The Function And Mechanism Of YAP-mediated Tumor Immune Tolerance In Breast Cancer

Objective: To determine the relationship between YAP expression and prognosis of the breast cancer or the function of immune cells.Methods: First,clinical database(http://hgserver1.amc.nl/cgi-bin/r2/main.cgi)was used to analyze the relationship between the YAP expression in breast cancer and the prognosis of the breast cancer patients.Then,immunohistochemistry(IHC)examination and hematoxylin-eosin staining was performed to evaluate the correlation of YAP expression in breast cancer and tumor Infiltrating lymphocytes(TIL)in breast cancer tissue samples.Furthermore,we verified the transfect efficiency of YAP overexpression plasmid and si RNAs via western blot.Next,lymphocytotoxicity test and Lymphocyte activation test was performed to determine whether YAP expression could influence the cytotoxicity and the activation of lymphocyte.Finally,the mouse breast cancer cell line 4T1 YAP KO stable cell line,which was obtained by CRISPR/Cas9 technology,and control cell line were injected subcutaneously into BALB/c mice by xenograft model.Flow cytometry was performed to determine the relationship between the YAP expression and the function of infiltrating lymphocyte of mouse breast cancer tumor,and the effect of YAP on tumor growth was detected by comparing tumor size.Results: Through clinical database analysis,we found that patients with high YAP expression in breast cancer had a significantly poorer prognosis.Immunohistochemistry and H&E staining suggest that the expression level of YAP in the tumor tissue of clinical breast cancer patients was positively correlated with the local lymphocyte infiltration in the tumor.Overexpression of YAP in HCC-1937 could significantly decrease the cytotoxicity of lymphocyte and the Granzyme B expression of CD8 T cells while knockdown YAP expression in MDA-MB-231 could significantly increase the cytotoxicity of lymphocyte and the Granzyme B expression of CD8 T cell after co-culture with PBMC.By subcutaneous xenograft model in mice,the tumor size of 4T1 YAPKO group less than the control group on average,and by flow cytometry,the expression of IFN-γ,Granzyme B in CD8+ cells of 4T1 YAPKO group were higher than the control group.Conclusions: The high expression of YAP can be used as an important indicator of poor prognosis in breast cancer patients.The infiltrating lymphocytes in tumor tissues with high expression of YAP are increased,suggesting that the infiltrating lymphocytes with high expression of YAP lose their function.In vitro and in vivo,YAP inhibited the activation and cytotoxicity of lymphocytes.Objective: To investigate the mechanism how YAP mediate tumor tolerance.Methods: Firstly,Human Cytokine Array was performed to detect cytokines,chemokines and acute phase proteins in sample,the changes of immune-related cytokines caused by the change of YAP expression level in tumor cells were detected and statistically analyzed to screen the related cytokines.Secondly,the gene expression microarray of breast cancer cell line MDA-MB-231 was obtained from GEO database to analyze the relationship between the immune-related cytokines and YAP expression.Thirdly,western blot and RT-q PCR were performed to determine the correlation between YAP and ICAM1.Fourthly,cell adhesion test was performed by co-culture breast tumor cell with PBMC or Jurkat T cell to evaluate the relationship between YAP expression and the ability of lymphocyte adhesion.Then,cell adhesion test was performed after using si RNAs of ICAM1 or Human ICAM1Antibody to determine whether ICAM1 expression could influence lymphocyte adhesion.Finally,lymphocytotoxicity test was performed to determine affected lymphocyte cytotoxicity through ICAM1.Results: By statistical analysis of the Human Cytokine Array,we found that the expression levels of 8 cytokines,such as CCl2,G-CSF and ICAM1,were changed after YAP knockdown in MDA-MB-231.The results of gene expression microarray analysis in GEO database indicated that after YAP knockdown in MDA-MB-231,the changes of ICAM1 mRNA levels in the above-mentioned cytokines were the most significant.The negative correlation between YAP and ICAM1 was verified by western blot and RT-q PCR at the protein level and m RNA level.Through cell adhesion test,we found that PBMC or Jurkat T cell had more significant adhesion ability to of YAP-knockdown MDA-MB-231,suggesting that the expression of YAP in breast cancer cell lines could inhibit the adhesion ability of PBMC or Jurkat.We found that the adhesion ability of PBMC to breast cancer cell lines was decreased by knocking down ICAM1 by si RNAs or blocking ICAM1 by anti-ICAM1 monoclonal antibody,suggesting that YAP inhibited lymphocyte adhesion through ICAM1.Lymphocytotoxicity test showed that lymphocyte cytotoxicity ability of PBMC was decreased when co-culture PBMC with breast cancer cell line which was deal with anti-ICAM1 monoclonal antibody,suggest that YAP inhibited lymphocyte killing through ICAM1.Conclusion: YAP and ICAM1 were negatively correlated at protein level and m RNA level;YAP can affect the adhesion and cytotoxicity ability of lymphocytes through ICAM1.