The Role And The Underlying Molecular Mechanisms Of TBK1 In The Regulation Of Tumor Cell Migration

Malignant tumor is one of the greatest challenges for human health at present.Metastasis,the key characteristics of malignant tumor,is the most destructive stage of advanced tumors and also the leading cause of recurrence and death in cancer patients.Metastasis is currently lack of efficient therapeutics and thus it is urgently requires basic and clinical research to strengthen the in-depth exploration of the regulation of tumor cell metastasis,in order to provide new molecular insights and potential targets for tumor metastasis therapy.Tumor metastasis is a continuous,progressive,multifactorial and multistep dynamic processes,during which tumor cells often change their migration ability.Therefore,exploring the regulation and mechanism of tumor cell migration plays a crucial role in the intervention of tumor metastasis.TBK1(Tank-binding kinase 1),a serinethreonine protein kinase,not only have an significant impact on the processes of innate immune response,inflammation,energy metabolism and autoimmune diseases,but also plays an important role in the occurrence and development of tumors.Besides,it is proved to be involved in multiple biological processes of tumors,such as cell mitosis,autophagy,proliferation,survival and tumor immune response.Despite that there are few studies on the regulation of tumor cell migration by TBK1,the molecular mechanisms for TBK1-mediated regulation of tumor cell migration remain elusive.IKKε(inhibitor of κB kinase ε),the homologous molecule of TBK1 and a recognized breast cancer oncogene,also plays an important role in the occurrence and development of tumors,especially in breast cancer.For TBK1,there are several key scientific questions need to be addressed: whether TBK1 plays a role in the regulation of tumor cell migration;whether TBK1 elicits its role in the regulation of tumor cell migration through regulating transcription of gene expression or through modulating phosphorylation and activation of new protein substrates;whether TBK1 regulates tumor cell migration through a similar or identical manner to its homologue IKKεeither at the phenomena level or at the molecular mechanisms level.This study focuses on the mechanism of TBK1’s regulation of tumor cell migration.In view of the fact that there are only few studies on the role of TBK1 in breast cancer,and also there is no specific researches referring to the regulation of TBK1 in breast cancer cell migration,we choose breast cancer as the object of our research.Meanwhile,parallel studies are carried out with the homologous molecule IKKε,in order to provide a new theoretical understanding of the migration regulation of tumor cells and a new potential molecular target for clinical treatment of breast cancer metastasis.Firstly,we searched the clinical databases TCGA(The Cancer Genome Atlas)and CPTAC(The National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium)for the data of TBK1 and IKKε in human breast cancer samples.We found that the expression of TBK1 was elevated in breast cancer both in transcription level and protein level,and the expression level of TBK1 might be positively correlated with tumor lymphatic metastasis but negatively correlated with overall survival of patients.The immunohistochemical examination of TBK1 in tissue microarray(breast cancer)showed that the levels of TBK1 and phosphorylated TBK1(p-TBK1)were increased in breast cancer tissues,and the expression level of TBK1 might be positively correlated with the pathological grade of breast cancer.Besides,the expression level of p-TBK1 was negatively correlated with patients’ survival.Analysis of the TBK1 homologous molecule IKKε using the same methods showed that the expression level of IKKε in breast cancer tissues was increased in TCGA and CPTAC database samples and was mainly reflected in triplenegative breast cancer.Whereas the expression level of IKKε was irrelevant to patient survival.Tissue microarray detection showed that the level of IKKε and phosphorylated IKKε(p-IKKε)might be positively associated with the pathological grade of breast cancer samples.However,there was also no significant difference in the levels of IKKε and p-IKKεwith patient survival.Therefore,the data derived from clinical breast cancer samples suggest that the amounts and activation of TBK1 are closely correlated with the lymphatic metastasis and overall survival of human breast cancer patients,indicating that TBK1 is expected to play a role in the regulation of tumor metastasis.Meanwhile,the amounts and activation of IKKε and p-IKKε were potentially correlated with the development and progression of breast cancer.Next,we confirmed the role of TBK1 and IKKε in the regulation of tumor cell migration by in vitro and in vivo experiments.We constructed a mouse mammary carcinoma cell line 4T1-Luc(4T1 cells genetically labelled with luciferase)with TBK1 knockout by CRISPR(Clustered regularly interspaced palindromic repeats)/Cas9(CRISPR-associated protein 