Cooperative TRAIL production mediates IFNα/Smac mimetic-induced cell death in TNFα-resistant solid cancer cells.

Smac mimetics antagonize IAP proteins, which are highly expressed in several cancers. Recent reports indicate that Smac mimetics trigger a broad cytokine response and synergize with immune modulators to induce cell death. Here, we identify a differential requirement of TRAIL or TNF alpha as mediators of IFNa/Smac mimeticinduced cell death depending on the cellular context. Subtoxic concentrations of Smac mimetics cooperate with IFN alpha to induce cell death in various solid tumor cell lines in a highly synergistic manner as determined by combination index. Mechanistic studies show that IFN alpha/BV6 cotreatment promotes the formation of a caspase-8activating complex together with the adaptor protein FADD and RIP1. Assembly of this RIP1/FADD/caspase-8 complex represents a critical event, since RIP1 silencing inhibits IFN alpha/BV6-induced cell death. Strikingly, pharmacological inhibition of paracrine/autocrine TNF alpha signaling by the TNF alpha scavenger Enbrel rescues HT-29 colon carcinoma cells, but not A172 glioblastoma cells from IFN alpha/BV6-induced cell death. By comparison, A172 cells are significantly protected against IFN alpha/BV6 treatment by blockage of TRAIL signaling through genetic silencing of TRAIL or its cognate receptor TRAIL receptor 2 (DR5). Despite this differential requirement of TNF alpha and TRAIL signaling, mRNA and protein expression is increased by IFN alpha/BV6 cotreatment in both cell lines. Interestingly, A172 cells turn out to be resistant to exogenously added recombinant TNF alpha even in the presence of BV6, whereas they display a high sensitivity towards TRAIL/BV6. In contrast, BV6 efficiently sensitizes HT-29 cells to TNF alpha while TRAIL only had limited efficacy. This demonstrates that a differential sensitivity towards TRAIL or TNF alpha determines the dependency on either death receptor ligand for IFN alpha/Smac mimetic-induced cell death. Thus, by concomitant stimulation of both death receptor systems IFN alpha/Smac mimetic combination treatment is an effective strategy to induce cell death in TNF alpha-or TRAIL-responsive cancers.