Study On The Role And Mechanism Of FAK In Anti-PD1 Treatment Of Primary Liver Cancer Formation Resistance

BackgroundIn recent years,immunotherapy has achieved remarkable curative effect in the treatment of some tumors,which has opened a new chapter for the treatment of tumors.Programmed cell death protein 1(PD1)has become the focus of tumor immunotherapy.However,when it is used in the treatment of Hepatocellular carcinoma(HCC),the effect is not ideal.A few patients had significant efficacy,while most patients had little efficacy,and the proportion of PD1 antibody effect was less than 20%,which greatly affected the application of PD1 antibody in HCC treatment.Most studies have shown that the poor therapeutic effect of HCC may be related to the insensitivity or resistance of HCC to PD1 antibody.Therefore,how to enhance the sensitivity of PD1 antibody and reduce its resistance is the key to improve the therapeutic effect.Focal adhesion kinase(FAK),an intracellular Protein Tyrosine kinase(PTK),is overexpressed and activated in a variety of cancer cells and is closely related to the occurrence,development,invasion and metastasis of tumors.FAK also regulates the expression of inflammatory genes,releases chemokines and cytokines,changes the Tumor microenvironment(TME),and promotes immune evasion and immunotherapy resistance.FAK can be used as a potential target for Tumor immunotherapy.FAK inhibitors(VS-4718)can inhibit the phosphorylation of FAK at Y397 and improve the tumor immune microenvironment,possibly synergistic with PD1 antibody.Therefore,the combination of FAK inhibitor and PD1 antibody may be an effective combination in the treatment of HCC.ObjectivesThe correlation between the expression of FAK in human HCC tissues and the prognosis and survival of patients as well as the immune microenvironment was analyzed.The possible mechanism of FAK resistance in PD1 antibody treatment of HCC was analyzed.The efficacy of FAK inhibitor(VS-4718)combined with PD1 antibody in the treatment of primary HCC in mice was also studied,providing new ideas and new targets for improving the efficacy of PD1 antibody in the treatment of liver cancer.Methods1.The relationship between FAK and prognosis and immune microenvironment was analyzed by bioinformatics method using TCGA database.The expression of FAK in clinical samples of HCC patients from TCGA database was analyzed.Then,according to the median expression level of FAK,The HCC samples were divided into FAK-high and FAK-low groups,and the relationship between The expression of FAK and the prognosis and survival of patients was studied.Differential gene expression(DEG)analysis was performed in FAK-high and FAK-low groups.DEG analysis results of FAK-high and FAK-low patients with HCC were analyzed by gene function enrichment analysis(GO).Then,FAK and immune cells(CD8+ T,Tregs,M0,M2,CAFs and MDSCs)were analyzed by TIMER2.0immunoassay website.2.The expression changes of FAK inhibitors and PD-L1 were observed at the cellular and molecular level.Two human HCC cell lines CLC1 and CLC5 were treated with FAK inhibitors(VS-4718).Then,the expression of PD-L1 in cell line 2was detected by q RT-PCR,Western-blot and immunofluorescence.3.To observe the synergistic effect of FAK inhibitor alone or combined with PD1 antibody in the treatment of HCC in mice and explore the related mechanisms.Modeling group and tumor observation: Primary mouse HCC model was induced by C-Met /β-catenin.Then,HCC mice were randomly divided into two groups and treated with corresponding drugs for 2 weeks.After sampling,the liver weight and the ratio of liver weight to body weight of mice were detected and counted,and the growth of liver tumor of mice in each group was observed.Tumor growth and immune cell infiltration were verified at tissue cell level: the proliferation,apoptosis,fibrosis and immune cell infiltration of liver tumor in mice in each group were detected by immunohistochemistry,immunofluorescence and flow cytometry.q RT-PCR was used to detect the expression of recruitment or polarization molecules in macrophages and Tregs cells in HCC tissues of mice in each group.Protein and RNA levels were used to verify the effect of FAK inhibitor on PD-L1 expression: The expression of PD-L1 in HCC tissues of mice in each group was detected by q RT-PCR,Western-blot and immunofluorescence.Statistical analysis: Graph Pad Prism 8.0.2 software was used for statistical analysis.Data were expressed as mean ± standard deviation(SD).Student’s T test was used to compare the two groups.Multiple groups were compared using ANOVA.p<0.05 was considered statistically significant.Results1.FAK is overexpressed in human HCC tissues,and is associated with over survival(OS)(p =0.049)and Progress free interval(PFI)(p =0.027).The higher the expression of FAK,the worse the prognosis of HCC patients.Both OS and PFI are short;Functional enrichment analysis of FAK-related genes showed that FAK was mainly related to immune response and antibacterial response.The expression of FAK in human HCC was inversely proportional to the number of CD8+ T cell infiltrates(Rho=-0.17;p=1.85e-2).It is proportional to the number of infiltrates of Tregs(Rho=0.28;p=1.16e-7);M0(Rho=0.292;p=3.16e-8);M2(Rho=0.233;p=1.19e-5);CAFs(Rho=0.22;p=3.67e-5)and MDSCs(Rho=0.363;p=3.02e-12).2.The expression of PD-L1 in both HCC cell lines CLC1 and CLC5 was significantly increased after treatment with FAK inhibitor.3.The liver weight of the FAK inhibitor group was relatively lower than that of the placebo group(P<0.01)and lower liver weight/body weight(p<0.001);HE staining showed that the liver structure was relatively normal and the tumor area was small.The number of PCNA positive cells in HCC tissues was small.The number of inhibitory immune cells(Tregs and macrophages)was small,but the number of immune effector cells(CD8+ T cells)was increased.4.The liver weight and liver weight/body weight of mice in the FAK inhibitor and PD1 antibody combination group were significantly lower than those in the placebo group and the single-drug group;The number of PCNA positive cells was less,Sirius red staining and a-SMA positive expression were also less.The number of immunoeffector cells(CD8+ T cells)increased,the number of inhibitory immune cells(Tregs and macrophages)decreased,and the proportion of M2/M1 macrophages decreased.Macrophage recruitment/polarization molecules and Tregs recruitment molecules decreased;PD-L1 expression was increased in HCC in the FAK inhibitor monotherapy and combination groups compared with the placebo group and PD1 monotherapy group.Conclusion1.The FAK expression is increased in human HCC tissues relative to adjacent and normal liver tissues and is associated with poor prognosis in HCC patients.2.The expression of FAK is associated with immune cell infiltration in HCC,and the number of effector T cells in patients with increased F A K expression.This may be a possible cause for the resistance of FAK in PD1 antibody treatment in HCC.3.The experimental results in mice show that the combination of FAK inhibitor and PD1 antibody has some synergistic effect in HCC treatment,which can more effectively promote the apoptosis of HCC cells and inhibit the proliferation of mouse HCC cells.FAK inhibitor were able to enhance the therapeutic sensitivity of mouse HCC to PD1 antibodies,probably because FAK inhibitors enhanced PD 1 antibody treatment for HCC effects by improving immune cell infiltration in HCC,reducing liver fibrosis,and induction of P D-L1 expression.