Synergistic effect of smac-mimetic and poly (I:C) on apoptosis of cholangiocarcinoma cells

Abstract

Cholangiocarcinoma (CCA), a malignancy transformed from cholangiocytes in the bile ducts is more common in Asia and has the highest incidence rate in Thailand. CCA is an aggressive malignancy which has high mortality, high recurrence rate and poor prognosis due to late diagnosis and lack of effective treatment, therefore identification of novel therapeutic targets could lead to the development of more efficient therapy. CCA is associated with chronic inflammation that could upregulate Toll-like receptor 3 (TLR3) in CCA cells. TLR3 agonist, poly(I:C) has been reported to directly induce apoptosis in selected cancers and also activates anti-tumor immunity. However, in some cancers including CCA, dysregulated NF-κB signaling which upregulates the expression of cellular inhibitor of apoptosis proteins (cIAPs) 1 and 2 has been demonstrated to confer resistance to poly(I:C)-induced apoptosis. This led us to ask the research question whether the combination treatment of TLR3 ligand, poly(I:C) and IAPs antagonist, Smac mimetic could synergistically induce apoptosis in CCA cells. Here, we showed that TLR3 expression was differentially expressed in 6 CCA cell lines but not in a non-tumor cholangiocyte cell line. However, stimulation with poly(I:C) alone had no effect on CCA cell death. We showed for the first time that targeting cIAP1 and cIAP2 degradation by Smac mimetic, SM-164 in the combination with poly(I:C) treatment synergistically and specifically induced apoptosis in two representative CCA cell lines, but not in a non-tumor cholangiocyte cell line. Mechanistically, poly(I:C) and Smac mimetic treatment activates caspase-8 and induces apoptosis through a receptor-interacting protein kinase-1 (RIPK1)-dependent manner which was confirmed by a pharmacological inhibitor of RIPK1, necrostatin-1, and a deletion of RIPK1 gene using CRISPR/Cas9 technology. In conclusion, our findings demonstrated for the first time that TLR3 ligand, poly(I:C) and Smac mimetic synergistically induced apoptosis in CCA cells that have important implications for the development of a novel therapeutic strategy which could lead to increase survival rate of CCA patients.