6(20): 3625-8, 2004 Sep 30.
Article in English | MEDLINE | ID: mdl-15387564

ABSTRACT

[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.


Subject(s)
Aldose-Ketose Isomerases/metabolism , Hydrocarbons, Fluorinated/chemical synthesis , Multienzyme Complexes/metabolism , Oxidoreductases/metabolism , Substrate Specificity/physiology , Catalysis , Hydrocarbons, Fluorinated/metabolism
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