EZH2 Suppresses NK Cell Anti-tumor Activity Through Interaction With Aiolos

Objectives:Histone methyltransferase EZH2 catalyzes chromosomal histone modification H3K27me3,regulates the transcriptional network in numerous kinds of cells by affecting the epigenetic modification status of downstream genes,and plays a key role in maintaining the normal development and functional status of the cells.The abnormal expression of EZH2 is closely related to a variety of tumor occurrence.Besides the tumor cells themselves,the occurrence and development of tumors are largely dependent on the tumor microenvironment.However,the relationship between the expression level of EZH2 in tumor microenvironment and the development of tumor remains to be studied.We attempted to analyze the association between EZH2 in the tumor microenvironment and the occurrence and metastasis of lung cancer based on the sample analysis of lung cancer patients and Ezh2 knockout transgenic mouse model,and molecular biological experiments for the mechanism investigation.We hope that this study can provide new insights for the diagnosis and treatment of tumor.Methods:1.We used immunohistochemistry staining to analyze clinical tumor samples from patients and analyze the relationship between the expression of EZH2 in tumor microenvironment and the clinical grade of lung cancer as well as the prognosis of patients.2.We established inducible EZH2 knockout mice model and analyzed the effect of EZH2 systemic knockout on the ratio of mice NK cells and T lymphocytes.We constructed a mice lung cancer metastasis model and analyzed the effect of EZH2 knockout on distant metastasis of tumor cells.We prepared NK cell-specific neutralizing antibodies from mice ascites,specifically removed NK cells in mice,in order to analyze the effect of EZH2 knockout on the anti-tumor activity of NK cells in mice.We used immunofluorescence staining to analyze the distribution of NK cells and T lymphocytes in mice tumor tissue sections,in-situ observed the influence of EZH2 knockout to mice tumor microenvironment.3.We applied IP-Mass spectrometry to identify whether Aiolos exist in the elution co-preciptated with EZH2 in 293T cells.We used Co-IP to verify the interaction between EZH2 and Aiolos in mice spleen NK cells.We applied the Duolink method to in-situ detect the interaction of EZH2 with Aiolos in mice spleen NK cells and NK92 cell line respectively.We constructed a series of truncated mutant type of Aiolos,and used Co-IP and Duolink experiments to identify the critical domain in which Aiolos interacts with EZH2 in 293T cells.4.Using the data of NK cell gene expression profiles in Ikzf3 and Ezh2 knockdown mice reported in the literatures,we identified several potential co-regulatory genes by Aiolos and EZH2,and performed functional analysis.We used chromosomal co-immunoprecipitation to identify the enrichment of Aiolos and EZH2 at the transcriptional start site of the above genes.We used Aiolos inhibitor Thalidomide to treat mice spleen NK cells,and observed whether the above genes' expression levels are affected or not after Aiolos has been downregulated.Meanwhile,we examined the effect of inhibition of Aiolos on the enrichment of EZH2 and histone-modification H3K27me3 at the transcriptional start sites.After treatment with Aiolos inhibitor,we tested the in vitro tumor killing activity of mice NK cells.Results:1.We analyzed clinical samples from patients with lung cancer and found that the number of tumor infiltrating lymphocytes with low EZH2 expression in the tumor microenvironment positively correlated with the patient's survival,negatively correlated with the occurrence of tumor lymph node metastasis,but did not obviously correlated with clinical tumor grading.2.In this study,we crossed ERT2-Cre and Ezh2fl/fl transgenic mice,and successfully generated the EZH2 inducible knockout mice model ERT2creEzh2fl/fl.We found that EZH2 knockout can result in proportion increasement of the NK cells by using the inducible EZH2 knockout mice model,and that the ability of mice to resist lung metastasis was significantly enhanced after EZH2 knockout.NK1.1 antibody-mediated NK cell depletion compromised the resistence of EZH2 knockout mice in inhibiting the pulmonary metastasis by LLC1 cells.3.After IP-Mass spectrometry we found that the proteins can be co-precipitated with EZH2 includes Aiolos.Co-IP in mice splenic NK cells showed that EZH2 interacts with Aiolos.Duolink experiments in mice NK cells and NK92 cell lines indicated that EZH2 interacts with Aiolos in situ.In 293T cells we transfected truncated mutant Aiolos with EZH2,and Co-IP experiments indicated that the missing of the seventh exon of Aiolos leads to disappearance of interaction between them.Duolink experiments showed that when the seventh exon of Aiolos is deleted,the intracellular co-localization between EZH2 and Aiolos disappears.4.Analysis of mice NK cell gene expression profile data revealed that 25 genes are upregulated after both Ikzf3 and Ezh2 knockout,among which Tcf7,Icos,Gpr56 and Marcks have been reported to be associated with NK cell development and function.CHIP assay showed the enrichment status of Aiolos and EZH2 at the above genes'promoters.Inhibition of Aiolos in mice NK cells using Thalidomide resulted in the down-regulation of the above genes,meanwhile the enrichment EZH2 and histone modification H3K27me3 were reduced at the transcriptional start site of the above genes.There was no significant change in the cytotoxicity of NK cells in mice after Thalidomide treatment.Conclusions:1.Tumor infiltrating Natural killer cells in the tumor microenvironment of lung cancer patients are beneficial to patients to obtain a more positive prognosis,and are beneficial to prolong patients' survival time.2.EZH2 knockout increase the proportion of NK cell in transgenic mice by affecting NK cell development,which enhance the resistence to tumor metastasis.3.Aiolos partially mediated the critical impact of EZH2 on NK cell's development and function,promotes the anti-tumor activity of NK cell.