Expression Of TANK-binding Kinase 1 Research Articles

CDK12 loss inhibits cell proliferation by regulating TBK1 in non-small cell lung cancer cells

Lung cancer is one of the most common malignant tumors and has a poor prognosis and a low survival rate. Traditional treatments, such as radiotherapy and chemotherapy, still face some challenges because of high drug resistance and toxicity. Therefore, it is necessary to discover a new kind of targeted drug with low toxicity and high efficiency. CDK12 is a cell cycle-dependent kinase whose main function is to activate RNA polymerase II (RNAPII) and promote the transcriptional extension of RNA. However, the role and molecular mechanism of CDK12 in lung cancer are still unclear.In this study, the mutation and RNA-Seq data of CDK12 in lung adenocarcinoma and squamous cell carcinoma were downloaded from The Cancer Genome Atlas (TCGA) database and analyzed with the custom scripts. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and cell colony formation assays. A subcutaneous tumor experiment in nude mice was used to examine the effects of CDK12 knockdown on the in vivo tumor growth of NSCLC cells. The cell cycle distribution and the apoptosis rate of lung cancer cells were assessed by flow cytometry. Regulation of TANK-binding kinase 1 (TBK1) by CDK12 was evaluated by quantitative PCR, immunoprecipitation and Western blot analysis.In this study we have analyzed the mutation and expression data of The Cancer Genome Atlas (TCGA) database and found that CDK12 is highly expressed in lung cancer tissues. Clinical correlation analysis showed that high expression of CDK12 in NSCLC reduces patient survival, but its high expression is only related to early tumor progression and has no significant correlation with late tumor progression and metastasis. Furthermore, we present evidence that CDK12 depletion in lung cancer cell lines not only leads to the inhibition of cell growth and induces apoptosis but also inhibits tumor growth of NSCLC cells in vivo. CDK12 positively regulates the expression of the oncogene TBK1 in lung cancer cells. These results revealed that CDK12 affects the progression of non-small cell lung cancer through positive regulation of TBK1 expression, suggesting that CDK12 might be a potential molecular target for the treatment of non-small cell lung cancer.

Serine/threonine kinase TBK1 promotes cholangiocarcinoma progression via direct regulation of β-catenin

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.

TBK1 promotes thyroid cancer progress by activating the PI3K/Akt/mTOR signaling pathway.

Thyroid cancer has received increasing attention; however, its detailed pathogenesis and pathological processes remain unclear. We investigated the role of TANK-binding kinase 1 (TBK1) in the progression of thyroid cancer. The expression of TBK1 in thyroid cancer and normal control tissues was analyzed using real-time quantitative polymerase chain reaction. The function of TBK1 on thyroid cancer cells was detected using MTT, colony formation, wound healing, and Transwell assays. The xenograft assay was carried out to check on the role of TBK1 in thyroid cancer. TBK1 was highly expressed in thyroid tumors. High expression of TBK1 raised viability, proliferation, migration, and invasion of thyroid cancer cells. Gene set enrichment analysis revealed that TBK1 activated the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway. In addition, Myc-associated zinc finger protein (MAZ) was overexpressed in thyroid cancer and transcriptionally activated BK1. MAZ silence reversed the effects of TBK1 overexpression on thyroid cancer progression. Cotransfection with MAZ small-interfering RNA(siRNA) and TBK1 siRNA did not strengthen the inhibitory effect of TBK1 silencing on the thyroid cancer cells. The xenograft tumor assay showed that TBK1 short hairpinRNA inhibited tumor growth. MAZ silencing inhibited tumor progress of thyroid cancer cells, whereas this inhibitory effect was reversed by TBK1 overexpression.

TBK1-medicated DRP1 phosphorylation orchestrates mitochondrial dynamics and autophagy activation in osteoarthritis.

