| Research Background and Purpose Liver cancer is the fifth most common cancer worldwide,the prevention and treatment of liver cancer remain a major health issue closely related to human’s wellbeing.The liver has a complex structure,with staggered distribution of parenchymal cells and non-parenchymal cells,which together maintain the normal physiological functions of the liver.Among them,macrophages account for the largest proportion of non-parenchymal cells.As the first defense line of immune system,macrophages have strong plasticity,high heterogeneity,and exhibit different activation phenotypes in response to complex microenvironments,thus playing multiple roles in tumorigenesis and cancer development.On the one hand,the inflammatory response,phagocytosis,and antigen presentation functions of macrophages make them have anti-tumor effects;on the other hand,excessive activation of macrophages can disrupt the homeostasis of the inflammatory microenvironment and trigger a chronic inflammatory state.In the study of immunosuppressive mechanisms of tumor microenvironment,highly plastic macrophages have undergone phenotypic and functional changes,transformed into tumor-associated macrophages(TAM),and promoted the malignant progression of tumors.However,the key molecular nodes and networks that regulate phenotypic transformation of macrophages in the tumor microenvironment need to be further studied.Therefore,in-depth exploration of driving factors and mechanisms of macrophages in tumor progression,and the development of drugs that selectively regulate the immune remodeling of macrophages may become a new strategy of targeted therapy for tumor.Behind the diverse and complex etiology of liver cancer,the transformation of chronic inflammation to cancer is the common road for most tumorigenesis.Inflammation is one of the ten basic characteristics of tumors.Uncontrollable inflammation affects the evolution of tumors and can be regarded as the initiating factor and driving engine of tumor development.In uncontrolled inflammation,the maintenance of inflammatory microenvironment homeostasis is inseparable from many immune cells.The innate immune system,like macrophages and NK cells,is the first cell types to respond to stimulatory signals.These cells interact with each other and with surrounding stromal cells,and influence the inflammation state by balancing the two opposing forces of pro-inflammatory and anti-inflammatory.Macrophages are a key cell type that regulates inflammatory homeostasis.Due to high heterogeneity and plasticity,their regulation of inflammation not only exerts a positive pro-inflammatory function,but also acts as a "main force" to inhibit excessive inflammatory activation.When the autoregulatory mechanism is damaged,under the hit of external pathogenic factors,the homeostasis of inflammation is broken,and uncontrollable inflammation drives the malignant transformation to form tumors.However,in-depth understanding of the pathological mechanisms and node molecules of the malignant transformation of liver inflammation remains indefinite,and the specific functions and regulatory mechanisms of macrophages in it are still unclear.Oncoprotein Gankyrin(also known as PSMD10,p28)is a key node regulating uncontrollable inflammatory malignant transformation.It was originally discovered in primary liver cancer tissue and plays an important driving role in the formation of liver cancer.Gankyrin is a protein with a molecular weight of 27 KD,which contains seven ankyrin repeats(ANK).It can bind to the 26 S proteasome regulatory subunit and the 19 S subunit,accelerating ubiquitination and degradation of p53 and phosphorylation of Rb to inhibit apoptosis and promote cell cycle,thereby accelerating the malignant progression of tumors.Based on the previous research of our group,Gankyrin in hepatocytes could sense upstream IL1β signals in the microenvironment,activate downstream AKT,JNK,VEGF and other tumor-related pathways,causing tumor angiogenesis,epithelial-mesenchymal transition,invasion and migration,and drug resistance,then promote the tumorigenesis and liver cancer development.However,the above studies are based on the results of cytology.The role of Gankyrin in vivo is not well understood,especially in non-parenchymal cells.Therefore,our laboratory constructed Gankyrin conditional knockout mice cooperating with Nanjing University.Based on this,we successively cultivated systemic Gankyrin knockout and hepatocyte-specific Gankyrin knockout mice.Through chemically induced liver cancer models,we analyzed the two different roles of Gankyrin in liver parenchyma cells and non-parenchymal cells,respectively,during the occurrence and development of liver cancer.Then,myeloid knockout mice were constructed.Combined with cytological experiments,we deeply explored the regulation of Gankyrin on macrophage function,and thoroughly elucidated the role and mechanism of Gankyrin in regulating in vivo inflammation and hepatitis-carcinoma transformation,which would provide new therapeutic strategies and molecular targets for the precise prevention and treatment of liver cancer.Research Methods 1.Construct Gankyrin knockout mouse in liver cells,systemic cells and myeloid cells,respectively,and comprehensively utilize various HCC models induced by DEN alone,DEN combined with CCl4 and oncogenes to study the effect of Gankyrin knockout in different cell types on HCC formation and progression.2.Isolate liver immune cells from HCC-bearing mice,and compare the changes in immune cell populations in the tumor microenvironment caused by Gankyrin knockout in myeloid cells by flow cytometry 