Identification of DR5 as a critical, NF-κB-regulated mediator of Smac-induced apoptosis

Smac mimetic promotes apoptosis by neutralizing inhibitor of apoptosis (IAP) proteins and is considered as a promising cancer therapeutic. Although an autocrine/paracrine tumor necrosis factor-alpha (TNF alpha) loop has been implicated in Smac mimetic-induced cell death, little is yet known about additional factors that determine sensitivity to Smac mimetic. Using genome-wide gene expression analysis, we identify death receptor 5 (DR5) as a novel key mediator of Smac mimetic-induced apoptosis. Although several cell lines that are sensitive to the Smac mimetic BV6 die in a TNF alpha-dependent manner, A172 glioblastoma cells undergo BV6-induced apoptosis largely independently of TNF alpha/TNFR1, as the TNF alpha-blocking antibody Enbrel or TNFR1 knockdown provide little protection. Yet, BV6-stimulated nuclear factor-kappa B (NF-kappa B) activation is critically required for apoptosis, as inhibition of NF-kappa B by overexpression of dominant-negative I kappa B alpha superrepressor (I kappa B alpha-SR) blocks BV6-induced apoptosis. Unbiased genome-wide gene expression studies in I kappa B alpha-SR-overexpressing cells versus vector control cells reveal that BV6 increases DR5 expression in a NF-kappa B-dependent manner. Importantly, this BV6-stimulated upregulation of DR5 is critically required for apoptosis, as transient or stable knockdown of DR5 significantly inhibits BV6-triggered apoptosis. In addition, DR5 silencing attenuates formation of a RIP1/FADD/caspase-8 cytosolic cell death complex and activation of caspase-8, -3 and -9. By identifying DR5 as a critical mediator of Smac mimetic-induced apoptosis, our findings provide novel insights into the determinants that control susceptibility of cancer cells to Smac mimetic.