FXR Modulates Non-small Cell Lung Cancer Carcinogenesis And Its Clinical Implications

Objective This study was aimed to investigate the expression and oncogenic role of farnesoid X receptor(FXR)in non-small cell lung cancer(NSCLC),as well as to explore the regulation of FXR on PD-L1 expression and potential role for targeted therapy.Methods1.Immunohistochemistry(IHC)was performed to detect FXR expression in NSCLC specimens.Correlations between FXR and overall survival(OS),as well as clinicopathological characteristics were analyzed.2.FXR antagonist Z-guggulsterone and specific si RNAs were used to suppress protein expression;FXR sh RNA or c DNA-expressing vectors were utilized to generate cells with FXR stably knockdown or overexpression.The effects of FXR on in vitro proliferation,cell cycle distribution,apoptosis,and in vivo tumor growth in nude mice were examined.Western blot was performed to evaluate the impacts of FXR on cell cycle regulators.Specific si RNAs to small heterodimer partner(SHP),a downstream target of FXR,were applied to suppress protein expression.Whether or not SHP is involved in the regulation of in vitro proliferation or cell cycle regulators was explored.3.Chromatin immunoprecipitation(Ch IP)assay and luciferase bioluminescence method were performed to examine the effects of FXR in directly transactivating cyclin D1.Rescue experiments were performed to evaluate the functional relevance of cyclin D1 and FXR in NSCLC cells.IHC was performed to detect cyclin D1 expression in the same cohort of NSCLC samples,and correlation between cyclin D1 and FXR expression was analyzed.4.The impact of FXR on PD-L1 expression in NSCLC cells was examined,and the correlation between FXR and PD-L1 expression was analyzed in the same NSCLC samples.The expression levels of mus FXR in mouse lung cancer,colorectal cancer,and melanoma,as well as the impact of mus FXR on mus PD-L1 expression in mouse cancer cells were evaluated.Whether or not SHP is involved in the regulation of PD-L1 expression was explored;Ch IP assay was performed to examine the binding of FXR to PD-L1 promoter;the impact of FXR on EGFR phosphorylation was evaluated, and whether or not phosphorylated EGFR mediates the effect of FXR on PD-L1 expression was explored.Results1.A total of 160 NSCLC patients were included in this study.IHC scoring showed that FXR protein was significantly upregualted in carcinous,compared to pericarcinous,lung tissues(P < 0.001).High FXR expression was positively associated with a more advanced pathological stage and T status,as well as a larger tumor size(all P < 0.05).Kaplan-Meier survival curves showed that the OS was significantly worse in “FXR high” NSCLC patients than in those with low expression(P = 0.0032).Multivariate Cox regression analysis revealed that “FXR high” pattern was an independent predictor for poor OS in NSCLC(Hazard ratio [HR] 1.71,95%CI 1.108-2.637;P = 0.015).2.NSCLC cells H1975 and H1299 highly express FXR protein and m RNA,while HCC4006 has relatively low FXR expression.FXR suppression led to significantly decreased in vitro proliferation and Ki-67 expression in H1975 and H1299 cells,whereas enforced FXR in HCC4006 significantly promoted cell proliferation and Ki-67 expression.Tumorigenicity assays in nude mice showed that FXR knockdown significantly inhibited the growth of xenografts.FXR suppression led to cell cycle G0/G1 arrest,while had no impact on apoptosis.Western blot results showed that the cyclin D1 and phosphorylated Rb(p-Rb)protein levels were remarkably reduced in FXR-suppressed cells,whereas ectopic FXR expression in HCC4006 increased the cyclin D1 and p-Rb protein levels.SHP suppression induced by specific si RNAs had no impact on either in vitro proliferation or cyclin D1 and p-Rb expression,implying that FXR promotes NSCLC cell proliferation and cell cycle regulators expression through a mechanism that is independent of SHP.3.Ch IP results indicated that FXR can directly bind to the IR-0 motif in cyclin D1 promoter in NSCLC cells.Luciferase reporter assay showed that FXR contributes to transactivating cyclin D1.Moreover,overexpression of cyclin D1 reversed the impaired in vitro proliferation and the delayed cell cycle progression induced by FXR-si RNAs.IHC staining showed that there was a positive correlation between FXR and cyclin D1 expression in NSCLC specimens(P < 0.001).Kaplan-Meier survival curves showed that a combinatorial expression pattern of “FXR high” and“cyclin D1 high” predicted the worst OS in NSCLC patients(P = 0.0077).4.FXR suppression significantly increased PD-L1 expression in NSCLC cells.Conversely,enforced FXR decreased PD-L1 expression.IHC staining showed that there was a negative correlation between FXR and PD-L1 expression in NSCLC specimens(P < 0.05).Mouse melanoma cell S91 had relatively high mus FXR expression;in addition,mus FXR-si RNA significantly unregulated mus PD-L1 expression in S91 cells.SHP suppression had no impact on PD-L1 expression in NSCLC cells,suggesting that FXR inhibited PD-L1 expression through a mechanism that was independent of SHP.Ch IP results showed that FXR can directly bind to the promoter of PD-L1 in NSCLC cells.At last,we found that FXR decreased PD-L1 expression via repressing EGFR phosphorylation(Tyr1068).Conclusions This study is the first to comprehensively investigate the expression and functional role of FXR in NSCLC.We found that FXR was upregulated in NSCLC specimens and independently predicted poor clinical outcomes.FXR contributes to NSCLC cell cycle progression and tumor growth via directly transactivating cyclin D1.Moreover,FXR inhibited PD-L1 expression in tumor cells through either directly transrepressing PD-L1 or suppressing EGFR phosphorylation.This study unravelled the biological role of FXR in NSCLC carcinogenesis,providing a promising prognostic biomarker and therapeutic target.