TBK1 is upregulated in CCA
To clarify the underlying role of TBK1 in ICC, Tumor Immune Estimation Resource was used to analyze the transcriptome sequencing data from the TCGA data set. The results revealed that TBK1 expression was up-regulated in nine types of cancer, including ICC. (Fig. 1A, and Supplementary. Fig. 1A), suggesting that TBK1 may participate in the tumorigenesis and progression of several solid tumors. In addition, upregulated expression of TBK1 in ICC was confirmed by tissue microarray (cohort 1, n=91, Outdo Biotech, Shanghai, China) (P < 0.001; Fig. 1B, C). Next, we examined TBK1 expression status in hepatic tissues obtained from patients. IHC analysis showed a high level of cytosol-localized TBK1 in 76.4%（139/182）of the ICC and 85.0% (34/40) of the ECC compared to the non-tumor tissues (P < 0.001; Fig. 1D, E, and Supplementary. Fig. 1B, C). Similar results were also observed in the immunoblot analyses (Fig. 1F). Besides, RT-qPCR and western blot found that the expression of TBK1 was significantly higher in several ICC cell lines than in human normal bile duct HiBEPIC cells (Fig. 1G). Consistently, the TBK1 expression was dynamically upregulated during the different stages of CCA carcinogenesis (hyperplasia, dysplasia, and CCA) in Spontaneous ICC models induced by rats and mice (Supplementary. Fig. 2A, B). Supplementary Fig. 1D, E revealed that TBK1 was primarily expressed in ICC tissues. Then far, we examined the expression levels of TBK1 in the same patient's liver tissue by immunofluorescence staining. Our results showed that TBK1 expression was higher in the bile ducts with ICC, lower in the adjacent bile ducts with tumor invasion, and no TBK1 expression was detected from adjacent non-tumor tissues (including the left hepatic duct, interlobular bile duct, and capillary bile duct) (Fig. 1H).
High expression of TBK1 was associated with aggressive clinicopathological characteristics
To further investigate the clinical significance of TBK1 expression in ICC, all 182 ICC patients were divided into two groups based on the overall expression level of TBK1; the high TBK1 expression group (n = 139) and the low TBK1 expression group (n = 43). As shown in Supplementary Table 1, the upregulation of TBK1 was significantly correlated with several aggressive clinicopathological characteristics, such as high serum CEA (P ＜ 0.001) and CA199 (P = 0.008), larger tumor diameter (P = 0.009), lymph node metastasis (P ＜ 0.001), and advanced TNM stage (P = 0.001) (Fig. 2A). The correlation between TBK1 and tumor size, lymph node metastasis, or TNM stages suggested that TBK1 may be involved in tumor progression in ICC (Fig. 2B-D).
Upregulation of TBK1 promotes ICC cell growth, motility, and metastasis both in vitro and in vivo.
To elucidate the functions of TBK1 in ICC progression, we knocked out TBK1 in HuCCT1 cells, which exhibited relatively high endogenous TBK1 levels. In addition, we overexpressed TBK1 in TFK1 cells, which exhibited relatively low endogenous TBK1 levels (Fig. 3A). The CCK8 assay, wound healing migration, transwell migration, and matrigel invasion assays revealed that the overexpression of TBK1 enhanced the growth migration and invasion ability, vice versa. (Fig. 3B and Supplementary. Fig. 3A, B). Similarly, knockdown of TBK1 by specific siRNAs repressed cell growth, migration, and invasion in TBK1 highly expressed HuCCT1 and RBE cells (Supplementary Fig. 4 A-D).
Subsequently, a mouse subcutaneous xenograft model was developed to evaluate the effect of TBK1 on ICC progression in vivo. The tumor growth curve and tumor weight showed that tumors from TBK1 overexpression cells grew significantly faster (Figure 3C-E). Subsequently, we assessed Ki-67 expression by immunohistochemistry and found more positive cells in xenografts from TBK1 overexpression cells (Supplementary Fig. 5 A). These results were further validated in human ICC tissues (Supplementary Fig. 5 B). Furthermore, the orthotopic ICC model was established to determine whether TBK1 had the same effect on ICC metastasis in vivo. As shown in Fig. 3F, depletion of TBK1 significantly inhibited liver colonization of HuCCT1 cells, whereas overexpression of TBK1 significantly promoted liver colonization of TFK1 cells. Next, the cells mentioned above were used to establish a lung metastasis mouse model by tail vein injection. More metastatic nodules were observed in mice injected with TBK1 overexpression cells (Fig. 3G). These results indicate that TBK1 can promote the growth, invasion, and metastasis of ICC cells both in vitro and in vivo.
