The Role And Mechanisms Of SLC7A11 In Radiation-induced Ferroptosis In Breast Cancer Cells

Background:Breast cancer is the most common type of cancer in the world,with high heterogeneity and remarkable diversity in genetics,genotypes,and biological behaviors.Molecular subtyping of breast cancer plays a major role in its treatment response.Estrogen receptor α(ERα),encoded by the ESR1 gene,is the most widely used molecular marker for breast cancer,and its activity involves multiple genes transcription,which are associated with breast cancer cell proliferation.ER(α)positive patients account for about 70-80% of breast cancer patients.Radiation therapy is a crucial adjuvant therapy in breast cancer treatment,which can significantly reduce the local recurrence rate after breast-conserving or total mastectomy,and prolong diseasefree survival and overall survival in patients with lymph node metastasis.Ferroptosis is a tightly regulated form of programmed cell death and is characterized by irondependent accumulation of lipid peroxidation,and distinct from apoptosis,necrosis,and autophagy.Extensive studies have shown that ferroptosis plays a key role in tumor suppression and can combat drug resistance in cancer therapy,and is providing new opportunities for cancer therapy.Reactive oxygen species(ROS)induced by ionizing radiation(IR)can attack cellular lipids and cause peroxidation of the plasma membrane,thereby inducing ferroptosis.Therefore,there is a strong relationship between radiotherapy and ferroptosis.Metastasis and drug resistance are the biggest challenges in breast cancer treatment at present.Whether IR can induce ferroptosis in breast cancer cells,improve the effectiveness of radiotherapy,and reduce the risk of recurrence needs to be further clarified.Objective:To explore the role and mechanism of SLC7A11,one of the key regulators of ferroptosis,in radiation-induced ferroptosis in breast cancer cells,and to find new clues and theories for sensitization of breast cancer radiotherapy.Methods:1.Differential expression analysis was performed to identify significantly altered genes between 1191 tumor and 112 normal tissues from breast cancer patients in the TCGA database.2.The patients were divided into two groups(high and low SLC7A11 expression levels),and the relationship between SLC7A11 expression status and clinicopathologic characteristics of breast cancer was analyzed by Chi-square test.Combined with the survival data,the relationship between SLC7A11 and the prognosis of breast cancer patients was analyzed by univariate and multivariate Cox regression models.The prognostic relationship between SLC7A11 and molecular subtypes of breast cancer was analyzed by K-M method.3.ER-positive cell lines MCF-7 and ZR-75-1 and the ER-negative cell line MDAMB-231 were treated with IR(8Gy).Levels of RNA and protein were analyzed using q PCR and western blotting.4.Cells were treated with IR,and the levels of cellular lipid peroxidation and death were detected by flow cytometry at 48 and 72 hours,respectively.5.The interaction between ESR1,NDEE4 L and NDFIP1 and SLC7A11 was detected by protein immunoprecipitation(IP).6.The transcriptional regulation effect of ESR1 on SLC7A11 and EGR1 on MALAT1 was detected by dual luciferase assay.7.By co-transfection with ubiquitin(Ub)plasmids,the ubiquitination level of SLC7A11 was detected by IP assay.Results:1.SLC7A11 was highly expressed in breast cancer tissues and associated with poor prognosis of patients.Gene differential expression analyses were performed for breast cancer patients in TCGA database,and results showed that SLC7A11 was highly expressed in cancer tissues,and lower in ER-positive patients than in ER-negative patients.Chi-square test results showed that the expression level of SLC7A11 was related to the age,histological type,molecular type,ER status,PR status and N stage type of breast cancer patients.Univariate and multivariate Cox regression analysis showed that the expression level of SLC7A11 was an independent factor affecting the prognosis of breast cancer patients.K-M survival analysis of different breast cancer molecular subtypes revealed that high SLC7A11 expression predicted poor prognostic outcomes in ER-positive patients.2.SLC7A11 was involved in the regulation of IR-induced ferroptosis in ERpositive breast cancer cells.IR treatment of different breast cancer cell lines showed that IR could effectively induce ferroptosis in ER-positive breast cancer cells MCF-7 and ZR-75-1,while IRinduced ferroptosis in ER-negative cells MDA-MB-231 was not obvious.After the knockdown of SLC7A11,the levels of IR-induced lipid peroxidation and cell death were significantly increased.After being treated by IR,the m RNA level of SLC7A11 was significantly increased and then recovered;the protein level of SLC7A11 increased first and then decreased below the basal expression.3.IR participated in radiation resistance by activating SLC7A11 via ESR1.Bioinformatics analysis showed that ESR1 was positively correlated with SLC7A11 in patients with ER-positive breast cancer,and high expression of ESR1 predicted poor prognosis.After IR treatment of ER-positive cells,the m RNA levels of ESR1 and SLC7A11 were both increased.But at the protein level,ESR1 was elevated and not decreased with SLC7A11.After inhibiting ESR1,the m RNA and protein levels of SLC7A11 were also significantly reduced,and overexpression of ESR1 could significantly enhance the transcriptional activity and protein expression of SLC7A11.In addition,knockdown of ESR1 was able to suppress the IR-induced adaptive elevation of SLC7A11 expression.In terms of cell death,knockdown of ESR1 enhanced IR-induced cellular lipid peroxidation and death,and this process was reversed by overexpression of SLC7A11.Overexpression of ESR1 in ER-negative cells MDA-MB-231 also reduced IR-induced levels of cellular lipid peroxidation and death.4.IR induced the degradation of SLC7A11 through the ubiquitination pathway.Treatment of cells with the proteasome inhibitor MG132 blocked IR-induced degradation of SLC7A11.After knockdown of the E3 ubiquitin ligase NEDD4 L,IRinduced degradation of SLC7A11 could be inhibited.IP and ubiquitination IP experiments also confirmed that IR enhanced the interaction between NEDD4 L and SLC7A11,and increased the ubiquitination level of SLC7A11.5.IR induced ubiquitin-mediated degradation of SLC7A11 via the EGR1-MALAT1-NDFIP1-NEDD4 L axis.Overexpression of LncRNA MALAT1 degraded SLC7A11 through the ubiquitination pathway and increased the level of ferroptosis in breast cancer cells.Under the induction of IR,EGR1 was activated firstly,which in turn promoted the transcription of MALAT1,and then caused the ubiquitin-mediated degradation of SLC7A11,which finally led to ferroptosis in breast cancer cells.Co-transfection of the NDFIP1 with NEDD4 L significantly increased the ubiquitination level of SLC7A11.In addition,inhibition of NDFIP1 simultaneously inhibited the ubiquitin-mediated degradation of SLC7A11 induced by MALAT1 and IR,and also reduced the level of cellular lipid peroxidation and death.Conclusion:1.SLC7A11 is highly expressed in breast cancer tissues and associated with poor prognosis in ER-positive patients.2.In patients with ER-positive breast cancer,the expression of ESR1 is positively correlated with that of SLC7A11,and the high expression of ESR1 indicates a poor prognosis.3.SLC7A11 is involved in the regulation of IR-induced ferroptosis in ER-positive breast cancer cells.4.IR activates the transcriptional activity of SLC7A11 through ESR1,inhibits cell ferroptosis,and participates in radiation resistance.5.IR induces ferroptosis by inducing NEDD4L-mediated ubiquitination of SLC7A11.6.IR activates the transcription of MALAT1 via EGR1,promotes the degradation of SLC7A11,and leads to radiation sensitization.7.MALAT1 induces the ubiquitin-mediated degradation of SLC7A11 through the adaptor protein NDFIP1 of NEDD4 L,which causes ferroptosis and plays a role in radiosensitivity.