HSP90 inhibitors 17-AAG and 17-DMAG target wild-type B-Raf signaling for the proliferation of human uveal melanoma cell lines
Narjes Babchia 1,
Armelle Calipel 1,
Frederic Mouriaux 2,
Anne-Marie Faussat 3,
and
Frederic Mascarelli 4*
1 UMRS 872 EQUIPE 17, INSERM, Paris, France
2 Service Universitaire d'Ophtalmologie, CHRU, Caen, France
3 UMRS 872, INSERM, Paris, France
4 UMRS 872 EQUIPE 17, INSERM, PARIS, France
* To whom correspondence should be addressed. E-mail: mascarelli{at}idf.inserm.fr.
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Abstract |
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Purpose. The HSP90 inhibitor 17-AAG has been shown to have promising results in antitumor activity through the degradation of the activated V600E mutant of B-Raf (V600EB-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (WTB-Raf), according to the WTB-Raf activation levels in the tumor cells. Uveal melanoma cells express WTB-Raf and only rarely V600EB-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 HSP90 inhibitors 17-AAG and 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. The effects of the downregulation of the co-chaperone of HSP90, cdc37 was investigated by si-RNA strategy.
Results. Inhibition of HSP90 downregulated B-Raf, decreased cell proliferation and reduced activation of MEK/ERK in uveal melanoma cell lines expressing WTB-Raf. HSP90 inhibition also reduced the expression of Akt, but inhibition of Akt had no effect on cell proliferation, ruling out a role of Akt in the 17-AAG-induced inhibition of cell proliferation. Downregulation of cdc37 did not affect the MEK/ERK signalling and cell proliferation. C-Kit was also downregulated following HSP90 inhibition. The combination of 17-DMAG with imatinib mesylate, the inhibitor of c-kit, had synergistic inhibitory effects on cell proliferation.
Conclusions. Therefore our study suggests that targeting HSP90 in tandem with c-Kit inhibition may be a promising therapeutic approach to uveal melanoma.
Key Words:
melanoma, signal transduction, heat shock protein
