Mechanism Study Of The Novel Fusion Peptide UM-6 In Anti-tumor And Tumor Immune Microenvironment Of Cervical Cancer

Background:Cervical cancer is one of the most common gynecological malignancies in the world,accounting for the second largest population of female cancer deaths.High risk human papillomavirus(HR-HPV)is the main cause of cervical cancer.Currently,management of cervical cancer depends on the staging of the disease.Briefly,treatment of cancer confined to the uterus is based on surgery,while concurrent radiochemotherapy is considered the standard management for locally advanced cancer.A platinum–paclitaxel combination chemotherapy,with the possible addition of bevacizumab,is considered the treatment options for advanced cervical cancer,but it has only a palliation intent.Therefore,it is urgent to improve the treatment status and bring survival benefits to patient.Bee venom(BV)is a kind of biotoxin,the main active components include melintin(MEL),enzymes,bioactive amines and so on.It has been reported that MEL has anti-inflammatory,antiviral and anti-tumor effects.In recent years,it has also been found that MEL can regulate tumor immune.However,whether MEL can inhibit the progression of cervical cancer and regulate tumor immunity of cervical cancer and its specific mechanism are still unclear.The application of MEL in cancer is limited by its toxicity,nonspecificity,easy degradation,limited bioavailability and hemolysis.We previously synthesized a new amino acid fusion peptide UM-6 with the basis of MEL and conducted a preliminary study.It was found that UM-6 not only played a good anti-tumor effect,but also made up for the shortcomings of MEL.Previous studies have confirmed that UM-6 has a good anti-cervical cancer effect,but the mechanism of action remains unclear.Objective:To explore the specific mechanism of action of the new amino acid fusion peptide UM-6 against cervical cancer and the specific mechanism of action of anti-tumor immunity of cervical cancer,so as to provide a new direction for drug research and development and treatment of cervical cancer.Methods:1.The study on the progression and mechanism of a novel fusion peptide UM-6against cervical cancer1)The effect of UM-6 on the proliferation and migration of cervical cancer.Cervical cancer cell lines He La and Caski were treated with different concentrations of UM-6 for 24 and 48 h.The cell activity was detected by CCK8 kit,and the IC50 of UM-6 was calculated.The effect of UM-6 on the proliferation of cervical cancer cells was detected by plate cloning experiment.The effect of UM-6 on the migration and invasion of cervical cancer cells was detected by wound healing assay,Transwell assay and three dimensional(3D)tumor sphere invasion assay.2)High throughput proteomics screened the UM-6 associated PI3K-AKT pathway and target protein Eph A2.The protein and m RNA expressions were verified by Western blot,immunofluorescence(IF)and q PCR.3)The study of UM-6 on inhibition the interaction between P-AKT and Eph A2 and Eph A2 degradation.The interaction of P-AKT with Eph A2 was detected by IF and immunoprecipitation(Co-IP).AKT activator(SC79)and inhibitor(LY294002)were used to further detect the effect of UM-6 on Eph A2 protein expression.4)The study of UM-6 on Eph A2 degradation by lysosomal pathway.The effect of UM-6 on the half-life and stability of Eph A2 was determined by protein biosynthesis inhibitor cycloheximide(CHX).The effect of UM-6 on Eph A2 protein sublocalization was detected by IF and Co-IP.The effect of UM-6 on Eph A2 ubiquitination and Eph A2 related ubiquitin ligase c-Cbl protein expression was detected by Western blot and IF.5)We constructed cervical cancer subcutaneous tumor-bearing mouse model and monitored the tumor growth rate,tumor volume and weight after treatment with intraperitoneal injection of UM-6.Western blot,immunohistochemical staining(IHC)and IF were used to detect the effect of UM-6 on P-AKT and Eph A2 expression in vivo.2.The study on tumor immune and mechanism of