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.

胆管癌 (CCA) 是一种高度异质性和转移性恶性肿瘤，即使在根治性肝切除术后预后也很差。迫切需要探索其发病机制和确定有效治疗靶点的研究。在这项研究中，我们发现 TANK 结合激酶 1 (TBK1) 是一种丝氨酸/苏氨酸蛋白激酶，在小鼠自发性 CCA 癌变（增生、发育不良和 CCA）的不同阶段表现出动态增加。TBK1 在人体组织中上调，包括肝内 ( n  = 182) 和肝外 ( n = 40) CCA 组织，与非肿瘤组织相比，TBK1 的表达升高与较大的肿瘤直径、淋巴结转移和晚期 TNM 分期呈正相关。功能研究表明 TBK1 在体外和体内促进 CCA 生长和转移。TBK1 直接与 β-catenin 相互作用，促进其在 S552 位点的磷酸化及其核转位，从而进一步激活 EMT 相关的转录重编程。GSK-8612 是一种 TBK1 抑制剂或激酶失活突变体，可有效抑制上述过程。此外，我们发现介导胆固醇内吞作用的低密度脂蛋白受体 (LDLR) 在 CCA 中上调。因此，我们设计了一种靶向 TBK1 (Cho-TBK1-HDO) 的胆固醇缀合的 DNA/RNA 异源双链寡核苷酸，可通过LDLR在CCA细胞内蓄积，降低TBK1 mRNA水平，抑制CCA肝内转移。此外，在182例ICC患者的实验组中，TBK1高表达结合核内β-catenin高表达预示着预后较差。总之，TBK1 可能作为 CCA 患者的潜在预后生物标志物和治疗靶点。