Effect of inhibition of heat-shock protein 90 on mutant c-Kit protein as modulated by mRNA expression and protein degradation through both proteasome and autophagy-mediated pathways.

Abstract

54 Background: c-Kit is a known client protein of heat-shock protein 90 (Hsp90). Inhibition of Hsp90 can reduce the expression of c-Kit protein in human mastocytoma and gastrointestinal stromal tumor (GIST) cells, which is conceived through the proteasome degradation pathway. Methods: COS-1 cells transfected with vectors containing CMV promoter-driven various c-Kit mutants (imatinib- sensitive or imatinib-resistant) and human GIST882 cells (with endogenous exon 13 mutant c-Kit) were used to investigate the efficacy NVP-AUY922, a new class of Hsp90 inhibitor, on suppressing the constitutive activation of mutated c-Kit. Detail mechanisms of protein regulation, as RNA transcription, RNA stability, and protein degradation were studied. Results: NVP-AUY922 is more potent than 17-AAG to inhibit the proliferation of imatinib-sensitive GIST882 cells. Further study showed that NVP-AUY922 down-regulated both total and phosphorylated c-Kit proteins in dose- and time-dependent manners in both GIST882 cells and mutant c-Kit (including imatinib-resistant exon11/17 double mutants) expressed in COS-1 cells. Surprisingly, the NVP-AUY922-induced c-Kit degradation in both cell models could be reversed by proteasome-degradation inhibitor and autophagy inhibitor. The involvement of autophagy in NVP-AUY922-induced c-Kit protein degradation was further confirmed by co-localization of c-Kit and autophagosome by immuno-staining. In addition, NVP-AUY922 treatment resulted in a reduction of c-Kit mRNA level in GIST882 cells but not c-Kit expressing COS-1 cells, which could be explained by the use of CMV promoter in artificial COS-1 cell model. Conclusions: Taken together, these findings provide the first evidence that down-regulation of mutant c-Kit protein by Hsp90 inhibitor involved both transcription and post-translation levels. Of the latter, autophagy- as well as proteasome-mediated degradation pathways were involved. These observations highlight the use of NVP-AUY922 either alone or in combination with autophagy modulators in imatinib-refractory, c-Kit-expressing GIST. Supported by 99D1-TC-CADOH03. [Table: see text]