Cyclin G2 In Macrophages Triggers CTL-mediated Antitumor Immunity And Antiangiogenesis Via Interferon-gamma

Objective:In the tumor microenvironment,the interaction between tumor cells,stromal cells,and immune system cells causes the cellular components of the tumor microenvironment to change in a direction that promotes tumor malignancy.The tumor microenvironment plays a key role in the progression of cell malignancy,immune escape and therapeutic resistance.As the most abundant cell type in tumor microenvironment,tumor-associated macrophages are the central mediators regulating tumorigenesis and tumor microenvironment.Macrophages affect vascular endothelial cells,dendritic cells,fibroblasts and T cells by producing cytokines,chemokines and growth factors.The immunosuppressive microenvironment caused by macrophages is a key cause of tumor treatment failure.Therefore,remodeling tumor-associated macrophages to change tumor microenvironment has become an effective strategy for cancer treatment.IFN-γis a key mediator to promote anti-tumor immunity and plays a key role in anti-tumor immunity.IFN-γcan induce M1-type polarization of macrophages and inhibit tumor growth,but the specific mechanism remains to be clarified.Cyclin G2 is an abnormal cyclin encoded by CCNG2 gene,which can negatively regulate cell cycle and hinder the process of cell cycle.Cyclin G2 acts as a tumor suppressor in various cancer cells,but its role in immune cells has not been reported.In view of the central regulatory role of macrophages in the tumor microenvironment,this study aims to investigate the role of cyclin G2 in macrophages on tumor progression in vitro and in vivo and its molecular mechanism under IFN-γ,to provide a theoretical basis for targeted cancer therapy.Methods:1.Detection of cyclin G2 expression in different types of macrophages:THP-1 cells,human peripheral blood monocytes and mice bone marrow derived monocytes were induced into different types of macrophages,and the expression of cyclin G2 was detected by RT-q PCR and Western blot.2.To determine the effect of cyclin G2 in macrophages on tumor growth in vivo:MC38 or LLC cells were mixed with bone marrow-derived macrophages from WT or Ccng2^-/-C57BL/6 mice and subcutaneously inoculated into the right flank of the mice.Appropriate time intraperitoneal injection of IFN-γ,measurement of tumor size on a regular basis.3.To evaluate the effect of cyclin G2 in macrophages on CTL chemotaxis and cytotoxic function:Immunohistochemical staining,immunofluorescence staining and flow cytometry were used to detect the number of CTL in mice tumor tissues.In addition,the biological effect of cyclin G2 in macrophages on CTL chemotaxis was evaluated by CTL chemotaxis assay,and the expressions of CTL granzyme B and perforin were detected by flow cytometry.4.To evaluate the effect of cyclin G2 in macrophages on angiogenesis:Immunohistochemical staining was used to detect the number of blood vessels in mice tumor tissues.Tube formation assay was used to investigate the effect of cyclin G2 in macrophages on tube formation ability of vascular endothelial cells.5.Verify that CXCL9 is the downstream effector molecule of cyclin G2:The secretion level of CXCL9 after cyclin G2 knockout was detected by enzymed-linked immunosorbent assay（ELISA）,and recombinant protein CXCL9 was added to macrophage conditioned medium,and verified by CTL chemotaxis assay and vascular endothelial cell tube formation assay.6.Explore the mechanism of cyclin G2 regulating CXCL9 secretion:CXCL9 m RNA expression in macrophages was detected by RT-q PCR.Protein expression of STAT1 and p-STAT1（Y701）was detected by Western blot.STAT1 expression in cytoplasm and nucleus was evaluated by cytoplasmic separation assay and immunofluorescence staining.7.Confirm that PP2Ac is a key molecule involved in cyclin G2 regulation of STAT1content:The relationship between cyclin G2,STAT1 and PP2Ac was studied by immunoprecipitation（IP）.After PP2Ac knockdown,protein expression of STAT1 and p-STAT1（Y701）was detected by Western blot.STAT1 expression in cytoplasm and nucleus was evaluated by cytoplasmic separation assay and immunofluorescence staining.Results:1.Cyclin G2 expression was upregulated in macrophages after IFN-γtreatment.2.IFN-γinhibited lung cancer and colon cancer via cyclin G2:Cancer cells were mixed with bone marrow-derived macrophages from WT or Ccng2^-/-mice and subcutaneously injected into the right flank of mice to establish a subcutaneous tumor model.compared with WT group,tumor weights and volumes were significantly increased in Ccng2^-/-group.There was an increase in the staining for Ki-67 in the tumor tissues from the Ccng2^-/-group compared to the WT group.Results showed that after the knockout of cyclin G2 in macrophages could attenuate the tumor suppressive effects of IFN-γ.3.Loss of cyclin G2 in macrophages could effectively reduce CTL recruitment:compared with tumor tissues from WT group,the number of CTLs in the tumor tissues from Ccng2^-/-group was significantly lower.CTL chemotaxis experiments showed that the chemotactic ability of CTL in Ccng2^-/-group was weakened.Results showed that knockout of cyclin G2 in macrophages could effectively reduce CTL recruitment,attenuating the tumor suppressive effects of IFN-γ.4.Loss of cyclin G2 in macrophages could effectively promote tumor angiogenesis:compared with tumor tissues from WT group,Ccng2^-/-group significantly increased the number of blood vessels.The tube formation experiments showed that the tube formation of vascular endothelial cells in Ccng2^-/-group was promoted.Results showed that knockout of cyclin G2 in macrophages could effectively promote tube formation,attenuating the tumor suppressive effects of IFN-γ.5.Cyclin G2 regulates CTL recruitment and vascular endothelial cell tube formation by CXCL9:compared with the WT group,CXCL9 secretion in Ccng2^-/-group decreased.Restore experiment verify that CXCL9 was the downstream effector of cyclin G2.6.Cyclin G2 upregulated CXCL9 m RNA expression by promoting STAT1 nuclear translocation:compared with the WT group,CXCL9 m RNA expression was also downregulated in Ccng2^-/-group after IFN-γtreatment.In addition,after cyclin G2knockdown,the protein expression of p-STAT1（Y701）was downregulated,and the nuclear content of STAT1 was decreased.The results showed that cyclin G2 regulated CXCL9 by STAT1.7.STAT1 nuclear translocation mediated by cyclin G2 is dependent on PP2Ac:Both STAT1 and cyclin G2 interact with PP2Ac under IFN-γstimulation.And when cyclin G2is knocked down,STAT1 can bind more PP2Ac.In addition,compared with the control group,knockdown PP2Ac upregulated the expression of p-STAT1（Y701）and restored the nuclear STAT1 content.The results showed that cyclin G2 increased the nuclear content of STAT1 in a PP2Ac dependent manner.Conclusion:1.IFN-γupregulates the expression of cyclin G2 in macrophages and exerts its inhibitory effect on colon and lung cancer through cyclin G2.2.Cyclin G2 promotes the nuclear translocation of STAT1 in a PP2Ac-dependent manner to upregulate the m RNA expression of CXCL9,which in turn increases the secretion of CXCL9,leading to the recruitment of CTL and the inhibition of angiogenesis to suppress tumor growth.