Abstract 1824: Functional characterization of DOG1 in Gastrointestinal Stromal Tumors (GIST)

Abstract

Abstract Introduction: DOG1, a calcium-dependent chloride channel, has been shown to be strongly and specifically expressed in GIST and is commonly used as diagnostic marker. Notably, KIT-negative GIST cell lines do not express DOG1. We have therefore sought to investigate the role of DOG1 for KIT expression and GIST growth and its potential as therapeutic target. Methods: GIST cell lines (GIST882: K642E, IM-sensitive, GIST-T1: del558-614, IM-sensitive, GIST48: V560D/D820A, IM-resistant) were transduced with lentiviral particles containing DOG1 sh-RNA. Expression of KIT and DOG1 was measured by immunoblotting. Effects of DOG1 knock-down on proliferation (with/without imatinib) were studied by BrdU incorporation assays and effects on chloride currents using patch clamp technique. Xenografts from transduced cells (and scrambled control) were generated and evaluated for tumor growth and expression of DOG1, KIT and Ki-67. Expression profiles from cell lines and xenograft tumors were compared using microarrays. Results: Lentiviral knock-down of DOG1 resulted in a near-complete inhibition of DOG1 while expression of KIT or the KIT-depending signalling pathways were not affected. Patch clamp measurements showed a strong inhibition of chloride currents in the majority of cells (DOG1-knock-down) tested compared to scrambled control. No difference was found in the growth rate of cells with DOG1-knockdown alone or in the presence of imatinib compared to cells transduced with scrambled sh-RNA. Xenografts generated from transduced cell lines retained DOG-1 knockdown as well as strong KIT-expression. Notably, xenografts with knock-down of DOG1 showed a lower proliferative activity (Ki-67: immunoreactivity score 12 (strongly positive) versus 6 (moderately positive)) with a reduction of tumor size of 43% after 19 days of tumor growth (n=8; p=0.003) when compared with the scrambled control. Expression analyses revealed MNK1 (MAP kinase-interacting serine/threonine-protein kinase 1) to be down-regulated in DOG1 negative GIST xenografts compared to scrambled control. Conclusion: DOG1 knock-down results in a reduction of intracellular chloride currents but does not affect KIT-expression or sensitizes cells to imatinib treatment. Proliferation is not affected by DOG1 knock-down in vitro but DOG1 appears to contribute to tumor growth in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1824. doi:1538-7445.AM2012-1824