Mitochondrial dynamics, including mitochondrial fission and fusion, are critical for maintaining mitochondrial functions. Evidence shows that TANK-binding kinase 1 (TBK1) regulates mitochondrial fusion and fission and then mitophagy. Since a previous study demonstrates a strong correlation between mitophagy and osteoarthritis (OA), we herein investigated the potential role of TBK1 in OA process and mitochondrial functions. We demonstrated a strong correlation between TBK1 and OA, evidenced by significantly downregulated expression of TBK1 in cartilage tissue samples of OA patients and in the chondrocytes of aged mice, as well as TNF-α-stimulated phosphorylation of TBK1 in primary mouse chondrocytes. TBK1 overexpression significantly attenuated TNF-α-induced apoptosis and abnormal mitochondrial function in primary mouse chondrocytes. Furthermore, TBK1 overexpression induced remodeling ofmitochondrial morphology by directly phosphorylating dynamin-related protein 1 (DRP1) at Ser637, abolishing the fission of DRP1 and preventing its fragmentation function. Moreover, TBK1 recruitment and DRP1 phosphorylation at Ser637 was necessary for engulfing damaged mitochondria by autophagosomal membranes during mitophagy. Moreover, we demonstrated that APMK/ULK1 signaling contributed to TBK1 activation. In OA mouse models established by surgical destabilization of the medial meniscus, intraarticular injection of lentivirus-TBK1 significantly ameliorated cartilage degradation via regulation of autophagy and alleviation of cell apoptosis. In conclusion, our results suggest that the TBK1/DRP1 pathway is involved in OA and pharmacological targeting of the TBK1-DRP1 cascade provides prospective therapeutic benefits for the treatment of OA.

Knockdown of TANK-Binding Kinase 1 Enhances the Sensitivity of Hepatocellular Carcinoma Cells to Molecular-Targeted Drugs.

The protein kinase, TANK-binding kinase 1 (TBK1), not only regulates various biological processes but also functions as an important regulator of human oncogenesis. However, the detailed function and molecular mechanisms of TBK1 in hepatocellular carcinoma (HCC), especially the resistance of HCC cells to molecular-targeted drugs, are almost unknown. In the present work, the role of TBK1 in regulating the sensitivity of HCC cells to molecular-targeted drugs was measured by multiple assays. The high expression of TBK1 was identified in HCC clinical specimens compared with paired non-tumor tissues. The high level of TBK1 in advanced HCC was associated with a poor prognosis in patients with advanced HCC who received the molecular-targeted drug, sorafenib, compared to patients with advanced HCC patients and a low level of TBK1. Overexpression of TBK1 in HCC cells induced their resistance to molecular-targeted drugs, whereas knockdown of TBK1 enhanced the cells’ sensitivity to molecular-targeted dugs. Regarding the mechanism, although overexpression of TBK1 enhanced expression levels of drug-resistance and pro-survival-/anti-apoptosis-related factors, knockdown of TBK1 repressed the expression of these factors in HCC cells. Therefore, TBK1 is a promising therapeutic target for HCC treatment and knockdown of TBK1 enhanced sensitivity of HCC cells to molecular-targeted drugs.

QKI deficiency in macrophages protects mice against JEV infection by regulating cell migration and antiviral response

Japanese encephalitis (JE) is a major reason to cause viral encephalitis, with 50% patients suffering from severe neuro-inflammation and permanent neural injury. Effective anti-viral treatment is urgently needed. Here, we found RNA binding protein quaking (QKI) was involved in the progression of JE by regulating migration and anti-viral response of macrophages. After JE virus (JEV) infection, QKI-deficient mice had lower viral loads in the brain and fewer neurological symptoms. In comparison with control mice, proinflammatory cytokines in the brain of QKI-deficient animals revealed distinct patterns, with lower levels of IL-6 (interleukin-6) and IFN-β (interferon-β) at the early stage but higher levels at the end of JE. Then we found infiltration of CCR2 positive ((C-C motif) receptor 2) peripheral macrophages and CCR2 expression on macrophages were inhibited in QKI-deficient mice, while the expression of CCR2 ligands was not changed. Bioinformatical analysis showed that a QRE (quaking response element) located on 3′UTR (untranslated region) of Ccr2. We further verified that QKI was able to interact with Ccr2 mRNA and regulate its degradation in vitro. Additionally, since the IFN-β production was increased in QKI-ablation mice after JEV infection, the anti-viral response was analyzed. Results in QKI-silenced N9 cells showed that the expression of RIG-I (retinoic acid-inducible gene-I) and TBK1 (TANK binding kinase 1) was increased, thus further inducing IRF3 (interferon regulatory factor 3) phosphorylation and interferon activation. Overall, these results revealed QKI mediated the anti-viral process via interfering migration of macrophages to CNS (central nervous system) and enhancing RIG-I/IRF3/IFN-β pathway to restrict virus dissemination.

