The Mechanism Of CD155/TIGIT Signaling Pathway In The Development Of Cervical Cancer

BackgroundCervical cancer is a common female malignant tumor.Cervical cancer had the fourth highest incidence rate（13.1%）and death rate（6.9%）among women’s malignancies worldwide in 2018,according to the World Health Organization’s（WHO）global cancer statistics report.Persistent infection with a high-risk human papillomavirus（HPV）is the main risk factor for cervical cancer.According to the natural course of cervical cancer,60%of low-grade squamous intraepithelial lesions（LSIL）resolved,30%of LSIL persisted,10%of LSIL progressed to high-grade squamous intraepithelial lesions（HSIL）,and 1%of LSIL developed into cervical cancer.From the time HPV infection begins until cervical cancer develops,it takes more than ten years.The majority of HPV infections are just transient.HPV infection is not the only cause of cervical cancer.In cervical adenosquamous carcinoma,the HPV negative rate is 14%,and the HPV negative rate in cervical adenocarcinoma is 30%.All these suggest that besides HPV infection,there are other factors involved in the occurrence and development of cervical cancer.Surgical treatment is currently the commonly used method for the treatment of cervical cancer.Radiotherapy and chemotherapy are used for postoperative adjuvant treatment and conventional treatment of metastatic/recurrent cervical cancer.However,this treatment has limited effect in advanced disease.The dysfunction of immune cells in the tumor immune microenvironment is one of the reasons for the uncontrolled progression of tumors.A healthy immune system must balance immune checkpoint blockade（ICB）to maintain immune balance.In recent years,immunomodulatory drugs on the ICB pathway have become an effective treatment for malignant tumors.In cervical cancer clinical trials,the overall response rate of programmed cell death 1（PD1）inhibitor Pembrolizumab was 14%.The remission rate of nivolumab,another monoclonal antibody against PD-1,was 26.3%.From the currently existing data,the effect of immunotherapy that inhibits the PD-L1/PD-1 pathway was limited.Therefore,relevant research on combined immunotherapy for patients with metastatic/recurrent cervical cancer is underway based on immune checkpoint inhibitors.A clinical trial study of Bintrafusp alfa（a dual-target antibody against PD-L1 and TGF-β）in metastatic/recurrent cervical cancer showed that Bintrafusp alfa could increase the total remission rate of patients to 30.8%.In addition,the effective rate of PD-1 inhibitor combined with cytotoxic T lymphocyte-associated antigen-4（CTLA-4）dual target antibody in advanced/recurrent cervical cancer has also increased to 31.6%-45.8%.It can be seen that the combined blockade of dual immune checkpoints can allow more patients to obtain clinical remission and significantly improve the prognosis of patients.CD 155 is a member of the immunoglobulin superfamily.CD 155 was first discovered as a poliovirus receptor（PVR）.CD 155 can initiate signal transduction through immunoreceptor tyrosine-based inhibitory motif（ITIM）,thereby regulating cell adhesion,movement,proliferation and survival.The expression of CD 155 in healthy tissues is relatively low,but the expression is elevated in various malignant tumors.Aside from its high expression in malignant tumors and cancer promotion,CD 155 can also interact with T cell immunoreceptors with immunoglobulin and ITIM domains with immunoglobulin and immunoreceptor tyrosine inhibitory motif domains（TIGIT）.TIGIT is an inhibitory receptor mainly expressed in NK cells（natural killer cells,NKs）,CD8+T cells,CD4+T cells,and Treg cells（regulatory cells,Tregs）.The combination of CD 155 and TIGIT can directly activate the ITIM of TIGIT,thereby inhibiting the function of NK and T lymphocytes.This topic studies the elevated expression of CD 155 in patients with cervical cancer.It investigated the molecular mechanism by which CD 155 promotes the occurrence and development of cervical cancer and the immunosuppressive effect of the CD155/TIGIT signaling pathway on cervical cancer CD8+T lymphocytes.The content of this research includes the following three parts:Part Ⅰ:Study on the expression,clinical significance and biological function of CD155 in cervical cancerObjective:1.To detect the expression