3.Clarify the effect of Gankyrin in different immune cell groups on the occurrence and development of liver cancer by the method of tumor subcutaneous implant with depletion of neutrophils,macrophages and CD8+ T cells,respectively.4.The mouse bone marrow-derived macrophage BMDM was induced in vitro into TAM and co-subcutaneously implanted with hepatoma cells hep1-6 into WT mice,to further study the effect of Gankyrin deletion in macrophages on tumor growth.5.The expression patterns of PD-1/PD-L1 in macrophages were measured by flow cytometry in HCC mice,and the regulation effect of Gankyrin on PD-1 expression in macrophages was further clarified in BMDM combined with Gankyrin rescue experiment.6.Using LPS combined with D-Gal N-induced acute liver injury model and the autoinflammatory state of aging mice,to study the effect of Gankyrin deletion in mouse macrophages on inflammation in vivo.7.Construct Gankyrin overexpressing and knockdown cells in BMDM and human monocyte cell line THP1,respectively,and illustrate the regulation of Gankyrin on the macrophages inflammatory response in vitro induced by inflammatory stimulation and co-culture with tumor cells.8.Transcriptome sequencing was used to analyze the expression of inflammatory factors and related pathway gene transcription levels in macrophages after Gankyrin knockout,and GSEA was used to analyze the activation of NF-k B signal pathway.9.Co-immunoprecipitation was used to study the physical interaction of Gankyrin and p65 in BMDM.Sub-localization of p65 in macrophages was shown by IF.Combined with the verification of the overexpression of Gankyrin and its mutant,the regulatory effect of Gankyrin on the NF-k B pathway was clarified.10.Tomatidine,a p65 nuclear translocation blocker,was applied in vitro and in vivo to further clarify the mechanism of Gankyrin deficiency in the regulation of inflammation in macrophages.11.Tomatidine inhibits the activation of NF-k B pathway.The relationship between the regulation of Gankyrin on PD-1 expression in macrophages and the activation of NF-k B pathway was studied by utilizing Tomatidine.12.PBMC from healthy people and HCC patients,was used to analyze the correlation between Gankyrin and IL1 b.13.Isolate TAM from clinical HCC samples,and co-subcutaneously implanted into NSG mice with human HCC cell line Hep G2,and analyze the correlation between the level of Gankyrin in macrophages and tumor growth;change the level of Gankyrin in TAM in vitro,and verify that the level of Gankyrin in TAM has a significant effect on tumor growth.Research Result 1.During hepatocarcinogenesis,hepatocyte-specific knockout of Gankyrin exerts a tumor suppressor effect,while Gankyrin systemic knockout exhibits a tumor-promoting effect.2.Gankyrin deletion in myeloid cells altered the composition of immune cells in the tumor microenvironment,with a significant increase in neutrophils and macrophages,and a decrease in the proportion of CD8+ T cells.3.Through the elimination of neutrophils,macrophages and CD8+ T cells in mice,it was found that the Gankyrin in macrophage mainly plays a tumor suppressor role.Co-subcutaneously implant of Gankyrin-deficient BMDM with hepatoma cells significantly promoted tumor growth.Gankyrin could inhibit the phagocytic function of macrophages.4.Gankyrin deletion could promote the expression of PD-1 in macrophages.5.The inflammation level in Gankyrin myeloid knockout mice was enhanced in vivo,and the level of inflammatory response in macrophages with Gankyrin deletion or knockdown was significantly increased in vitro.Gankyrin in macrophages could inhibit the excessive activation of inflammation.6.Transcriptome and GSEA analysis revealed that Gankyrin-deficient macrophages significantly activated NF-k B pathway,suggesting that Gankyrin in macrophages could inhibit the activation of NF-k B pathway.7.Gankyrin in macrophages could inhibit the activation of NF-k B pathway by binding with p65 to prevent nuclear translocation.Tomatidine inhibited inflammatory hyperactivation induced by Gankyrin deletion in macrophages in vivo and in vitro.8.Inhibition of NF-k B pathway activation can reduce the level of PD-1 in Gankyrin-deficient macrophages,suggesting that the Gankyrin-NF-k B-PD-1 regulatory axis in macrophages plays an important role in the development of hepatocellular carcinoma in mice.9.On the clinical level,Gankyrin expression in PBMC of HCC patients was significantly lower than that of healthy people.Gankyrin levels in tumor-associated macrophages of HCC tissues were negatively correlated with tumor growth.By altering Gankyrin levels in TAMs,TAMs with low Gankyrin expression significantly promoted tumor growth.Research Conclusion Gankyrin in hepatocytes and myeloid cells plays different roles in hepatocarcinogenesis.Gankyrin in macrophages plays a major tumor suppressor function and regulates the expression of PD-1 in macrophages.Macrophage Gankyrin negatively regulates the inflammatory response of macrophages,thereby inhibiting the malignant transformation of hepatitis-carcinoma.Mechanistically,macrophage Gankyrin can bind to NF-k B p65 subunit to prevent its entry into the nucleus,inhibit the expression of its downstream inflammatory factors and PD-1,and then inhibit tumor progression.This study is the first to discover the tumor suppressor mechanism of the oncogene Gankyrin,revealing the inhibitory effect of Gankyrin in the innate immune inflammatory response of macrophages.The discovery of the Gankyrin-NF-k B-PD-1 axis in macrophages provides new insight and promising therapeutic targets for the prevention and treatment of liver cancer. |
PDF Full Text Request