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Oxidative Stress Plays a Critical Role in Inactivating Mutant BRAF by Geldanamycin Derivatives

¹ Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri; ² Department of Paracitology, Kurume University Medical School, Kurume, Japan; ³ Department of Molecular Cellular Oncology and Microbiology, Tokyo Medical and Dental University, Tokyo, Japan; ⁴ Department of Endocrinology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; and ⁵ Department of Radiation Oncology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee

Requests for reprints: Nobuo Horikoshi, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park Boulevard, St. Louis, MO 63108. Phone: 314-362-9774; Fax: 314-362-9790; E-mail: nhorikoshi{at}radonc.wustl.edu.

Key Words: BRAF • MAP kinase • geldanamycin • HSP90 • ROS

The geldanamycin derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) are promising chemotherapeutic drugs that inhibit heat shock protein 90 (HSP90) function. Previous studies have shown that 17-AAG/DMAG treatment induces the degradation of mutant BRAF (V600E) and inhibits the activation of mitogen-activated protein/extracellular signal-regulated kinase 1/2 (MEK1/2). We have found, however, that HSP90 inhibition alone is not sufficient for efficient BRAF(V600E) degradation in some cells. HSP90 inhibitors structurally unrelated to geldanamycin, radicicol and novobiocin, while inducing the degradation of the HSP90 client protein RAF-1 fail to induce BRAF(V600E) degradation or inhibit MEK1/2 activation in HT29 human colon cancer cells. Moreover, after treatment with 17-DMAG, the kinase activity of residual, undegraded BRAF(V600E) was also lost. Incubation of cells with a reactive oxygen species (ROS) scavenger, N-acetyl cysteine, partially restored kinase activity and also partially prevented BRAF(V600E) degradation due to 17-DMAG treatment. Conversely, treatment with the ROS producing drug menadione clearly inhibited MEK1/2 and reduced BRAF(V600E). These results suggest that in addition to direct inhibition of HSP90, the antitumor effect of geldanamycin and its derivatives is also mediated though the production of ROS, which may directly inactivate tumorigenic mutant BRAF(V600E). [Cancer Res 2008;68(15):6324–30]