TBK1 Mediates Innate Antiviral Immune Response against Duck Enteritis Virus.

Duck enteritis virus (DEV) can infect several types of waterfowl can cause high mortality and huge economic losses to the global waterfowl industry. Type I interferons (IFN) are important for host defense against virus infection through induction of antiviral effector molecules. TANK-binding kinase 1 (TBK1) is a key kinase required for the induction of type I IFNs; however, the role of TBK1 on DEV infection remains unclear. Here, we observed that the expression levels of TBK1 and IFN-β were upregulated during DEV infection in vivo and in vitro. Thus, the function of TBK1 on DEV infection was determined. The results showed that overexpression of TBK1 reduced DEV infection and knockdown of TBK1 resulted in the increased of DEV infection. Additionally, TBK1 overexpression upregulated the expression of IFN-β and a few interferon-stimulated genes (ISGs), which thus inhibited the synthesis of DEV glycoprotein B. On the other hand, the TBK1 inhibitor Amlexanox down-regulated the expression levels of IFN-β and IRF3. Interestingly, the expression levels of MAVS and GSK-3β were decreased in the cells treated with Amlexanox. Furthermore, overexpression of TBK1 activated the expression of upstream molecules MAVS and GSK-3β. Whereas, the expression of TBK1, IRF3 and IFN-β was inhibited by the GSK-3β inhibitor SB216763. Our findings suggest that DEV–stimulated TBK1 may be involved in defense against DEV infection.

Novel Intronic Mutations of TBK1 Promote Aberrant Splicing Modes in Amyotrophic Lateral Sclerosis.

TANK-binding kinase 1 (TBK1) has been identified as a causative gene of amyotrophic lateral sclerosis (ALS) in the Caucasian population in 2015. Here, we sequenced for TBK1 variants in a cohort of 15 familial ALS (fALS) and 275 sporadic ALS (sALS) of Chinese origin by targeted next-generation sequencing. We identified one likely benign missense variant (p. Ser398Pro), two missense variants of uncertain significance (p. Ile37Leu and p. Tyr677Asn), and two novel heterozygous variants in introns of TBK1, c.1522-3T > G and c.2066 + 4A > G. We performed splicing assays through minigene plasmids and RNA pull-down assay to determine that the two substitutions of nucleotides disrupted the binding of the important splicing regulator hnRNPA1 and promoted aberrant pre-mRNA splicing modes. The c.1522-3T > G variant promoted nearly 50.0% of abnormal transcripts (3 different types of insertions and deletions (indels) in junction of intron 13-exon 14) and the c.2066 + 4A > G variant inhibited about 75.0% inclusion of exon 19, both causing premature stop codon and producing TBK1 protein without CCD2. Immunofluorescence analysis showed that the expression of TBK1 with intronic variants was lower since less TBK1 distribution was observed in HEK293T cells. Both patients carrying TBK1 c.1522-3T > G and c.2066 + 4A > G variants developed a rapidly progressive ALS, with a survival of 31 and 10 months, respectively. The frequency of loss of function (LoF) variants in TBK1 was 0.73% in sALS in our cohort. We emphasize that intronic sequencing and pre-mRNA splicing analysis cannot be ignored to demonstrate the complex mutational spectrum and pathogenesis of ALS.