novel fusion peptide UM-6against cervical cancer1)Effect of UM-6 on tumor immune of cervical cancer.The mouse cervical cancer U14 cells homologous subcutaneous tumor-bearing C57BL/6 mouse model was established,and the tumor growth was monitored after intraperitoneal injection of UM-6.IHC and IF were used to detect the expression levels of PD-L1,Cleaved caspase3(CCA3)and Ki67 in tumor grafts after UM-6 treatment.Flow cytometry was used to detect the changes in the levels of killing CD8+ T cells,depleted T cells,effective CD4+T cells and the number of Treg cells after UM-6 treatment.2)Correlation between Eph A2 and T cell-related immune checkpoint molecule PD-L1 in cervical cancer.Gene Set Enrichment Analysis(GSEA)from the TCGA database screened T cell immune checkpoint molecules associated with Eph A2.The correlation between Eph A2 and immunosuppressive signals of cervical cancer was studied by TIMER database.Westernblot,flow cytometry and IF were used to detect the effect of Eph A2 on PD-L1 expression.The expression and correlation of Eph A2 and PD-L1 in cervical cancer were evaluated by TCGA database and IHC.3)The study on the mechanism of UM-6 affecting PD-L1 expression.Westernblot,flow cytometry and IF were used to detect the expression of total and membrane expression of PD-L1.Tunicamycin(TM),an N-glycosylation inhibitor,was co-treated with UM-6 to detect the glycosylation of PD-L1.The effect of UM-6 on the half-life and turnover of PD-L1 was detected by CHX analysis.The effect of UM-6 on PD-L1 sublocalization and stability was detected by IF and Co-IP.Results:1.The study on the progression and mechanism of a novel fusion peptide UM-6against cervical cancer.1)UM-6 effectively inhibited the proliferation,migration and invasion of cervical cancer cell lines in a dose-dependent and time-dependent manner,but had little effect on the proliferation and toxicity of HaCaT cells.2)UM-6 downregulated the expressions of P-AKT and Eph A2 in cervical cancer,but did not affect the m RNA levels of Eph A2.IF showed that before UM-6 treatment,Eph A2 was mainly localized in the cell membrane,after UM-6 treatment,Eph A2 was translocated to the cytoplasm while the expression of the cell membrane was significantly reduced.3)UM-6 significantly reduced the phosphorylation level of Eph A2 at Ser897 and the interaction of Eph A2 with P-AKT.AKT inhibitor LY294002 inhibited phosphorylation of Eph A2 at Ser897,and AKT agonist SC79 significantly increased phosphorylation of Eph A2 at Ser897 and restored UM-6-mediated phosphorylation of Eph A2 at Ser897.4)UM-6 significantly increased the turnover of Eph A2 and shortened the half-life.When UM-6 was co-incubated with the proteasome inhibitor MG-132 or the lysosome inhibitor chloroquine(CQ),only CQ could significantly reverse the UM-6-induced degradation of Eph A2.IF showed that UM-6 significantly increased and prolonged the localization of Eph A2 in early and late endosomes,while significantly decreased the localization of Eph A2 in circulating endosomes and increased the localization of Eph A2 in lysosomes.UM-6 also significantly increased the phosphorylation of c-Cbl and its association with Eph A2 leading to increased ubiquitination of Eph A2 and interaction of Eph A2 with the ESCRT complex.5)UM-6 inhibited the growth,volume and tumor weight of tumor xenografts.In addition,the expression of Eph A2 and P-AKT was significantly reduced in the UM-6treatment group.2.The study on tumor immune and mechanism of novel fusion peptide UM-6against cervical cancer.1)UM-6 significantly inhibited the growth of U14 xenografts,decreased the expression of PD-L1 protein in tumor grafts,and increased the amount of granzyme B(GB)released by CD8+ T cells.As anti-tumor immune was associated with apoptosis in tumor tissues,a strong apoptotic signal occurred in the UM-6 treatment group,and the expression of proliferation marker Ki67 was also significantly reduced.Flow cytometry showed that UM-6 enhanced the secretion of IFN? by CD8+ T cells and reduced the proportion of exhausted PD-1+ T cells.UM-6 also increased the proportion of effector IFN?