Upregulation of TBK1 promoted EMT in ICC cells
To further explore the potential mechanism of TBK1 in promoting tumor progression, we performed RNA-sequencing and detected approximately 3101 differentially expressed genes (DEGs) after TBK1 knockdown (Fig. 4A). The gene rank of these differential genes indicated that epithelial-associated genes were significantly upregulated, while mesenchymal-associated genes were downregulated. (Fig. 4B). Moreover, Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA) revealed that the TBK1 knockdown regulated genes associated with several EMT-related signaling pathways, such as the Wnt signaling pathway and the β-catenin nucleic translocation pathway (Figure.4C, D). These data suggested that TBK1knockdown affected EMT remarkably in CCA cells.
Next, the mRNA expression levels of E-cadherin and a series of EMT inducers, including Vimentin, ZEB1, Snail, and Twist1, were measured in both TBK1-overexpressing and TBK1 knockdown cells. Overexpression of TBK1 stimulates the EMT process (Fig. 4E). Western blot analysis also confirmed the above results (Fig. 4F). We then assessed the clinical relationship between TBK1 and EMT in ICC tissues. Immunohistochemical staining displayed that ICC patients with low TBK1 expression showed higher E-cadherin and lowered Vimentin expression levels than ICC patients with high TBK1 expression (Fig. 4G). Moreover, correlation analyses showed that the expression of TBK1 correlated with the expression of E-cadherin (r = -0.3799,P ＜ 0.001) and Vimentin (r = 0.3818, P ＜ 0.001). Taken together, these results demonstrated that TBK1 plays a vital role in EMT in ICC (Fig. 4H).
TBK1 promotes the EMT process through β-catenin activation
β-Catenin plays a critical role in the induction of EMT during ICC metastasis . To determine whether β-catenin is involved in the TBK1-mediated EMT process, we assessed the clinical relationship between TBK1 and β-catenin in ICC tissues. IHC showed that ICC patients with low TBK1 expression displayed lower nuclear β-catenin expression levels than ICC patients with high TBK1 expression (Fig. 5A). Consistent with this finding, correlation analyses showed that the protein expression of TBK1 was closely associated with that of nuclear β-catenin (r = 0.4807, P < 0.001) (Fig. 5B). These results suggested that TBK1 might promote the nuclear translocation of β-catenin.
Similar to the above findings, β-catenin was localized primarily in the cytoplasm of cells, which have relatively low levels of TBK1 expression. However, nuclear β-catenin was significantly enhanced in cells, showing relatively high levels of TBK1 expression (Fig. 5C). In addition, the cytosolic and nuclear fractions of cell lysates were separated to verify this finding. Neither overexpression nor knockdown of TBK1 significantly affected the total β-catenin level. However, TBK1 overexpression decreased the cytosolic β-catenin and increased the level of nuclear β-catenin, while the knockdown of TBK1 showed the opposite effect (Fig. 5D).
To determine whether the activation of β-catenin is essential for the TBK1-mediated promotion of EMT, XAV939  was used to inhibit nuclear entry of β-catenin in TBK1-overexpressing cells and observed that inhibited nuclear entry of β-catenin eliminated the TBK1-mediated upregulation of Vimentin, as well as the downregulation of E-cadherin (Fig. 5E). In line with this finding, the EMT-mediated upregulation of cell invasion and migration was abolished by β-catenin inhibited (Fig. 5F, G), suggesting that β-catenin plays a pivotal role in the TBK1-induced EMT process.
To further confirm the role of TBK1 in the EMT process, TGFβ1 was used to induce EMT in ICC cells . As shown in Supplementary Fig. 6A, treatment with TGFβ1 promoted the nuclear translocation of β-catenin, elevated the transcriptional activity of β-catenin, decreased the expression of E-cadherin, and increased the levels of Vimentin, indicating that EMT was induced by TGFβ1 treatment. However, TGFβ1-induced EMT was significantly attenuated by inhibition of TBK1 (Supplementary Fig. 6A, B). Similarly, we found that inhibition of TBK1 abrogated the increase in cell motility induced by TGFβ1 (Supplementary Fig.6C, D). These results demonstrated that TBK1 overexpression leads to β-catenin activation, promoting the EMT process.
