The Mechanism Of O-GlcNAcylation Modified YAP Regulating FOXM1 To Promote The Progression Of Lung Adenocarcinoma

Background and objectiveLung cancer is one of the major cancers threatening human health.Lung adenocarcinoma is the most common histological type of lung cancer.O-linked-β-N-acetylglucosamine(O-GlcNAc)modification(O-GlcNAcylation)is a unique form of protein modification,which widely exists in the serine or threonine residues of nuclear,cytoplasmic and mitochondrial proteins.O-GlcNAcylation is a highly dynamic process.O-GlcNAc transferase(OGT)will transfer O-GlcNAc from uridine diphosphate-N-acetylglucosamine(UDP-GlcNAc)to target proteins,while O-GlcNAcase(OGA)will remove O-GlcNAc from glycosylated proteins.OGT and OGA are the only and highly conserved pair of enzymes in O-GlcNAcylation cycle.The O-GlcNAcylation of target proteins is closely related to the modification of nearby histone or non-histone proteins,protein biological function,protein stability,intracellular localization,protein recruitment and the activation of specific functions of downstream proteins,and the O-GlcNAcylation and phosphorylation of proteins have extensive and complex interactions.Overexpression of OGT,elevated level of O-GlcNAcylation and increased O-GlcNAcylation of proteins have been proved to be closely related to proliferation,migration and invasion of various malignant tumors.Hippo signaling pathway is a highly conserved signaling pathway in mammals,which plays a key role in development and tissue internal environment stability.Its downstream effector is Yes-associated protein(YAP).When the Hippo pathway is turned on,YAP is phosphorylated at the serine 127(Ser127)site and binds with14-3-3 to remain in the cytoplasm,and cannot enter the nucleus as a transcription coactivator to start the transcription of downstream target genes;when the Hippo pathway is closed,the unphosphorylated YAP enters the nucleus and combines with transcriptional enhancer associate domain(TEAD)to form a transcriptional coactivation complex,which activates and regulates the transcription of downstream target genes.In many tumors,Hippo pathway is dysfunctional and YAP is abnormally expressed.Studies have shown that the expression of YAP in the nucleus is usually associated with poor prognosis,while the expression of YAP in the cytoplasm often predicts a good prognosis.Forkhead box(FOX)protein family is a large and widely functional transcription factor family.FOXM1,as a member of FOX family,is almost undetectable in stable cells,while FOXM1 expression is increased in proliferative cells.FOXM1 plays an important role in a variety of biological processes,including proliferation,cell differentiation,angiogenesis,apoptosis,redox signal transduction and DNA damage repair.Studies have shown that FOXM1 is highly expressed in a variety of tumors and plays a role in tumor promotion.At present,some studies have found that YAP O-GlcNAcylation in tumor can regulate the function of YAP,and regulate its intracellular localization by changing the phosphorylation state of YAP,thus affecting the biological behavior of tumor.Some studies have also found that YAP can regulate the expression of FOXM1 and promote the malignant phenotype after it enters the nucleus.Some studies have shown that the level of O-GlcNAcylation is increased in lung adenocarcinoma,and there is a high expression of YAP and FOXM1.However,the mechanism of the interaction between O-GlcNAcylation and YAP and FOXM1 proteins,as well as the synergistic regulation of lung adenocarcinoma progression,is still unclear.The purpose of this study is to explore the effect of O-GlcNAcylation on the intracellular localization of YAP and the regulation of FOXM1 expression,thus affecting the cell function,by means of clinical pathological research,cell experiment and animal experiment,and to provide theoretical basis for a new target for targeted treatment of lung adenocarcinoma.MethodsIn this study,the clinicopathological characteristics and survival data of lung adenocarcinoma cases were collected and analyzed.The expression of O-GlcNAc,OGT,YAP and FOXM1 in lung adenocarcinoma tissues was detected by immunohistochemical staining,and the correlation between their expression and clinicopathological characteristics and prognosis was analyzed.In the cell experiment,small molecule inhibitors and transfection technology were used to change the expression of genes or proteins.The effects of O-GlcNAc,YAP and FOXM1 on the proliferation,migration,invasion and protein expression of A549 and H1299 cells were detected by CCK8 assay,colony formation assay,wound healing assay,transwell assay and western blot,and the correlation between their effects was analyzed.At the same time,protein immunoprecipitation and chromatin immunoprecipitation were used to study the regulatory effect of O-GlcNAcylation on YAP intracellular localization and FOXM1 expression,and to explore its mechanism.In the animal experiment,the nude mice xenotransplantation A549 tumor model was established,and the effect of YAP inhibitor on the growth of the transplanted tumor,the expression of related proteins and the treatment of the transplanted tumor was detected by measuring the volume and weight of the tumor body,western blot and immunohistochemical staining.Results1.In the 223 cases of lung adenocarcinoma,114 cases(51.1%)had high expression of O-GlcNAc,122 cases(54.7%)had high expression of OGT,196 cases(87.9%)had high expression of YAP in the nucleus or cytoplasm,and 114 cases(51.1%)had high expression of FOXM1,all of which were significantly higher than those in paracancerous lung tissues(80 cases).2.In lung adenocarcinoma,the expression of O-GlcNAc was positively correlated with the expression of OGT,YAP in the nucleus and FOXM1,and negatively correlated with the expression of YAP in the cytoplasm.The expression of OGT was positively correlated with the expression of YAP in the nucleus and FOXM1.The expression of YAP in the nucleus was negatively correlated with the expression of YAP