Mechanism Of FXR Involvement In NSCLC Metastasis Via JAK2/STAT3 Signaling Pathway

Background:Non-small cell lung cancer(NSCLC),which accounts for about 85%of all new lung cancers,represents the leading cause of cancer-related death worldwide.Despite recent therapeutic advances,the clinical outcomes for NSCLC patients remain dismal,with the 5-year survival rate only 18%,which can be largely attributed to metastatic spread.It has been acknowledged that invasion-metastasis cascade mainly involves local migration and invasion,intravasation,survival in circulation,extravasation and colonization in distant organs.In addition,the primary tumors can also stimulate the formation of new blood vessels,termed neo-angiogenesis,to assist tumor metastasis.Nevertheless,to data,our knowledge regarding the biological underpinnings that underlie metastatic process is still lacking.Signal transducer and activator of transcription-3(STAT3)is a member of the STAT family of cytoplasmic transcription factors that regulated gene expression involved in cell proliferation,differentiation,anti-apoptosis,angiogenesis,metastasis and immune responses.etc.When activated,STAT3 translocates to nucleus and binds specific DNA sequences as a homodimer to induce target gene transcription.As a canonical stimuli,the cytokine IL-6 binds to its a-receptor subunit,IL-6R,triggering an activated signaling function of another receptor subunit,IL-6ST,also known as gp130,which recruits and elicits the phosphorylation of Janus kinases-1(Jak1)and Jak2.The activated Jak2 in turn phosphorylates the cytoplasmic tyrosine residues on IL-6 receptors,which act as a dock for the SH2 domain of STAT3,resulting in the phosphorylation(at residue of Tyr705)and activation of STAT3 homodimer.Aberrant activation of STAT3 signals in tumor cells is tightly associated with tumor metastasis and angiogenesis.Chang et al reported that IL-6 is increased at the tumor leading edge of invasive breast tumors.IL-6 induces metastases and angiogenesis and promotes myeloid cell recruitment in breast cancer via activating IL-6/Jak1/2/STAT3 signaling pathway.Another study emphasized a pivotal role for IL-6/Jak2/STAT3 signaling pathway in carcinogen Bisphenol A-induced osteosarcoma metastasis both in vitro and in vivo.Therefore,targeting the IL-6/Jak2/STAT3 pathway has recently emerged as an appealing approach for cancer therapy.Farnesoid X Receptor(FXR)belongs to a nuclear receptor superfamily that is highly expressed in enterohepatic tissues.Once being activated by ligands,for example bile acids(BAs),FXR translocate from cytoplasm to nucleus,and transcriptionally regulates downstream target genes related to bile acid,cholesterol,glucose and lipid metabolism.Recently,FXR has been implicated either as an oncogene or as a tumoricidal gene in multiple malignancies,including liver,intestinal,breast,and pancreatic cancers.We previously found that FXR is upregulated in NSCLC than in pericarcinous lung tissues,and that its ligands deoxycholic acid,ursodeoxycholic acid and chenodeoxycholic acid are increased in NSCLC serum than in healthy controls.FXR promotes NSCLC cell proliferation via transcriptionally activating CCND1.Additionally,we detected an immunosuppressive role for FXR in tumor microenvironment,which should also facilitate local tumor growth in NSCLC.However,as a multiple functional transcription factor,whether or not FXR contributes to NSCLC metastasis is still unclear.In this study,we demonstrated that FXR promotes the migration,invasion and angiogenic ability of NSCLC cells in vitro,and increases NSCLC metastasis in mouse models in vivo.Importantly,we discovered a new mechanism that FXR promotes NSCLC metastasis via binding specifically to the promoters of IL-6ST and IL-6 to increase their transcription and activating the downstream Jak2/STAT3 signaling pathway.Clinically,FXR was confirmed to be positively correlated with phosphorylated STAT3 and IL-6ST in NSCLC patients,which indicate poor prognosis.At last,the anti-metastasis activity of FXR inhibitor against FXR-high NSCLC was verified in mouse models in vivo.Our findings suggested that FXR-induced IL-6/IL6ST/Jak2/STAT3 signals promotes NSCLC metastasis,which may open up a novel therapeutic option for treating advanced or metastatic NSCLC patients.Objective:To investigate the effect of FXR on the migration and invasion and angiogenic ability of NSCLC in vivo and in vitro experiments,and whether FXR is involved in the metastasis of NSCLC through the IL-6/IL-6ST/Jak2/STAT3 signaling pathway.Methods:1.Clinical data collection and TCGA database analysis:Immunohistochemistry(IHC)was performed on the adj acent tissues of 160 NSCLC patients and follow-up was conducted.Spearman correlation analysis and Mann-Whitney U test were performed on immunohistochemical results to clarify the correlation between FXR and p-STAT3(Tyr705).The Kaplan Meier analysis was used to investigate the overall survival rate(OS)of patients with follow-up results.In the TCGA public database,Spearman correlation analysis was used to determine the correlation between FXR and the mRNA expressions of STAT3,IL-6,and IL-6ST in NSCLC patients,and Kaplan Meier method was used for survival analysis.2.In vitro experiments,A549 stable transfection strains with high FXR expression(abbreviated as A549 FXR)and blank control stable transfection strains(abbreviated as A549 Lock)were constructed by transfecting