Abstract
Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.
Highlights
Kit, a cell-surface receptor for stem cell factor (SCF), belongs to the type III receptor tyrosine kinase family that includes platelet-derived growth factor receptor α/β (PDGFRα/β), Flt3, and Fms [1,2,3,4]
The proliferation of HMC-1.2, which heterozygously express KitV560G,D816V [12,13,27] (Fig 1A), was unaffected by imatinib treatment (Fig 1B, right, closed circles)
These results indicate that imatinib does not have an inhibitory effect on growth of these neoplastic mast cells
Summary
A cell-surface receptor for stem cell factor (SCF), belongs to the type III receptor tyrosine kinase family that includes platelet-derived growth factor receptor α/β (PDGFRα/β), Flt, and Fms [1,2,3,4]. It plays a pivotal role in the development of mast cells, interstitial cells of Cajal (ICC), hematopoietic cells, germ cells, and melanocytes [3,4,5]. This activates the Akt-Bad pathway and the Ras-Mek-Erk cascade, which regulate gene expression and cytoskeletal structures, resulting in cell proliferation and survival [7,8,9,10,11]
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