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Original Article
CNS cancer Elevated SLC3A2 Expression Promotes the Progression of Gliomas and Enhances Ferroptosis Resistance through the AKT/NRF2/GPX4 Axis
Yuqian Zheng1,2orcid, Shaolong Zhou1, Yiran Tao1,2, Zimin Shi1,2, Xiang Li1,2, Xudong Fu1, Jian Ma1, Weihua Hu1, Wulong Liang1orcid, Xinjun Wang1,2,3,4,5orcid
Cancer Research and Treatment : Official Journal of Korean Cancer Association 2026;58(1):71-94.
DOI: https://doi.org/10.4143/crt.2024.933
Published online: March 10, 2025

1Department of Neurosurgery, The Fifth Affiliated Hospital of Zhengzhou University, Zhengzhou, China

2Department of Neurosurgery, The Third Affiliated Hospital of Zhengzhou University, Zhengzhou, China

3Henan International Joint Laboratory of Glioma Metabolism and Microenvironment Research, Zhengzhou, China

4Maternal and Child Neurological Disorders International Joint Research Center, Zhengzhou, China

5Tianjian Laboratory of Advanced Biomedical Sciences, School of Life Sciences, Zhengzhou University, Zhengzhou, China

Correspondence: Xinjun Wang, Department of Neurosurgery, The Fifth Affiliated Hospital of Zhengzhou University, 03-Kangfuqian Street, Erqi District, Zhengzhou 450000, Henan, China
Tel: 86-13700883378 E-mail: wangxj@zzu.edu.cn
Co-correspondence: Wulong Liang, Department of Neurosurgery, The Fifth Affiliated Hospital of Zhengzhou University, 03-Kangfuqian Street, Erqi District, Zhengzhou 450000, Henan, China
Tel: 86-15929300106 E-mail: fenghua880725@126.com
• Received: October 31, 2024   • Accepted: March 7, 2025

