The Mechanism Of FXR-mediated Ferroptosis Inhibiting Invasion And Metastasis In Breast Cancer

Background:The primary reasons for death from breast cancer are metastasis and diffusion,and a critical process for the invasion and metastasis of tumor cells is epithelial-mesenchymal transition（EMT）.The analysis revealed ferroptosis a novel cell death defined by iron ion and lipid peroxide accumulating,which opens a new prospective for the therapy of breast cancer patients.Prior studies have demonstrated that farnesoid X receptor（NR1H4/FXR）plays a key role in EMT and ferroptosis,but the mechanism of how FXR inhibits breast cancer invasion and metastasis by regulating ferroptosis remains unclear.Objectives:This study aims to study the effects of FXR-regulated ferroptosis on EMT and lung metastasis of breast cancer cells through breast cancer clinical samples,breast cancer cell and xenografted model and give new suggestions for clinical breast cancer treatment.Methods:1.The expression of FXR and EMT-related marker Vimentin in clinical breast cancer samples were both shown using immunohistochemical,and the relationship between them and clinicopathological features as well as the correlation between FXR and Vimentin were analyzed.2.TGF-β1 was used to establish the EMT model of breast cancer cells,and the small molecule inhibitor Z-Guggulsterone（Z-GS）and si RNA technology were used to inhibit the expression of FXR.CCK8 was utilized to identify how Z-GS affected the growth of breast cancer cells MDA-MB-231 and MCF-7 and normal mammary epithelial cells MCF-10A.To examine how FXR affects breast cancer cells’capability to invade and metastasize through cell wounding healing,Transwell migration and invasion experiments.Western blotting is utilized to identify EMT-related and ferroptosis related protein,and the intracellular ROS, MDA,and Fe²⁺content of the lipid ROS,MDA,and Fe²⁺are detected by the commercial kit.3.The effect of FXR on the growth of transplanted tumor and lung metastasis in nude mice was seen,and the level of MDA,Fe²⁺and GSH in tumor were examined.Pathological changes of tumor,lung,liver and kidney were observed by HE staining,and the expressions of FXR,EMT related index E-Cadherin and ferroptosis related index GPX4 were observed by IHC.Results:1.Our clinical sample results showed that FXR and Vimentin expressions were higher in breast cancer tissues of patients with breast cancer metastasis than in paracancer tissues.Furthermore,cancer tissues of patients with breast cancer metastasis as compared to those without metastasis,FXR and vimentin expression was elevated.In addition,we observed that FXR and Vimentin had a positive correlation.2.In breast cancer cell,we discovered that 20μM and 40μM Z-GS may dramatically reduce the growth of MDA-MB-231 and MCF-7 cells.In cell wound healing,Transwell migration and invasion experiment,compared with 20μM Z-GS,40μM Z-GS more significantly inhibit the invasion and metastasis in breast cancer cells.The addition of ferroptosis antagonist Fer-1,apoptosis antagonist Z-VAD-FMK,necrosis antagonist necrosulfonamide（NSA）and autophagy antagonist 3-methyladenine（3-MA）displayed Z-GS mainly acted an anti-tumor growth impact through ferroptosis.Western blotting showed that FXR antagonists Z-GS and si FXR could drastically decrease the expression of FXR and EMT-related proteins N-cadherin and Vimentin,and increased E-cadherin.The expressions of SLC7A11,FTH1 and GPX4,which negatively regulate ferroptosis,were significantly down-regulated.Compared with TGF-β1 group,the intracellular ROS,MDA,and Fe²⁺level of TGF-β1+Z-GS treatment group are significantly increased.Ferroptosis inducer erastin can enhance the effect of FXR antagonist Z-GS to further inhibit the invasion and metastasis of breast cancer cells,while Fer-1 has the opposite effect.3.In the nude mice xenograft experiment,our findings demonstrated that FXR antagonist Z-GS could dramatically slow down xenograft tumor development and lung metastasis,and the ferroptosis inducer erastin could enhance the effect of FXR antagonist and further inhibit the tumor growth and lung metastasis,while the ferroptosis inhibitor Fer-1 had the opposite effect.Interestingly,we also found that FXR antagonist Z-GS significantly up-regulated MDA and Fe²⁺levels in transplanted tumor tissue,while down-regulated GSH levels.Meanwhile western blotting and IHC results also revealed Z-GS dramatically upregulating E-cadherin expression and decreased FXR and GPX4 expression.In addition,HE staining of liver and kidney showed that Z-GS had no adverse effects on both liver and kidney of nude mice.Conclusions:1.Clinical breast cancer tissues has more expression of FXR and Vimentin than paracancer tissues,and the more expression of FXR and Vimentin is related to lymph node metastasis and late TNM stage.2.FXR antagonist Z-GS and si FXR may prevent the invasion and metastasis on breast cancer cells MDA-MB-231 and MCF-7 through regulating ferroptosis.3.FXR antagonist Z-GS inhibits the growth of transplanted tumors and lung metastasis in nude mice by inducing ferroptosis.