A549 and H1975 cells with FXR siRNAs to knock down FXR expression,and by infecting A549 cells with lentivirus.We then tested the effects of FXR on the metastasis,invasion,and angiogenesis of NSCLC through Transwell and angiogenesis experiments.Western blot and real-time fluorescence quantitative polymerase chain reaction(q-PCR)were used to detect the effects of FXR on proteins and IL-6,IL-6ST,and IL-6R mRNA in the Jak2/STAT3 signaling pathway,and to examine whether FXR triggers the Jak2/STAT3 signaling pathway,as well as the effects of FXR on classic upstream triggers of the Jak2/STAT3 signaling pathway such as IL-6,IL-6ST,and so on.3.In vitro experiments,STAT3 siRNAs were introduced or STAT3 inhibitors(statics)were added to inhibit the expression of STAT3.After that,the effects of the Jak2/STAT3 signaling pathway on the metastasis of NSCLC were detected through Transwell experiments and angiogenesis experiments.IL-6ST siRNAs were introduced to inhibit the expression of IL-6ST.Western blot was used to test the interference efficiency of IL-6ST siRNAs,and the effect of IL-6ST on the metastasis of NSCLC was detected by Transwell and angiogenesis experiments.4.In order to further explore the regulatory mechanism of FXR participating in the Jak2/STAT3 signal transduction pathway,we used chromatin immunoprecipitation assay(CHIP)to detect the regulation of FXR on IL-6/IL-6ST DNA,and dual luciferase assay(luciferase assay)to detect the targeted transcriptional regulation of FXR on IL6/IL-6ST promoter.5.In vivo experiments,a stable A549 cell line with high FXR expression(A549FXR)and its blank control(A549-Mock),a stable H1975 cell line with low FXR expression(H1975 FXR shRNA)and its blank control(H1975-Mock)were constructed using lentivirus.Nude mice were randomly divided into groups and injected with lung adenocarcinoma cells through the tail vein to establish a mouse model of lung metastasis,further verifying the effect of FXR on the metastasis of NSCLC.The surface nodules of dissected lung tissue were counted,and the specimens were subjected to H&E staining and immunohistochemical staining to further verify the correlation between FXR and the expression of IL-6,IL-6ST,p-STAT3(Tyr705).Results:1.In vitro experiments revealed that FXR could promote the migration,invasion and angiogenic ability of NSCLC cells.In contrast,inhibition of FXR expression decreased the migratory and invasive ability of NSCLC cells and reduced the tubeforming ability of HUVEC.2.By interfering with the expression of FXR,it was found that FXR can promote the expression of p-Jak2(Tyr1007/100)and p-STAT3(Tyr705),indicating that FXR mediates the activation of the JAK2/STAT3 signaling pathway.After inhibiting the transduction of Jak2/STAT3 signal pathway in A549 cells with high expression of FXR,it was found that the migration,invasion,and angiogenesis abilities of A549 cells decreased.It has been verified that FXR promotes migration,invasion,and angiogenesis of NSCLC by influencing the Jak2/STAT3 signaling pathway.3.When exploring the effect of FXR on upstream targets of the Jak2/STAT3 pathway,it was found that IL-6 and IL-6ST mRNA and protein expressions were downregulated after FXR knockdown,but there was no statistically significant impact on IL-6R.Inhibition of IL-6ST expression in A549 cells with high FXR expression showed that IL-6ST downregulation inhibited the Jak2/STAT3 signaling pathway,and decreased cell metastasis,invasion,and angiogenesis were observed.4.Targeted activation of IL-6 and IL-6ST promoters by FXR resulted in a corresponding increase in the activity of IL-6 and IL-6ST promoters overexpressed by FXR,and a corresponding increase in the content of IL-6 and IL-6ST DNA in immunoprecipitation.5.In vivo experiments,the number of lung metastases in the A549-FXR group was significantly higher than that in the A549-Mock control group,and it was found that the number of metastatic tumors decreased with the increase in the dose of intraperitoneal injection of Z-GS;Similarly,the number of lung metastases in the H1975-FXRshRNA group was significantly lower than that in the H1975-NC group.In vivo,these results indicate that FXR affects the metastasis of NSCLC,and the higher the expression of FXR,the stronger the metastasis ability.6.Immunohistochemical analysis of adjacent tissues from patients with NSCLC showed that there was a positive correlation between FXR and the expression of pSTAT3(Tyr705),and the high expression of FXR and p-STAT3(Tyr705)suggested a poor prognosis.TCGA public database analysis showed that FXR was positively correlated with the expression of IL-6ST,p-STAT3(Tyr705),and the survival rate of patients with high expression of FXR,IL-6ST,p-STAT3(Tyr705)decreased.Conclusion:1.In vitro,FXR promotes the invasion,migration,and angiogenesis of NSCLC cells.2.FXR is involved in the invasion,migration,and angiogenesis of NSCLC by activating the Jak2/STAT3 signaling pathway.3.FXR promotes tumor metastasis and angiogenesis mediated by Jak2/STAT3 by upregulating cytokines such as IL-6 and IL-6ST in NSCLC cells.4.In NSCLC cells,FXR directly transactivates the transcription of IL-6ST and IL6.5.FXR promotes NSCLC metastasis in vivo,and Z-GS reduces NSCLC metastasis of FXR-high in mouse models by inhibiting the FXR/IL-6/IL-6ST/p-STAT3 axis.6.FXR is positively correlated with the expression of p-STAT3(Tyr705)and IL6ST,indicating a poor prognosis in patients with NSCLC.