YAP Mediates The Migration Of MDSCs By Regulating CXCL5/CXCR2 Resulting In The Inhibition Of T Cell Proliferation In EGFR Mutant Lung Adenocarcinoma

Background Non-small cell lung cancer(NSCLC)is the most common histological subtype of lung cancer,accounting for more than 85% of all lung cancer.The prognosis of advanced NSCLC is poor.NSCLC patients with epidermal growth factor receptor(EGFR)mutation respond well to corresponding molecularly targeted drug therapy with few adverse reactions.However,despite a good initial response to targeted therapy,virtually all EGFR-mutated NSCLC treated with targeted therapy will inevitably progress over time due to acquired drug resistance.For NSCLC,where resistance to tyrosine kinase inhibitors(TKIs)contributes to disease progression,further treatment strategies are currently limited.In recent years,immunotherapy has become a first-line treatment strategy for patients with NSCLC without driver gene mutation,with small adverse reactions,which has greatly improved the quality of life and survival of patients.However,patients with targeted gene mutations,including EGFR-mutant NSCLC,have poor responses to anti-PD-1/PD-L1 immunotherapy.The mechanisms that mediate the poor efficacy of immunotherapy in patients with EGFR-mutant NSCLC remain unclear.Therefore,in-depth study of the resistance mechanism of EGFR-mutant lung adenocarcinoma and TKIs-resistant NSCLC to immunotherapy is expected to provide new ideas for improving the efficacy of immunotherapy.Hippo pathway effector YAP(yes-associated protein)acts as an oncogene in many cancers.When the Hippo pathway is turned on,YAP is phosphorylated and retained in the cytoplasm.When the Hippo pathway is closed,YAP is transported to the nucleus to form a transcription complex with TEADs,playing a role in cell proliferation,apoptosis inhibition and other pathways.A growing number of studies have shown that the Hippo/YAP pathway can regulate the immune system.Prostate cancer studies have shown that YAP may contribute to cancer progression and immunotherapy resistance by regulating the infiltration of myeloid derived suppressor cells(MDSCs).Simultaneous inhibition of YAP may inhibit MDSCs recruitment,thereby activating effector T cell activity to enhance tumor sensitivity to immunotherapy.Although further mechanistic and preclinical studies are needed,the combination of YAP inhibitors and immunotherapy may be a novel therapeutic strategy.At present,there is no relevant study on the regulation of MDSCs migration by YAP in lung adenocarcinoma.Therefore,we designed this project to explore the effect of YAP on MDSCs migration its underlying mechanism in EGFR-mutant lung adenocarcinoma.Methods Proteomic analysis was used to identify pleural effusion in patients with EGFR-sensitive and TKIs-resistant lung adenocarcinoma.RNA expression data of lung adenocarcinoma in The Cancer Genome Atlas database were analyzed by UALCAN and GEPIA.192 cases of lung adenocarcinoma with EGFR mutation and 60 cases of lung adenocarcinoma without driver gene mutation were retrospectively analyzed.Immunohistochemical methods were used to analyze the expression of YAP and CXCL5 in tumor cells and their correlation with clinicopathological features and prognosis of lung adenocarcinoma.CD33 and CD8 monoclonal antibody were used to identify MDSCs and T cells,respectively.The number of CD33+MDSCs and CD8+T in the tumor nests was evaluated,and to investigate the relationship with YAP expression.PD-L1 IHC 22C3 pharm Dx antibody was used to evaluate the expression of PD-L1 in tumor cells.In vitro cell experiment,Western blot,ELISA,and RT-q PCR were used to detect the expression of YAP and CXCL5 in human NSCLC cell lines A549 and H1299,PC9 and PC9/GR.Human peripheral blood mononuclear cells were isolated,and CD33+MDSCs and CD8+T cells were obtained by magnetic beads.Through the establishment of co-culture system,to verify the effect of YAP expression on MDSCs migration in PC9 cells,and the inhibitory effect of MDSCs on proliferation of CD8+T cells was detected.Chromatin immunoprecipitation(Ch IP)and dual luciferase reporter gene assays were used to detect the regulatory effect of YAP and CXCL5 promoter.Results 1.Proteomic analysis revealed that YAP,CXCR2 and CXCL5 proteins were significantly different in gefitinib-sensitive and TKIs-resistant patients and were associated with many important functions in cells.2.UALCAN and GEPIA database analysis results showed that patients with lung adenocarcinoma with high expression of YAP m RNA had poor prognosis,while CXCL5 m RNA expression had no significant correlation with