The role of autophagy in cytotoxicity induced by new oncogenic B-Raf inhibitor UI-152 in v-Ha-ras transformed fibroblasts

In human cancers, B-Raf is the most frequently mutated protein kinase in the MAPK signaling cascade, making it an important therapeutic target. We recently discovered a potent and selective B-Raf inhibitor, UI-152, by using a structure-based drug design strategy. In this study, we examined whether B-Raf inhibition by UI-152 may be an effective therapeutic strategy for eliminating Cancer cells transformed with v-Ha-ras (Ras-NIH 3T3). UI-152 displayed selective cytotoxicity toward Ras-NIH 3T3 cells while having little to no effect on non-transformed NIH 3T3 cells. We found that treatment with UI-152 markedly increased Autophagy and, to a lesser extent, Apoptosis. However, inhibition of Autophagy by addition of 3-MA failed to reverse the cytotoxic effects of UI-152 on Ras-NIH 3T3 cells, demonstrating that Apoptosis and Autophagy can act as cooperative partners to induce growth inhibition in Ras-NIH 3T3 cells treated with UI-152. Most interestingly, cell responses to UI-152 appear to be paradoxical. Here, we showed that although UI-152 inhibited ERK, it induced B-Raf binding to Raf-1 as well as Raf-1 activation. This paradoxical activation of Raf-1 by UI-152 is likely to be coupled with the inhibition of the mTOR pathway, an intracellular signaling pathway involved in Autophagy. We also showed for the first time that, in multi-drug resistant cells, the combination of UI-152 with verapamil significantly decreased cell proliferation and increased Autophagy. Thus, our findings suggest that the inhibition of Autophagy, in combination with UI-152, offers a more effective therapeutic strategy for v-Ha-ras-transformed cells harboring wild-type B-Raf.