Silence of TANK-binding kinase 1 (TBK1) regulates extracellular matrix degradation of chondrocyte in osteoarthritis by janus kinase (JAK)-signal transducer of activators of transcription (STAT) signaling

ABSTRACT TANK-binding kinase 1 (TBK1) was previously reported to be critical for the regulation of osteoclast differentiation. However, its function in osteoarthritis (OA) has not yet been determined. This study aims to reveal the role of TBK1 in the extracellular matrix (ECM) degradation in OA. C57BL/6 J mice were subjected to anterior cruciate ligament transection (ACLT) surgery to establish an OA animal model. ATDC5 cells were treated with IL-1β to construct a cell model of OA. Changes in the expression of TBK1 were analyzed by qRT-PCR, Western blotting, and immunohistochemistry. Safranin O-fast green staining, ELISA, and Western blotting were performed to evaluate the ECM degradation. By searching GSE75181 and GSE6119 datasets, TBK1 was found to be highly expressed in the OA model. Its upregulation was also confirmed in ACLT mice and in a cell model of OA. Silencing of TBK1 reduced cartilage degradation, OARSI score, and serum levels of CTX-II and COMP. Silencing of TBK1attenuated ECM degradation, as ADAMTS-4, MMP3, and MMP13 were downregulated, whilst SOX9, collagen II, and aggrecan were upregulated. Furthermore, TBK1 activates the JAK/STAT signaling pathway. Transfection of cells with the STAT3 overexpression plasmid blocked the beneficial effects of TBK1 silencing. In conclusion, TBK1 is highly expressed in OA. Silencing of TBK1 inhibited ECM degradation.

A Nanoparticle-Conjugated Anti-TBK1 siRNA Induces Autophagy-Related Apoptosis and Enhances cGAS-STING Pathway in GBM Cells.

Background Gene therapy shows considerable clinical benefit in cancer therapy, in which single-stranded ribonucleic acid (siRNA) is a promising strategy in the treatment of glioblastoma (GBM). TANK-binding kinase 1 (TBK1) is critical in tumorigenesis and development, which lays a foundation for an ideal target for tumor therapy. However, the practical application of free siRNA is limited. It is urgent to develop novel strategies to deliver TBK1 siRNA to activate apoptosis and cGAS-STING pathway as a therapeutic strategy for GBM. Methods The expression and prognostic value of TBK1 were evaluated in the TCGA, CGGA, and GTEx databases. A novel gene delivery system was designed here via PEGylated reduced graphene oxide (rGO-PEG) to targeted delivery of anti-TBK1 siRNA efficiently. The efficacy of TBK1si/rGO-PEG was evaluated in GBM cells. The underlying pathways were explored by Western blot. Results TBK1 was highly expressed in glioma samples, and its high expression indicated poor prognoses in glioma patients. The rGO-PEG presented great efficiency in targeted delivery of TBK1si RNA into GBM cells with up to 97.1% transfection efficiency. TBK1si/rGO-PEG exhibited anti-GBM activities by inhibiting TBK1 and autophagy, as well as activating apoptosis and cGAS-STING pathway. Conclusion The rGO-PEG could be an efficient system facilitating the delivery of specific siRNA. TBK1si/rGO-PEG could be a novel strategy for the treatment of GBM.

TANK-Binding Kinase 1 (TBK1) Serves as a Potential Target for Hepatocellular Carcinoma by Enhancing Tumor Immune Infiltration.