Copyright © 2026 by the Korean Cancer Association

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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  • Purpose
    The aim of this study is to determine the impact of solute carrier family 3 member 2 (SLC3A2) on the malignant phenotype of gliomas and its role in regulating ferroptosis sensitivity.
  • Materials and Methods
    The malignant phenotype of glioma was assessed by cell proliferation assay, colony formation assay, EdU assay, wound healing, and Transwell experiments. We further validated the impact of reduced SLC3A2 expression on the sensitivity to ferroptosis in glioma cells through Cell Counting Kit-8 assays, flow cytometry, western blotting, and transmission electron microscopy. Western blot was used to explore how SLC3A2 affects glioma sensitivity to ferroptosis through the AKT/NF-E2-related factor 2 (NRF2)/glutathione peroxidase 4 (GPX4) axis. By establishing a subcutaneous xenograft tumor model in BALB/c-nude mice, we investigated the growth of tumors following the knockout of SLC3A2 in glioma cells.
  • Results
    Downregulation of SLC3A2 suppressed the malignant phenotype of glioma by blocking the cell cycle and epithelial-mesenchymal transition processes. On the other hand, loss of SLC3A2 not only downregulated SLC7A11 but also prevented the activation of the AKT/NRF2/GPX4 axis. These lead to increased accumulation of reactive oxygen species and lipid peroxides, ultimately enhancing the susceptibility of glioma to ferroptosis.
  • Conclusion
    Our findings suggest that SLC3A2 is an oncogene in gliomas, promoting their occurrence and development. It plays a critical role in ferroptosis resistance through the AKT/NRF2/GPX4 axis.
  • Key words: Glioma, Ferroptosis, SLC3A2, Therapeutic sensitivity
Glioma is the most common malignant tumor of the central nervous system, accounting for approximately 30% of primary brain tumors [1]. Glioblastoma (GBM) is the most malignant tumor among gliomas, and the median survival post-treatment is only 14.6 months [2]. Standard-of-care therapy for glioma consists of safe maximum resection followed by adjuvant chemoradiotherapy [3]. The residual tumor burden and therapy resistance of glioma lead to a high recurrence rate and unfavorable prognosis [4]. Although many targeted therapies and immunological therapies were under clinical trials [5,6], patients with glioma have limited treatment options. Therefore, the exploration of new effective therapeutic targets for glioma is urgently needed.
Ferroptosis is a form of cell death that depends on iron ions and is characterized by the accumulation of lipid peroxides [7]. Accelerated metabolism of tumor cells can lead to increasing of reactive oxygen species (ROS) loads in many kinds of cancers including glioma. Breaking the lipid redox balance and inducing ferroptosis in tumor cells become a potentially effective strategy for cancer treatment [8]. Ferroptosis is the most enriched programmed cell death process in glioma [9]. Studies which used ferroptosis inducers to inhibit glioma have shown promising anti-tumor effect in vitro and in vivo [10,11]. Hence, increasing susceptibility to ferroptosis may be an ideal way for glioma treatment.
Solute carrier family 3 member 2 (SLC3A2, also known as 4F2hc or CD98hc) is a type II transmembrane glycoprotein belonging to the SLC3 family of solute carriers. It forms the Xc- system together with solute carrier family 7 member 11 (SLC7A11), which is responsible for transporting cystine into cells and promoting the synthesis of glutathione, that acts as a substrate for the reduction of lipid peroxidase [12]. In addition to SLC7A11, SLC3A2 can also act as a heavy chain to form various heteromeric amino acid transporters with other members of the SLC7 family, mediating the transmembrane transport of various essential amino acids and polyamine substances in cells [13,14]. SLC3A2 has a wide range of regulatory effects on cellular processes such as growth, differentiation, metabolism, and adipogenesis [15,16]. In tumor cells, studies have shown that SLC3A2 is involved in the proliferation, migration, invasion and chemoradiotherapy sensitivity of different kinds of tumors [17-19]. However, the specific regulatory roles of SLC3A2 in the malignant phenotype and ferroptosis sensitivity of gliomas have not been reported.
In this study, the role of SLC3A2 in the malignant progression and ferroptosis sensitivity of gliomas was explored by using glioma cell lines, pathological tissue samples, and animal models. We found that SLC3A2 is a promising target for glioma treatment.
1. Cell culture and reagents
Human astrocytes (NHA) and human glioma cell lines (U87, U118, U251, LN18, LN229, and T98G) were purchased from iCell Bioscience Inc. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biosharp) supplemented with 10% fetal bovine serum (ExCell) and penicillin-streptomycin solution (Solarbio) at a 37℃ CO2 incubator. The antibodies used in this study included anti-CD98 (PA5-96401, Invitrogen), anti-SLC7A11 (12691S, Cell Signaling Technology), anti-GPX4 (ab125066, Abcam), anti-AKT1/2/3 (T55561F, Abmart), anti–phospho-AKT (Ser473) (T40067F, Abmart), EMT Marker/Epithelial to Mesenchymal Transition Marker (ab216833, Abcam), anti-Nrf2 (ab62352, Abcam), anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6000-1-lg, Proteintech), anti–β-tubulin (M20005, Abmart). The AKT activator SC79 (SF2730) was purchased from Beyotime (Beijing). The ferroptosis inducer RSL-3 (HY-100218A) and Erastin (HY-15763) were purchased from MCE. The apoptosis inhibitor Z-VAD-FMK (C1202) and the necrosis inhibitor necrosulfonamide (SC4359) were purchased from Beyotime. The ferroptosis inhibitor ferrostatin-1 (17729-10) was purchased from Cayman Chemical.
2. Sample collection
All human tissue specimens used in this study were obtained from surgical patients at the Fifth Affiliated Hospital of Zhengzhou University, from 2016 to 2019. According to the World Health Organization (WHO) tumor classification, 11 cases were low-grade gliomas (grades I-II) and 17 cases were high-grade gliomas (grades III-IV). None of the patients received radiotherapy or chemotherapy before surgery. Detailed clinical data were collected for all specimens.
3. RNA sequencing data acquisition and differential gene expression (differentially expressed genes) analysis
RNA sequencing data from the TCGA-GBM and TCGA-LGG (lower grade glioma) projects were downloaded from The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer.gov) and processed to extract TPM-formatted data and clinical data. The median SLC3A2 expression was regarded as the cut-off value to identify differentially expressed genes (DEGs) between the two groups (low- and high-expression) of SLC3A2 in GBM/LGG samples. And we used the DESeq2 R package ver. 1.36.0 according to the standard procedure for analysis [20-22].
4. Functional enrichment analysis and gene set enrichment analysis
The clusterProfiler R package ver. 4.4.4 was used for functional enrichment analysis [23] and gene set enrichment analysis (GSEA) [24]. The threshold of DEGs performed for functional enrichment analysis was defined as |logFC| > 1 and adjusted p-value < 0.05. Gene Ontology, consisting of biological processes (BP), cellular components (CC), and molecular functions (MF), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were analyzed. For GSEA, the number of permutations was set to 1,000 for each analysis. Enrichment results were considered statistically significant when p.adj < 0.05 and false discovery rate q-value < 0.25.
