Proapoptotic signalling through Toll-like receptor-3 involves TRIF-dependent activation of caspase-8 and is under the control of inhibitor of apoptosis proteins in melanoma cells

Toll-like receptor-3 (TLR3), a member of an immune recognition receptor family, is widely expressed in tumour cells and has been shown previously to have the capacity to not only activate immune signalling pathways, but also to exert proapoptotic activity in some cells. We show here that HaCaT human keratinocytes are susceptible to apoptosis induction by the TLR3 ligand poly I:C, and use these cells as a model to analyse the apoptotic signalling pathway. Although the BH3-only protein Noxa was transcriptionally induced by poly I:C and translocated to mitochondria, RNAi experiments showed that the BH3-only proteins Noxa, Bim and Puma were individually dispensable for poly I:C-induced apoptosis. Instead, poly I:C-induced activation of caspase-8 via TLR3 and its adapter TRIF was required for apoptosis. In human melanoma cell lines poly I:C failed to induce apoptosis unless protein synthesis was blocked. Significantly, sensitisation towards poly I:C- dependent caspase-8 activation and apoptosis in melanoma cells was also achieved by the synthetic Smac mimetic/inhibitor of apoptosis protein (IAP) antagonist, LBW242, or by specific downregulation of cIAP1 by siRNA. Inactivation of caspase-8 by CrmA overexpression reduced poly I:C/LBW242-induced apoptosis. These results indicate that the proapoptotic activity of TLR3/TRIF/caspase-8 in melanoma cells is under the control of IAPs, and the use of novel Smac mimetics might be a feasible approach to target melanoma. Cell Death and Differentiation (2010) 17, 942-951; doi:10.1038/cdd.2009.190; published online 18 December 2009