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Down-regulation of c-FLIP Enhances Death of Cancer Cells by Smac Mimetic Compound

¹ Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and ² Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada

Requests for reprints: Robert G. Korneluk, Apoptosis Research Centre, Children's Hospital of Eastern Ontario, Research Institute, 401 Smyth Road, Ottawa, Ontario, Canada K1H 8L1. Phone: 613-738-3281; Fax: 613-738-4833; E-mail: bob{at}arc.cheo.ca.

Key Words: Cancer • Apoptosis • Smac Mimetic Compounds • TNF • TRAIL • c-IAPs • drug resistance • c-FLIP • JNK • complex II

Smac mimetic compounds (SMC) are novel small molecules being developed for cancer therapy. The mechanism of SMC-induced sensitivity in cancer cells depends on autocrine release of tumor necrosis factor (TNF); however, potential mechanisms of resistance remain unknown. Here, we investigated the molecular profile and cytotoxic responsiveness of a diverse panel of 51 cancer cell lines to combinations of a dimeric SMC (AEG40730), death ligand TNF, and tumor necrosis factor-related apoptosis-inducing ligand. Synergy was seen in combination with death receptor agonists in some cells, although single-agent activity was limited to a fewsensitive lines. Unexpectedly, the majority of cell lines resistant to combinations of SMC-AEG40730 and death ligands expressed caspase-8, FADD, RIP1, and ligand receptors necessary for apoptosis execution. Furthermore, TNF-mediated ubiquitination of RIP1 was repressed by SMC-AEG40730 treatment, leading to the formation of the proapoptosis complex II. However, in resistant cancer cells, SMC-AEG40730 repressed TNF-mediated c-jun-NH₂-kinase activation and the levels of caspase-8 inhibitor c-FLIP were persistently elevated, in contrast to SMC-responsive cancer cells. Importantly, the silencing of c-FLIP restored SMC sensitivity in previously resistant cancer cells by allowing ligand-mediated activation of caspase-8 and caspase-3 to proceed. Together, these results provide mechanistic insight into the action of SMCs, demonstrating that the deciphering of the relevant molecular signature in cancer cells leads to the prediction of cancer cell responsiveness to SMC treatment. Furthermore, a majority of resistant cancer cells were sensitized to SMC-AEG40730 and TNF by down-regulating c-FLIP, suggesting novel approaches in the use of SMCs and c-FLIP antagonists in treating cancer. [Cancer Res 2009;69(19):7729–38]