The Effect And Mechanism Of TRIM59-regulated Macrophages In Inhibiting Tumor Growth And Metastasis

As an important member of the body’s innate immunity,macrophages play a key role in all stages of health or disease states.In tumors,the infiltration rate of macrophages is as high as 70%,so the role of macrophages in tumors is critical.Macrophages kill tumors in a variety of ways.Firstly,macrophages kill tumor cells by releasing TNF-α,IL-6 and other pro-inflammatory factors.Secondly,macrophages can directly engulf and kill tumor cells.In addition,macrophages,as important antigen-presenting cells,can activate adaptive immune responses.However,the mechanism of macrophages’killing effect on tumors is still unclear.Therefore,the mechanism of macrophages killing tumor has become a key scientific problem to be solved in the field of tumor immunity.Previous research by our group found that BCG-activated macrophages have obvious antitumor effects,especially on fibrosarcoma MCA207 cells,and this process is closely related to certain protein molecules unique to the surface of cell membranes.After proteomics detection and comparison of membrane proteins of BCG and thioglycolate（TG）-induced activated macrophages,we obtained 454 proteins that were up-regulated on the surface of BCG-activated macrophage membranes.Based on bioinformatics predictions,we selected several proteins that may be involved in the directional differentiation of macrophages and cell-to-cell contact and whose functions are not yet clear.TRIM59 is one of them.TRIM59,also known as mouse ring finger protein 1（Mrf1）,belongs to the evolutionarily conserved tripartite motif（TRIM）family of proteins.The TRIM family is an evolutionarily conserved gene family that involves many key processes,such as resistance to viral infections,cell differentiation,and cancer.However,the functions of most TRIM family members have not been determined to date.They usually have the typical RING finger,B box and coiled-coiled（RBCC）domains,which have the function of mediating protein-protein interaction.In addition to the RBCC domain,TRIM13 and TRIM59 also have transmembrane regions.These structural features potentiate TRIM59 as a possible mediator in molecular interactions between BAM and target cells.Studies have shown that TRIM59 was up-regulated in a variety of tumors,played a role in promoting tumor cell proliferation and migration,and has long been regarded as a candidate tumor promoter gene.However,some literatures have shown that TRIM59 can regulate the innate immune response.Our laboratory also found that TRIM59 is involved the cytotoxicity of BCG-activated macrophages.Although TRIM59 regulates the innate immune response,the function of TRIM59 in macrophages has not yet been studied.In order to reveal the role and significance of TRIM59 expression in macrophages,we carried out research from the following three parts:Part 1:Effect of TRIM59-regulated macrophages on tumorigenesis and developmentFirstly,we analyzed the expression of TRIM59 in various tumor cells.It was found that not all tumor cells had a high expression of TRIM59,and the expression of TRIM59 was low in fibrosarcoma and melanoma cells.Macrophage-specific TRIM59knockout mice（TRIM59-CKO）revealed that tumors were significantly enlarged in the absence of TRIM59 in macrophages.In addition,the absence of TRIM59 in macrophages leads to a reduction in tumor local macrophage infiltration,and macrophages are mainly M2 type.In addition,cytotoxic T cells and B cells in the spleen and lymph nodes were not affected by TRIM59.These results indicated that TRIM59-expressing macrophages exhibited a major cytotoxic effect on tumors.In vitro,we co-cultured macrophages with highly expressing TRIM59 fixed with 1%paraformaldehyde and various tumor cells.Macrophages with highly expressing TRIM59 had a significant killing effect on MCA207 and this effect was significantly weakened after TRIM59 antibody treatment.The macrophage culture supernatant with high expression of TRIM59 was co-cultured with various tumor cells.We found that the culture supernatant of macrophages with high expression of TRIM59 had little killing effect on MCA207 fibrosarcoma cells,but we also saw more obvious cytotoxicity to tumors such as melanoma.This indicates that macrophages with TRIM59 may kill different tumors in different modes.Part 2:Direct killing effect and mechanism of TRIM59-regulated macrophages on fibrosarcomaThe results showed that BCG could indeed cause high expression of TRIM59 in macrophages,and that TRIM59 was mainly expressed on the macrophage membrane.Based on the results of the first part,we further explored the killing effect of TRIM59-regulated macrophages on MCA207 fibrosarcoma cells.It was found that macrophages overexpressing TRIM59 significantly inhibited the growth of MCA207fibrosarcoma and could induce tumor cell apoptosis.In addition,the PI3K-Akt pathway of MCA207 co-cultured with macrophages highly expressing TRIM59 was significantly inhibited,and the activation of the PI3K-Akt pathway in MCA207 was not affected after co-culture with TRIM59-CKO macrophages.These results indicate the important role of TRIM59 as an antitumor effector molecule for BCG-activated macrophages,and propose new therapeutic targets for the treatment of fibrosarcoma.Part 3:Effect and mechanism of TRIM59-regulated macrophages on melanoma metastasisIn the first part of the results,we found that melanomas in TRIM59-CKO mice increased distinctly,and that macrophage culture supernatants that overexpressed TRIM59 had significant cytotoxic effects on melanoma,but the mechanism is still unclear.We first tested the phenotype of macrophages in tumors of TRIM59-CKO mice,and found that macrophages in B16 tumors also mainly showed M2 phenotype.To mimic the tumor microenvironment in mice,we polarized macrophages from control mice and TRIM59-CKO mice to M2 phenotype in vitro and collected their culture supernatants conditioned media（CM）,respectively.The results showed that the supernatant of TRIM59^-/--M2 phenotype macrophages significantly promoted the migration and invasion of B16-F0 and B16-F10 cells,and the level of TNF-αin the supernatant of TRIM59^-/--M2 phenotype macrophages increased about 15 times.Through transcriptome sequencing and analysis,we found that TRIM59^-/--M2macrophage supernatant promoted the expression of Mmp-9 and Madcam1 in melanoma cells,which is TNF-αdependent.By analyzing the possible downstream pathways,it was found that they activated the tumor’s ERK signaling pathway.In addition,knockdown of Mmp9 and Madcam1 in B16-F10 cells inhibited the epithelial-mesenchymal transition process,and reduced the growth of subendothelial tumors and the formation of metastatic lung nodules.These data indicate that the weakened expression of TRIM59 in tumor-associated macrophages plays a role in promoting melanoma proliferation,migration,and invasion.In addition,these results also suggest the key role of TRIM59 as a potential regulator of tumor metastasis in macrophages,making TRIM59 a new target for cancer immunotherapy.