in the cytoplasm,and positively correlated with the expression of FOXM1.3.In lung adenocarcinoma,the high expression of O-GlcNAc was related to the larger diameter of tumor invasion area and the higher T stage;the high expression of OGT was associated with lymphatic invasion,tumor necrosis,lymph node metastasis and the higher TNM stage;the high expression of YAP in the nucleus was associated with larger total tumor diameter,larger tumor invasion area diameter,vascular invasion,lymphatic invasion,tumor necrosis,higher tumor grade,lymph node metastasis,and the higher T stage and TNM stage;there was no significant difference in the expression of YAP in the cytoplasm between different clinicopathological characteristics;the high expression of FOXM1 was associated with larger tumor invasion area diameter,vascular invasion,lymphatic invasion,tumor necrosis,spread through air spaces,higher tumor grade,lymph node metastasis,and the higher T stage and TNM stage.4.In lung adenocarcinoma,the high expression of O-GlcNAc,OGT,YAP in the nucleus and FOXM1 were associated with the shortened progression-free survival(PFS);the high expression of YAP in the nucleus and FOXM1 was significantly associated with the shortening of overall survival(OS),and the high expression of YAP in the cytoplasm was associated with the prolongation of OS.The high expression of FOXM1 was an independent adverse prognostic factor for PFS and OS.5.The expression of O-GlcNAc,YAP and FOXM1 protein in human non-small cell lung cancer cell line A549 and human lung adenocarcinoma cell line H1299 was higher than that in human bronchial epithelioid cell line 16 HBE.6.After the A549 and H1299 cells were treated with OSMI-1(OGT inhibitor),the cell proliferation,migration and invasion ability decreased,while the overall O-GlcNAcylation level of the cells decreased,the total YAP content did not change significantly,the YAP in the nucleus decreased,the proportion of p-YAP(Ser127)to the total YAP content increased,and the expression of FOXM1,Cyclin A2,Cyclin B1,Snail and MMP-2 decreased.7.After the A549 and H1299 cells were treated with Thiamet G(TMG,OGA inhibitor),the cell proliferation,migration and invasion ability increased,while the overall O-GlcNAcylation level of the cells increased,the total YAP content did not change significantly,the YAP in the nucleus increased,the proportion of p-YAP(Ser127)to the total YAP content decreased,and the expression of FOXM1,Cyclin A2,Cyclin B1,Snail and MMP-2 increased.8.After knockdown of OGT,the proliferation,migration and invasion ability of A549 and H1299 cells decreased,while the expression of OGT in cells decreased significantly,the overall O-GlcNAcylation level of the cells decreased,the total YAP content did not change significantly,the YAP in the nucleus decreased,the proportion of p-YAP(Ser127)to the total YAP content increased,and the expression of FOXM1,Cyclin A2,Cyclin B1,Snail and MMP-2 decreased.9.After overexpression of OGT,the proliferation,migration and invasion ability of A549 and H1299 cells increased,while the expression of OGT in cells increased,the overall O-GlcNAcylation level of the cells increased,the total YAP content did not change significantly,the YAP in the nucleus increased,the proportion of p-YAP(Ser127)to the total YAP content decreased,and the expression of FOXM1,Cyclin A2,Cyclin B1,Snail and MMP-2 increased.10.After knockdown of OGT,the overall O-GlcNAcylation level of A549 and H1299 cells decreased,while the proportion of YAP modified by O-GlcNAcylation to the total YAP content decreased significantly.At the same time,after overexpression of OGT,the overall O-GlcNAcylation level of A549 and H1299 cells increased,and the proportion of YAP modified by O-GlcNAcylation to the total YAP content increased significantly.YAP O-GlcNAcylation reduced p-YAP(Ser127)and nuclear YAP increased.11.After knockdown of YAP,the proliferation,migration and invasion ability of A549 and H1299 cells decreased.The total YAP content and the expression of YAP in the nucleus were significantly decreased,the expression of FOXM1,Cyclin A2,Cyclin B1,Snail and MMP-2 decreased.TMG treatment could not reverse the above effects.12.After the A549 and H1299 cells overexpressing OGT were treated with thiostrepton(FOXM1 inhibitor),the cell proliferation,migration and invasion ability decreased,and the expression of FOXM1 in the cells decreased,and the expression of Cyclin A2,Cyclin B1,Snail and MMP-2 decreased.13.After the A549 and H1299 cells were treated with verteporfin(YAP inhibitor),the cell proliferation,migration and invasion ability decreased,and the expression of YAP in the cells decreased,and the expression of FOXM1,Cyclin A2,Cyclin B1,Snail and MMP-2 decreased.14.Through chromatin immunoprecipitation,it was found that the concentration of FOXM1 DNA in the products of anti-YAP antibody precipitation in A549 and H1299 cells was significantly higher than that in the negative control,which confirmed that YAP directly regulated the transcription of FOXM1 gene.15.Verteporfin significantly delayed the growth of xenotransplantation A549 tumor in nude mice,reduced the expression of YAP,FOXM1,Cyclin A2,Cyclin B1,Snail and MMP-2 in the tumor,and tumor metastasis occurred in the lungs of two nude mice in the control group.Conclusions1.This study confirms that O-GlcNAc,YAP and FOXM1 are highly expressed in lung adenocarcinoma tissues and correlated with the survival of patients.At the same time,the expression of the three in lung adenocarcinoma tissues is significantly correlated.2.The results of this study in vitro show that YAP O-GlcNAcylation reduces the phosphorylation of YAP and increases YAP entry into the nucleus;intranuclear YAP promotes the expression of FOXM1 by activating the transcription of FOXM1 gene and plays a role in tumor promotion.3.This study preliminarily reveals the mechanism of O-GlcNAcylation,YAP and FOXM1 in the progression of lung adenocarcinoma,providing a theoretical basis for further study of lung adenocarcinoma and targeted therapy.