of CD 15 5 in plasma and paraffin tissue specimens of patients with cervical cancer.2.CD 155 affects the biological functions of cervical cancer cells.Methods:1.We analyzed the differential expression of CD 155 in cervical cancer and normal cervical tissues through TCGA and GEO databases and collected data for survival analysis.The high expression of CD 155 affected patient survival.2.We detected the expression level of CD 155 in plasma and paraffin tissue specimens of cervical cancer patients,HSIL patients and normal cervix patients by enzyme-linked immunosorbent assay（ELISA）and immunohistochemistry（IHC）.The diagnostic efficacy of soluble or secreted（sCD155）in cervical cancer was analyzed based on the expression level of sCD155 in plasma.3.We regulated the expression level of CD155 in cervical cancer cell lines CaSki and HeLa.The effect of CD 155 on the proliferation of cervical cancer cells was determined using the CCK-8 proliferation assay,cell cycle test,and nude mouse subcutaneous tumor formation test.The effect of CD 155 on cell apoptosis was detected using an apoptosis test.The effect of CD 155 on cell migration and invasion was determined using the Transwell assay.Western Blot was used to detect the effect of CD 155 on the expression of cell proliferation and apoptosis-related molecules.Results:1.Plasma and paraffin tissue specimens from cervical cancer patients show a high level of CD 155 expressionWe downloaded the database containing TCGA and GEO of cervical cancer tissue and normal cervical tissue and analyzed the expression difference of CD 155 in each database.The results showed that in the databases GSE29570,GSE52903,GSE67522 and TCGA-GTEx,the expression level of CD 155 in cervical cancer was higher than that in normal cervical tissues.Survival analysis was performed on the databases with survival information,and the results showed that in the databases GSE44001,GSE52903 and TCGA,high expression of CD 155 is associated with poor patient survival.We took plasma samples from 30 patients with cervical cancer,20 patients with HSIL,and 17 patients with normal cervix.According to ELISA results,the expression level of sCD155 in cervical cancer patients and HSIL patients was significantly higher than in normal cervical patients.In addition,we also applied a Logistic retrospective analysis of the diagnostic efficacy of sCD155 for cervical cancer and HSIL+（cervical cancer+HSIL）patients.By analyzing the expression level of sCD155 between patients with cervical cancer and those with the normal cervix,sCD155 has higher diagnostic efficiency for patients with cervical cancer,with an area under the curve（AUC）0.727,a sensitivity of 0.63,and a specificity of 0.76.By analyzing the expression level of sCD155 between HSIL+（cervical cancer+HSIL）patients and normal cervix patients,the results showed that sCD155 also has excellent diagnostic performance for HSIL+patients with an AUC of 0.769,a sensitivity of 0.6,and a specificity of 0.88.To further study the expression level of CD 155 in cervical cancer patients,we collected 66 tissues from cervical cancer patients,16 tissues from HSIL patients,and 18 tissues from the normal cervix.The results of the analysis showed that the expression level of CD 155 in patients with the normal cervix,HSIL and cervical cancer gradually increased.2.CD155 promotes the proliferation,invasion,migration and anti-apoptosis of cervical cancer cellsAfter knocking down CD 155 in CaSki and HeLa,cell proliferation,invasion and migration capabilities decreased,and apoptosis ability increased.After overexpressing CD 155 in CaSki and HeLa cells,cell proliferation,invasion and migration increased,and apoptosis ability decreased.We constructed tumor xenograft models by injecting PCMV-NC or PCMV-CD155 transfected CaSki cells into nude mice.Tumor weight and volume were significantly higher in the PCMV-CD155 than in the PCMV-NC group.In order to further verify the effect of CD 155 on the proliferation and apoptosis of cervical cancer cells,we used Western Blot to detect the expression of proteins that affect the cell cycle and apoptosis-related molecules.The results showed that after CaSki and HeLa cells down-regulated CD 155,the expression of cell proliferationrelated molecules CDK2,CyclinD1,P27KIP1,C-myc,E2F1,and Ki67 decreased,while