17-AAG and 17-DMAG-induced inhibition of cell proliferation through B-Raf downregulation in WTB-Raf -: Expressing uveal melanoma cell lines

PURPOSE. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) has been shown to have promising results in antitumor activity through the degradation of the activated V600E mutant of B-Raf (B-V600E-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (B-WT-Raf), according to the B-WT-Raf activation levels in the tumor cells. Uveal melanoma cells express B-WT-Raf and only rarely express B-V600E-Raf. This study was conducted to investigate the effects of HSP90 inhibition on uveal melanoma cell lines. METHODS. Human uveal melanoma cell lines were treated with the HSP90 inhibitors 17-AAG and 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG). Cell proliferation was assessed by MTT staining, and apoptosis was quantified by flow cytometry. Analysis of the expression of HSP90 and activation of the MEK/ERK downstream signaling of B-Raf was performed by Western blot. Effects of the downregulation of the HSP90 cochaperone, cdc37, on cell proliferation and activation of MEK/ERK was investigated by siRNA strategy. RESULTS. The inhibition of HSP90 downregulated B-Raf, decreased cell proliferation, and reduced activation of MEK/ERK in uveal melanoma cell lines expressing B-WT-Raf. HSP90 inhibition also reduced the expression of Akt, but the inhibition of Akt had no effect on cell proliferation, ruling out a role of Akt in the 17-AAG-induced inhibition of cell proliferation. The downregulation of cdc37 did not affect MEK/ERK signaling and cell proliferation, demonstrating that the cochaperone was not required for HSP90-controlled stability of B-Raf. c-Kit was also downregulated after HSP90 inhibition. The combination of 17-DMAG with imatinib mesylate, the inhibitor of c-kit, had synergistic inhibitory effects on cell proliferation in B-WT-Raf uveal melanoma cell lines. CONCLUSIONS. These results suggest that targeting HSP90 in tandem with c-Kit inhibition may be a promising therapeutic approach to uveal melanoma.