Receptor-interacting protein kinase 1 is akey mediator in TLR3 ligand and Smac mimetic-induced cell death and suppresses TLR3ligand-promoted invasion in cholangiocarcinoma

Abstract

BackgroundToll-like receptor 3 (TLR3) ligand which activates TLR3 signalinginduces both cancer cell death and activates anti-tumor immunity. However, TLR3signaling can also harbor pro-tumorigenic consequences. Therefore, we examinedthe status of TLR3 in cholangiocarcinoma (CCA) cases to better understand TLR3signaling and explore the potential therapeutic target in CCA.MethodsThe expression of TLR3 and receptor-interacting protein kinase 1(RIPK1) in primary CCA tissues was assayed by Immunohistochemical staining andtheir associations with clinicopathological characteristics and survival datawere evaluated. The effects of TLR3 ligand, Poly(I:C) and Smac mimetic, an IAPantagonist on CCA cell death and invasion were determined by cell deathdetection methods and Transwell invasion assay, respectively. Both genetic andpharmacological inhibition of RIPK1, RIPK3 and MLKL and inhibitors targetingNF-κB and MAPK signaling were used to investigate the underlyingmechanisms.ResultsTLR3 was significantly higher expressed in tumor than adjacentnormal tissues. We demonstrated in a panel of CCA cell lines that TLR3 wasfrequently expressed in CCA cell lines, but was not detected in a nontumorcholangiocyte. Subsequent in vitro study demonstrated that Poly(I:C)specifically induced CCA cell death, but only when cIAPs were removed by Smacmimetic. Cell death was also switched from apoptosis to necroptosis whencaspases were inhibited in CCA cells-expressing RIPK3. In addition, RIPK1 wasrequired for Poly(I:C) and Smac mimetic-induced apoptosis and necroptosis. Ofparticular interest, high TLR3 or low RIPK1 status in CCA patients wasassociated with more invasiveness. In vitro invasion demonstrated thatPoly(I:C)-induced invasion through NF-κB and MAPK signaling. Furthermore, theloss of RIPK1 enhanced Poly(I:C)-induced invasion and ERK activation in vitro.Smac mimetic also reversed Poly(I:C)-induced invasion, partly mediated by RIPK1.Finally, a subgroup of patients with high TLR3 and high RIPK1 had a trend towardlonger disease-free survival (p = 0.078,28.0 months and 10.9 months).ConclusionRIPK1 plays a pivotal role in TLR3 ligand, Poly(I:C)-induced celldeath when cIAPs activity was inhibited and loss of RIPK1 enhancedPoly(I:C)-induced invasion which was partially reversed by Smac mimetic. Ourresults suggested that TLR3 ligand in combination with Smac mimetic couldprovide therapeutic benefits to the patients with CCA.A6ojaa_YT1EY3titiz6eLsVideoabstractGraphical abstract