the expression of apoptosis-related molecules CL-Caspase3,CL-Caspase9,and CL-PARP increased.When CD 155 is up-regulated in CaSki and HeLa cell lines,the expressions mentioned above cycle-influencing proteins and apoptosis-related molecules showed opposite trends.Conclusions:1.CD 155 is highly expressed in patients with HSIL and cervical cancer.2.CD 155 affects the proliferation,apoptosis,migration and invasion of cervical cancer cells.Part Ⅱ:The study of the molecular mechanism of CD155 in the occurrence and progression of cervical cancerObjective:In the first part,we have made it clear that CD 155 is highly expressed in cervical cancer and promotes the malignant behavior of cervical cancer.In this part,we are trying to study the upstream and downstream mechanisms of CD155 in cervical cancer.Methods:1.Signal pathway antibody chip results in analysis,CD 155 inhibited autophagy of cervical cancer cells and activated AKT/mTOR and NF-κB pathways.2.Reverse verification by suppressing AKT.AKT was down-regulated in cervical cancer cells overexpressing CD 155 and their controls.Western Blot was used to detect changes in AKT/mTOR and NF-κB related pathway molecules,and flow cytometry was used to detect changes in the proportion of apoptosis.3.After down-regulating CD 155 in cervical cancer cells,the autophagy inhibitor bafilomycin A1（Baf A1）/chloroquine（CQ）was used to explore the relationship between autophagy and cervical cancer cell apoptosis.4.Through database prediction and analysis,the CD 155 promoter region has the binding site of transcription factor YY1.RT-PCR and Western Blot verified the regulatory relationship between CD 155 and YY1.Chromatin immunoprecipitation assay and dual-luciferase experiments proved that YY1 directly binds to the promoter region of CD 155.Results:1.Result of signal pathway antibody chipThe signal pathway antibody chip is a tumor signal pathway chip developed by Shanghai Huaying Biotechnology Co.,Ltd.The experiment was performed after CaSki cells down-regulated CD 155.The analysis results showed that compared with the control group,161 protein molecules were up-regulated and 127 protein molecules were down-regulated.We screened and compared 16 signal pathways related to tumorigenesis and development.Pathway analysis results showed that the differential protein molecules are mainly concentrated in autophagy,AKT/mTOR,and NF-κB pathways.2.CD155 inhibits autophagy of cervical cancer cells and activates AKT/mTOR and NF-κB pathwaysWe performed Western Blot studies to look for changes in the protein expression of autophagy-related molecules,the AKT/mTOR,and the NF-κB signaling pathways after modulating CD155.The results showed that downregulating CD155 in CaSki and HeLa cells reduced p-AKT,p-mTOR,p-P70S6K,p-4EBP1,p-IKKγ,p-IKKα/β,p-NFκB p65,and elevated Beclin-1,LC3-II/LC3I.After CD155 was overexpressed,the result was the opposite.Cellular immunofluorescence was used to detect the effect of CD 155 on the level of autophagy.After CaSki and HeLa cell down-regulated CD155,the number of LC3B fluorescent plaques increased.After CaSki and HeLa cells upregulated CD155,the results were opposite.The effect of CD155 on the expression of cytoplasmic p-IκBa and nuclear p-NF-κBp65 was detected by cellular immunofluorescence.After CaSki and HeLa cells down-regulated CD 155,the cytoplasmic p-IκBα and nuclear p-NF-κBp65 expression levels decreased.After CD 155 was up-regulated,the result was the opposite.The above results indicated that CD 155 inhibited autophagy in cervical cancer cells and activated the AKT/mTOR and NF-κB pathways.3.The autophagy induced by knocking down CD155 is protective.To determine whether down-regulation of CD155-mediated autophagy is protective autophagy or death autophagy.After down-regulating CD 155,we applied autophagy inhibitor Baf Al or CQ.After 48 hours,we evaluated the changes in apoptosis-related molecules and apoptosis ratios in CaSki and HeLa cells.Western Blot experiment results showed that the expression of apoptosis-related molecules CLCaspase3,CL-Caspase9 and CL-PARP increased.In addition,the apoptosis experiment showed that the number of apoptotic cells increased.The above results indicated that down-regulation of CD 155-induced autophagy has a protective effect,and inhibition of autophagy can