BackgroundNumerous cancer types present the aberrant TANK-binding kinase 1 (TBK1) expression, which plays an important role in driving inflammation and innate immunity. However, the prognostic role of TBK1 and its relationship with immune cell infiltration in hepatocellular carcinoma (HCC) remain unclear.MethodsThe expression and prognostic value of TBK1 was analyzed by Tumor Immune Estimation Resource (TIMER), Kaplan-Meier plotter and Gene Expression Profiling Interactive Analysis (GEPIA), Clinical Proteomic Tumor Analysis Consortium (CPTAC) and further confirmed in the present cohort of patients with HCC. The association between TBK1 and HCC immune infiltrates, and its potential mechanism were investigated via analyses of the Tumor Immune Estimation Resource, tumor-immune system interactions database (TISIDB), CIBERSORT, STRING, and Metascape. The effect of TBK1 on immune infiltrates and the therapeutic value of targeting TBK1 were further investigated in a HCC mouse model by treatment with a TBK1 antagonist.ResultsThe level of TBK1 expression in HCC was higher than that measured in normal tissues, and associated with poorer overall survival (GEPIA: hazard ratio [HR]=1.80, P=0.038; Kaplan–Meier plotter: HR=1.87, P<0.001; CPTAC: HR=2.23, P=0.007; Our cohort: HR=2.92, P=0.002). In addition, high TBK1 expression was found in HCC with advanced TNM stage and identified as an independent poor prognostic factor for overall survival among patients with HCC. In terms of immune infiltration, tumor tissues from HCC patients with high TBK1 expression had a low proportion of CD8+ T cells, and TBK1 expression did not show prognostic value in HCC patients with enriched CD8+ T cells. Furthermore, TBK1 expression was positively correlated with the markers of T cell exhaustion and immunosuppressive cells in the HCC microenvironment. Mechanistically, the promotion of HCC immunosuppression by TBK1 was involved in the regulation of inflammatory cytokines. In vivo experiments revealed that treatment with a TBK1 antagonist delayed HCC growth by increasing the number of tumor-infiltrating CD8+ T cells.ConclusionsThe up-regulated expression of TBK1 may be useful in predicting poor prognosis of patients with HCC. In addition, TBK1, which promotes the HCC immunosuppressive microenvironment, may be a potential immunotherapeutic target for patients with HCC.

A Novel Transcript Isoform of TBK1 Negatively Regulates Type I IFN Production by Promoting Proteasomal Degradation of TBK1 and Lysosomal Degradation of IRF3.

TANK-binding kinase 1 (TBK1), an IKK-related serine/threonine kinase, is pivotal for the induction of antiviral type I interferon (IFN) by TLR and RLR signaling pathways. In a previous study, we demonstrated that TBK1 spliced isoforms (TBK1_tv1 and TBK1_tv2) from zebrafish were dominant negative regulators in the RLR antiviral pathway by targeting the functional TBK1–IRF3 complex formation. In this study, we show that the third TBK1 isoform (namely TBK1_tv3) inhibits zebrafish type I IFN production by promoting TBK1 and IRF3 degradation. First, ectopic expression of TBK1_tv3 suppresses poly(I:C)- and Spring viremia of carp virus-induced type I IFN response, and also inhibits the up-regulation of IFN promoter activities stimulated by RIG-I, MDA5, MAVS, TBK1, and IRF3. Second, TBK1_tv3 targets TBK1 and IRF3 to impair the formation of TBK1 dimer, TBK1–IRF3 complex, and IRF3 dimer. Notably, TBK1_tv3 promotes the degradation of TBK1 through the ubiquitin–proteasome pathway and the degradation of IRF3 through the lysosomal pathway. Further analysis demonstrates that TBK1_tv3 promotes the degradation of TBK1 for K48-linked ubiquitination by targeting the K251, K256, and K271 sites of TBK1. Collectively, our results suggest a novel TBK1 isoform-mediated negative regulation mechanism, which serves to balance the production of type I IFN and ISGs.

