Early recurrence (within 2 years after resection) is the primary cause of poor outcomes among hepatocellular carcinoma (HCC) patients, and liver cancer stem cells are the main contributors to postsurgical HCC recurrence. Nucleolar and spindle‐associated protein 1 (NUSAP1) has been reported to be involved in tumor progression. We investigated the function and clinical value of NUSAP1 in early recurrence of HCC. Data from public datasets and our cohort were used to assess the association between NUSAP1 expression and early HCC recurrence. Gain‐ and loss‐of‐function experiments were carried out in vivo and in vitro. The predictive effect of NUSAP1 on early HCC recurrence was further evaluated by a validation cohort. We found that elevated NUSAP1 expression in HCC specimens was correlated with poor outcome, especially in cases with postoperative early recurrence. Functional studies indicated that NUSAP1 significantly promotes HCC progression. A postsurgical recurrence murine model further revealed that upregulated NUSAP1 dramatically increased the likelihood of HCC early recurrence. RNA sequencing data revealed that the gene sets of cancer stemness and the signal transducer and activator of transcription 3 (STAT3) pathway were enriched by NUSAP1 overexpression. Mechanistically, NUSAP1 enhanced cancer stemness through stimulating STAT3 nuclear translocation and activation through receptor of activated protein C kinase 1 (RACK1). In a validation cohort with 112 HCC patients, NUSAP1 effectively predicted HCC early recurrence. Our results indicated that NUSAP1 promotes early recurrence of HCC by sustaining cancer stemness and could serve as a valuable predictive indicator for postsurgical intervention in HCC patients.

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.