9)gene-editing technology.The in vitro transwell experiments and in vivo mouse model(intravenous metastasis)experiments showed that the in vitro migration and in vivo metastasis of mouse mammary carcinoma cells were significantly inhibited after TBK1 knockout.When TBK1 was overexpressed,the in vitro migration and in vivo metastasis of mouse mammary carcinoma cells were promoted.The TBK1 inhibitors Amlexanox and MRT67307,which can inhibit the activity of TBK1 in mammary carcinoma cells,could effectively inhibit the in vitro migration and in vivo metastasis of mammary carcinoma cells.In the parallel study on IKKε,the in vitro migration and in vivo metastasis of mouse mammary carcinoma cells were also significantly inhibited after IKKε knockout.To further investigate whether there is a synergistic effect between TBK1 and IKKε,we constructed a mouse mammary carcinoma cell line 4T1-Luc with TBK1 and IKKε double knockout(DKO).Further in vitro and in vivo experiments revealed that the migration and metastasis of mouse mammary carcinoma cells were inhibited more significantly in DKO cell lines than in either TBK1 knockout or IKKε knockout cell lines,which suggested that the two kinases may elicit a synergistic effect in regulating the migration of mammary carcinoma cells.We then constructed a human mammary carcinoma cell line MDA-MB-231-Luc with TBK1 knockout by CRISPR/Cas9 technology,or knocked down the expression level of TBK1 in human mammary carcinoma cell lines MDA-MB-468 and MCF-7 by RNA interference.After TBK1 knockout or knockdown,the in vitro migration and in vivo metastasis of human mammary carcinoma cells were suppressed.The in vitro migration of human mammary carcinoma cells was also significantly inhibited after the treatment with the TBK1 inhibitor Amlexanox.We also carried out the above experiments on IKKε and found that the in vitro migration of the cancer cells was suppressed after knockdown of IKKε in human mammary carcinoma cells.The TBK1 knockdown combined with IKKε knockdown inhibited the migration of cells more significantly,as compared with single-molecule knockdown,indicating that TBK1 and IKKε also have a synergistic effect in human mammary carcinoma cells.We also preliminarily verified the regulation of migration by TBK1 in human hepatocellular carcinoma cell lines Hep G2 and HCCLM3 and human colon cancer cell lines RKO.After TBK1 knockdown in Hep G2 and RKO cells,the in vitro migration ability of both were inhibited.The metastasis model for RKO cell lines after TBK1 knockout depicted that TBK1 knockout also inhibited the in vivo metastasis.In addition,the in vitro migration ability of HCCLM3 cells was significantly decreased after treatment with Amlexanox.The above experiments indicate that TBK1 participates in the regulation of tumor cell migration and TBK1 inhibition attenuates tumor cell migration.The homologous molecule IKKε of TBK1 also elicits similar function,which can be synergistically enhanced by targeting TBK1.To explore the molecular mechanisms involved in TBK1-and IKKε-mediated regulation of tumor cell migration,we used multi-omics techniques,including transcriptome sequencing,proteomic analysis of TBK1-interacting partners and proteomic analysis of phosphorylated proteins,to search for the potential key molecules responsible for TBK1-mediated regulation of tumor cell migration.We also verified the contribution of the identified molecules to TBK1-mediated regulation of tumor cell migration at the molecular,cellular and living animal levels.For the TBK1 homologue IKKε,similar experiments were performed.In the 4T1-Luc cells with TBK1 knockout,IKKε knockout or TBK1 plus IKKεdouble knockout(DKO),we performed RNA-sequencing to examine the transcriptome changes.We found that TBK1 knockout affected the expression of Rho family molecules and AMPK [Adenosine 5‘-monophosphate(AMP)-activated protein kinase,AMPK]subunits,which may contribute to TBK1-mediated regulation of tumor cell migration.Whereas IKKε knockout affected the expression of integrins,possibly through which IKKεpromoted the migration of tumor cells.In the DKO cells,the differential genes involved in the regulation of cell motility and adhesion as well as tumor-promoting microenvironment,including growth factors and immune cell infiltration,were affected,indicating that TBK1 and IKKε may regulate tumor cell migration via diverse mechanisms.We then analyzed the TBK1-interacting partners by using mass spectrometry analysis after immunoprecipitation of TBK1.The data indicated that TBK1 may interact with AMPKα and Cdc42(cell division cycle 42),which play crucial roles in regulating cell motility and actin dynamics and have been implicated in the processes such as metabolisms of carbohydrate and lipid,functional maintenance of mitochondria and energy metabolism.Further Western blotting experiments confirmed that TBK1 could complex with AMPKα and Cdc42.By using the proteomic analysis of phosphorylated proteins in the knockout cell lines,we found that TBK1 could affect the phosphorylation of AMPK and Rac1(Ras-related