increase cell apoptosis.4.AKT knockdown reverses the anti-apoptosis induced by CD155 overexpression and the activation of AKT/mTOR and NF-κB pathwaysThe previous results indicated that overexpression of CD 155 inhibits autophagy and apoptosis of cervical cancer cells and activates the AKT/mTOR and NF-κB pathways.AKT is upstream of the mTOR and NF-κB pathways,so we knock down the expression of AKT for reverse verification.Subsequent flow cytometry and western blot analysis showed that knocking down AKT in CaSki and HeLa cells overexpressing CD 155 reduced the anti-apoptotic effect.Knockdown of AKT significantly reduced the expression of p-mTOR,p-IKKα/β and p-IKKγ induced by CD 155 overexpression.At the same time,the expression of Beclin-1 and the LC3II/LC3I ratio increased.Finally,knockdown of AKT reversed the increases in CL-caspase9 and Ki67 induced by overexpression of CD 155.5.YY1 is highly expressed in cervical cancer and regulates the expression of CD155We searched the CD 155 promoter sequence through the NCBI website and then used JASPAR software and the Cistome website to predicted the transcription factor and binding site of the CD 155 promoter.We analyzed the expression levels of YY1 mRNA in cervical cancer and normal cervical tissues in GEO,TCGA and GTEx databases and found that the expression of YY1 mRNA in cervical cancer patients’tissues was higher than that in normal cervical tissues.Spearman correlation analysis showed that CD 155 is positively correlated with YY1.We studied whether YY1 has a regulatory relationship with CD 155 from the level of RNA and protein.After knocking down YY1 in CaSki and HeLa cells,the expression of CD 155 was significantly reduced,and after overexpressing YY1 in CaSki and HeLa cells,the expression of CD 155 increased.YY 1 could regulate the expression of CD 155 at the mRNA and protein levels.6.YY1 transcription activates the CD155 promoter to regulate its expressionIn order to verify whether YY1 can directly regulate the expression of CD 155,we designed plasmids and primers based on the previously predicted binding sites for subsequent experiments.First,we conducted dual-luciferase studies to confirm that overexpression of YY1 boosted the luciferase activity of the wild-type CD 155 promoter considerably.The mutant CD 155 promoter’s luciferase activity was drastically lowered when the site1 and site2 binding sites were altered.In addition,we designed primers covering site1 and site2 and verified the binding of YY1 to CD 155 through chromatin immunoprecipitation.7.YY1 knockdown rescues the promotion of overexpressing CD155 in cervical cancerWe have verified the regulatory effect of YY1 on CD 155.Next,we will further study the biological functions of YYl on CD 155 regulation.We carried out a "Rescue"experiment to observe whether YY1 has a rescue effect on the proliferation and metastasis of cervical cancer cells caused by CD 155 overexpression.We knocked down YY1 in cervical cancer cells overexpressing CD 155 and performed follow-up experimental verification after 48 hours.CCK-8 experiment results showed that the cell proliferation rate decreased after down-regulating YY1 in CD 155 overexpressing cervical cancer cells.Transwell experiments showed that after knocking down YY1 in cervical cancer cells overexpressing CD 155,the migration and invasion ability of the cells decreased.The apoptosis experiments showed that knocking down YY1 in CD 155 overexpressing cervical cancer cells increases the number of apoptotic cells.In conclusion,we found that knocking down YY1 reverses the promotion of CD 155 overexpression in cervical cancer.Conclusions:1.CD 155 inhibits autophagy of cervical cancer cells and activates AKT/mTOR and NF-κB pathways to promote the malignant behavior of cervical cancer cell.2.YY1 is the direct upstream gene of CD 155,which activates CD 155 through transcription and regulates cervical cancer cell proliferation,invasion,metastasis and apoptosis.Part Ⅲ:CD155/TIGIT regulates the function and mechanism of CD8+T lymphocytesObjective:1.To detect the expression of TIGIT and TIGIT+CD8+T lymphocytes in the peripheral blood mononuclear cell（PBMC）and tissue of patients with cervical cancer.2.Clarify the immunosuppressive effect of CD155/TIGIT immune checkpoint on cervical cancer CD8+T lymphocytes.Methods:1.We