O-GlcNAcylation Level Of Lung Adenocarcinoma Promotes Tumor Invasion And Metastasis By Regulating YAP

BackgroundNon-small cell lung cancer(NSCLC)is one of the major cancers threatening human health.Although targeted therapy and immune checkpoint inhibitors have improved the prognosis of NSCLC to a certain extent,the prognosis of most patients,especially those in advanced stage,is still poor.Adenocarcinoma is the most frequently diagnosed subtype of NSCLC,and its incidence is increasing year by year.Thus,it is of great significance to study the mechanism of pathogenesis and progress of lung adenocarcinoma and find new therapeutic targets to improve the prognosis of patients.O-Glc NAcylation is an important posttranslational modification of proteins.The cycling of O-Glc NAc on and off of protein substrates is catalyzed by the actions of O-Glc NAc transferase(OGT)and O-Glc NAcase(OGA/MGEA5),respectively.Multiple proteins can be regulated by O-Glc NAcylation;this modification widely exists at serine/threonine residues of proteins,and highly overlaps with phosphorylation residues,which plays an important role in regulating protein nucleocytoplasmic distribution and protein stability.Elevated O-Glc NAcylation and aberrant expression of OGT and OGA have been observed in a variety of human solid tumors and cell lines.Increased O-Glc NAcylation is associated with tumor progression,and can promote malignant phenotype of tumors through multiple mechanisms.Hippo signaling pathway plays an important role in a variety of tumors.Yes-associated protein(YAP)is the target factor of Hippo pathway.As a transcription coactivator,YAP is mainly regulated by phosphorylation of upstream kinases.When Hippo pathway is activated,YAP is phosphorylated at Ser127,which facilitates its binding to 14-3-3 protein and sequestration in the cytoplasm.At this circumstance,YAP cannot be translocated to the nucleus and the transcriptional activity of downstream target genes is inhibited.When Hippo pathway is turned off,YAP is translocated to the nucleus,specifically binds with multiple transcription factors,and displays a promoting or suppressing role in different types of tumors.Previous studies have found that the level of O-Glc NAcylation and YAP expression are both elevated in NSCLC,and the subcellular location of YAP demonstrates different prognostic significance in patients.Recently,O-Glc NAcylation modification has been found to regulate YAP activity directly or indirectly in some tumor and non-tumor cell lines,but the number of studies is limited,and current studies mainly focus on cytologic experiments and animal experiments with an absence of detailed clinicopathologic analysis.Besides,the effect of O-Glc NAcylation on YAP has not been explored in lung cancer.The purpose of this study is to analyze the relationship between O-Glc NAcylation and YAP in lung adenocarcinoma,and explore the regulation of global O-Glc NAcylation level on YAP and its effect on biological behavior of tumor using online database,clinicopathologic analysis and in vitro cytologic experiments,in order to provide theoretical support for OGT or YAP targeted therapy of lung adenocarcinoma.MethodsIn this study,RNA expression data of lung adenocarcinoma in The Cancer Genome Atlas database were analyzed by UALCAN and GEPIA.The expression of YAP and O-Glc NAcylation related proteins in histologic specimens of lung adenocarcinoma were detected by immunohistochemical staining.Correlation between the expression of these proteins and clinicopathologic parameters and the significance on prognosis were explored.Meanwhile,correlation between YAP intracellular expression and O-Glc NAcylation related proteins were also analyzed.In addition,si RNA transfection,Western Blot,CCK8 cell proliferation assay,wound healing assay,and cell migration and invasion assay were used to explore the regulation of global O-Glc NAcylation level on YAP expression and their effect on cell proliferation,migration and invasion.Results1.Survival analysis using UALCAN found that patients with high expression of YAP m RNA had shorter survival time,while the m RNA expression of OGT and OGA showed no significance on the prognosis of patients.2.GEPIA was used to analyze the correlation of m RNA expression of YAP,OGT and OGA in lung adenocarcinoma.It was found that the expression levels of OGT and OGA,YAP and OGT,YAP and OGA were positively correlated,respectively.3.The expression of