C3 Botulinum toxin substrate1)/Cdc42-associated molecules while IKKε may regulate tumor cell migration via Rap1 or proteins involved in epithelial-mesenchymal transition(EMT).In TBK1 and IKKε DKO cells,proteins involved in regulating tumor cell growth and tumor microenvironment were identified,which may contribute to the observed inhibition of tumor cell migration.It was noteworthy that TBK1 and IKKε DKO could potentially regulate the endocrine resistance of breast cancer and the aberrations of PD1 immune checkpoint pathway.We went further to verify the findings predicted by the multi-omics analysis by performing molecular,cellular and living animal imaging experiments.We found that TBK1 knockout promoted the activation of AMPKα(Thr172)and decreased the GTP-bound levels of Rac1 and Cdc42 small GTPases.The AMPK agonist Metformin could potentiate the inhibitory effects of TBK1 knockout on the in vitro migration of breast cancer cells while the AMPK inhibitor Compound C could reverse the inhibitory effects of TBK1 knockout.In the in vivo metastatic models,Metformin treatments could potentiate the inhibition of metastasis of breast cancer cells and significantly prolong the survival of mice.When the cells were treated with the AMPK inhibitor Compound C,the levels of GTP-bound Rac1 and Cdc42 were significantly enhanced in wild type 4T1-Luc cells while they were partially increased in TBK1 knockout 4T1-Luc cells,indicating that TBK1 promoted the in vitro migration and in vivo metastasis of breast cancer cells through inhibiting the activation of AMPK and,in turn,enhancing the activation of Rac1 and Cdc42.In IKKε knockout 4T1-Luc cells,the activation of AMPK and Rac1/Cdc42 was minimally altered,as compared to that in wild type 4T1-Luc cells,which indicated that IKKε,unlike TBK1,may not regulate tumor cell migration via modulating the activity of AMPK and Rac1/Cdc42.After IKKε knockout,the amounts of NCadherin were decreased,indicating that IKKε may promote the tumor cell migration through affecting the EMT process of mouse mammary carcinoma cells.Meanwhile,the levels of phosphorylated ERK1/2(extracellular signal-regulated kinase 1 and 2)(p-ERK1/2)were decreased in IKKε knockout cells.So we inferred that IKKε promoted the activation of ERK1/2 and thus the EMT process,leading to enhanced migration and metastasis of tumor cells.In summary,our study reveals the important role of TBK1 and IKKε in regulating the in vitro migration and in vivo metastasis of breast cancer cells and possibly other tumor cells.Moreover,our study elucidates the underlying mechanisms responsible for TBK1-and IKKε-mediated regulation of tumor cell migration by taking advantages of multi-omics analysis combined with verification experiments.In the aspect of clinical significance,we have somehow systematically examined the expression and activation status of TBK1 and IKKε in tumor samples,and identified the close relationship of TBK1 and IKKε with the progression and metastasis of human breast cancer.In the aspect of scientific findings,we have verified the positive role of TBK1 and IKKε in regulating tumor cell migration by using in vitro cell models and in vivo mouse models,thus highlighting TBK1 and IKKε as potential targets for preventing tumor metastasis.In the aspect of molecular mechanisms,we have revealed that TBK1 and IKKε may regulate multiple biological processes of tumor cells through affecting gene transcription and through their enzymatic activity-dependent modulation of substrates phosphorylation.Our data support the proposal that TBK1 regulates tumor cell migration through the TBK1-AMPKα-Rac1/Cdc42 signaling pathway while IKKε regulates tumor cell migration through modulating ERK1/2 activation and EMT process.The novelty of our study may lie in that TBK1 and IKKε regulate tumor cell migration through different molecular pathways,although both kinases share with similar structure as well as similar function in innate immune response and in regulating tumor cell migration.Our study verifies that inhibiting TBK1 and IKKε can be regarded as potential strategy to prevent tumor metastasis,which is of great value in tumor practice and of great significance in clinical tumor treatments.Our experiences in searching molecular mechanisms for TBK1 and IKKε by using multi-omics methods may benefit to similar researches in the future.However,our study has not yet addressed many other scientific questions: whether TBK1 and IKKε are also aberrantly expressed in other clinical tumor samples;how TBK1 inhibits the activation of AMPKα and what are the detailed molecular events;how AMPKα regulates the GTP-bound status of Rac1/Cdc42 and what are the intermediate molecules;how IKKε modulates the process of EMT in tumor cells;whether there are unidentified molecular mechanisms contributing to TBK1-and IKKε-mediated regulation of tumor cell migration;and whether TBK1 and IKKε can influence the remodeling of tumor immune microenvironment and facilitating the metastasis of tumors.All these scientific questions await future investigations.