ALS-associated TBK1 variant p.G175S is defective in phosphorylation of p62 and impacts TBK1-mediated signalling and TDP-43 autophagic degradation

Mutations affecting SQSTM1 coding for p62 and TANK-Binding Kinase 1 (TBK1) have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TBK1 is a serine-threonine kinase that regulates p62's activity as an autophagy receptor via phosphorylation and also has roles in neuroinflammatory signalling pathways. The mechanisms underlying ALS and FTLD pathogenesis as a result of TBK1 mutations are incompletely understood, however, loss of TBK1 function can lead to dysregulated autophagy and mitophagy. Here, we report that an ALS-associated TBK1 variant affecting the kinase domain, p.G175S, is defective in phosphorylation of p62 at Ser-403, a modification critical for regulating its ubiquitin-binding function, as well as downstream phosphorylation at Ser-349. Consistent with these findings, expression of p.G175S TBK1 was associated with decreased induction of autophagy compared to wild type and reduced degradation of the ALS-linked protein TDP-43. Expression of wild type TBK1 increased NF-κB signalling ~300 fold in comparison to empty vector cells, whereas p.G175S TBK1 was unable to promote NF-κB signalling above levels observed in empty vector transfected cells. We also noted a hitherto unknown role for TBK1 as a suppressor of oxidative stress (Nrf2) signalling and show that p.G175S TBK1 expressing cells lose this inhibitory function. Our data suggest that TBK1 ALS mutations may broadly impair p62-mediated cell signalling, which ultimately may reduce neuronal survival, in addition TDP-43 was not efficiently degraded, together these effects may contribute to TBK1 mutation associated ALS and FTLD pathogenesis.

Intestinal Epithelial TBK1 Prevents Differentiation of T-helper 17 Cells and Tumorigenesis in Mice

Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice. We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1IEC-KO), Tbk1IEC-KO mice crossed with ApcMin/+ mice, and Mt-/- mice crossed with ApcMin/+ mice. Some mice were given intraperitoneal injections of a neutralizing antibody against interleukin 17 (IL17) or IL1β. Intestine tissues were collected from mice and analyzed by histology, for numbers of adenomas and Th17 cells, and expression of inflammatory cytokines by real-time PCR. IECs were isolated from wild-type and Tbk1IEC-KO mice, stimulated with lipopolysaccharide, co-cultured for with bone marrow-derived macrophages, and analyzed by RNA sequencing and biochemical analyses. Compared to ApcMin/+Tbk1WT mice, ApcMin/+Tbk1IEC-KO mice had significant increases in number and size of intestinal polyps, and significantly more Th17 cells in lamina propria. Administration of an antibody against IL17 reduced the number of intestinal polyps in ApcMin/+Tbk1IEC-KO mice to that observed in ApcMin/+Tbk1WT mice. In culture, TBK1-deficient IECs promoted expression of IL1β by macrophages, which induced differentiation of naïve CD4+ T cells into Th17 cells. RNA sequencing analysis revealed that the TBK1-deficient IECs had increased expression of metallothionein 1 (MT1), an immune regulator that promotes intestinal inflammation. Intestine tissues from ApcMin/+Mt-/- mice had significant fewer Th17 cells than ApcMin/+Mt+/+ mice, and a significantly lower number of polyps. Analyses of colorectal tumors in the Cancer Genome Atlas found colorectal tumors with high levels of MT1 and IL17 mRNAs to be associated with reduced survival times of patients. Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1β by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

Expression and prognostic role of IKBKE and TBK1 in stage I non-small cell lung cancer.

BackgroundThe inhibitors of nuclear factor kappa-B kinase subunit epsilon (IKBKE) and TANK-binding kinase 1 (TBK1) are important members of the nonclassical IKK family that share the kinase domain. They are important oncogenes for activation of several signaling pathways in several tumors. This study aims to explore the expression of IKBKE and TBK1 and their prognostic role in stage I non-small cell lung cancer (NSCLC).Patients and methodsA total of 142 surgically resected stage I NSCLC patients were enrolled and immunohistochemistry of IKBKE and TBK1 was performed.ResultsIKBKE and TBK1 were expressed in 121 (85.2%) and 114 (80.3%) of stage I NSCLC patients respectively. IKBKE expression was significantly associated with TBK1 expression (P=0.004). Furthermore, multivariate regression analyses showed there was a significant relationship between patients with risk factors, the recurrence pattern of metastasis and IKBKE+/TBK1+ co-expression (P=0.032 and P=0.022, respectively). In Kaplan–Meier survival curve analyses, the IKBKE+/TBK1+ co-expression subgroup was significantly associated with poor overall survival (P=0.014).ConclusionsThis is the first study to investigate the relationship between IKBKE and TBK1 expression and clinicopathologic characteristics in stage I NSCLC patients. IKBKE+/TBK1+ co-expression was significantly obvious in patients with risk factors and with recurrence pattern of distant metastasis. Furthermore, IKBKE+/TBK1+ is also an effective prognostic predictor for poor overall survival.