A website (https://cn.string-db.org/) was used to predict the mutual relationship between SLC3A2 and other proteins such as SLC7A11, serine/threonine kinase (AKT), NF-E2-related factor 2 (NRF2), and glutathione peroxidase 4 (GPX4).
5. Immunohistochemistry
Tumor tissue samples were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and cut into 5 μm sections. The sections were deparaffinized by xylene and rehydrated in a graded series of ethanol. Antigen retrieval was performed using sodium citrate antigen repair solution, endogenous peroxidase was blocked with 3% hydrogen peroxide, and non-specific binding was blocked with goat serum. The sections were incubated with primary antibodies at 4℃ overnight and then incubated with peroxidase-conjugated secondary antibodies at room temperature for 20 minutes. The sections were then subjected to colorimetric detection using 3,3’-diaminobenzidine (DAB) and counterstained with hematoxylin. The staining results were evaluated by assessing the proportion and intensity of stained cells, and the immunoreactivity score (IRS) and Histochemistry score (H-Score) were calculated. The IRS was calculated as SI (staining intensity)×PP (percent of positive cell). SI was divided into four levels: 0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining. PP was divided into four levels: 0, 0%-5%; 1, 6%-25%; 2, 26%-50%; 3, 51%-75%; 4, > 75%. The H-Score was calculated as Σ(pi×i)=(percentage of weak intensity cells×1)+(percentage of moderate intensity cells×2)+(percentage of strong intensity cells×3), where i represents the grading of positive cells and pi represents the corresponding percentage of positive cells.
6. Western blot
Total proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology) with protease inhibitors and phosphatase inhibitors (Solarbio). The concentration of the extracted proteins was determined using the bicinchoninic acid protein assay kit (Beyotime). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes (0.22 μm) (Millipore) using a wet transfer system (200 mA, 2 hours). The membranes were blocked with blocking buffer (NCM Biotech) for 10 minutes and then incubated with primary antibodies overnight at 4°C. After incubation with secondary antibodies (1:5,000, Abcam) at room temperature for 1 hour, the immunoblots were detected using a chemiluminescence detection kit (Thermo Fisher Scientific). GAPDH or β-tubulin was used as the internal control.
7. Cell viability and proliferation assays
The cell counting kit-8 (CCK8) (C0043, Beyotime) was used for cell viability and proliferation tests. For the cell viability assay, 3,000 cells were seeded in each well of a 96-well plate. After treatment with the indicated reagents, the culture medium was replaced with DMEM containing CCK8 solution and incubated in a cell culture incubator for 2 hours. The optical density was measured at 450 nm using a microplate reader (Tecan). For the cell proliferation assay, 1,500 cells were seeded in each well of a 96-well plate and measured on days 1, 2, 3, 4, and 5.
8. Colony formation assay
One thousand cells were seeded in a 6-well plate and grown for 11-14 days. Then, the cells were fixed with 4% paraformaldehyde (Biosharp) and stained with crystal violet (Solarbio).
9. EdU-DNA synthesis assay
Cell proliferation and DNA synthesis activity were measured using the BeyoClick EdU Cell Proliferation Kit with Alexa Fluor 555 (C0075S, Beyotime). The cells were labeled with the pre-warmed EdU working solution for 2 hours, fixed with 4% paraformaldehyde (Biosharp) for 15 minutes, permeabilized with 0.3% Triton X-100 in phosphate buffered saline (PBS) for 15 minutes, and then replaced with 500 μL click reaction mixture. After incubation at room temperature in the dark for 30 minutes, Hoechst 33342 was added to each well and incubated at room temperature in the dark for 10 minutes. Images were captured using a fluorescence microscope (Nikon).
10. Wound healing and Transwell assay
Cell migration ability was assessed using the wound healing assay. When the cells reached approximately 70% confluence, a straight scratch was made using a 200 μL pipette tip, and the cells were washed with PBS and replaced with fresh medium. Photographs were taken at 0, 24, and 48 hours. Cell invasion ability was measured using the Transwell assay (Corning). Matrigel Matrix (Corning) was added to the upper chamber (100 μL/well), then 100 μL of serum-free culture medium containing 1.5×104 cells was added to the upper chamber. The lower chamber was filled with 800 μL of complete medium containing 10% fetal bovine serum to attract cell migration and invasion. After incubation for 48 hours, the cells on the upper surface were removed using a cotton swab, fixed with 4% paraformaldehyde (Biosharp) for 30 minutes, stained with 1% crystal violet for 15 minutes, and washed with PBS to remove the excess crystal violet. Five random fields were selected and photographed under microscope (Nikon).
11. Detection of lipid peroxide levels
The level of lipid peroxides in glioma cells was detected using the Lipid Peroxide Probe Kit (L248, Dojindo). The cells were labeled with Lipperfluo for 30 minutes and analyzed using a flow cytometer (BD FACSAria II). The results were analyzed using FlowJo X ver. 10.0.7 software.
12. Detection of ROS levels
The ROS levels in glioma cells were detected using the Reactive Oxygen Species Assay Kit (S0033S, Beyotime). The cells were labeled with fluorescence probe DCFH-DA for 20 minutes and observed directly using a fluorescence microscope or collected after trypsinization for flow cytometry analysis.
13. Cell cycle analysis
Glioma cells were fixed overnight in 70% ethanol and stained with propidium iodide at room temperature for 30-60 minutes. The cells were then analyzed by flow cytometry, and the data were analyzed using ModFit LT 5.0 software (Verity Software House).
14. Transmission electron microscopy
The treated cells were fixed with electron microscope fixative (Servicebio), embedded in resin, and sliced with an ultra-thin slicer. The slices were stained with 2% uranyl acetate and 2.6% lead citrate. Then, the slices were observed and imaged using a transmission electron microscope (Hitachi, HT7800/HT7700).
15. Regulation of SLC3A2 expression using siRNA and CRISPR/Cas9 technology
Lipofectamine 3000 (Invitrogen) was used to transfect small interfering RNA (siRNA). The specific target sequences were as follows: siRNA-SLC3A2-001: GTCCTTGCATCCGA-CTAA; siRNA-SLC3A2-002: GTGCCAACATGACTGTGA; siRNA-SLC3A2-003: CCGGACTCTTCTCCTATA; siRNA-SLC3A2-004: GGATGAGATTGGCCTGGAT. To stably knock out the SLC3A2 gene in glioma cells, we constructed a lentiviral vector carrying sgRNA (lentiCRISPR v2-puro) and infected tumor cells using a lentivirus. The sgRNA sequence was GAAGGTCGCCGATGCGGTAG. After 24 hours of virus infection, the culture medium containing the virus was removed and replaced with medium containing puromycin (1 μg/mL) for selection. The knockout efficiency was verified using western blotting.
16. Xenograft tumor experiment
BALB/c-nude mice (6-8 weeks old, 18-21 g) were purchased from Gempharmatech Co., Ltd. LN229 cells transfected with either NC or KO-SLC3A2 were injected subcutaneously into the mice at a dose of 1×106 cells per mouse. Tumor growth was measured every 7 days, and tumor volume was calculated using the formula: length×width2×Π/6. On day 35, the mice were euthanized, and the tumors were removed and weighed. The tumor tissues were sectioned and stained for SLC3A2 and Ki-67. Three random fields were selected for photography, and the proportion and intensity of positive cells were calculated.
