A specific Hsp90 small molecular inhibitor demonstrates anti-tumor activity in melanoma cells through V600E B-Raf protein degradation

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The molecular chaperone heat shock protein 90 complex (Hsp90) is required for the conformational maturation and stability of a variety of key signaling proteins, including the ErbB family, AKT, Raf, p53, cdk4, etc.. These Hsp90 “clients” are involved in the pathways regulating proliferation and transformation of tumor cells. Geldanamycin derivatives, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), exhibit potent anti-tumor activity against cancer cells and have entered clinical trails. Here we describe a novel small molecule compound, pyrrolidine resorcinol amide PF-4470296, a specific Hsp90 inhibitor which is able to induce Hsp90 client protein degradation and inhibit cell proliferation and transformation in human melanoma cells. Using this Hsp90 specific inhibitor, we demonstrate a differential sensitivity in B-Raf protein degradation and anti-tumor activity in human melanoma cell lines harboring WT vs. mutant B-Raf (V600E). Using total cell lysates extracted from a variety of melanoma cell lines harboring either WT or mutant B-Raf, we measured Hsp90 client protein degradation either by western blot analysis or ELISA. PF-4470296 treatment induced differential B-Raf degradation with lower IC50 values in cell lines containing mutant B-Raf than those containing WT B-Raf, while there was no such differentiation with other client protein degradation (such as AKT, Her2, and EGFR1). Cell proliferation and soft agar assays were performed to determine anti-tumor activity. PF-4470296 was able to inhibit cell proliferation and the anchorage independent growth of all melanoma cell lines tested with a correlation of the IC50 value to the B-Raf mutation status of the melanoma cell lines. These results suggest that V600E B-Raf mutation plays an important role in tumor progression in melanoma cells and the inhibition of Hsp90 activity may have therapeutic advantages in melanoma cells harboring mutant vs. WT B-Raf.