TBK1 activated β-catenin through direct interaction
The phosphorylate β-catenin at S552 has been demonstrated to promote β-catenin activation and nuclear translocation . As we know, various kinases are activated, leading to the phosphorylation and activation of transcription factors. Thus, we hypothesize that TBK1, a serine/threonine-protein kinase, would stimulate β-catenin activation. To explore the mechanism of TBK1 promoting β-catenin activation, we first examined the specific S552 phosphorylation of β-catenin by immunoblot analyses. The levels of S552 phosphorylation were significantly reduced in HuCCT1-shTBK1 cells, and the opposite results were obtained in TBK1-overexpressing TFK1 cells, suggesting that TBK1 contributes to β-catenin activation via phosphorylation of S552 (Fig. 6A). The experimental findings are confirmed by molecular simulations (Fig. 6B). The interactions were further validated by immunoprecipitation with a TBK1 antibody (Fig 6C, D). Altogether, these findings identify that TBK1 overexpression stimulated the nuclear translocation and activation of β-catenin through direct interaction.
As shown in Fig. 6E, F, we constructed a TBK1 mutant in S172 and found the mutation eliminated the interaction between TBK1 and β-catenin and the nuclear translocation of β-catenin. Similarly, the TBK1-S172A mutant did not induce the EMT process and cell motility (Fig. 6I and Supplementary Fig. 8A, B).
We further confirm that GSK-8612 (2.0 μM), a TBK1 S172 specific inhibitor (Supplementary Fig.7A), did not affect proliferation but effectively inhibited TBK1 phosphorylation at S172 and increased E-cadherin and decreased Vimentin expression in both HuCCT1 and TFK1 cells (Fig. 6G, H and Supplementary Fig. 7B). Immunofluorescence analysis shows that GSK-8612 notably inhibits the nucleic expression of β-catenin in indicated cells (Supplementary Fig. 7C). Taken together, these results demonstrated that the active site of TBK1 is crucial for TBK1-mediated β-catenin activation.
TBK1-HDO and Pharmacological inhibition of TBK1 reduce CCA cells growth both in vitro and in vivo
Given that depletion of TBK1 by shRNA and siRNA inhibited human CCA cell growth, we further evaluated the effect of TBK1 inhibitors in our system. Here, we designed TBK1-HDO, a short DNA/RNA heteroduplex oligonucleotide (Fig. 7A). Notably, the HDO carried cholesterol leading to ASO accumulation in the liver and was more potent at reducing the expression of TBK1 mRNA in the liver [27, 28] (Fig. 7B). Immunofluorescence staining shows the nucleic accumulation after treated TBK1-HDO at 0 min, 30 min, 60 min, and 90 min (Fig. 7C). AS shown in Fig. 7D, 7E, theTBK1-HDO significantly decreased TBK1 protein expression in indicated cells in a dose-dependent way. A relatively selective accumulation of fluorescence-labeled TBK1-HDO in the liver and other mice organs was detected by histological analysis (Fig. 7F) and quantifying the signal intensities (Fig. 7G). Furthermore, the orthotopic ICC model was established to determine whether TBK1 inhibitor had the same effect on ICC metastasis in vivo. As shown in Fig. 7H and 7I, GSK-8612 and TBK1-HDO remarkably inhibited liver colonization of HuCCT1 cells. Besides, GSK-8612 significantly inhibited the subcutaneous xenograft growth, which showed the same effection as gemcitabine, the first-line treatment for ICC. (Supplementary Fig. 9A-D). Taken together, these findings indicate that pharmacological inhibition of TBK1 decreases CCA cell growth both in vitro and in vivo.
Upregulation of TBK1 predicts poor survival in ICC patients
The prognostic implication of TBK1 in ICC was explored next. Kaplan–Meier survival analysis revealed that patients with a high level of TBK1 expression exhibited a significantly poorer overall survival (hazard ratio (HR), 3.78; 95% confidence interval (CI), 2.48–5.75) and disease-free survival (hazard ratio (HR), 2.97; 95% confidence interval (CI), 2.11–4.16) in our cohorts (Fig. 8A). Similar results were observed in cohort 1 (Supplementary. Fig. 10C). Unfortunately, it has no significance in TCGA and might be related to the small sample size. (Supplementary. Fig. 10A, B).
Consistently, nuclear β-catenin overexpression exhibited a significantly poorer overall survival and disease-free survival (Fig. 8B). Interestingly, we found that patients with low expression of both TBK1 and nuclear β-catenin had the best prognosis (Fig. 8C). Subsequently, TBK1 expression status and prognostic clinicopathological parameters identified by univariate analysis (P < 0.01) were entered into a multivariate model to identify independent predictors of overall survival. We found that TBK1 upregulation was an independent statistically significant risk factor for overall survival (P < 0.001, Fig. 8D), suggesting that upregulation of TBK1 may play a pivotal role in the overall survival of ICC patients. Taken together, these results indicated that the combination of TBK1 and nuclear β-catenin could serve as a biomarker in ICC for evaluating the metastatic potential and predicting the prognosis of ICC patients.