detected the expression of TIGIT and TIGIT+CD8+T lymphocytes in PBMC and paraffin tissue specimens of patients with cervical cancer by flow cytometry and immunohistochemistry.2.The CD8+T lymphocytes separated from the peripheral blood of healthy people were activated by anti-CD3/CD28 antibody stimulation in vitro.Set the groups:CD8+T lymphocytes+Isotype control（IgG）group,CD8+T lymphocytes+recombinant protein CD 155（CD 155-Fc）group,CD8+T lymphocyte+TIGIT blocking antibody,CD8+T lymphocyte+PD-1 blocking antibody and CD8+T lymphocyte+TIGIT blocking antibody+PD-1 blocking antibody.After 24 hours of co-cultivation,flow cytometry was used to detect the secretion of functional factors of CD8+T lymphocytes,and Western Blot detected the changes of ERK and NF-κB pathway-related molecules in CD8+T lymphocytes.3.U14-KO-CD155 and U14-NC-CD155 cell lines were constructed by CRISPR/Cas9 technology.C57BL/6 mice were used to construct U14-KO-CD155 and U14-NCCD 155 cervical cancer xenograft models to study the immunosuppressive effect of CD155 on CD8+T lymphocytes in vivo experiments.We used wild-type U14 cells to established a C57BL/6 mouse cervical cancer transplantation tumor model,intraperitoneal injection of TIGIT blocking antibody or PD-1 blocking antibody,to studied the blocking of TIGIT and the combined blocking of TIGIT and PD-1 on CD8+T lymphocytes Immune function.Results:1.TIGIT is highly expressed in PBMC and tissues of patients with cervical cancerWe collected peripheral blood from 16 patients with cervical cancer,15 patients with HSIL,and 16 patients with normal cervix from Qilu Hospital of Shandong University.RT-PCR results showed that the expression level of TIGIT in PBMC of cervical cancer patients was significantly higher than that of patients with HSIL and normal cervix.In addition,we also performed Spearman correlation tests on PBMC mRNA levels of TIGIT and PD-1,TIGIT and Lymphocyte-activation gene 3（LAG3）,TIGIT and T cell immunoglobulin and mucin domain-containing protein 3（Tim3）in cervical cancer patients.The results showed that:TIGIT and PD-1,TIGIT and LAG3,TIGIT and Tim3 expression are positively correlated.We collected cancer tissues and adjacent tissues from 11 patients with cervical cancer and performed immunohistochemical analysis.We found that the expression level of TIGIT in cervical cancer tissues was significantly higher than that in adjacent tissues.2.TIGIT+CD8+T lymphocyte is highly expressed in PBMC and tissues of patients with cervical cancerWe collected peripheral blood from 20 patients with cervical cancer,20 patients with HSIL and 20 patients with normal cervix from Qilu Hospital of Shandong University and detected CD8+T lymphocyte surface molecules.The results showed that the ratio of TIGIT+CD8+T lymphocytes in patients with cervical cancer was significantly higher than that of patients with HSIL and the normal cervix.In addition,we also collected five pairs of cervical cancer and adjacent tissues.Multiple immunofluorescence histochemistry experiments showed that TIGIT is located on CD8+T lymphocytes.Furthermore,the expression of TIGIT+CD8+T lymphocytes in cervical cancer tissues is significantly higher than in adjacent tissues.We performed lymphocyte function analysis on TIGIT+CD8+T lymphocytes cells and TIGIT+CD8+T lymphocytes cells.The results showed that the ability of TIGIT-CD8+T lymphocytes cells to secrete cytokines TNF-α and IFN-y was significantly higher than that of TIGIT+CD8+T lymphocytes cells.3.CD155/TIGIT inhibits the function of CD8+T lymphocytesThe CD8+T lymphocytes isolated from the peripheral blood of healthy people were co-cultured with CD155-Fc after anti-CD3/CD28 and IL-2 activation stimulation.Three groups were set:CD8+T lymphocytes+IgG,CD8+T Lymphocytes+5μg/mL CD155-Fc,CD8+T lymphocytes+10μg/mL CD155-Fc.In addition,the activated CD8+T lymphocytes and blocking antibodies were taken to establish a co-culture system,and four groups were set up:CD8+T lymphocytes+IgG,CD8+T lymphocytes+blocking antibody TIGIT,CD8+T lymphocytes+blocking antibody PD-1,CD8+T lymphocytes+blocking antibody TIGIT+blocking antibody PD-1.After 24 hours of co-culture,the flow cytometry results showed that CD 155-Fc inhibited the production of TNF-α、IFN-γ and GranzymeB in CD8+T lymphocytes.Blocking TIGIT increased the production