Abstract
Background
Toll-like receptor 3 (TLR3) ligand which activates TLR3 signaling
induces both cancer cell death and activates anti-tumor immunity. However, TLR3
signaling can also harbor pro-tumorigenic consequences. Therefore, we examined
the status of TLR3 in cholangiocarcinoma (CCA) cases to better understand TLR3
signaling and explore the potential therapeutic target in CCA.
Methods
The expression of TLR3 and receptor-interacting protein kinase 1
(RIPK1) in primary CCA tissues was assayed by Immunohistochemical staining and
their associations with clinicopathological characteristics and survival data
were evaluated. The effects of TLR3 ligand, Poly(I:C) and Smac mimetic, an IAP
antagonist on CCA cell death and invasion were determined by cell death
detection methods and Transwell invasion assay, respectively. Both genetic and
pharmacological inhibition of RIPK1, RIPK3 and MLKL and inhibitors targeting
NF-κB and MAPK signaling were used to investigate the underlying
mechanisms.
Results
TLR3 was significantly higher expressed in tumor than adjacent
normal tissues. We demonstrated in a panel of CCA cell lines that TLR3 was
frequently expressed in CCA cell lines, but was not detected in a nontumor
cholangiocyte. Subsequent in vitro study demonstrated that Poly(I:C)
specifically induced CCA cell death, but only when cIAPs were removed by Smac
mimetic. Cell death was also switched from apoptosis to necroptosis when
caspases were inhibited in CCA cells-expressing RIPK3. In addition, RIPK1 was
required for Poly(I:C) and Smac mimetic-induced apoptosis and necroptosis. Of
particular interest, high TLR3 or low RIPK1 status in CCA patients was
associated with more invasiveness. In vitro invasion demonstrated that
Poly(I:C)-induced invasion through NF-κB and MAPK signaling. Furthermore, the
loss 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 toward
longer disease-free survival (p = 0.078,
28.0 months and 10.9 months).
Conclusion
RIPK1 plays a pivotal role in TLR3 ligand, Poly(I:C)-induced cell
death when cIAPs activity was inhibited and loss of RIPK1 enhanced
Poly(I:C)-induced invasion which was partially reversed by Smac mimetic. Our
results suggested that TLR3 ligand in combination with Smac mimetic could
provide therapeutic benefits to the patients with CCA.
Graphical abstract