17. Statistical analysis
Data were analyzed using GraphPad Prism 9 software (GraphPad Software Inc.). Measurement data are presented as the mean±standard deviation. Two-group comparisons were performed using the t test, and the p-value was calculated using a two-tailed test. A p-value < 0.05 was considered statistically significant. Cox proportional hazards regression analysis was used for univariate and multivariate analysis.
1. Upregulation of SLC3A2 in glioma is associated with poor prognosis and clinical features
The expression of SLC3A2 in cancer tissues and corresponding normal tissues was analyzed based on TCGA and GTEx data sets. The results showed that the expression of SLC3A2 is significantly increased in most types of cancer, including LGG and GBM (Fig. 1A). The SLC3A2 expression went up with the grades of glioma (Fig. 1B). Compared with patients with isocitrate dehydrogenase (IDH) mutation, tumor tissues from patients with IDH wild-type contained more SLC3A2 transcript (Fig. 1C). Higher SLC3A2 level was detected in patients with 1p/19q co-deletion than in wild-type patients (Fig. 1D). Similarly, the patients in SLC3A2 higher expression group contained higher proportions of WHO grade IV (p < 0.001), IDH1 wild-type (p < 0.001), and 1p/19q co-deletion (p < 0.05) than the low expression group (Table 1). The SLC3A2 protein expression was higher in glioma cells than astrocytic cells (Fig. 1E and F) and the histopathologic slides from high-grade patients had a higher IRS than the low-grade group (Fig. 1G). The risk factor plot showed individuals with higher SLC3A2 expression possessed high-risk scores (Fig. 1H), and Kaplan-Meier analysis demonstrated that upregulation of SLC3A2 expression is associated with poor prognosis in glioma patients across different clinical subgroups (Fig. 1I). Univariate Cox regression analysis showed that SLC3A2 expression level is a reliable prognostic factor for glioma overall survival. In the multivariate analysis, SLC3A2 shows a p-value of 0.05, indicating borderline statistical significance (Table 2). The time-dependent receiver operating characteristic (ROC) curves were developed based on SLC3A2 expression to predict the probability of 1-year, 3-year, and 5-year overall survival rates. The ROC curves plotted over time predicted the probabilities of 1-year, 3-year, and 5-year overall survival based on SLC3A2 expression. The area under curves for 1-year, 3-year, and 5-year survival were 0.569, 0.655, and 0.623, respectively (Fig. 1J).
2. SLC3A2 promotes the malignant progression of glioma
To investigate the role of SLC3A2 in glioma, SLC3A2-knockout LN229 and LN18 cells were constructed (Fig. 2A). The proliferation and clone formation were attenuated after SLC3A2 knockout in both cell lines (Fig. 2B and C). Similarly, siRNA-mediated downregulation of SLC3A2 expression significantly delayed the proliferation of glioma cells (Fig. 2B). Changes in cell cycle of the SLC3A2-knockout cells were found by flow cytometry. There was a decrease in the proportion of cells in the G2/M phase in SLC3A2-knockout cells compared with the control group (Fig. 2D and E). EdU staining results showed a significant reduction in DNA replication activity after SLC3A2 knockout (Fig. 2F-H). These results indicate that the proliferation activity of glioma cells is significantly reduced after downregulation of SLC3A2 expression. Transwell and wound healing assays showed that SLC3A2 knockout significantly decreased the invasion and migration ability of LN229 and LN18 cells (p < 0.01) (Fig. 3A-C). Analysis of TCGA database revealed that the expression of SLC3A2 in glioma patients is positively related to the expression of epithelial-mesenchymal transition (EMT) markers CDH2 (N-cadherin), Snail, vimentin, and FN1 (Fig. 3D). In glioma cells, the downregulation of SLC3A2 also led to a decrease in the expression of N-cadherin and SNAIL+SLUG (Fig. 3E). These results suggest that SLC3A2 can regulate the invasion and migration ability of glioma cells.
3. Knockout of SLC3A2 increases the ferroptosis susceptibility of glioma
Erastin and RSL-3 were used to induce ferroptosis in glioma cells. The results showed that the cell survival rates significantly declined after SLC3A2 knockout when undergoing the treatment of Erastin or RSL-3 in both LN229 and LN18 cells (Fig. 4A and B). In addition, the ferroptosis inhibitor ferrostatin-1 (Fer-1) significantly rescued the cell survival decrement caused by Erastin and RSL-3 treatment, while apoptosis inhibitor (Z-VAD-fmk) or necroptosis inhibitor necrostatin-1s (Nec-1) did not show the same viability (Fig. 4A). Notably, the cellular ROS and lipid peroxidation levels didn’t alter with SLC3A2 expression level. However, the levels of ROS and lipid peroxidation in SLC3A2 knockout cells were significantly higher than those in the control group after RSL-3 treatment (Fig. 4C-E). The mitochondrial double membrane structure and the mitochondrial cristae were integrality under transmission electron microscopy both in cells with or without SLC3A2 knockout. After RSL-3 treatment, mitochondrial volume reduction, decrease or disappearance of mitochondrial cristae, and increased density of mitochondrial double membrane were observed in SLC3A2 knockout cells, while the decrease of mitochondrial cristae was observed only in part of mitochondria in control cells (Fig. 4F). These results suggest that SLC3A2 knockout couldn’t directly trigger ferroptosis, but significantly increases the ferroptosis susceptibility of cells.
4. SLC3A2 regulates the activation of AKT pathway and expression of anti-ferroptosis proteins
Here, we explored the signaling pathways and key proteins regulated by SLC3A2 behind the results observed above in glioma. Firstly, the differentially expressed genes were analyzed by using the transcriptomic data of glioma patients which were divided into two cohorts based on the expression level of SLC3A2. There were 137 upregulated genes and 529 downregulated genes acquired with |logFC| > 1 and adjusted p-value < 0.05 as a standard (Fig. 5A). The activation of phosphoinositide 3-kinase–AKT signaling pathway was detected by KEGG enrichment analysis based on differentially expressed genes (Fig. 5B). GSEA enrichment analysis revealed that these differentially expressed genes involved in biological functions and signaling pathways including ferroptosis, DNA damage repair, cell cycle, apoptosis, p53 signaling pathway, mitogen-activated protein kinase signaling pathway, transforming growth factor β signaling pathway, extracellular matrix receptor interaction, metabolism of glycine, serine, and threonine, cell-cell junctions and glutamate signaling pathway (Fig. 5C). The interaction proteins of SLC3A2 were analyzed based on STRING database and the result showed that the AKT, and ferroptosis-related proteins SLC7A11, NRF2 and GPX4 were closely related to SLC3A2 (Fig. 5D). In glioma cells, the p-AKT, SLC7A11 and NRF2 expression were inhibited when SLC3A2 was downregulated by siRNA (Fig. 5E). The decrease of GPX4 was more obvious in SLC3A2 knockout glioma cells than control on the condition of RSL-3 treatment (Fig. 5F). These results suggest that SLC3A2 may regulate the sensitivity of glioma cells to ferroptosis by affecting the stability of the Xc- transporter complex and the AKT-NRF2-GPX4 pathway.
5. SLC3A2 inhibits ferroptosis by activating the AKT pathway
