ABSTRACT
KIT or alpha-platelet-derived growth factor receptor (alpha-PDGFR) activating mutations are the pathogenic mechanisms that characterize gastrointestinal stromal tumors (GIST). Despite excellent responses to imatinib mesylate (IM), patients are relapsing. We developed an IM-resistant GIST cell line (GIST-R) from the IM-sensitive GIST882 cell line (GIST-S) by growing these cells in IM. Gene expression profiling (GEP) of GIST-S, GIST-R cells and two IM resistant GIST patients demonstrated that KIT is downregulated implying a major role in IM resistance. Instead, GIST-R cells have acquired IM resistance by overexpressing the oncogenic receptor tyrosine kinase - AXL - in a 'kinase switch'. Further, the two IM resistant GIST patients express AXL and not c-Kit, seen by immunohistochemistry (IHC). Real time reverse transcriptase-polymerase chain reaction and Western blotting of the GIST-S and GIST-R cells confirmed the switch from Kit to AXL. In GIST-R, AXL is tyrosine phosphorylated and its ligand growth-arrest-specific gene 6 is overexpressed implying autocrine activation. The kinase switch is associated with a morphological change from spindle to epithelioid. Molecular modeling of the kinase domain of mutant c-Kit (V654A) and AXL showed no binding to IM but efficient binding to MP470, a novel c-Kit/AXL kinase inhibitor. MP470 synergizes with docetaxel (taxotere) and is cytotoxic to GIST cells.
Subject(s)
Drug Resistance, Neoplasm/genetics , Gastrointestinal Stromal Tumors/genetics , Oncogene Proteins/genetics , Piperazines/pharmacology , Proto-Oncogene Proteins c-kit/genetics , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/genetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Benzamides , Blotting, Western , Cell Line, Tumor , Cell Shape/drug effects , Cell Survival/drug effects , Docetaxel , Drug Synergism , Gastrointestinal Stromal Tumors/drug therapy , Gastrointestinal Stromal Tumors/pathology , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Imatinib Mesylate , Models, Molecular , Mutation , Oligonucleotide Array Sequence Analysis , Oncogene Proteins/chemistry , Oncogene Proteins/metabolism , Phosphorylation/drug effects , Piperazines/therapeutic use , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Proto-Oncogene Proteins , Proto-Oncogene Proteins c-kit/chemistry , Proto-Oncogene Proteins c-kit/metabolism , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Taxoids/pharmacology , Thiourea , Axl Receptor Tyrosine KinaseABSTRACT
During metamorphosis in the hawkmoth, Manduca sexta, identified larval leg motoneurons survive the degeneration of their larval targets to innervate new muscles of the adult legs. The dendrites and axon terminals of these motoneurons regress at the end of the larval stage and then regrow during adult development. Previous studies have implicated the insect steroid, 20-hydroxyecdysone (20-HE), in similar examples of dendritic reorganization during metamorphosis. The present studies were undertaken to test whether 20-HE acts directly on the leg motoneurons to regulate dendritic growth. Larval leg motoneurons were labeled with a fluorescent dye to permit their identification in culture following the dissociation of thoracic ganglia at later stages of development. Leg motoneurons isolated from early pupal stage animals (just before the normal onset of dendritic regrowth) survived in vitro and grew processes regardless of whether 20-HE was added to the culture medium. The extent of process outgrowth, however, as measured by the total length of all processes and the number of branches, was significantly greater for motoneurons maintained in the presence of 20-HE. The enhancement could be blocked by the addition of a juvenile hormone analog. By contrast, larval leg motoneurons that were isolated just before the normal period of dendritic regression did not show enhanced growth of neurites in the presence of 20-HE. The results suggest that 20-HE acts directly on the leg motoneurons to regulate the growth of processes during metamorphosis.
