17-AAG and 17-DMAG-induced inhibition of cell proliferation through B-Raf downregulation in WT B-Raf-expressing uveal melanoma cell lines.
OBJECTIVE
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 (V600E B-Raf) in cutaneous melanoma cell lines. It has different effects, however, on the wild-type form of B-Raf (WT B-Raf), according to the WT B-Raf activation levels in the tumor cells. Uveal melanoma cells express WT B-Raf and only rarely express V600E B-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 WT B-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 WT B-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.
Babchia
Narjes
N
Centre de Recherche des Cordeliers, Université Pierre et Marie Curie, Paris, France.
Calipel
Armelle
A
Mouriaux
Frédéric
F
Faussat
Anne-Marie
AM
Mascarelli
Frédéric
F
D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides; D016227:Benzoquinones / Q000494:pharmacology*; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D056741:Cyclin D; D016213:Cyclins / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D020868:Gene Silencing; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D006801:Humans; D000068877:Imatinib Mesylate; D047029:Lactams, Macrocyclic / Q000494:pharmacology*; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000008:administration & dosage; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D048493:Proto-Oncogene Proteins B-raf / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D011743:Pyrimidines / Q000008:administration & dosage; D034741:RNA, Small Interfering / Q000494:pharmacology; D014604:Uveal Neoplasms / Q000378:metabolism / Q000473:pathology*
D001549:Benzamides; D016227:Benzoquinones; D056741:Cyclin D; D016213:Cyclins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D010879:Piperazines; D011743:Pyrimidines; D034741:RNA, Small Interfering; C448659:17-(dimethylaminoethylamino)-17-demethoxygeldanamycin; C112765:tanespimycin; D000068877:Imatinib Mesylate; D019009:Proto-Oncogene Proteins c-kit; C482119:BRAF protein, human; D017346:Protein Serine-Threonine Kinases; D048493:Proto-Oncogene Proteins B-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3