With treatment of ferroptosis inducers, the cell survival rate of SLC3A2 knockout glioma cells was elevated when the AKT activator SC79 was added (Fig. 6A). The agent SC79 could reduce the accumulation of intracellular ROS (Fig. 6B and D) and lipid peroxidation (Fig. 6C) in SLC3A2 knockout cells with RSL-3 treatment. The mitochondrial morphological changes caused by ferroptosis inducers, such as volume reduction, cristae decrease or disappearance, and disappearance of the double membrane, were significantly alleviated with the addition of SC79 (Fig. 6E). The expression of NRF2 and GPX4 in glioma cells increased when SC79 was added to cell culture medium containing RSL-3 (Fig. 6F). These results indicate that SLC3A2 can regulate the antioxidant capacity of cells by affecting the AKT-NRF2-GPX4 pathway, thereby affecting the sensitivity of glioma cells to ferroptosis.
6. SLC3A2 is essential for glioma growth in vivo
To validate the biological impact of SLC3A2 in vivo, nude mice were divided in two groups which were injected subcutaneously with LN229 or SLC3A2 knockout LN229 cells respectively. Downregulation of SLC3A2 reduced tumor growth, as shown by the decreased tumor volume and weight in the SLC3A2 KO group compared with the SLC3A2 wild-type group. (Fig. 7A-D). Immunohistochemical analysis of the mice tumor tissue showed a significant decrease in Ki-67 expression after SLC3A2 knockout (p < 0.05) (Fig. 7E and F). The expression levels of SLC7A11, p-AKT, and NRF2 were also reduced following the knockout of SLC3A2 (Fig. 7G and H).
Malignant glioma is among the deadliest types of brain cancer. Patients with glioma have limited treatment options in the clinic. There is an urgent need for novel therapeutic targets for glioma treatment. In this study, we found that the expression of SLC3A2 was elevated in glioma which promoted the malignant proliferation and ferroptosis resistance. The expression of SLC3A2 was upregulated in many types of tumors, including lung cancer, oral squamous cell carcinoma, and head and neck squamous cell carcinoma, and was associated with poor prognosis of patients [25-27]. Here, we found that SLC3A2 was upregulated in glioma by database analysis and pathology test. The prognosis of patients with higher SLC3A2 expression was significantly worse, particularly in those with grade 3 and 4 gliomas, astrocytomas and 1p/19q non-codeleted gliomas. The expression of SLC3A2 was closely related to glioma risk factors, such as tumor grade, IDH mutation and 1p/19q co-deletion. This suggests that SLC3A2 is a prognostic indicator for glioma. Interestingly, patients with 1p19q co-deletion exhibit higher levels of SLC3A2 expression; however, this expression does not result in a statistically significant difference in patient survival. The underlying mechanisms of this observation warrant further investigation.
SLC3A2 protein promoted the proliferation of tumor cells through various ways. As an auxiliary subunit, SLC3A2 combined with various members of the solute carrier family, such as SLC1A5 and SLC7A5, enhancing the transmembrane transport of amino acids. In laryngeal cancer, the upregulation of SLC3A2 promoted tumor cell proliferation by activating the mammalian target of rapamycin pathway [28]. Downregulation of SLC3A2 in colorectal cancer significantly inhibited cell proliferation efficiency through the AKT/glycogen synthase kinase 3 beta pathway [29]. Loss of SLC3A2 in osteosarcoma arrested the cell cycle at G2/M phase [30]. Consistent with these findings, our study found that downregulation of SLC3A2 expression significantly inhibited the proliferation ability of glioma cells, and resulted in cell cycle arrest at the G1 phase.
The SLC3A2 also involved in regulation of tumor migration and invasion through multiple pathways. The integrin-dependent cell spreading and migration was activated by SLC3A2 in renal cancer [17]. Cell migration and invasion were enhanced by upregulated SLC3A2 which promoting the process of EMT in colorectal cancer [29]. Additionally, SLC3A2 promoted the aggressive phenotype of gastric cancer by upregulating several mucin genes expression [31]. Our study found that the migration and invasion of glioma cells were inhibited and the expression of EMT markers were decreased after SLC3A2 knockout. This indicates that SLC3A2 regulates the migration and invasion ability of glioma cells by regulating the EMT process.
The Xc– system is comprised of subunits SLC7A11 and SLC3A2, which contribute to glutathione synthesis and ferroptosis inhibition. Studies have reported that PARP inhibitors and metformin can effectively induce ferroptosis in BRCA-proficient ovarian cancer and breast cancer by downregulating the expression of SLC7A11 [32,33]. As a partner subunit, SLC3A2 stabilizes the transport function of SLC7A11 [34], and the loss of SLC3A2 leads to a decrease in the expression level of SLC7A11 [35]. In line with these results, both in vivo and in vitro, the expression of SLC7A11 declined in glioma cells when SLC3A2 was knocked down in our study. In addition, we found that SLC3A2 could enhance antioxidant metabolism to prevent the ferroptosis in glioma cells. Reports showed that the activation of the AKT pathway can affect the expression of NRF2 in cells, thereby affecting the cellular redox balance [36]. In IDH1-mutant glioma, activation of AKT activated NRF2, reduced the cellular ROS and inhibited ferroptosis, thus promoted tumor progression [37]. The AKT-NRF2 pathway inhibition after SLC3A2 downregulation in glioma was also observed in our experiment. As a transcription factor, NRF2 can promote the expression of GPX4, and alleviated cellular lipid peroxidation [38]. We detected that the depletion of GPX4 was more pronounced in SLC3A2 knockout cells compared to wild-type glioma cells, on the condition of ferroptosis inducer RSL-3 treatment. These results demonstrate that SLC3A2 can regulate the expression of NRF2-GPX4 by modulating the activation status of AKT, thereby regulating the balance of intracellular redox and maintaining the steady state of lipid peroxidation. Our study suggests that the regulatory mechanism of SLC3A2 on ferroptosis sensitivity is not limited to the regulation of Xc- transporter stability, but also involves the regulation of AKT-NRF2-GPX4 axis.
Therapies targeting SLC3A2 have shown promising prospects in various cancers. The SLC3A2 antibody designated IGN523 demonstrated robust inhibition effect in acute myeloid leukemia and non–small cell lung carcinoma xenograft models [39]. The SLC3A2-specific chimeric antigen receptor T cell immunotherapies reduced the tumor cell growth and increased overall survival in subcutaneous xenografts model of human prostate cancer [40]. In this study, the SLC3A2 knockout glioma cell line was constructed and the mouse xenograft model was established based on the cell line. We observed a significant reduction in the growth rate of glioma in vivo after the knockout of SLC3A2. This result confirmed the value of SLC3A2 as a therapeutic target in glioma.
In conclusion, downregulation of SLC3A2 suppressed the malignant phenotype of glioma by blocking the cell cycle and EMT processes. On the other hand, loss of SLC3A2 not only downregulated SLC7A11 but also prevented the activation of the AKT/NRF2/GPX4 axis. These lead to increased accumulation of ROS and lipid peroxides, ultimately enhancing the susceptibility of glioma to ferroptosis (Fig. 7I). The overexpression of SLC3A2 in glioma promotes the malignant phenotype and resistance to ferroptosis, thus promoting tumor progression.

