YAP-induced CD24 Expression Drives Immune Evasion In Esophageal Squamous Carcinoma

BackgroundAccording to the 2020 global cancer burden data,the incidence and total mortality rate of esophageal cancer ranked in the top 10,the fatality rate continued to rise,and there were obvious geographical differences,Lin Zhou in northern Henan Province and its adjacent areas had the highest incidence and mortality rate of esophageal cancer in the world.Unlike Western esophageal cancer,which is mostly adenocarcinoma,more than90% of esophageal cancers in China are ESCC.The pathogenesis of ESCC is unclear,the lack of specific symptoms in the early stage and the advanced diagnosis of patients make the overall survival rate of middle and advanced patients at 5 years still hovering at about15%,which seriously threatens people’s lives and health.The previous research of our group has confirmed that YAP is involved in regulating the occurrence and development of ESCC,after knocking out the YAP gene in ESCC cells,RNA-Seq data analysis was carried out,and we unexpectedly found that "don’t eat me" signal CD24 is the key downstream gene of YAP,and whether YAP promotes immune escape of ESCC by regulating the expression of CD24 has yet to be confirmed.ObjectiveTo illustrate whether YAP drives immune evasion of esophageal squamous cell carcinoma by regulating CD24,providing theoretical support for the development mechanism of esophageal squamous cell carcinoma and new and immunotherapy targets.Methods1.Use CRISPR/Cas9 gene editing technology to knock out YAP genes in ECA9706 and ECA109,and evaluate the YAP knockout effect by PCR experiments and gene sequencing.2.After knockout YAP in ECA9706 and ECA109,the phenotypic changes of ESCC cells after YAP knockout were assessed by CCK8 cell proliferation experiment,plate cloning experiment,transwell experiment.3.RNA-seq analysis of wild-type cells and YAP knockout ECA9706 and ECA109 cells was performed to explore the mechanism of YAP influencing proliferation,migration and invasion of ESCC cells.4.After knockout YAP in ECA9706 and ECA109,it was assessed whether YAP gene knockout would downregulate the expression of CD24 in ESCC cells by flow cytometry,immunofluorescence experiment,Western blot experiment,and q PCR experiment.5.Evaluate whether YAP gene knockout is upregulating the phagocytic ability of macrophages to ESCC cells by flow cytometry and confocal imaging.6.Construct lentiviral stable strains of YAP continuous activation（YAP-5SA）and lentiviral stable strains of TEAD binding site mutation（YAP-5SA-S94A）in ECA9706 and ECA109.Flow cytometry,Immunofluorescence experiment,Western blot experiment,q PCR experiment to assess whether YAP sustained activation upregulated CD24 expression in ESCC cells was evaluated.7.Flow cytometry and confocal imaging were used to verify whether continuous YAP activation could down-regulate the phagocytosis of macrophages to ESCC cells.8.Both ChIP experiments and dual luciferase reporting experiments showed that YAP was able to regulate transcriptional activation of CD24.9.The correlation between the expression levels of YAP and CD24 and the clinical stage and TNM stage of ESCC was analyzed in 223 cases of esophageal squamous cell carcinoma tissue chips with complete clinical follow-up data.10.Immunohistochemical staining was performed on 223 cases of esophageal squamous cell carcinoma tissue microarrays with complete clinical follow-up data.Univariate and multivariate analysis was performed using Cox proportional risk regression model to evaluate whether YAP and CD24 overexpression were independent risk factors for poor prognosis of ESCC patients.11.Construct a mouse esophageal squamous cell carcinoma induction model,and use the YAP inhibitor verteporfin intervention to evaluate the effect of YAP inhibitor on the incidence of ESCC in mice and the survival of mice.Results1.PCR experiments and gene sequencing results showed that YAP gene knockout was successful in ESCC cells ECA9706 and ECA109.2.The results of CCK8 cell proliferation experiment,plate cloning experiment and Transwell experiment show that the proliferation,migration and invasion ability of ESCC cells after YAP gene knockout are significantly reduced.3.RNA-seq analysis of wild-type cells and YAP-knockout esophageal squamous cell carcinoma cells revealed that CD24 is a key downstream molecule regulated by YAP.4.The results of flow cytometry,immunofluorescence experiment,Western blot experiment and q PCR experiment showed that the expression level of CD24 was significantly down-regulated after YAP gene knockout.5.Flow cytometry and confocal imaging results showed that the phagocytic ability of macrophages on ESCC cells after YAP gene knockout was significantly enhanced.6.Flow cytometry,Immunofluorescence experiment,Western blot experiment,q PCR experiment results show that YAP continuous activation can upregulate CD24 expression level in ESCC cells.7.Flow cytometry and confocal imaging results show that YAP sustained activation can downregulate the ability of macrophages to phagocytosis of ESCC cells.8.Both ChIP experiments and dual luciferase reporting experiments showed that YAP was able to regulate transcriptional activation of CD24.9.The results of YAP and CD24 immunohistochemical staining of tissue chips for esophageal squamous cell carcinoma with complete clinical follow-up data showed that the expression of YAP and CD24 was significantly related to the TNM staging and clinical staging of esophageal squamous cell carcinoma.10.The YAP and CD24 immunohistochemical staining results of 223 esophageal squamous cell carcinoma tissue chips with complete clinical follow-up data showed that both YAP and CD24 were independent risk factors for the prognosis of esophageal squamous cell carcinoma patients.11.After the Verteporfin intervention,the incidence of esophageal cancer in mice decreased significantly,and the survival period was significantly prolonged.ConclusionsYAP gene knockout can significantly weaken the proliferation,migration and invasion of ESCC.The expression of YAP and CD24 was significantly correlated with TNM stage of esophageal squamous cell carcinoma,and the patients with high expression of YAP and CD24 had poor prognosis.YAP gene can regulate the expression of "don’t eat me" signal CD24 and promote the immune escape of ESCC.