YAP,OGT,OGA and O-Glc NAc in 148 cases of lung adenocarcinoma and 32 cases of adjacent normal lung tissues was detected using immunohistochemical staining.The expression of YAP,OGT,OGA and O-Glc NAc in lung adenocarcinoma was all found to be significantly higher than those in normal lung tissues.4.In 148 cases of lung adenocarcinoma,84 cases(56.8%)showed high expression of YAP in the nucleus,and 68 cases(45.9%)showed high expression of YAP in the cytoplasm.Tumors with high expression of YAP in the nucleus had larger diameters and more advanced stages,and were more prone to show recurrence or metastasis;tumors with low expression of YAP in the cytoplasm were more prone to show lymph node metastasis and necrosis,and were more likely to compound solid pattern.5.In 148 cases of lung adenocarcinoma,25 cases(16.9%)showed high expression of OGT,55 cases(37.2%)showed high expression of OGA,and 55 cases(37.2%)showed high expression of O-Glc NAc.Tumors with high expression of OGT were more likely to occur in female patients and compound lepidic pattern without necrosis;tumors with high expression of OGA were more likely to contain solid components;tumors with high expression of O-Glc NAc were more likely to show necrosis and compound solid pattern.6.Univariate analysis showed that high expression of YAP and O-Glc NAc were associated with worse disease-free survival(DFS),but there was no significant correlation between their expression and overall survival(OS);no significance was observed between the expression of other proteins and DFS or OS.Multivariate analysis showed that YAP,OGT,OGA and O-Glc NAc protein expression were not independent risk factors for DFS.7.Nuclear expression of YAP was positively correlated with OGT,OGA and O-Glc NAc expression;cytoplasmic YAP was negatively correlated with OGA and O-Glc NAc expression.At the same time,there was a positive correlation between the expression of OGT,OGA and O-Glc NAc.8.The global level of O-Glc NAcylation in A549 cells was significantly increased after treated with OGA inhibitor PUGNAc.The expression of total YAP protein increased with the elevated O-Glc NAcylation level,and the proportion of phosphorylated(Ser127)YAP in total YAP decreased.Meanwhile,nuclear YAP was increased significantly with the increase of O-Glc NAcylation.9.Treatment of A549 cells with PUGNAc had no significant effect on proliferation,but significantly facilitated cell migration and invasion.10.Treatment of A549 cells with PUGNAc could up-regulate the expression of vimentin.11.Knockdown of OGT expression in H1299 cells by si RNA significantly decreased its global O-Glc NAcylation level.Total protein expression of YAP decreased and the proportion of phosphorylated(Ser127)YAP in total YAP increased significantly after down-regulation of O-Glc NAcylation level.Meanwhile,nuclear YAP was decreased significantly.12.Knockdown of OGT expression by si RNA could significantly inhibit cell proliferation,migration and invasion in H1299 cells.13.Knockdown of OGT expression by si RNA could down-regulate the expression of vimentin in H1299 cells.14.After knockdown of YAP expression by si RNA in A549 cells,the migration and invasion ability of A549 cells decreased significantly.PUGNAc could not reverse the decline of migration and invasion by increasing O-Glc NAcylation level.Conclusions1.In the histological specimens of lung adenocarcinoma,elevated O-Glc NAcylation level and high YAP nuclear expression are associated with adverse clinicopathologic parameters and shorter DFS;high YAP nuclear expression is associated with tumor recurrence and metastasis.2.In the histological specimens of lung adenocarcinoma,elevated O-Glc NAcylation level and high YAP nuclear expression are positively correlated.3.In A549 cells,increased global O-Glc NAcylation level promotes cell migration and invasion;in H1299 cells,decreased global O-Glc NAcylation level suppresses cell proliferation,migration and invasion.4.Cellular O-Glc NAcylation level has effect on epithelial-mesenchymal transition.5.Cellular global O-Glc NAcylation level is able to regulate YAP in A549 and H1299 cells.Increased level of O-Glc NAcylation up-regulates the expression of YAP and promotes its nuclear translocation;vice versa,decreased O-Glc NAcylation level suppresses YAP expression and its nuclear translocation,and promotes YAP phosphorylation on Ser127.6.Global O-Glc NAcylation level promotes cell migration and invasion by regulating YAP.