| Lung cancer is one of the most serious cancer-related death diseases at present.The rate of morbidity and mortality is the highest among malignant tumors in China,and it is growing rapidly.Therefore,lung cancer is a serious threat to human health and life.Non-small cell lung cancer(NSCLC)is the most important histological type of lung cancer,accounting for about 85%of lung cancers,and about 80%of patients have metastasis in the middle and late stages.In recent years,with the in-depth exploration of the pathogenesis of lung cancer,molecular targeted therapy and immunotherapy therapy have become research hotspots in the treatment of NSCLC.In particular,the advanced methods,the inhibitors of EGFR,ALK and NTRK and immune checkpoint inhibitor,such as PD-1 and PD-L1,have made a breakthrough in the therapy field of NSCLC.However,there is still a lack of effective treatment for most patients who are in the middle or late stage of diagnosis,because of the high complexity and heterogeneity of NSCLC.The efficacy of targeted therapy and immunotherapy in improving the survival of patients with advanced NSCLC remains limited.Therefore,in-depth research on the molecular mechanism of NSCLC development is of great significance to further explore key diagnostic and therapeutic targets and achieve individualized "precise" treatment for NSCLC patients.The G protein coupled receptor(GPCRs)family plays a central role in physiological functions and is closely related to many human diseases.In particular,G protein-mediated signal transduction plays an increasingly important role in tumor angiogenesis,immune escape,tumor metastasis and drug resistance.Therefore,GPCRs is considered as one of the most effective therapeutic targets for tumors.G protein-coupled bile acid receptor(GPBAR)is the membrane receptor for bile acids and a driving force of the liver-bile acid-microbiota-organ axis to regulate metabolism and other pathophysiological processes such as metabolism and inflammation.GPBAR has become an important therapeutic target for a series of metabolic and inflammatory diseases.Current studies have found that GPBAR plays an important role in the occurrence and development of a variety of tumors.For example,GPBAR can promote the proliferation of colorectal cancer,cholangiocarcinoma,pancreatic cancer,esophageal carcinoma and endometrial cancer by regulating different signaling pathways.Interestingly,the roles of GPBAR in carcinogenesis has also been reported to inhibit the cancer development.For example,GPBAR activity might suppress liver and kidney cancer cell proliferation and migration and promote cancer cell apoptosis.Importantly,several groups have reported that even in the same cancer types,GPBAR activated by different agonists may exert different pro-or anti-tumour effects.Therefore,GPBAR plays different regulatory roles in different tumor tissue types or cell environments.So far,studies on the role of GPBAR in NSCLC are limited.It has been reported that GPBAR is highly expressed in NSCLC,and interference with GPBAR can inhibit the proliferation,migration and invasion of NSCLC cells.However,the specific functions,molecular mechanisms and potential diagnostic value of GPBAR activated by different ligands in NSCLC have not been clarified.This study conducted an in-depth study on the role,mechanism and structural basis of GPBAR activation by different ligands in NSCLC.The main methods and results are as follows:1.Different ligands activate GPBAR to differentially regulate malignant phenotypes of NSCLCIn order to explore the role of GPBAR in the occurrence and development of NSCLC,we first detected the expression of GPBAR in tissues of NSCLC patients and paracancer tissue samples by immunohistochemistry experiments,and the results showed that GPBAR was highly expressed in NSCLC tissues.It is also positively correlated with clinical TNM(Tumor node metastasis)stage.Meanwhile,we intervened GPBAR expression in NSCLC cell lines with different levels of GPBAR expression by MTT(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide).The influence of GPBAR on the proliferation and apoptosis of NSCLC cells was detected by flow cytometry and TUNEL(Terminal dUTP nick-end labeling).Overexpression of GPBAR can significantly promote the proliferation of NSCLC cells,Silencing GPBAR inhibits NSCLC cell proliferation and promotes cell apoptosis.We used NSCLC cell lines and NSCLC patient tissues with different GPBAR expression levels to analyze the correlation between GPBAR expression and clinicopathological features of NSCLC patients,and further explore the possible net effect of GPBAR in NSCLC,The expression of GPBAR in tissues of NSCLC patients and adjacent tissues was detected by immunohistochemistry.The results further confirmed the high expression of GPBAR in NSCLC tissues,and it was positively correlated with the clinical TNM stage of NSCLC.In vitro cell function experiments showed that GPBAR overexpression could significantly promote the proliferation of NSCLC cells.On the contrary,GPBAR silencing inhibits NSCLC cell proliferation and promotes cell apoptosis.In order to determine the effect of changes in GPBAR activity on the phenotype of NSCLC cells,we treated NSCLC cell lines with different concentrations of endogenous bile acids of GPBAR and exogenous synthetic bile acid derivatives,respectively,and detected the difference in proliferation ability of NSCLC cells stimulated by different ligands.The findings:Cholic.acid(CA),Lithocholic