Blocking TBK1 alleviated radiation-induced pulmonary fibrosis and epithelial-mesenchymal transition through Akt-Erk inactivation

As a common serious complication of thoracic radiotherapy, radiation-induced pulmonary fibrosis (RIPF) severely limits radiation therapy approaches. Epithelial–mesenchymal transition (EMT) is a direct contributor to the fibroblast pool during fibrogenesis, and prevention of EMT is considered an effective strategy to inhibit tissue fibrosis. Our previous study revealed that TANK-binding kinase 1 (TBK1) regulates EMT in lung cancer cells. In the present study, we aimed to investigate the therapeutic potential of targeting TBK1 to prevent RIPF and EMT progression. We found radiation-induced EMT and pulmonary fibrosis in normal alveolar epithelial cells and lung tissues. TBK1 knockdown or inhibition significantly reversed EMT in vivo and in vitro and attenuated pulmonary fibrosis and collagen deposition. Moreover, we observed that TBK1 was elevated in a time- and dose-dependent manner by radiation. Meanwhile, radiation also induced time- and dose-dependent activation of AKT and ERK, each of whose inhibitors suppressed radiation-induced EMT. Intriguingly, silencing of TBK1 with shRNA also blocked the radiation-induced activation of AKT and ERK signaling. The ERK inhibitor did not obviously affect the expression of TBK1 or phosphorylated AKT, while AKT inhibition suppressed activation of ERK without changing the expression of TBK1. Finally, we found that a TBK1 inhibitor inhibited inflammatory cytokine expression in a RIPF model and Amlexanox protected normal cells and mice from ionizing radiation. In conclusion, our results indicate that the TBK1–AKT–ERK signaling pathway regulates radiation-induced EMT in normal alveolar epithelial cells, suggesting that TBK1 is a potential target for pulmonary fibrosis prevention during cancer radiotherapy.

LDK378 improves micro- and macro-circulation via alleviating STING-mediated inflammatory injury in a Sepsis rat model induced by Cecal ligation and puncture

BackgroundSepsis is a systemic inflammatory response syndrome caused by severe infections. LDK378, a second-generation ALK inhibitor, exhibits a potential anti-inflammatory function against sepsis. Micro- and macro-circulatory dysfunctions are pivotal elements of the pathogenesis of severe sepsis and septic shock. We hypothesized that LDK378 can improve micro- and macro-circulation of septic rats, therefore improving the outcome of survival via blocking the ALK-STING pathway to attenuate inflammatory injuries.MethodsA septic rat model was established by the cecal ligation and puncture (CLP) method. A total of 60 rats were randomized into three groups: a sham group, CLP group, and CLP + LDK378 group (n = 20 in each group). Five rats were randomly selected from each group for the mechanism study; the remaining 15 rats in each group were involved in a survival curve examination. A sidestream dark field video microscope was used to record sublingual microcirculation and mean arterial pressure (MAP) and levels of inflammatory cytokine secretion were examined at 6 h, 30 h, and 54 h after CLP surgery. Expressions of TANK binding kinase 1 (TBK1) and its downstream targets were determined, and histological alterations to the heart, lungs, and kidneys were examined at 54 h after CLP surgery.ResultsWe found the group that received LDK378 treatment showed increased MAP levels compared to the CLP group at 30 h and 54 h. Meanwhile, LDK378 ameliorated the perfused small vessel density and microvascular flow index, decreased the expression of TNF-a and IL-6, and upregulated the expression of IL-10 in comparison with the CLP group. LDK378 injections also downregulated the expression of TBK1 and its downstream targets. Furthermore, LDK378 treatment significantly reduced sepsis-induced organ injuries, therefore improving survival rates.ConclusionsThese findings demonstrate that LDK378 treatment can improve microcirculation and reduce organ injuries in CLP-induced septic rats via the regulation of inflammatory cytokine secretion and the downstream signaling components of the ALK-STING pathway.

