The Mechanism Of Targeting SLC7A11 Induces Ferroptosis And Immunogenic Death In Nasopharyngeal Carcinoma

Background and Purpose:Nasopharyngeal carcinoma(NPC)is a malignant tumor originating from the epithelium of the nasopharyngeal mucosa,with high incidence in Guangdong,Hunan of China,and Southeast Asia.Genetic,racial,and environmental factors have been implicated in the etiology and pathogenesis of NPC.Epidermal growth factor receptor(EGFR)is a 170k Da transmembrane receptor,and over 80%of NPC patients have EGFR overexpression,which is associated with poor prognosis.However,the involvement of EGFR in the pathogenesis of NPC has not yet been fully elucidated.Research has found that the cytoplasmic domain of EGFR(ΔN-term)can bind to the central part of member 11 of the solute carrier family 7(SLC7A11)and stabilize its protein expression with non-kinase dependent activity.SLC7A11 is one of the components of the Xc~-system,which can specifically mediate the uptake of extracellular cysteine in a1:1 ratio and pump out intracellular glutamate.The cysteine transported into cells is reduced to cysteine,which is then synthesized into reduced glutathione(GSH).As a cofactor of ROS detoxification enzymes,GSH can protect cells from ROS induced damage,including resistance to excessive intracellular ROS accumulation leading to ferroptosis in cancer cells.On the other hand,SLC7A11 also affects the tumor microenvironment by outputting glutamic acid,inhibiting immune activity and inducing tumor escape.In tumors with high expression of SLC7A11,it is often observed that there is a significant decrease in tumor infiltration of activated T cells and a low immune response to treatment.The SLC7A11 protein is highly expressed in various tumors such as lung cancer,pancreatic ductal adenocarcinoma,and esophageal squamous cell carcinoma,and can serve as a poor prognostic indicator for tumor patients.However,research on its role in NPC is limited and worthy of further investigation.Ferroptosis is a cell death mode caused by the accumulation of iron dependent ROS and the depletion of plasma membrane polyunsaturated fatty acids,which has attracted much attention in the field of anti-tumor.SLC7A11 has been reported to induce ferroptosis resistance in various tumors,and targeting SLC7A11 can induce ferroptosis in tumors,thereby exerting therapeutic effects.Tumor immune surveillance requires CD8~+T cells to recognize major histocompatibility complex I(MHC-I)class molecules.Cancer can reduce the expression of MHC-I cell membrane by downregulating or losing the processing and presentation of tumor specific or associated antigens,thereby avoiding immune monitoring of CD8~+T cells.In many cancers,including esophageal cancer,colorectal cancer,head and neck squamous cell carcinoma,melanoma,etc.,the loss of MHC-I is associated with a worse clinical prognosis.Therefore,understanding the potential mechanisms of MHC-I deficiency is crucial for understanding the pathogenesis of NPC and evaluating the possibility of restoring MHC-I expression to improve treatment methods.This study aims to elucidate the molecular mechanism of SLC7A11in the malignant progression of NPC,provide more sufficient evidence and new perspectives for anti-SLC7A11 treatment,and provide new treatment ideas for NPC patients with low or no response to immunotherapy.Methods:1.Immunohistochemical assay was used to detect the correlation between the expression of EGFR and SLC7A11 in NPC tissues;Western blot(WB)was used to detect the effect of EGFR on the expression of SLC7A11;Co-IP experiment and immunofluorescence assay to detect whether EGFR can bind to SLC7A11;Determination of glutathione content to evaluate the effect of si EGFR and tyrosine kinase inhibitor gefitinib treatment on the expression and transport function of SLC7A11;MG132,Chloroquine combined with WB experiment to determine the way EGFR stabilizes SLC7A11 protein.2.Bioinformatics analysis of the expression of SLC7A11 in various tumors and its relationship with clinical prognosis;Bioinformatics and immunohistochemical analysis of the expression of SLC7A11 m RNA and protein in NPC tissues and its relationship with clinical and pathological characteristics of patients.3.Cystine deficiency experiment confirms the effect of cystine on the proliferation of NPC cells;si SLC7A11 combined with CCK8 and clone formation confirmed the effect of SLC7A11 on the proliferation of NPC cells;The effects of si SLC7A11 and Sorafenib on ROS levels in NPC were detected by DCF cell fluorescence and flow cytometry;RNA-seq combined with GSEA enrichment analysis was used to investigate the effects of SLC7A11 on the ferroptosis pathway and the levels of important ferroptosis genes;WB validation of the effects of si SLC7A11 and Sorafenib on the expression of ferroptosis marker proteins;Transmission electron microscope was used to detect changes in the subcellular structure of NPC after treatment with si SLC7A11 and Sorafenib;Monitoring the in vivo therapeutic effect of Sorafenib on NPC through subcutaneous tumorigenesis in nude mice.4.Immunohistochemical assay to detect the expression of CD8 in NPC tissues;Bioinformatics analysis and RNA-seq combined with GO and KEGG enrichment analysis of signal pathways involved in SLC7A11;si SLC7A11 was co cultured with T cells in NPC cells,and combined with CCK8 and clone formation to confirm the protective effect of SLC7A11 on NPC