Ethical Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Fifth Affiliated Hospital of Zhengzhou University (KY2023038). All patients provided informed consent. All animal experiments were performed in accordance with the guidelines of the Ethics Committee of the Fifth Affiliated Hospital of Zhengzhou University (KY2023038).

Author Contributions

Conceived and designed the analysis: Liang W, Wang X.

Collected the data: Zheng Y, Tao Y, Shi Z.

Contributed data or analysis tools: Hu W, Liang W.

Performed the analysis: Zheng Y.

Wrote the paper: Zheng Y, Li X.

Funding acquisition: Zhou S, Fu X, Wang X.

Supervision: Zhou S, Fu X, Ma J.

Conflicts of Interest

Conflict of interest relevant to this article was not reported.

Funding

This research was sponsored by the National Natural Science Foundation of China (No. 81972361 & 82303029); Henan Province key research and development and promotion special (No. 222102310039); Henan Zhongyuan Talent Program (Talent Education Series); Science and technology of Henan Province (No. 231111521000; No. 232102311183; LHGJ20230390); Henan Provincial Health Commission (No. SBGJ202301008). Henan Provincial Department of Science and Technology (No. 232102311134), Health Commission of Henan Province (No. YXKC2022020). The Youth Cultivation Program of the Fifth Affiliated Hospital of Zhengzhou University (No. YQ2024020).

Fig. 1.
Solute carrier family 3 member 2 (SLC3A2) expression and clinical significance in glioma patients. (A) The Cancer Genome Atlas database showed that SLC3A2 was upregulated in various tumor tissues. (B-D) Association between SLC3A2 expression and clinical features. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; Codel, codeleted; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; FPKM, fragments per kilobase of transcript per million mapped reads; GBM, glioblastoma multiforme; LGG, brain lower grade glioma; HNSC, head and neck squamous cell carcinoma; IDH, isocitrate dehydrogenase; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; Mut, mutant; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; TPM, transcripts per million; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma; WHO, World Health Organization; WT, wild type. (E, F) The protein expression of SLC3A2 by immunoblot analysis. (G) Lower grade glioma (LGG) and glioblastoma (GBM) tissue were stained with immunohistochemistry. (H) SLC3A2 expression, risk score, and survival time distribution. *p < 0.05, **p < 0.01, ***p < 0.001. (I) Kaplan-Meier survival analyses of glioma with different SLC3A2 expression levels and clinical variables. (J) Time-dependent receiver operating characteristic curves and area under curve (AUC) values of SLC3A2 in glioma. CI, confidence interval; FPR, false positive rate; HR, hazard ratio; TPR, true positive rate.
crt-2024-933f1.jpg
Fig. 2.
Altered expression of solute carrier family 3 member 2 (SLC3A2) in glioma leads to changes in cell proliferation. (A) Western blot detected SLC3A2 levels in glioma cells after knockout of SLC3A2. (B) Cell proliferation rates evaluated by Cell Counting Kit-8 assay after downregulation of SLC3A2 in LN229 and LN18 cells. (C) Proliferation of glioma cells was assessed by colony-forming assay after knockout of SLC3A2. (D, E) Flow cytometry detected the cell cycle distribution of glioma cells. (F-H) DNA replication activity through the EdU assay. *p < 0.05, **p < 0.01, ***p < 0.001, n=3.
crt-2024-933f2.jpg
Fig. 3.
Altered expression of solute carrier family 3 member 2 (SLC3A2) in glioma leads to changes in cell migration and invasion. (A, B) Cell migration evaluated by wound healing assay after knockout of SLC3A2 in glioma cells. (C) Cell invasion evaluated by Transwell assay after knockout of SLC3A2 in glioma cells. (D) Correlation between SLC3A2 expression and four epithelial-mesenchymal transition markers. (E) Expression of N-cadherin and SNAIL+SLUG was measured by western blotting after knockout of SLC3A2 in glioma cells. FPKM, fragments per kilobase of transcript per million mapped reads; TPM, transcripts per million. **p < 0.01, ***p < 0.001, n=3.
crt-2024-933f3.jpg
Fig. 4.
Solute carrier family 3 member 2 (SLC3A2) regulates ferroptosis susceptibility in glioma cells. (A) Cell viability was determined in LN229 and LN18 cells with SLC3A2-knockout treated with the 7.5 μM erastin (Era) or 150 nM RSL-3 combined with 5 μM Z-VAD-fmk (Z-VAD), 2 μM necrostatin-1 (Nec-1), or 1 μM ferrostatin-1 (Fer-1). (B) Cell viability was evaluated in SLC3A2-knockout glioma cells treated with different concentrations of erastin or RSL-3. (C, D) Flow cytometry detected reactive oxygen species (ROS) and lipid peroxidation levels in NC and Knockout-SLC3A2 LN229 cells after 150 nM RSL-3 treatment. (E) The ROS levels of LN229 and LN18 cells after RSL-3 treatment detected by fluorescent probe. (F) After 150 nM RSL-3 treatment, NC and knockout-SLC3A2 LN229 cells were prepared for transmission electron microscopy observation. Arrows indicate mitochondria exhibited characteristic morphological features following ferroptosis. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance; n=3.
crt-2024-933f4.jpg
Fig. 5.
Functional enrichment analysis of differentially expressed genes (DEGs) and expression of anti-ferroptosis proteins. (A) A total of 137 upregulated and 529 downregulated genes were identified between solute carrier family 3 member 2 (SLC3A2) high-expression and low-expression groups. (B) Gene Ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs. (C) Gene set enrichment analysis between the SLC3A2 high-expression and low-expression groups. BP, biological process; CC, cellular component; ECM, extracellular matrix; GPCR, G protein–coupled receptor; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; MF, molecular function; PI3K, phosphoinositide 3-kinase; TGF-β, transforming growth factor β. (D) The relationship between SLC3A2 and SLC7A11, AKT, NF-E2-related factor 2 (NRF2), glutathione peroxidase 4 (GPX4) was analyzed by STRING database (https://string-db.org). (E) Western blot analysis was used to detect the expression of SLC7A11, AKT, p-AKT, NRF2, and GPX4 after downregulation of SLC3A2 in LN229 and LN18 cells. (F) Western blot analysis was used to detect the expression of SLC7A11, AKT, p-AKT, NRF2, and GPX4 in NC and knockout-SLC3A2 LN229 cells after RSL-3 treatment.
crt-2024-933f5.jpg
Fig. 6.
Activation of the AKT pathway reverses ferroptosis susceptibility caused by solute carrier family 3 member 2 (SLC3A2) knockout. (A) In LN229 and LN18 cells, the changes in cell viability after treatment of ferroptosis inducers were reversed by SC79. (B-D) SC79 reversed the changes in reactive oxygen species and lipid peroxidation levels caused by RSL-3 in knockout-SLC3A2 LN229 cells. (E) After DMSO, SC79, RSL-3, or RSL-3+SC79 treatment, knockout-SLC3A2 LN229 cells were prepared for transmission electron microscopy observation. Arrows indicate mitochondria exhibited characteristic morphological features following ferroptosis. (F) The changes in expression of p-AKT, NF-E2-related factor 2 (NRF2), and glutathione peroxidase 4 (GPX4) in knockout-SLC3A2 LN229 cells treated with RSL-3 (150 nM) were reversed by SC79. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance; n=3.
crt-2024-933f6.jpg
Fig. 7.
Knockout of solute carrier family 3 member 2 (SLC3A2) reduces tumor growth in vivo. (A) Nude mice injected subcutaneously with NC and SLC3A2 knockout LN229 cells on day 35. (B) Harvested xenografts from nude mice. (C) Tumors injected with NC and SLC3A2 knockout LN229 cells were measured every 7 days from 1 week after injection and the tumor growth curves were analyzed over the course of 35 days. (D) Tumor weight of the NC and knockout SLC3A2 of LN229 groups was evaluated. (E, F) Paraffin sections of tumor samples were used for immunohistochemical staining with antibodies against SLC3A2 or Ki-67. *p < 0.05, **p < 0.01; n=3. (G, H) Paraffin sections of tumor samples were used for immunohistochemical staining with antibodies against SLC7A11, p-AKT and NRF2. (I) A schematic illustration of the proposed model, showing that downregulation of SLC3A2 inhibited the malignant proliferation and enhanced ferroptosis susceptibility through the AKT/NRF2/GPX4 axis. EMT, epithelial-mesenchymal transition; GPX4, glutathione peroxidase 4; GSH, reduced glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species; NRF2, NF-E2-related factor 2; SLC7A11, solute carrier family 7 member 11.
crt-2024-933f7.jpg
Table 1.
Correlations between SLC3A2 expression and clinicopathological characteristics in GBM patients
Characteristic Low expression of SLC3A2 (n=349) High expression of SLC3A2 (n=350) p-value
Sex
 Female 150 (21.5) 148 (21.2) 0.853
 Male 199 (28.5) 202 (28.9)
Age (yr)
 ≤ 60 289 (41.3) 267 (38.2) 0.033
 > 60 60 (8.6) 83 (11.9)
WHO grade
 G2 133 (20.9) 91 (14.3) < 0.001
 G3 124 (19.5) 121 (19)
 G4 59 (9.3) 109 (17.1)
IDH status
 WT 81 (11.8) 165 (23.9) < 0.001
 Mut 261 (37.9) 182 (26.4)
1p/19q co-deletion
 Non-codel 273 (39.5) 247 (35.7) 0.022
 Codel 73 (10.5) 99 (14.3)

