Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation

Abstract

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts potent cytotoxic activity against transformed keratinocytes, whereas primary keratinocytes are relatively resistant. In several cell types, inhibition of the proteasome sensitizes for TRAIL-induced apoptosis by interference with NF-κB activation. Here we describe a novel intracellular mechanism of TRAIL resistance in primary cells and how this resistance is removed by proteasome inhibitors independent of NF-κB in primary human keratinocytes. This sensitization was not mediated at the receptor-proximal level of TRAIL DISC formation or caspase 8 activation but further downstream. Activation of caspase 3 was critical, as it only occurred when mitochondrial apoptotic pathways were activated, as reflected by Smac/DIABLO, HtrA2, and cytochrome c release. Smac/DIABLO and HtrA2 are needed to release the X-linked inhibitor-of-apoptosis protein (XIAP)-mediated block of full caspase 3 maturation. XIAP can effectively block caspase 3 maturation and, intriguingly, is highly expressed in primary but not in transformed keratinocytes. Ectopic XIAP expression in transformed keratinocytes resulted in increased resistance to TRAIL. Our data suggest that breaking of this resistance via proteasome inhibitors, which are potential anticancer drugs, may sensitize certain primary cells to TRAIL-induced apoptosis and could thereby complicate the clinical applicability of a combination of TRAIL receptor agonists with proteasome inhibitors.

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Proteasome Inhibition Results in TRAIL Sensitization of Primary Keratinocytes by Removing the Resistance-Mediating Block of Effector Caspase Maturation

ACKNOWLEDGMENTS

We thank P. H. Krammer for monoclonal antibodies to caspase 8 (C-15) and cFLIP (NF-6), D. Nicholson for caspase 3 and XIAP antisera as well as biotin-DEVD-aomk, M. MacFarlane for rabbit serum to Smac/DIABLO, X. Wang for rabbit serum to Bid, R. Takahashi for rabbit serum to HtrA2/Omi, S. Roy for the protocol to perform affinity labeling experiments, and C. S. Duckett for the XIAP expression vector. We are also grateful to Evi Horn for excellent technical assistance and Heiko Stahl for generating monoclonal antibodies to TRAIL-R1 to -R4.

Part of this study was funded by grants from the IZKF Würzburg (01 K5 9603), Sander-Stiftung (2000.092.1), and Deutsche Krebshilfe (10-1951-Le1) to Martin Leverkus. Henning Walczak is supported by a BioFuture grant from the Bundesministerium für Bildung und Forschung (BMBF). Manfred Neumann and Bernd Baumann were supported by grants from the Deutsche Forschungsgemeinschaft (NE 608/1-3 and/2-2 to M.N. and SFB 497/B1 to B.B.).

The first two authors contributed equally to this paper.

The last two authors share senior authorship of this paper.