cells against T cell killing;The effects of SLC7A11on the expression levels of TAP1 and FAF2 and the membrane localization of MHC-I were detected by q PCR and WB;The effect of Does WB detection affect the expression of MHC-I after regulating TAP1expression;Verification of high SLC7A11 expression and glucose-dependent regulation of GR nucleocytoplasmic heterotopy through nucleocytoplasmic protein isolation;Double Luciferase reporter gene experiment combined with CHIP experiment confirmed that GR started TAP1 transcription;The effect of ERAD inhibitors combined with ubiquitination detection on MHC-I ubiquitination levels after regulating FAF2;Monitoring the in vivo therapeutic effect of Sorafenib on NPC through subcutaneous tumorigenesis in nude mice combined with adoptive T cell therapy.Results:1.High EGFR expression in NPC stabilizes the expression of SLC7A11 protein in a kinase independent manner.(1)The expression level of EGFR in NPC tissues is higher than that in paired adjacent cancer tissues,and the EGFR expression is associated with distant tumor metastasis and survival status(P<0.001,P=0.002).Patients with high EGFR expression have significantly shorter survival time(P=0.03);(2)The expression of EGFR and SLC7A11 proteins was significantly positively correlated in NPC tissues(r=0.289,P<0.001),and the survival time of patients with high EGFR and SLC7A11 expression was significantly shortened(P<0.01);(3)In NPC cells,EGFR can positively regulate the protein level and transport function of SLC7A11,while the regulation of EGFR phosphorylation level by gefitinib does not affect the protein level and transport function of SLC7A11;(4)In NPC cells,EGFR binds to SLC7A11 and stabilizes the protein level of SLC7A11 by inhibiting its proteasome degradation.2.SLC7A11 is highly expressed in most malignant tumors and NPC and is associated with poor prognosis in NPC patients.(1)In most malignant tumors,the expression of SLC7A11 m RNA expression is higher in cancer tissues than in non-cancerous tissues,and the high expression of SLC7A11 protein indicates poor prognosis in patients;(2)SLC7A11 m RNA and protein are highly expressed in NPC patients,and high expression of SLC7A11 protein is associated with T stage,distant tumor metastasis,and survival status(P=0.018,P<0.001,P<0.001).Patients with high expression of SLC7A11 have significantly shorter survival time(P<0.001),and SLC7A11 protein can be used as a molecular indicator to evaluate the prognosis of NPC patients.3.Targeting SLC7A11 induces ferroptosis in NPC cells.(1)The deficiency of cystine has a significant inhibitory effect on the proliferation of NPC cells,and the demand for cystine is higher in SLC7A11 overexpressing 6-10B and SUNE1 cells;(2)Knocking down SLC7A11 inhibits NPC cell proliferation and clone formation;(3)Knocking down SLC7A11 induces ROS accumulation in NPC cells,and upregulates the expression of ferroptosis protein and changes in the ferroptosis subcellular structure after knocking down SLC7A11;(4)Sorafenib targets SLC7A11 to inhibit the proliferation and clone formation of NPC cells,upregulate the expression of ferroptosis protein,and alter the subcellular structure of ferroptosis;(5)Sorafenib inhibits the growth of NPC cells,induces cell death,and upregulates the expression of ferroptosis protein in vivo.4.Targeting SLC7A11 promotes antigen presentation and activation of T cell immunity in NPC cells.(1)The survival time of patients with high CD8~+T infiltration in NPC tissues was significantly prolonged(P=0.015),and SLC7A11expression was negatively correlated with tumor infiltrating lymphocyte(r=-0.144,P=0.003);(2)Knocking down SLC7A11 can enhance the killing ability of T cells against NPC cells;(3)The downstream genes of si SLC7A11 in NPC are enriched during MHC-I antigen presentation and ERAD pathway.Among them,SLC7A11 inhibits TAP1 and weakens the expression of MHC-I membrane in NPC cells,while SLC7A11 induced high glucose dependent inhibition of transcription factor GR into the nucleus inhibits TAP1transcription;meanwhile,SLC7A11 promotes FAF2 expression,activates the ERAD pathway,and promotes MHC-I degradation;(4)Sorafenib targeting SLC7A11 induces NPC cells to be killed by T cells,and Sorafenib combined with T cell adoptive therapy shows the maximum therapeutic effect in vivo.Conclusion:1.There is a positive correlation between the expression of EGFR and SLC7A11 in NPC tissues and cells;after binding to EGFR and SLC7A11,the SLC7A11 protein expression is stabilized in a kinase independent manner,avoiding its degradation through the proteasome pathway.2.In most malignant tumors,SLC7A11 is highly expressed,and its high expression indicates poor prognosis for patients;SLC7A11 is highly expressed in NPC cells and tissues,and is associated with poor prognosis in NPC.3.Knocking down SLC7A11 can induce ferroptosis in NPC cells;Sorafenib targeting SLC7A11 can induce ferroptosis in NPC cells in vitro and in vivo.4.SLC7A11 mediates high glucose dependent inhibition of GR nuclear translocation in cancer cells,downregulates TAP1 transcription,and weakens MHC-I membrane expression in NPC cells.At the same time,SLC7A11 upregulates FAF2 expression and activates the ERAD pathway to promote MHC-I degradation,ultimately promoting immune escape in NPC cells by inhibiting the antigen presentation process of cancer cells;Sorafenib targets SLC7A11 to promote immunogenic death of NPC cells.