prognosis.YAP expression was positively correlated with CXCL5 expression.3.Immunohistochemical results in lung adenocarcinoma tissues showed that the high cytoplasmic YAP expression in EGFR mutation and non-mutation tumor cells were 62.0% and 48.3%,respectively,with no statistical significance(P=0.072).High nuclear YAP expression was 47.9% and 26.7%,respectively,and the difference was statistically significant(P=0.004).High cytoplasmic CXCL5 expression in tumors cells in the two groups was 51.0% and 31.7%,respectively,with statistically significant(P=0.009).Tumor-infiltrating immune cells,the high density of MDSCs in the two groups was 35.4% and 18.3%,respectively,the difference was statistically significant(P=0.013);the high density of CD8 was 44.3% and 63.3%,respectively,which was statistically significant(P=0.01).4.Clinicopathological analysis showed that nuclear YAP expression was positively correlated with histological grade(P=0.016),pathologic TNM stage(P<0.001)and tumor diameter(P=0.001).There was no correlation between cytoplasmic YAP expression and clinicopathological features;high cytoplasmic CXCL5 expression of 98 cases in EGFR mutated lung adenocarcinoma.Clinicopathological analysis showed that CXCL5 expression was positively correlated with TNM stage(P=0.001).5.Univariate survival analysis showed that tumor diameter>3cm(P<0.001),low histopathological differentiation(P<0.001),stage III-IV(P<0.001),high nuclear YAP expression(P<0.001),high expression of CXCL5(P<0.001)and high density of MDSCs(P<0.001)were associated with short overall survival(OS).High nuclear YAP expression(P<0.001),high expression of CXCL5(P=0.019),high density of MDSCs(P=0.001),tumor diameter>3cm(P=0.001),and stage III-IV(P<0.001)were associated with short disease-free survival(DFS).Multivariate analysis showed that tumor diameter>3cm(P<0.001),high expression of CXCL5(P=0.003),and high nuclear YAP expression(P=0.036)were independent prognostic factors for OS.High nuclear YAP expression(P=0.004)was independent prognostic factors for DFS.6.Analysis of the expression of CD33+MDSCs and CD8 in tumor-infiltrating immune cells and its correlation with the expression of YAP and CXCL5 in lung adenocarcinoma tissues demonstrated that the number of tumor-infiltrating CD33+MDSCs was negatively correlated with the number of CD8+T lymphocytes(R=-0.309,P=0.005).The number of tumor-infiltrating CD33+MDSCs was significantly positively correlated with CXCL5 expression(R=0.355,P=0.002),and nuclear YAP expression was positively correlated with CXCL5 expression(R=0.376,P= 0.001).7.The expression of PD-L1 was positively correlated with the nuclear YAP expression(P=0.001).The expression of PD-L1 was not correlated with EGFR mutation(P=0.916).8.The nuclear YAP expression in PC9 cells was higher than that in H1299 and A549 cells,P<0.01,PC9/GR was higher than that in PC9 cells,P<0.05.9.The expression of CXCL5 protein and m RNA in PC9 cells was higher than that in H1299 and A549 cells,P<0.01.The expression of CXCL5 protein and m RNA in PC9/GR cells was higher than that in PC9 cells,P<0.01.10.si RNA knockdown of YAP decreased CXCL5 protein and m RNA expression in PC9/GR cells,P<0.001.Overexpression of YAP significantly increased CXCL5 protein and m RNA expression in PC9 cells,P<0.01.11.In vitro co-culture experiments of PC9 and MDSCs showed that the CXCL5/CXCR2 pathway promoted the migration of MDSCs,and blocking this pathway significantly reduced MDSCs migration.YAP regulate the expression of CXCL5 and promotes the migration of MDSCs.MDSCs co-cultured with T cells significantly inhibited the proliferation of T cells.12.Through JASPAR database analysis,YAP/TEADs binding sites were found in the promoter region of CXCL5 gene.Ch IP confirmed that YAP/TEADs complex could bind to the CXCL5 promoter region.Dual luciferase reporter gene assay further verified the regulation of YAP/TEADs on CXCL5.Conclusions 1.Nuclear YAP and CXCL5 was highly expressed in EGFR mutant lung adenocarcinoma.CXCL5 expression was positively correlated with nuclear YAP expression.High nuclear expression of YAP and CXCL5 are independent prognostic factors of OS.High nuclear expression of YAP is an independent prognostic factor for DFS.2.YAP protein and CXCL5 protein was highly expressed in EGFR mutant and EGFR mutant gefitinib resistant lung adenocarcinoma cell lines.YAP can directly bind to the CXCL5 and regulates CXCL5 transcription.Blocking CXCL5/CXCR2 signaling pathway can significantly reduce MDSCs migration,and YAP mediated MDSCs migration through CXCL5/CXCR2 signaling pathway,resulting in inhibition of T cell proliferation.