Inhibition of TBK1 reduces choroidal neovascularization in vitro and in vivo

choroidal neovascularization (CNV), a characteristic of wet age-related macular degeneration (AMD), causes severe vision loss among elderly patients. TANK-binding kinase 1 (TBK1) is a ubiquitously expressed serine–threonine kinase and is found to induce endothelial cells proliferation, represent a novel mediator of tumor angiogenesis and exert pro-inflammatory effect. However, the role of TBK1 in choroidal neovascularization has not been investigated so far. In this study, we found that the expression of TBK1 and VEGF was up-regulated in RF/6 A cells chemical hypoxia model and laser-induced mouse CNV model. Silencing of TBK1 suppressed the proliferation and tube formation activity of RF/6 A cells. Intravitreal injection of anti-TBK1 monoclonal antibody ameliorates CNV formation. Taken together, these findings exhibit a proangiogenic role for TBK1 via upregulating the expression of VEGF, and may suggest that TBK1 inhibition offers a unique and alternative method for prevention and treatment of AMD.

Common and rare TBK1 variants in early-onset Alzheimer disease in a European cohort

TANK-binding kinase 1 (TBK1) loss-of-function (LoF) mutations are known to cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), often combined with memory deficits early in the disease course. We performed targeted resequencing of TBK1 in 1253 early onset Alzheimer's disease (EOAD) patients from 8 European countries to investigate whether pathogenic TBK1 mutations are enriched among patients with clinical diagnosis of EOAD. Variant frequencies were compared against 2117 origin-matched controls. We identified only 1 LoF mutation (p.Thr79del) in a patient clinically diagnosed with Alzheimer's disease and a positive family history of ALS. We did not observe enrichment of rare variants in EOAD patients compared to controls, nor of rare variants affecting NFκB induction. Of 3 common coding variants, rs7486100 showed evidence of association (OR 1.46 [95% CI 1.13–1.9]; p-value 0.01). Homozygous carriers of the risk allele showed reduced expression of TBK1 (p-value 0.03). Our findings are not indicative of a significant role for TBK1 mutations in EOAD. The association between common variants in TBK1, disease risk and reduced TBK1 expression warrants follow-up in FTD/ALS cohorts.

Genetic and functional analysis of TBK1 variants in Korean patients with sporadic amyotrophic lateral sclerosis

The TANK-binding kinase 1 (TBK1) gene has recently been identified as a novel causative gene of amyotrophic lateral sclerosis (ALS). This study aims to determine the frequency and spectrum of TBK1 variants and their functional implications in Korean patients with sporadic ALS (sALS). TBK1 sequences were analyzed in 129 consecutive patients with sALS using either multigene panel or exome sequencing. One frameshift (c.1414delA) and 3 missense variants of uncertain significance in TBK1 were found in 4 patients each. In vitro functional studies revealed that the c.1414delA (p.Ile472Serfs*8) variant was associated with reduced mRNA expression of TBK1. Moreover, protein expression of this variant in patient-derived fibroblasts disrupted binding to autophagy adapter proteins and inhibited the function of TBK1 in HEK293T cells. In contrast, the 3 other missense variants of uncertain significance showed normal mRNA expression and no abnormalities in protein function. Based on these findings, the frequency of pathogenic TBK1 variants in Korean sALS patients was estimated to be 0.8% (1/129). In conclusion, pathogenic variants in TBK1 are rare but could be responsible for sALS in a small number of Korean patients.