+CD4+T cells and decreased the number of Treg in the xenografts.2)GSEA showed that Eph A2 and adaptive immune response signaling pathway were highly correlated in cervical cancer,and CD274(PD-L1)score enriched in the pathway was higher,and Eph A2 and PD-L1 were positively correlated in cervical cancer.The TIMER database showed that Eph A2 was associated with the immunosuppressive signal characteristics of cervical cancer,such as depleted T cell signal and effector Treg cell signal.Eph A2 overexpression increased PD-L1 total and membrane expression and the binding of PD-1 to PD-L1 on tumor cell surface.Eph A2 Ser897 may also be involved in the regulation of PD-L1 and tumor immunity.The results of Westernblot and flow cytometry showed that the overexpression of Eph A2 wild type and Eph A2 S897 D significantly increased the total and membrane expression of PD-L1,while the overexpression of Eph A2 S897 A downregulated the total and membrane protein levels of PD-L1.The TCGA database and IHC showed that Eph A2 and PD-L1 were highly expressed in cervical cancer tissues,and PD-L1 expression was also increased in patients with high Eph A2 expression,and Eph A2 and PD-L1 showed obvious colocalization.3)Westernblot and flow analysis showed that UM-6 inhibited the membrane expression of PD-L1 and the binding of PD-1 to PD-L1 on the surface of tumor cells.IF showed that PD-L1 was mainly located on the cell membrane in the control group,and the signal on the cell membrane of PD-L1 was weakened after UM-6 treatment,accompanied by an increase in punctate staining patterns of the cytoplasm.Further detection showed that most PD-L1 was detected at ?45 k Da in untreated cells,while PD-L1 had obvious band shift after UM-6 treatment,with ?45 k Da significantly reduced and ?33 k Da bands increased,which confirmed that UM-6 inhibited the glycosylation of PD-L1.TM treatment resulted in the shift of most ?45-k Da fully glycosylated proteins of PD-L1 to the non-glycosylated ?33 k Da form.The transfer of PD-L1 to non-glycosylation was more obvious after cotreatment with UM-6 and TM.The CHX analysis showed that the UM-6-induced non-glycosylated ?33 k Da PD-L1 had a shorter half-life and a faster turnover rate,and the UM-6-induced ?33 k Da PDL1 showed more ubiquitination level.IF results showed that PD-L1 was mainly located on the cell membrane in the control group,while PD-L1 was co-located with the endoplasmic reticulum marker HSP90B1 after UM-6 treatment,and no colocalization with the golgi body marker TGN46,indicating that the non-glycosylated form of PD-L1 increased after UM-6treatment and remained in the endoplasmic reticulum.Westernblot and CO-IP confirmed that UM-6 reduced the protein level of STT3 A and the interaction with PDL1,and increased the binding of PD-L1 with the components of endoplasmic reticulum associated degradation pathway(ERAD),indicating that UM-6 promoted the degradation of PD-L1 through the ERAD pathway.Conclusion:1.UM-6 inhibited the proliferation and invasion of cervical cancer in vitro and in vivo,the main mechanism is that UM-6 inhibits the phosphorylation of Eph A2 on Ser897 mediated by the PI3K-AKT signaling pathway,then recruits and phosphorylates c-Cbl and increases Eph A2 ubiquitination,finally increases the number of Eph A2 to the lysosome and promotes Eph A2 degradation in lysosome.2.Eph A2 may be involved in tumor immune through PD-L1,and PI3K-AKT/Eph A2/PD-L1 may be a new pathway involved in tumor immune in cervical cancer.The blockade of Eph A2 may represent a new therapeutic approach for immunotherapy of cancers.3.UM-6 is involved in the regulation tumor immune microenvironment mediated by PD-L1/PD-1 axis in cervical cancer.The specific mechanism is that UM-6 inhibits the initial glycosylation of PD-L1,hinders the PD-L1 from the endoplasmic reticulum to the golgi apparatus leading to degradation of PD-L1 by ERAD pathway,eventually preventing PD-L1 interaction with PD-1.