| Abstract: | Background: Gastrointestinal stromal tumors (GISTs) mostly harbor oncogenic c-Kit mutations or, partially, with plate-let-derived growth factor receptor alpha (PDGFRA). Imatinib (Gleevec?; Novartis Pharmaceuticals Corporation, East hanover, New Jersey, USA) is a selective tyrosine kinase inhibitor (TKI) achieving a partial response or stable disease in nearly 80% of patients with metastatic GIST. However, imatinib resistance is an increasing clinical problem during chronic therapy due to secondary mutation of c-Kit, most on exons 13, 14, or 17. Several TKIs and universal inhibitors are studied to overcome imatinib-resistance. Hsp90 inhibitor was proven that it could inhibit c-Kit protein expression on several GIST cell lines, but double mutants of c-Kit that led to drug-resistance were not well-con rmed. Moreover, the mechanism of Hsp90 related c-Kit inhibition is not clari ed. Method: COS-1 cells transfected with mutated c-Kit protein and c-Kit endogenous GIST882 cells were used to validate a novel Hsp90 inhibitor, NVP-AUY922, e cacies on imatinib-sensitive and -resistant c-Kit mutants. Detail mechanisms of protein regulation, as RNA transcription, RNA stability, and protein degradation, were studied. Results: NVP-AUY922, the most potent Hsp90 inhibitor recently studied, inhibited the proliferation of imatinib-sensitive GIST882 cells and induced apoptosis. NVP-AUY922 also downregulated both total and phosphorylated c-Kit proteins in dose- and time-dependent manners in GIST882 cells. e COS-1 cells expressed c-Kit protein, acquiring single mutants on exon11 or 17 or imatinib-resistant double mutants on exon11/17, were also suppressed by NVP-AUY922. In addition, c-Kit RNA was reduced under NVP-AUY922 treatment on GIST882 cells through gene transcription, but not on the COS-1 cell model, which could be explained by the use of cytomegalovirus promoter in the arti cial COS-1 cell model. Further studies on the protein-degradation pathway indicated that proteosome and autophagy both played roles in NVP-AUY922–induced c-Kit inhibition on GIST882 and COS-1 cells. e involvement of autophagy in NVP-AUY922–induced c-Kit protein degradation was further con rmed by LC3 conversion and colocalization of c-Kit with autophagosome by immunostaining. Conclusion: Taken together, NVP-AUY922 could inhibit c-Kit expression, despite imatinib-sensitive and -resistant mutations. Our ndings rst proved that both transcription and autophagy were involved in NVP-AUY922–induced mutant c-Kit protein downregulation. ese observations highlight the use of NVP-AUY922 in imatinib-refractory, c-Kit-expressing GIST. |
