LIMK1 Nuclear Translocation Promotes Tumor Progression And Achieves The Susceptibility From Anti-EGFR Therapies In Hepatocellular Carcinoma

Background and Objective:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors of digestive system and the second leading cause of cancer mortality in the world.Although emerging molecular targeted therapy and immunotherapy have been used to the treatment of HCC,the efficacy are still limited.It is also urgent to study the molecular mechanism of HCC progression and find new therapeutic strategies.Method:The expression of LIM kinase 1(LIMK1)was upregulated in HCC tissues by analyzing HCC data from the Cancer Genome Atlas(TCGA)public database.The expression of LIMK1 in HCC tissues and HCC cell lines was detected by immunohistochemistry(IHC),immunofluorescence(IF)and Western blot analysis.Moreover,the relationship between the expression of LIMK1 in subcellular components and clinicopathological parameters such as tumor stage,metastasis and prognosis were also analyzed.Gain-and loss-of-function experiments were performed in vitro to evaluate the effects of nuclear LIMK1 on biological behaviour of HCC cells.Colony formation assays and transwell assays were used to evaluate the colony formation and migration abilities of HCC cells.Cholycystokinin-8(CCK-8)assays and wound healing assays were used to evaluate the proliferative capacity and cell motility potential of HCC cells.The mechanisms of LIMK1 nuclear translocation and the nuclear LIMK1-enhanced aggressive phenotypes of HCC were clarified by bioinformatics analysis,western blot analysis,immunofluorescence(IF)assay,coimmunoprecipitation(Co-IP)analysis,chromatin immunoprecipitation(ChIP)assays,dual luciferase reporter assay and Real-time PCR assays.The suppressive effects of cetuximab on LIMK1 nuclear translocation and HCC progression were studied by western blot analysis,immunofluorescence(IF),immunohistochemistry(IHC),subcutaneous tumorigenesis and tail vein injection in nude mice.Results:The expression of LIMK1 in HCC tissues was significantly higher than in adjacent tissues and paired normal tissues.The positive rate of nuclear LIMK1 expression in HCC tissues was higher than in normal tissues,while the positive rate in the cytoplasm was the complete opposite.Survival analysis revealed that compared with the total and cytoplasmic LIMK1,only nuclear LIMK1 had a correlation with the long-term prognosis of HCC patients.Nuclear LIMK1 enhances the aggressive phenotype of HCC cells in vitro.Bioinformatics analysis combined with functional research in vitro showed that LIMK1 and p-ERK interacted together and both went to the nucleus upon epidermal growth factor(EGF)stimulation.Chromatin immunoprecipitation(ChIP)assays and dual luciferase reporter assay showed that Nuclear LIMK1 transcriptionally activates the expression of c-Myc by directly binding to its promoter.Nuclear LIMK1 also promoted HCC progression by activating c-Myc downstream cancer related genes combined with western blot analysis and Real-time PCR assays.Functional research in vitro and in vivo indicated that cetuximab,an anti-EGFR monoclonal antibody,could suppress tumor progression by decreasing LIMK1 nuclear translocation.Conclusion:Upon EGF stimulation,LIMK1 and p-ERK interacts mutually to facilitate their nuclear translocation.Cetuximab,an anti-EGFR monoclonal antibody,could specifically decrease the nuclear LIMK1 mediated HCC progression,which provided a new clinical therapeutic strategy for HCC patients.