Role And Mechanism Of ANXA1-derived Peptide On Inhibiting Immune Evasion In Multiple Cancers

Background and objectives:The programmed cell death 1(PD-1)and programmed cell death ligand 1(PD-L1)interaction is an important immune checkpoint mediating tumor immunosuppression that has become one of the major targets in anticancer immunotherapy.Immune checkpoint inhibitors(ICIs)therapy using neutralizing antibodies against PD-1/PD-L1 reactivates the inactivated T cells,and recovers the response of cytotoxic T cells to tumors in the tumor microenvironment to kill tumor cells,and has displayed significant clinical benefits for treating multiple cancers.However,the accumulated data showed that fewer than 30%of cases across multiple types of cancers are responsive to ICIs therapy,and a significant portion of cancer patients do not benefit from ICIs therapy.Therefore,developing an alternative strategy to target PD-1/PD-L1immune checkpoint will bring new options for cancer immunotherapy.Our previous study showed that ANXA1-derived peptide(EYVQTVKSSKG),named as A11,dramatically suppresses nasopharyngeal carcinoma cell growth in vitro and in immune-deficient nude mice.To identify the novel A11 peptide-targeting proteins,we recently used biotin pull-down and mass spectrometry analysis to search proteins interacted with A11 peptide in the cancer cells,and found that PD-L1 interacted with A11.In this study,we investigated the effects of A11 peptide on PD-L1 protein levels and tumor immunosuppression in melanoma,and breast and lung cancer.Materials and Methods:(1)Biotin pull-down and MS analysis were performed to identify the proteins interacting with A11 peptide in cancer cells;(2)Western blot and flow cytometry were used to detect the expression level of PD-L1 in the human and mouse breast cancer,lung cancer and melanoma cells treated with A11 peptide respectively,and cell-penetrating peptide(YGRKKRRQRRR)(CPP)-treated cancer cells were served as control;(3)T cell-mediated tumor cell killing assay,flow cytometric analysis of cell apoptosis and ELISA were performed to analyze whether A11 peptide enhances the ablity of T cell killing human MDA-MB-231 breast cancer cells,human H460 lung cancer cells and human A375 melanoma cells,and the activity of T cells;(4)The subcutaneous xenografts of mouse 4T1,LLC and B16F10 cancer cells were established in the immune-deficient and immune-competent mice,which were treated with A11 peptide and/or PD-1 mAb,and the xenograft tumor growth and the survival time of mice were analyzed;(5)The effects of A11 and/or PD-1 mAb on CD8~+T cells and their activity were detected by flow cytometric analysis,and immunofluorescent and immunohistochemical staining.Results:(1)A11 peptide bound to PD-L1 protein in the human and mouse breast cancer cells,lung cancer cells,and melanoma cells,as well as exogenous PD-L1 in the HEK293 cells transfected with PD-L1expression plasmid.(2)A11 peptide dramatically decreased PD-L1 levels in human and mouse breast cancer cells,lung cancer cells,and melanoma cells.(3)A11 peptide sensitized human MDA-MB-231 breast cancer cells,H460 lung cancer cells and A375 melanoma cells to T cell-mediated killing as compared to control peptide;coculture with the three types of canncer cells reduced IFN-γlevels secreted by activated T cells,and its levels could be reversed by A11 peptide;coculture with the three types of cancer cells increased apoptotic rate of Jurkat T cells,which could be recovered by A11 peptide;coculture with the three types of cancer cells reduced IL-2 levels of secreted by activated Jurkat T cells,which could be reversed by A11 peptide.(4)The inhibitory effect of A11 peptide on the xenografts was significantly stronger in the immune-competent mice than that in the nude mice,and the survival time of immune-competent mice was also significantly longer than that of nude mice.(5)A11 peptide inhibited the xenograft growth of mouse 4T1 breast cancer cells,LLC lung cancer cells and melanoma B16F10 cells;PD-1mAb only inhibited the xenograft growth of LLC cells and extended the survival of mice with LLC cell xenografts,and the xenografts of 4T1 and B16F10 cells were resistant to PD-1 mAb treatment;cotreatment with A11 peptide and PD-1 mAb conferred better efficacy as compared to A11peptide or PD-1 mAb treatment alone.(6)A11 peptide significantly reduced PD-L1 levels,and significantly increased the numbers of tumor-infiltrating CD8~+T cells and CD8~+GZMB~+T cells in the xenografts of 4T1,LLC and B16F10 cells,whereas PD-1 mAb only increased tumor-infiltrating CD8~+T cell population and their GZMB levels in the xenografts of LLC cells.Cotreatment with CPP-A11 and PD-1 mAb exhibited a significant synergetic effect in terms of increasing tumor-infiltrating CD8~+T cells and CD8~+GZMB~+T cells populations.Conclusion:(1)A11 peptide downregulates PD-L1 levels in the breast cancer,lung cancer,and melanoma cells;(2)A11 peptide promotes T cell activation and T cell killing of breast cancer,lung cancer,and melanoma cells in vitro,(3)A11 peptide exerts a broad-spectrum anticancer effect by inhibiting tumor immune escape in vivo;(4)A11peptide synergizes with PD-1 mAb to inhibit the immune evasion of breast,lung and melanoma cancer cells in mice.