The Role And Mechanism Of UBC9 Inhibiting Tumor-associated Macrophage Activation And Subsequently Promoting Prostate Cancer Progression

PurposeProstate Cancer（PCa）is the most common malignant tumor in male.However,PCa has a poor response to immunotherapy.Tumor-associated macrophages（TAMs）are critical in balancing antitumor and pro-tumor responses,which affect the outcome of standard-of-care therapeutics such as chemotherapy,radiotherapy and immune checkpoint blockade（ICB）.The role of TAMs along with the regulatory mechanisms underlying distinct macrophage activation states remain poorly understood in PCa.SUMOylation（small ubiquitin modifier）is one type of post-translational modification that covalently adds SUMO into lysine residues of target proteins.However,whether SUMOylation can regulate the activation and functions of TAMs during PCa development remains unclear.Materials And MethodsBioinformatics and clinical samples were conducted to predict and verify the correlation between UBC9 and PCa pathological features,prognosis and immune function.A SUMOylation inhibitor and macrophage Ubc9-specific knockout mouse model to detect PCa subcutaneous tumor-bearing progression,immune cell infiltration levels and function.Macrophages adoptive transfer in B16-OVA melanoma and the co-culture assay of macrophages with OT-I mouse-derived CD8⁺T cells investigate the effects of UBC9-mediated macrophage activation and antigen presentation on T cell proliferation and activation.Flow cytometry screening,RNA-seq and co-immunoprecipitation were carried to verify the mechanism of UBC9 in tumor-associated macrophages.To that end,we compared the efficacies between the combinatorial therapy and 2-D08,anti-PD-1monotherapies in the prostate tumor model.ResultsHerein,we reported that PCa growth in SUMOylation inhibitor-treated and macrophage-specific Ubc9^-/-mouse was significantly reduced compared to the wild-type littermates,which was partially ascribed to the augmented CD8⁺T response.Molecular analysis revealed that the signal transducer and activator of transcription 4（STAT4）is a UBC9-mediated small SUMOylation target at lysine residue 350.Site-directed mutation of the SUMOylation site（K350R）enhanced STAT4 protein stability,its nucleus translocation and the transcription of downstream antitumor cytokines including IFN-γand TNF-α.Importantly,administration of SUMOylation inhibitor,2-D08,promoted the antitumor effect of TAMs,while adversely increasing the expression of PD-1 in CD8⁺T cells.We further demonstrated that a combination of 2-D08 and anti-PD-1 antibody remarkably suppressed PCa progression.ConclusionsOur data demonstrated that ablation of UBC9 can reverse the immunosuppressive effect of TAMs via promoting STAT4-mediated macrophage activation and macrophage/CD8⁺T cell crosstalk,suggesting that targeting UBC9 can reprogram TAMs into an anti-tumoral phenotype.The SUMOylation inhibitor had synergistic effect with anti-PD-1 for the inhibition of prostate tumor growth...