Study On The Role And Mechanism Of ANLN-modulated Cytokinesis In The Progression Of Hepatocellular Carcinoma

Background Hepatocellular carcinoma(HCC)is a common malignancy,which seriously endangers life and health.Surgery-based multi-discipline therapy is the main therapeutic option for HCC,but the curative effect is not ideal.Therefore,novel therapeutic strategies are urgently needed in the treatment of HCC.Continuous cell division is a characteristic feature of cancer.Cytokinesis is the final stage of cell division that generates two daughter cells.Accurate cytokinesis ensures the rapid proliferation of cancer cells,while cytokinesis failure leads to multinucleated cells and polyploidy,which affect cell proliferation.Thus,targeting cytokinesis-related elements is a promising strategy for HCC treatment.Objectives1.To investigate the role of Anillin actin binding protein(ANLN)in the tumorigenesis and progression of HCC.2.To elucidate the molecular mechanism of ANLN-modulated cytokinesis in promoting HCC proliferation.3.To explore the correlation between the expression of ANLN and the clinicopathological characteristics and prognosis of HCC patients;4.To investigate the relationship between ANLN-modulated cytokinesis and cell polyploidy in the progression of HCC.5.To study the role of ANLN in the efficacy of sorafenib treatment in HCC.6.To provide the theoretical basis on targeting cytokinesis for HCC therapy.Methods1.We screened the HCC-related genes based on TCGA database and clinical samples.The correlation between gene expression and HCC patients’ clinicopathological characteristics was studied.2.Liver conditional ANLN knockout mice were constructed to investigate the role of ANLN in the tumorigenesis of HCC by utilizing HCC hydrodynamic venous injection model.3.The effect of ANLN on cell proliferation was studied in vitro by cell proliferation,cell cycle,cell polyploidy,cell apoptosis and DNA damage detection experiments.4.The effect of ANLN on tumor growth was studied in vivo using HCC subcutaneous xenograft model and HCC orthotopic xenograft model.5.Immunofluorescence,co-immunoprecipitation assay,pull-down assay,and phosphorylation site mutation assay were used to study the molecular mechanism of ANLN in regulation of cytokinesis.6.RNA sequencing and HCC PDX models were performed to investigate the correlation between ANLN expression and sorafenib treatment efficacy in HCC.Results1.ANLN was highly expressed in HCC tissues compared with paracancerous tissues.The expression of ANLN in HCC tissues was correlated with tumor histological grade,tumor size,TNM stage and patients’ prognosis.The expression of ANLN in HCC tissues was negatively correlated with the proportion of binucleated polyploidy.2.Liver conditional ANLN knockout inhibits the growth of tumor during HCC tumorigenesis in mice.3.ANLN knockdown inhibits the proliferation of HCC cells in vitro.ANLN inhibition promotes G2/M phase arrest and leads to the formation of multinucleated cells and polyploidy due to cytokinesis failure,ultimately inducing DNA damage and apoptosis.4.ANLN interacts with RACGAP1 to promote its phosphorylation.ANLN can act as a scaffold to promote the interaction between PLK1 and RACGAP1.ANLN activates Rho A by promoting PLK1-mediated RACGAP1 phosphorylation in regulation of cytokinesis.5.ANLN knockdown inhibits ERK activation and enhances the sensitivity of HCC cells to sorafenib treatment.The level of ANLN in the tumors of HCC patients correlates with tumor response to sorafenib treatment.Conclusion1.The expression of ANLN is correlated with the clinicopathological characteristics of tumor,patients’ prognosis and the proportion of binucleated polyploidy in HCC.2.Liver conditional ANLN knockout can inhibit HCC tumorigenesis,and knockdown of ANLN inhibits the growth of HCC in vivo and in vitro.3.ANLN inhibition leads to cytokinesis failurer,multinucleated cell formation,DNA damage and apoptosis in HCC cells.4.ANLN can act as a scaffold to promote PLK1-mediated RACGAP1 phosphorylation,which then activates Rho A to ensure the fidelity of cytokinesis in HCC cell.5.ANLN knockdown increases the sensitivity of HCC cells to sorafenib,and ANLN can be used as a biomarker to predict tumor response to sorafenib in HCC treatment.