Values are presented as number (%). The dataset contains unaddressed missing values, resulting in discrepancies between the total counts of some clinical variables and the overall sample size. GBM, glioblastoma; IDH, isocitrate dehydrogenase; SLC3A2, solute carrier family 3 member 2; WHO, World Health Organization.

Table 2.
Univariate analysis and multivariate analysis for prognostic factors affecting overall survival
Characteristic Total Univariate analysis
 Multivariate analysis
HR (95% CI) p-value HR (95% CI) p-value
SLC3A2 698 1.846 (1.445-2.358) < 0.001 1.533 (1.001-2.347) 0.050
WHO grade 636
 G2 223 Reference Reference
 G3 245 2.967 (1.986-4.433) < 0.001 1.950 (1.238-3.071) 0.004
 G4 168 18.600 (12.448-27.794) < 0.001 5.946 (1.897-18.641) 0.002
IDH status 688
 WT 246 Reference Reference
 Mut 442 0.116 (0.089-0.151) < 0.001 0.555 (0.327-0.941) 0.029
1p/19q co-deletion 691
 Non-codel 520 Reference Reference
 Codel 171 0.225 (0.147-0.346) < 0.001 0.611 (0.353-1.056) 0.078
Age (yr) 698
 ≤ 60 555 Reference Reference
 > 60 143 4.696 (3.620-6.093) < 0.001 4.253 (2.582-7.003) < 0.001
Sex 698
 Female 297 Reference Reference
 Male 401 1.250 (0.979-1.595) 0.073 1.829 (1.163-2.878) 0.009
Primary therapy outcome 464
 PD 112 Reference Reference
 SD 148 0.440 (0.294-0.658) < 0.001 0.363 (0.217-0.607) < 0.001
 PR 65 0.167 (0.073-0.385) < 0.001 0.186 (0.067-0.520) 0.001
 CR 139 0.131 (0.063-0.273) < 0.001 0.175 (0.081-0.377) < 0.001

CI, confidence interval; CR, complete response; HR, hazard ratio; IDH, isocitrate dehydrogenase; PD, progressive disease; PR, partial response; SD, stable disease; SLC3A2, solute carrier family 3 member 2; WHO, World Health Organization.

