Identification Of L-cycloserine As A Ferroptosis-regulating Anti-breast Cancer Agent Based On TCGA Data

Objective:In recent years,with the clinical use of chemotherapy drugs,the survival rate of breast cancer has been improved.However,the acquired chemotherapy resistance in the process of medication leads to the high recurrence rate and distant metastasis of breast cancer patients after radical resection.As a key molecule in ferroptosis signaling,GPX4 is involved in the regulation of tumor recurrence,drug resistance and prognosis.Therefore,ferroptosis has the potential to become a therapeutic target for breast cancer.Unfortunately,to date,only 10 antitumor agents regulating ferroptosis have been identified.Therefore,based on bioinformatics and network pharmacology approaches,we aim to screen innovative anti-breast cancer drugs that can improve the poor prognosis of patients by regulating the ferroptosis pathway.Methods:1.The transcriptome data and clinical data of breast cancer patients in TCGA database were used as the research objects.limma package was used to analyze the differentially expressed genes of ferroptosis.Univariate Cox regression analysis and Lasso Cox regression analysis were used to screen prognostic genes and construct a prognostic model.2.The ferroptosis-regulating anti-breast cancer drug ——Cycloserine was selected by CMap based on ferroptosis differential genes and breast cancer genes suggesting poor prognosis.3.The viability of MDA-MB-231 and MCF-7 breast cancer cells and MCF-10 A and BEAS-2B normal cells incubated with different concentrations of L-cycloserine was determined by CCK8 assay.4.The levels of reactive oxygen species(ROS),iron ion and the expression of ferroptosis key protein GPX4 in two breast cancer cells MDA-MB-231 and MCF-7 were detected by relevant kits and Western Blot.Results:1.49 differentially expressed genes(28 up-regulated genes and 21 down-regulated genes)were screened by "limma" package.Five prognostic genes including CS,GCLC,EMC2,NQO1 and G6 PD were screened by univariate Cox regression analysis,and five prognostic genes were included in Lasso Cox regression analysis to construct prognostic models.Patients were divided into high and low risk groups according to the median score.In order to validate the model,we performed differential analysis and found that the expression levels of the five prognostic genes involved in the model were statistically different between the high and low risk groups(P<0.001).The chi-square test was used to analyze the clinical characteristics such as age,gender,stage,T,M,N between the high and low risk groups,and the results showed that the prognosis characteristics between the high and low risk groups were not affected by age and other clinical characteristics(P>0.05).In order to verify the validity of the risk score,Kaplan-Meier analysis and survival status evaluation were used,and it was found that the overall survival rate(OS)of patients in the high-risk group was worse,and with the increase of risk score,the number of deaths increased,and the expression of genes involved in model construction was up-regulated.These findings indicate that our model was successfully constructed and that the risk score can be used to evaluate the prognostic value.2.We screened Cycloserine,a ferroptosis-regulated anti-breast cancer drug that can inhibit the poor prognosis of breast cancer patients by CMap(enrichment =-0.762,P =0.00658 for values in the tumor suppression group;enrichment =-0.724,P = 0.0118 for values in the high-risk gene suppression group).3.L-cycloserine significantly inhibited the proliferation of MDA-MB-231 and MCF-7 cells in a concentration-dependent manner,and the inhibitory effect on MDA-MB-231 cells was stronger than that on MCF-7 cells.However,L-cycloserine did not significantly inhibit the viability of MCF-10 A breast epithelial cells and BEAS-2B lung epithelial cells.4.L-cycloserine significantly promoted the accumulation of reactive oxygen species(ROS)and iron ion in MDA-MB-231 and MCF-7 cells,and also significantly inhibited the expression of GPX4(P<0.05).Furthermore,in the presence of ferroptosis inhibitor Fer-1,the inhibitory effect of L-cycloserine on MDA-MB-231 and MCF-7breast cancer cells was abolished.Conclusion:L-cycloserine inhibits the proliferation of MDA-MB-231 and MCF-7 cells by inducing the accumulation of ROS and iron ions,down-regulating the expression of GPX4 protein.We propose L-cycloserine as a new potential drug for clinically treating breast cancer that acts by regulating the ferroptosis pathway.