B-Raf inhibitors have been used for the treatment of some B-Raf–mutated cancers. They effectively inhibit B-Raf/MEK/ERK signaling in cancers harboring mutant B-Raf, but paradoxically activates MEK/ERK in Ras-mutated cancers. Death receptor 5 (DR5), a cell surface pro-apoptotic protein, triggers apoptosis upon ligation with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or aggregation. This study focused on determining the effects of B-Raf inhibition on DR5 expression and DR5 activation-induced apoptosis in Ras-mutant cancer cells. Using chemical and genetic approaches, we have demonstrated that the B-Raf inhibitor PLX4032 induces DR5 upregulation exclusively in Ras-mutant cancer cells; this effect is dependent on Ras/c-Raf/MEK/ERK signaling activation. PLX4032 induces DR5 expression at transcriptional levels, largely due to enhancing CHOP/Elk1-mediated DR5 transcription. Pre-exposure of Ras-mutated cancer cells to PLX4032 sensitizes them to TRAIL-induced apoptosis; this is also a c-Raf/MEK/ERK-dependent event. Collectively, our findings highlight a previously undiscovered effect of B-Raf inhibition on the induction of DR5 expression and the enhancement of DR5 activation-induced apoptosis in Ras-mutant cancer cells and hence may suggest a novel therapeutic strategy against Ras-mutated cancer cells by driving their death due to DR5-dependent apoptosis through B-Raf inhibition.

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The candidate proteins that are involved in the cyclin-dependent kinase 2 (cdk2) signaling pathway were analyzed by comparing different proteins between dominant negative cdk2 overexpressed and control HeLa cells using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The 2-DE and MS indicated that stathmin and its monophosphorylated form were induced in etoposide-treated HeLa cells compared to untreated cells and this effect was inhibited by overexpression of dominant negative mutant form of cdk2. Further analysis showed that serine-25 (Ser-25), which comprises the conserved target motif for phosphorylation by mitogen-activated protein kinase (MAPK), was the major phosphorylation site of monophosphorylated form of stathmin. These findings indicate that etoposide-induced expression and phosphorylation at Ser-25 of stathmin might be mediated by activation of the MAPK signaling pathway, which is mediated by the cdk2 activation during the onset of the anticancer agent induced apoptotic events in the cancer cells.