The Mechanism Of Promotion Of Cancer Immunity By ISG12a Through Inhibiting The Canonical Wnt/?-catenin Signaling Pathway

Cancer seriously endangers human health.Through enhancing the anticancer effect of adaptive immunity dominated by effective T cells,inhibitors of immune checkpoints such as PD-1 are widely used in cancer therapy.However,due to cancer heterogeneity,only a small number of patients have a durable effect in immunotherapy.The activation of canonical Wnt/?-catenin signaling pathway leads to the occurrence and development of cancer,drug and treatment resistance,characteristic of cancer stem cells(CSC)and epithelial-mesenchymal transition(EMT),and may also subvert immunosurveillance.Innate immunity is the basis of cancer radiotherapy,chemotherapy and immunotherapy,which not only mediates the recognition to cancer cells as well as induction and expansion of immune response,but also exerts anticancer effect and alleviates immunosuppression in cancer microenvironment.Interferon(IFN)activates the JAK-STAT signal pathway to induce the expression of IFN stimulated-genes(ISGs),exerting anticancer effect.However,only a very small number of ISGs such as RIG-I have been found with anticancer effect,the function and mechanism of most ISGs are still unclear.Our previous studies have shown that IFN stimulated-gene ISG12 a reverses drug resistance of cancer cells,and promotes the innate immune response to viral infection,acting as an innate immune effector.We speculated that ISG12 a may regulate the occurrence and development of carcinomas and cancer immunity,and conducted the following r esearches.The chapter 2 uncoveres the low expression of ISG12 a in cancer cells and cancer tissues.To study the anticancer effect of ISG12 a,we chosed hepatocellular cancer(HCC)and gastric cancer(GC)as the research subjects.Through conducting immunoblots,quantitative real-time PCR(q RT-PCR)and immunohistochemistry(IHC)staining,we determined the responsive effect of ISG12 a to IFN stimulation,and the low expression of ISG12 a in hepatic and gastric cancer cells as well as that in cancer tissues of HCC and GC,suggesting that ISG12 a may inhibit the occurrence and development of cancer.The chapter 3 determines the anticancer effect of ISG12 a.We performed the following studies on hepatic and gastric cancer cells with ISG12 a knockdown: Cell growth curve,crystal violet staining and agarose colony formation assays uncovered the inhibitory effect of ISG12 a on cancer cell proliferation;Wound-healing and transwell assays showed the blockade of ISG12 a on cancer cell migration;Adhesion assay identified that ISG12 a decreases the adhesion bility of cancer cells;Subcutaneous and tail vein injection of cancer cells in BALB/c nude mice showed that ISG12 a inhibits cancer occurence,growth and metastasis;Cultivation of cancer stem spheres revealed that ISG12 a reduces CSC phenotype;Examining the expression changes of EMT-associated markers and F-actin cytoskeleton determined the negative regulatory effect of ISG12 a on EMT.Based on the above findings,we believe that ISG12 a is a tumor suppressor gene.The chapter 4 describes the mechanism of ISG12 a on inhibiting the canonical Wnt/?-catenin signaling pathway.Combining the expression change of EMT associated marker ?-catenin in cancer cells and RNA sequencing,we speculated that ISG12 a might regulate Wnt/?-catenin signaling pathway;Through conducting immunoblots,immunofluorescence staining,q RT-PCR and dual-luciferase reporter assay,we verified the inhibitory effect of ISG12 a on the activation of Wnt/?-catenin signaling pathway;Combining ubiquitin degradation an d co-immunoprecipitation experiments of protein with bioinformatics analysis,we clarified the mechanism that ISG12 a blocks the ubiquitin degradation of Axin and then promotes the ubiquitin degradation of ?-catenin,thus inhibiting Wnt/?-catenin signaling pathway.The chapter 5 reveals the role and mechanism of ISG12 a in promoting cancer immunity.We excluded the regulatory effect of ISG12 a on cancer immune infiltration by IHC staining,and confirmed that ISG12 a promotes anticancer immunity mediated by NK cells through flow cytometry.Interestingly,we found that ISG12 a inhibits the expression of immune checkpoint PD-L1 on the surface of cancer cells by flow cytometry,and unocovered that ?-catenin is a new transcription factor of PD-L1 through exerting chromatin immunoprecipitation and dual-luciferase reporter assay.Through combining NK cell killing and receptor-ligand blockade experiments,we revealed that ISG12 a promotes the anticancer immunity mediated by NK cells through reversing PD-1/PD-L1 axis.Based on the above findings,we believe that ISG12 a blockades PD-1/PD-L1 axis by suppressing Wnt/?-catenin signaling pathway in cancer cells,and then inhibits immune evasion,thus promoting NK cell killing.The chapter 6 defines the clinical significance of IS G12 a research.Firstly,we analyzed the expression of ISG12 a and Axin protein in clinical tissue samples to determine the specific regulation of ISG12 a on Wnt/?-catenin signal pathway in vivo.Then we analyzed the relationship between the expression level of ISG12 a and clinical prognosis,and found that low expression of ISG12 a in cancer tissue may predict poor prognosis.Finally,the relationship between the expression level of ISG12 a and clinicopathology was analyzed,and it was confirmed that low express ion of ISG12 a in cancer tissue may lead to malignant progress.The innovation and significance of this study are mainly embbedied in three aspects:(1)It was found that ISG12 a inhibits the proliferation,migration,metastasis,CSC and EMT of cancer cells,being defined as a new tumor suppressor gene,which is tightly associated with the clinical prognosis and malignant progress of HCC and GC.(2)It was first discovered that SKP2 is an E3 ubiquitin ligase that degrades the scaffold protein Axin of destruct ion complex of ?-catenin protein.(3)?-Catenin was found and confirmed as a transcription factor of immune checkpoint PD-L1 for the first time.All in all,the innate immune effector ISG12 a reverses the PD-1/PD-L1 axis by inhibiting the canonical Wnt/?-catenin signaling pathway in cancer cells,thus promoting the killing effect of NK cells toward cancer cells.Cancer cells with upregulated expression of PD-L1 through activating Wnt/?-catenin signaling pathway achieve immune evasion.The study on the antica ncer effect and mechanism of ISG12 a emphasizes the key role of IFN and innate immunity in promoting immunotherapy.ISGs such as ISG12 a may be targets for cancer diagnosis and treatment and play a role in cancer prevention and treatment.The relationship am ong innate immunity,Wnt/?-catenin signaling pathway and cancer immunity determined in this study will provide a new strategy for improving immunotherapy.