Cholangiocarcinoma (CCA) is a highly heterogeneous and metastatic malignancy with a poor prognosis even after curative hepatectomy. Studies exploring its pathogenesis and identifying effective therapeutic targets are urgently needed. In this study, we found that TANK-binding kinase 1 (TBK1), a serine/threonine-protein kinase, showed a dynamic increase during the different stages of murine spontaneous CCA carcinogenesis (hyperplasia, dysplasia, and CCA). TBK1 was upregulated in human tissues, including intrahepatic (n = 182) and extrahepatic (n = 40) CCA tissues, compared with nontumor tissues, and the elevated expression of TBK1 was positively correlated with larger tumour diameter, lymph node metastasis, and advanced TNM stage. Functional studies indicated that TBK1 promoted CCA growth and metastasis both in vitro and in vivo. TBK1 directly interacts with β-catenin, promoting its phosphorylation at the S552 site and its nuclear translocation, which further activates EMT-related transcriptional reprogramming. GSK-8612, a TBK1 inhibitor or a kinase-inactivating mutation, effectively suppresses the above processes. In addition, we found that low-density lipoprotein receptor (LDLR), which mediates the endocytosis of cholesterol, was upregulated in CCA. Therefore, we designed a cholesterol-conjugated DNA/RNA heteroduplex oligonucleotide targeting TBK1 (Cho-TBK1-HDO), which could accumulate in CCA cells via LDLR, reduce the TBK1 mRNA level and inhibit intrahepatic metastasis of CCA. Besides, in the experimental group of 182 ICC patients, high TBK1 expression combined with high nuclear β-catenin expression predicted a worse prognosis. In summary, TBK1 might serve as a potential prognostic biomarker and therapeutic target for patients with CCA.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver bile duct malignancy with limited therapeutic options. Metastasis is one of the main contributors to ICC progression and poor prognosis. However, the underlying mechanism of ICC metastasis remains largely unknown. Here, we showed that TANK-binding kinase 1 (TBK1), a serine/threonine-protein kinase, was significantly upregulated in tumor tissues of ICC patients with larger tumor diameter, lymph node metastasis, and advanced TNM stage. Consistently, during the different stages of cholangiocarcinoma (CCA) carcinogenesis (hyperplasia, dysplasia, and CCA), TBK1 showed a dynamic increase in spontaneous rat and mouse models. Functional studies showed that enforced expression of TBK1 promoted metastasis both in vitro and in vivo. Mechanistically, TBK1 directly interacts with β-catenin and stimulates its nuclear translocation, further activating the β-catenin-mediated epithelial-mesenchymal transition (EMT) process. Moreover, we demonstrate that the S172 site of TBK1 kinase domain was essential for the interaction between TBK1 and β-catenin as well as for TBK1 mediated β-catenin activation. In addition, high levels of TBK1 in clinical ICC tissues were correlated with elevated nuclear β-catenin levels and predicted worse overall and disease-free survival. A TBK1 inhibitor GSK-8612 and the liver-specific accumulation of DNA/RNA heteroduplex oligonucleotide (TBK1-HDO) significantly reduced TBK1 expression of ICC and inhibited its intrahepatic metastasis. In summary, our study demonstrated that TBK1 could activate β-catenin via protein-protein interaction, then promote EMT and ICC metastasis, which might serve as a potential therapeutic target for patients with cholangiocarcinoma.