acid(LCA),Deoxycholic acid(DCA),Ursodeoxycholic acid(UDCA)and INT-777(6 alpha-23(S)-methylcholic acid)inhibited cell proliferation after activation of GPBAR.CDCA(Chenodeoxycholic acid),TCA(Taurocholic acid),TDCA(Tauroursodeoxycholic acid),GCA(Glycocholic acid),P395 and R399 significantly promoted cell proliferation.INT-777 with the effect of tumor suppressive and R399 with the effect of tumor promoting were selected as research objects,and their effects on malignant phenotypes of NSCLC cells were detected by MTT assay,flow cytometry and TUNEL assay.The results showed that INT-777 inhibited cell proliferation and significantly induced cell apoptosis.But R399 stimulates cell proliferation.Xenograft tumor experiments in nude mice further confirmed the differential regulation of INT-777 and R399 on the growth and apoptosis of NSCLC cells.These results suggest that GPBAR activated by INT-777 and R399 play different biological roles in the regulation of malignant phenotypes in NSCLC cells.2.INT-777 and R399 regulate the malignant phenotype of NSCLC cells by bidirectional regulation of YAP signal differenceTo further investigate the downstream intracellular signaling modulated by R399 and INT-777,we performed transcriptome microarray analysis of H1299 cells exposed to R399,INT-777 and vehicle.We found transcriptome profiling identified YAP signaling to be activated by R399 but inhibited by INT-777.We further verified the differential regulation of the above ligands on YAP signaling pathway by YAP transcriptional activity,YAP phosphorylation level and subcellular localization.Meanwhile,cell function experiments showed that overexpression of YAP could significantly inhibit the apoptosis induced by ENT777,while silencing the expression of YAP or inhibiting its activity could reverse the promoting effect of R399 on H1299 cell proliferation.These results suggest that INT-777 activation of GPBAR plays a carcinogenic role by inhibiting YAP activity,while R399 activation of GPBAR plays a carcinogenic role by activating YAP activity.3.INT-777 and R399 regulate YAP activity and malignant phenotype of NSCLC cells through differential preference signal transduction(Gs vs.β-arrestin)In order to elucidate the specific function and molecular mechanism,we investigated the signal transduction pathways of different ligands and their functions.The Glo-SensorTM cAMP assay,G protein dissociation assay,and β-arrestin recruitment assay revealed that R399 was biased to activate the GPBAR-β-arrestin signaling pathway.On the contrary,similar to endogenous agonist INT-777 was biased to activate GPBAR-Gs signaling pathway.Subsequently,YAP activation assay and cell function experiments indicated that after silencing or inhibiting Gs,the inhibitory effect of INT-777 on YAP activity and cell proliferation was significantly decreased,while after interfering β-arrestinl,R399 could activate YAP and promote cell proliferation was inhibited.These results suggest that GPBAR preference signal transduction driven by INT-777 and R399 may differentially regulate YAP activity,thus playing distinct roles in malignant phenotypes of NSCLC cells.G protein coupled receptor kinase,GRK-induced the phosphorylation of GPCR plays an important role in the GPCR-β-arrestin signaling pathway,but the specific role of GRKs in regulating GPBAR function and phosphorylation remains unclear.This study explored the role of different GRKs subtypes in R399 driven GPBAR-β-arrestin signaling pathway through qRT-PCR and BRET(Bioluminescence resonance energy transfer).GRK2 and GRK5 play a key role in R399 induced GPBAR-β-arrestin 1 recruitment.Dual luciferase reporter gene assay and MTT assay showed that both GRK2 and GRK5 silencing reduced R399 induced YAP transcription activity and cell proliferation.In addition,the potential sites for R399 to induce GRK2 phosphorylation of GPBAR(S310,S321,S323 and S324)were identified by mass spectrometry.4.Structural basis of GPBAR mediated preference pathwayTo elucidate the structural basis of the GPBAR-mediated preference pathway,we recombined R399-GPBAR-Gs signal transduction complex in vitro and analyzed the highresolution structure of R399-GPBAR-Gs using single-particle cryo-electron microscopy.We further inspected the conformational differences between the R399-and INT-777-bound GPBAR structures via root-mean-square deviation(RMSD)over Cα atoms.The results show that conformational differences are concentrated in ECL1,ECL2 and ICL1.Subsequently,alanine mutation was performed on the amino acids with significant conformational differences to detect the effects on downstream Arrestin and cAMP.We found that mutations in R44ICL1,L45ICL1,Q77ECL1 and P151ECL2 led to a significant reduction in Arrestin recruitment.In addition,subsequent cell proliferation assay showed that the P151A mutation eliminated the promoting cell proliferation induced by R399,suggesting that the above amino acids play a key role in GPBAR recruitment of β-arrestin 1.In summary,our study reveals the molecular mechanism of the differential regulation of YAP activity by different ligands driven GPBAR preference signaling(Gs vs.β-arrestin),thus affecting the malignant phenotype of NSCLC.This study also clarified the structural basis of GPBAR preference transduction.All these have laid a foundation for enriching the GPBAR signal transduction mechanism and NSCLC genesis and development,and also provided a new direction for exploring the diagnosis and treatment targets of NSCLC. |