of TNF-α、IFN-γ,and GranzymeB in CD8+T lymphocytes.The combined blocking of TIGIT and PD-1 has a more vital secretion function than the blocking antibody TIGIT alone.4.CD155/TIGIT inhibits the function of CD8+T lymphocytes through the ERK and NF-κB pathwaysThe effect of CD155/TIGIT on the immunosuppressive effect of CD8+T cells was also investigated.Co-culture with CD 155-Fc dramatically reduced the protein expression levels of p-ERK,p-IκBα,and p-NF-κBp65 in CD8+T cells.Blocking TIGIT increased the protein expression levels of p-ERK,p-IκBα,and p-NF-κBp65 in CD8+T lymphocytes,and blocking TIGIT and PD-1 together increased the protein expression levels of p-ERK,p-IκBα,and p-NF-κBp65 in CD8+T lymphocytes even more than the blocking TIGIT group alone.We also confirm that TIGIT on CD8+T cells can bind to SHIP-1.TIGIT can recruit SHIP-1 and interact with it to form a TIGIT/SHIP-1 complex.According to co-immunoprecipitation and Western Blot data,the amount of TIGIT/SHIP-1 complex in CD8+T cells co-cultured with CD 155-Fc was dramatically increased.5.CD155/TIGIT inhibits the growth of cervical cancer transplanted tumors and the function of CD8+T lymphocytes in C57BL/6 miceBy measuring the volume of subcutaneously transplanted cervical cancer tumors in C57BL/6 mice,it was shown that compared with the U14-NC-CD155 group,the growth rate of the tumors in the U14-KO-CD155 group was significantly slower.The infiltration level of CD8+T lymphocytes and the ability of CD8+T lymphocytes to secrete functional factors TNF-α and IFN-γ were increased.In wild-type C57BL/6 mice,blocking TIGIT inhibited cervical cancer transplantation in C57BL/6 mice tumor growth and proliferation,combined block TIGIT and PD-1 to increased the anti-tumor function of the blocking antibody TIGIT.Combined block TIGIT and PD-1 tumor tissue infiltration level of CD8+T lymphocytes and the levels of cell-secreted functional factors TNF-α and IFN-γ>the blocking antibody TIGIT group alone>IgG group.Conclusions:1.TIGIT+CD8+T lymphocytes are highly expressed in PBMC and tissues of patients with cervical cancer,exerting immunosuppressive effects to inhibit the function of CD8+T lymphocytes.2.In vivo and in vitro experiments confirmed that CD 155 inhibits the function of CD8+T lymphocytes.Blocking TIGIT increases the function of CD8+T lymphocytes,blocking TIGIT and PD-1 further increases the function of CD8+T lymphocytes.3.CD155/TIGIT induces immunosuppression of CD8+T lymphocytes through ERK and NF-κB pathways.The innovation of this study1.In vivo and in vitro experiments have confirmed the role of CD 155 in the occurrence and development of cervical cancer.Clinical samples verified the feasibility of CD 155 as a molecular marker for cervical cancer tumors.2.CD 155 inhibited autophagy of cervical cancer cells,activated the AKT/mTOR and NF-κB pathways to promote the malignant behavior of cervical cancer cells,and provided new ideas for the pathogenesis of cervical cancer.3.For the first time,it was verified that YY1 directly regulates the expression of CD 155.YY1 activated the transcription of CD 155 by binding to the promoter region of CD 155,thereby promoting the cancer-promoting effect of CD 155.4.Discuss the negative immune regulation mechanism of CD155/TIGIT in cervical cancer patients.In vivo and in vitro experiments verified that CD155/TIGIT induced the immunosuppression of CD8+T lymphocytes,thereby promoting cervical cancer development.CD155/TIGIT is expected to become a new target for cervical cancer immunotherapy.Limitations of this study1.The number of clinical samples of cervical cancer included in this study is limited,and most of them are HPV-positive cervical squamous cell carcinomas.We still need larger samples for verification,especially for HPV-negative cervical cancer clinical samples.2.Humanized immunized mice have part of the human immune function,which can better simulate the immune state of humans.We lack the verification of the negative regulatory effect of CD155/TIGIT immune checkpoints in humanized immunized mouse models.3.CD155/TIGIT may have multiple negative immunoregulatory mechanisms in cervical cancer patients.In this study,we studied CD155/TIGIT to induce CD8+T lymphocytes to exert immunosuppressive effects,and there is a lack of research on other immune cells.