| Background & AimsPrimary liver cancer(also known as liver cancer)is one of the most common tumors in the world,which poses great challenges to global human health and disease burden.Due to the insidious onset and the lack of early effective diagnostic markers,the early detection rate of liver cancer is low,and the efficacy of existing advanced treatment drugs is still limited,resulting in a high mortality rate.Therefore,finding sensitive and reliable early diagnostic markers,exploring new effective treatment drugs and methods,and improving the efficacy of advanced treatment are crucial to the prevention and treatment of liver cancer.The Annexin superfamily is a class of calcium-dependent phospholipid-binding proteins.Annexin A1(ANXA1)is the first discovered member of its superfamily,and was initially widely recognized as an important "anti-inflammatory protein".In recent years,many studies have found that ANXA1 also plays an important role in the immune regulation of various inflammatory diseases,and participates in various processes necessary for tumors.However,previous tumor-related studies on ANXA1 have mainly focused on its direct role in tumor cells,and have obtained contradictory conclusions in various types of tumors,including liver cancer.Liver cancer is one of the malignant tumors most related to inflammation.The change of the microenvironment is closely related to the occurrence and development of liver cancer.However,the role of ANXA1 in the microenvironment of liver cancer is still unknown.Therefore,it is particularly necessary to fully understand the role of ANXA1 in liver cancer.Contents & MethodsWe performed bioinformatics analysis of ANXA1 expression level in liver fibrosis/cirrhosis and liver cancer tissues,and its correlation with clinicopathological features,immune cell infiltration,and immune checkpoint expression in TCGA-LIHC,GEO,and TIMER databases,mainly including:(1)The expression of ANXA1 in liver fibrosis/cirrhosis and liver cancer tissues was analyzed;(2)The correlation between ANXA1 expression and patient survival prognosis,clinicopathological features,immune cell infiltration,and immune checkpoints were analyzed;(3)The differentially expressed genes and potential pathways associated with ANXA1 enrichment in liver cancer were predicted.We constructed mouse models of liver fibrosis/cirrhosis and liver cancer,and used human liver cancer tissues to explore the expression level and localization of ANXA1,and its clinical significance in liver diseases:(1)The mouse models of liver fibrosis and orthotopic tumor implantation in the background of liver fibrosis were established to explore the expression and localization of ANXA1 in the progression of liver fibrosis/cirrhosis to liver cancer;(2)The localization of ANXA1 expression was verified by human liver cancer tissue microarray,and the relationship between ANXA1 expression and survival prognosis,immune checkpoints was analyzed.We used various cell experiments such as cytokine stimulation,sh RNA transfection,hr ANXA1 treament,and in vitro co-culture to investigate the effect of ANXA1 on macrophage polarization,the biological function of co-cultured liver cancer cells and CD8+ T lymphocytes with macrophages and its possible molecular mechanism :(1)The expression of ANXA1 in different subtypes of macrophages was explored;(2)The effect of ANXA1 expression on macrophage polarization was investigated;(3)The potential regulatory pathways of ANXA1 expression affecting macrophage polarization were explored;(4)The expression level of ANXA1 in various liver cancer cells,and the direct effect of ANXA1 expression changes on the biological behaviors of liver cancer cells such as proliferation and migration were explored;(5)The co-culture models of macrophage-liver cancer cell and macrophage-lymphocyte were established to explore the effects of ANXA1 expression changes in macrophages on biological behaviors of co-cultured liver cancer cells such as proliferation and migration,and the killing capacity of co-cultured CD8+ T lymphocytes;(6)The potential molecular regulatory mechanisms of ANXA1 expression in macrophages affecting the biological function of co-cultured liver cancer cells and co-cultured CD8+ T lymphocytes were investigated.We constructed different types of liver cancer mouse models,and used ANXA1 mimic petide Ac2-26 to explore the role of ANXA1 in the occurrence and development of liver cancer and its effect on the microenvironment of liver cancer.Further,the role of ANXA1 in the regulation of liver cancer microenvironment was clarified by depletion different types of cells in vivo:(1)The mouse models of in situ liver cancer implantation and orthotopic tumor implantation in the background of liver fibrosis were established to explore the effects of increased ANXA1 expression on the occurrence and development of liver cancer,and the enrichment of macrophages and lymphocytes in tumor tissues.(2)The reconstructed model after depletion of macrophages in mice orthotopically implanted with liver cancer was established to explore the the effect of the ANXA1 expression in macrophages on the progression of liver cancer;(3)The depletion models of macrophages and CD8+ T cells in mice with orthotopic implantation of liver cancer was established to explore the effect of ANXA1 on the microenvironment of liver cancer in vivo.Results(1)Bioinformatics analysis and histological validation showed that the expression of ANXA1 was increased in liver fibrosis/ cirrhosis and liver cancer tissues,especially in mesenchymal tissues,mainly localized in macrophages.(2)In liver cancer tissues,the expression of ANXA1 in mesenchymal cells was negatively correlated with the survival prognosis,and was positively correlated with the size of the primary tumor and the expression of PD-L1 in tumor cells.(3)The expression of ANXA1 in M2 macrophages was increased,and the increased expression of ANXA1 in macrophages promoted its polarization to M2 type.(4)The potential regulatory pathways of ANXA1 expression affecting macrophage polarization include NOTCH1,NF-κB,JAK-STAT,PI3K-AKT,ERK classical regulatory pathways.(5)The change of ANXA1 expression in liver cancer cells had no effect on proliferation and migration of liver cancer cells.(6)The down-regulation of ANXA1 expression in macrophages inhibits the proliferation and migration of co-cultured liver cancer cells,which possibly through the co-regulation of FOXO3 by the PI3K-AKT and MEK-ERK pathways.In vivo experiments demonstrated that the down-regulation of ANXA1 expression in macrophages inhibited the progression and metastasis of liver cancer.(7)The down-regulation of ANXA1 expression in macrophages promotes the killing capacity of co-cultured CD8+ T lymphocytes,which may affect the immune response by regulating the STAT3-PD-L1 pathway.(8)ANXA1 mimic peptide Ac2-26 accelerated the progression of liver fibrosis/cirrhosis to liver cancer in mice,promoted the progression of liver cancer,and led to the enrichment of M2 macrophages and the reduction of CD8+ T lymphocytes in tumor tissues.(9)hr ANXA1 promoted the progression of liver cancer in vivo,increased the accumulation of M2 TAMs and Foxp3+-Tregs cells,and decreased the ratio of CD8+T lymphocytes in tumor tissues;(10)In vivo depletion of macrophages impaired the effect of hr ANXA1 on the progression of liver cancer,while depletion of CD8+ T cells synergistically promoted the effect of hr ANXA1 on the progression of liver cancer,suggesting that ANXA1 regulates macrophages to affect liver cancer progression may be dependent on CD8+ T cells.ConclusionsANXA1 regulates macrophages to play a tumor-promoting role in the microenvironment of liver cancer.In liver cancer patients,ANXA1 expression in mesenchymal cells of adjacent tissues may be one of the predictive indicators for the clinical prognosis.Targeting ANXA1 in macrophages suppresses liver tumor growth by regulating macrophage polarization and activating the CD8+ T cell antitumor immune response.The results may provide a new target for early intervention,targeted therapy and immunotherapy of liver cancer.There are 60 pictures,6 tables and 310 references in full text(198references in main text,112 references in review)... |
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