Molecular Mechanism of SCFβ-TrCP Ubiquitination and Degradation of TFAP4 to Inhibit Epithelial-mesenchymal Transition in Colorectal Cancer
Objective To explore the effect of SCFβ-TrCP ubiquitination and degradation of transcription factor-activated enhancer-binding protein 4(TFAP4)on epithelial-mesenchymal transformation in colorectal cancer.Methods Human colorectal adenocarcinoma cells SW480 were cultured in vitro and treated with different concentrations of MLN4924 for 24h,and then Western blot assay was used to detect the changes of endogenous TFAP4 protein levels.On the basis of MLN4924 treatment,CHX was added to intervene at different times to detect the difference of TFAP4 protein half-life time and ubiquitination level.Flag-labeled TrCP-1 was overexpressed in CHX-interfered SW480 cells to explore the influence of β-TrCP on TFAP4 expression.The overexpression or knockdown of β-TrCP in co-immunoprecipitation(Co-IP)experiments did not change the level of TFAP4 because it affected other signaling pathways.The E(135)on TFAP4 was mutated to A,and the S(139)was mutated to A,and then transfected TFAP4 with mutated domain in SW480 cells treated with CHX to detect the changes in TFAP4half-life time and ubiquitination level.CK1/CK2/GSK3 phosphorylated key enzyme in-hibitors were used to intervene SW480 cells,Western blot assay was used to detect TFAP4 levels,and then the influence of CK2 inhibitors on TFAP4 ubiquitination level was detected.Cell migration performance was analyzed by scratch assay,and cell invasion ability was ana-lyzed by Transwell assay.Results TFAP4 increased in a dose-dependent manner with the concentration change of MLN4924 treatment.MLN4924 treatment could significantly prolong the half-life time of endogenous TFAP4 protein.There was an interaction between TFAP4 and CK2,CK2 inhibitor could reduce the ubiquitination level of TFAP4 and significantly prolong the half-life time of the protein.The knockdown of β-TrCP significantly inhibited the degradation of TFAP4.β-TrCP directly interacted with TFAP4 and promoted its ubiq-uitination.The mutation of E135A and S139A in TFAP4 prolonged its half-life time,and the silencing of β-TrCP promoted cell prolif-eration and migration.Conclusion SCFβ-TrCP may degrade TFAP4 through ubiquitination modification,thereby inhibiting the occurrence of colorectal epithelial-mesenchymal transformation,and then inhibiting tumor invasion and metastasis,which may provide a new idea for the treatment of colorectal cancer.
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