ABSTRACT
Thrombin cleavages of selective proteinase-activated receptors (PAR) as well as PAR-activating peptide ligands can initiate the phosphoinositide 3-kinase (PI3K) signaling cascade in platelets. Downstream to this event, fibrinogen receptors on platelets undergo conformational changes that enhance fibrinogen binding. In our study, we used this phenomenon as a surrogate biomarker for assessing effects on PI3K activity. Our method, using flow cytometric measurement of fluorescent ligand and antibody binding, uncovered a 16- to 45-fold signal window after PAR-induced platelet activation. Pretreatment (in vitro) with the PI3K inhibitors wortmannin and LY294002 resulted in concentration-dependent inhibition at predicted potencies. In addition, platelets taken from mice treated with wortmannin were blocked from PAR-induced ex vivo activation concomitantly with a decrease in phosphorylation of AKT from excised tumor xenografts. This surrogate biomarker assay was successfully tested (in vitro) on blood specimens received from volunteer cancer patients. Our results indicate that measurement of platelet activation could serve as an effective drug activity biomarker during clinical evaluation of putative PI3K inhibitors.
Subject(s)
Androstadienes/pharmacology , Blood Platelets/drug effects , Chromones/pharmacology , Enzyme Inhibitors/pharmacology , Morpholines/pharmacology , Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Platelet Activation/drug effects , Animals , Blood Platelets/enzymology , Female , Flow Cytometry , Humans , Mice , Mice, Nude , Neoplasms/enzymology , Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Receptor, PAR-1/metabolism , Signal Transduction , Transplantation, Heterologous , WortmanninABSTRACT
Although AKT1 (v-akt murine thymoma viral oncogene homologue 1) kinase is a central member of possibly the most frequently activated proliferation and survival pathway in cancer, mutation of AKT1 has not been widely reported. Here we report the identification of a somatic mutation in human breast, colorectal and ovarian cancers that results in a glutamic acid to lysine substitution at amino acid 17 (E17K) in the lipid-binding pocket of AKT1. Lys 17 alters the electrostatic interactions of the pocket and forms new hydrogen bonds with a phosphoinositide ligand. This mutation activates AKT1 by means of pathological localization to the plasma membrane, stimulates downstream signalling, transforms cells and induces leukaemia in mice. This mechanism indicates a direct role of AKT1 in human cancer, and adds to the known genetic alterations that promote oncogenesis through the phosphatidylinositol-3-OH kinase/AKT pathway. Furthermore, the E17K substitution decreases the sensitivity to an allosteric kinase inhibitor, so this mutation may have important clinical utility for AKT drug development.
Subject(s)
Blood Proteins/chemistry , Cell Transformation, Neoplastic/genetics , Mutation/genetics , Neoplasms/genetics , Phosphoproteins/chemistry , Proto-Oncogene Proteins c-akt/chemistry , Proto-Oncogene Proteins c-akt/genetics , Sequence Homology, Amino Acid , Animals , Breast Neoplasms/genetics , Colorectal Neoplasms/genetics , DNA Mutational Analysis , Enzyme Activation/genetics , Female , Humans , Leukemia/genetics , Mice , Models, Molecular , Neoplasms/pathology , Ovarian Neoplasms/genetics , Protein Structure, Tertiary/genetics , Protein Transport , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
With human volunteers inoculated at two sites with Haemophilus ducreyi, outcomes for a subject were not independent. In a reinfection trial, 2 of 11 previous pustule formers and 6 of 10 previous resolvers resolved all sites of infection. There was no correlation between serum bactericidal or phagocytic activity and outcome in the trial. These data indicate that different hosts are differentially susceptible to disease progression versus resolution in the model.
