Novel aspects of the molecular biology of gastrointestinal stromal tumors

Abstract: The gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in the gastrointestinal tract (GI). Historically, these tumors were commonly mistaken for myogenic and neurogenic masses, and then eventually came to be recognized as a distinct type of soft-tissue sarcoma through ultrastructural findings and specific immunomarkers. GISTs can arise anywhere along the GI tract, and are believed to originate from or share a common progenitor with the interstitial cell of Cajal. The majority of GISTs carry activating KIT or PDGFRA mutations, which form the molecular basis for the successful tyrosine kinase inhibitor therapy. Although genetic discoveries and treatment advances have greatly improved clinical outcomes, the significance of GIST neuroendocrine phenotype, the role of the relatively newly identified DOG1, and the impact of regional imatinib pharmacodynamics remain obscure. The aim of the overall thesis was to explore functional aspects of the human GIST biology. Evaluation of the presence of functional GIST cell stimulus-secretion coupling demonstrated an intact intracellular Ca2+-signaling pathway and an active ATP release that is dependent on [Ca2+]e levels and is augmentable by pharmacological stimuli. (Paper I) The existence and composition of a putative GIST secretome was assessed by shotgun proteomics. The findings demonstrate that GIST cells contain a secretome signature made up of classically and non-classically released proteins. The protein subsets and appurtenant functional clustering varied in the presence of drug stimulation. The types of released proteins, which significantly increased through cell stimulation, were consistent with the types of proteins found in other cancers. Moreover, the secretome overlapped extensively with exosomal proteins.(Paper II) A protocol to measure intracellular imatinib levels was developed for use in both in vitro and in vivo systems of GIST cells. The liquid-liquid extraction LC-MS TOF-based protocol offered a reliable way to determine intracellular imatinib levels with high recovery, good linearity, and low limit of detection, in both the experimental and clinical settings. The imatinib uptake differed between imatinib-sensitive and imatinib-resistant cell lines, and accumulated in tumors from three patients, with large intra- and inter-tumoral variations. (Paper III) The functional significance of DOG1 in GIST cells was addressed. DOG1 have different subcellular localizations in imatinib-sensitive and imatinib-resistant GIST cells. Specific inhibitors or activators modulated the DOG1 activity efficaciously. The overall effect on GIST cell viability and proliferation was small, but DOG1 inhibition induced late apoptosis among a small proportion of early apoptotic imatinib-resistant GIST cells. (Paper IV)