Smac mimetic sensitizes glioblastoma cells to Temozolomide-induced apoptosis in a RIP1-and NF-κB-dependent manner

Inhibitor of apoptosis (IAP) proteins are expressed at high levels in many cancers and therefore represent attractive targets for therapeutic intervention. Here, we report for the first time that the second mitochondria-derived activator of caspases (Smac) mimetic BV6 sensitizes glioblastoma cells toward Temozolomide (TMZ), the first-line chemotherapeutic agent in the treatment of glioblastoma. BV6 and TMZ synergistically reduce cell viability and trigger apoptosis in glioblastoma cells (combination index <0.4-0.8), which is accompanied by increased loss of mitochondrial-membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis. Analysis of the molecular mechanisms reveals that BV6 causes rapid degradation of clAP1, leading to stabilization of NF-kappa B-inducing kinase and NF-kappa B activation. BV6-stimulated NF-kappa B activation is critically required for sensitization toward TMZ, as inhibition of NF-kappa B by overexpression of the mutant l kappa B alpha super-repressor profoundly reduces loss of mitochondrial membrane potential, cytochrome c release, caspase activation and apoptosis. Of note, BV6-mediated sensitization to TMZ is not associated with increased tumor necrosis factor alpha (TNF alpha) production. Also, TNF alpha, CD95 or TRAIL-blocking antibodies or knockdown of TNFR1 have no or little effect on combination treatment-induced apoptosis. Interestingly, BV6 and TMZ cooperate to trigger the formation of a RIP1 (receptor activating protein 1)/caspase-8/FADD complex. Knockdown of RIP1 by small interfering RNA significantly reduces BV6- and TMZ-induced caspase-8 activation and apoptosis, showing that RIP1 is necessary for apoptosis induction. By demonstrating that BV6 primes glioblastoma cells for TMZ in a NF-kappa B- and RIP1-dependent manner, these findings build the rationale for further (pre)clinical development of Smac mimetics in combination with TMZ. Oncogene (2013) 32, 988-997; doi:10.1038/onc.2012.108; published online 2 April 2012