摘要
Abstract
Objective To investigate the regulatory role of Krüppel-like factor 4(KLF4)in targeting ferroptosis-related proteins to modulate tamoxifen resistance in estrogen receptor(ER)-positive breast cancer.Methods The tamoxifen-resistant subline,TAMR,of ER-positive breast cancer MCF-7 cells was validated for tamoxifen resistance using colony formation assay.Western blotting was per-formed to assess the expressions of ferroptosis-related proteins in MCF-7 and TAMR cells.For functional studies,MCF-7 and TAMR cells with the knockdown of solute carrier family 7 member 11(SLC7A11,also known as xCT),glutathione peroxidase 4(GPX4),or KLF by retroviruses carrying small interference RNA(siRNA)were treated with tamoxifen,and the cell viability was measured via Cell Count-ing Kit-8(CCK-8)assay.The overexpression of KLF4 in MCF-7 and TAMR cells was achieved by transfecting the plasmid of adenovirus KLF4(Ad-KLF4)into the cells.TAMR cells with KLF4 overexpression were treated with different concentrations of tamoxifen,ferroptosis inducer Erastin,and GPX4 inhibitor RSL3,and the cell viability was measured.PCR was used to analyze the knockdown/overexpression efficiency,and assess xCT expression changes upon KLF4 overexpression/knockdown in the cells.Lipid reactive oxygen species(ROS)and free Fe2+levels in MCF-7 and TAMR cells were quantified via flow cytometry using C11-BODIPY,Phen Green™ SK diacetate(PGSK),and FerroOrange probes.The JASPAR database predicted potential KLF4 binding sites on the xCT promoter.Chromatin immunoprecipi-tation(ChIP)assay was conducted to validate these interactions.Results CCK-8 assay and lipid ROS measurements revealed that TAMR cells exhibited significantly higher ferroptosis sensitivity than MCF-7 cells after 48-hour treatment with Erastin at 15,20,25,30,35,40,and 45 μmol/L,or RSL3 at 200,300,400,500,1 000,and 2 000 nmol/L(all P<0.01).Western blotting demonstrated the elevated expres-sions of KLF4,and ferroptosis-related proteins including xCT and GPX4 in TAMR cells compared to MCF-7 cells(all P<0.05).TAMR cells with KLF4 knockdown treated with tamoxifen at 5,10,20,and 30 μmol/L for 48 h had reduced cell viability,increased tamoxifen sensitivity,and elevated levels of free Fe2+and ferroptosis(all P<0.05),while TAMR cells with KLF4 overexpression had enhanced cell viability and decreased levels of lipid ROS and free Fe2+following Erastin(80 μmol/L)or RSL3(25 nmol/L)treatment for 48 h(all P<0.05),suggesting that KLF4 modulates tamoxifen sensitivity and ferroptosis in breast cancer cells.PCR analysis confirmed coordinated expression trends between KLF4 and xCT in MCF-7 and TAMR cells 48 h after KLF4 knockdown/overexpression.ChIP assay validated KLF4 binding to predicted promoter regions of xCT,establishing a direct regulatory mechanism for KLF4 in ferroptosis modulation.Conclusions KLF4 is upregulated in TAMR cells,and its silencing downregulates xCT expression,promotes ferroptosis,and thereby restores tamoxifen sensi-tivity in TAMR cells.关键词
乳腺癌/他莫昔芬/耐药/Krüppel样转录因子4/xCTKey words
breast cancer/tamoxifen/drug resistance/Krüppel-like factor 4/xCT