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        Elevated SLC3A2 Expression Promotes the Progression of Gliomas and Enhances Ferroptosis Resistance through the AKT/NRF2/GPX4 Axis
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      Elevated SLC3A2 Expression Promotes the Progression of Gliomas and Enhances Ferroptosis Resistance through the AKT/NRF2/GPX4 Axis
      Image Image Image Image Image Image Image
      Fig. 1. Solute carrier family 3 member 2 (SLC3A2) expression and clinical significance in glioma patients. (A) The Cancer Genome Atlas database showed that SLC3A2 was upregulated in various tumor tissues. (B-D) Association between SLC3A2 expression and clinical features. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; Codel, codeleted; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; FPKM, fragments per kilobase of transcript per million mapped reads; GBM, glioblastoma multiforme; LGG, brain lower grade glioma; HNSC, head and neck squamous cell carcinoma; IDH, isocitrate dehydrogenase; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; Mut, mutant; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; TPM, transcripts per million; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma; WHO, World Health Organization; WT, wild type. (E, F) The protein expression of SLC3A2 by immunoblot analysis. (G) Lower grade glioma (LGG) and glioblastoma (GBM) tissue were stained with immunohistochemistry. (H) SLC3A2 expression, risk score, and survival time distribution. *p < 0.05, **p < 0.01, ***p < 0.001. (I) Kaplan-Meier survival analyses of glioma with different SLC3A2 expression levels and clinical variables. (J) Time-dependent receiver operating characteristic curves and area under curve (AUC) values of SLC3A2 in glioma. CI, confidence interval; FPR, false positive rate; HR, hazard ratio; TPR, true positive rate.
      Fig. 2. Altered expression of solute carrier family 3 member 2 (SLC3A2) in glioma leads to changes in cell proliferation. (A) Western blot detected SLC3A2 levels in glioma cells after knockout of SLC3A2. (B) Cell proliferation rates evaluated by Cell Counting Kit-8 assay after downregulation of SLC3A2 in LN229 and LN18 cells. (C) Proliferation of glioma cells was assessed by colony-forming assay after knockout of SLC3A2. (D, E) Flow cytometry detected the cell cycle distribution of glioma cells. (F-H) DNA replication activity through the EdU assay. *p < 0.05, **p < 0.01, ***p < 0.001, n=3.
      Fig. 3. Altered expression of solute carrier family 3 member 2 (SLC3A2) in glioma leads to changes in cell migration and invasion. (A, B) Cell migration evaluated by wound healing assay after knockout of SLC3A2 in glioma cells. (C) Cell invasion evaluated by Transwell assay after knockout of SLC3A2 in glioma cells. (D) Correlation between SLC3A2 expression and four epithelial-mesenchymal transition markers. (E) Expression of N-cadherin and SNAIL+SLUG was measured by western blotting after knockout of SLC3A2 in glioma cells. FPKM, fragments per kilobase of transcript per million mapped reads; TPM, transcripts per million. **p < 0.01, ***p < 0.001, n=3.
      Fig. 4. Solute carrier family 3 member 2 (SLC3A2) regulates ferroptosis susceptibility in glioma cells. (A) Cell viability was determined in LN229 and LN18 cells with SLC3A2-knockout treated with the 7.5 μM erastin (Era) or 150 nM RSL-3 combined with 5 μM Z-VAD-fmk (Z-VAD), 2 μM necrostatin-1 (Nec-1), or 1 μM ferrostatin-1 (Fer-1). (B) Cell viability was evaluated in SLC3A2-knockout glioma cells treated with different concentrations of erastin or RSL-3. (C, D) Flow cytometry detected reactive oxygen species (ROS) and lipid peroxidation levels in NC and Knockout-SLC3A2 LN229 cells after 150 nM RSL-3 treatment. (E) The ROS levels of LN229 and LN18 cells after RSL-3 treatment detected by fluorescent probe. (F) After 150 nM RSL-3 treatment, NC and knockout-SLC3A2 LN229 cells were prepared for transmission electron microscopy observation. Arrows indicate mitochondria exhibited characteristic morphological features following ferroptosis. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance; n=3.
      Fig. 5. Functional enrichment analysis of differentially expressed genes (DEGs) and expression of anti-ferroptosis proteins. (A) A total of 137 upregulated and 529 downregulated genes were identified between solute carrier family 3 member 2 (SLC3A2) high-expression and low-expression groups. (B) Gene Ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs. (C) Gene set enrichment analysis between the SLC3A2 high-expression and low-expression groups. BP, biological process; CC, cellular component; ECM, extracellular matrix; GPCR, G protein–coupled receptor; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen-activated protein kinase; MF, molecular function; PI3K, phosphoinositide 3-kinase; TGF-β, transforming growth factor β. (D) The relationship between SLC3A2 and SLC7A11, AKT, NF-E2-related factor 2 (NRF2), glutathione peroxidase 4 (GPX4) was analyzed by STRING database (https://string-db.org). (E) Western blot analysis was used to detect the expression of SLC7A11, AKT, p-AKT, NRF2, and GPX4 after downregulation of SLC3A2 in LN229 and LN18 cells. (F) Western blot analysis was used to detect the expression of SLC7A11, AKT, p-AKT, NRF2, and GPX4 in NC and knockout-SLC3A2 LN229 cells after RSL-3 treatment.
      Fig. 6. Activation of the AKT pathway reverses ferroptosis susceptibility caused by solute carrier family 3 member 2 (SLC3A2) knockout. (A) In LN229 and LN18 cells, the changes in cell viability after treatment of ferroptosis inducers were reversed by SC79. (B-D) SC79 reversed the changes in reactive oxygen species and lipid peroxidation levels caused by RSL-3 in knockout-SLC3A2 LN229 cells. (E) After DMSO, SC79, RSL-3, or RSL-3+SC79 treatment, knockout-SLC3A2 LN229 cells were prepared for transmission electron microscopy observation. Arrows indicate mitochondria exhibited characteristic morphological features following ferroptosis. (F) The changes in expression of p-AKT, NF-E2-related factor 2 (NRF2), and glutathione peroxidase 4 (GPX4) in knockout-SLC3A2 LN229 cells treated with RSL-3 (150 nM) were reversed by SC79. *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance; n=3.
      Fig. 7. Knockout of solute carrier family 3 member 2 (SLC3A2) reduces tumor growth in vivo. (A) Nude mice injected subcutaneously with NC and SLC3A2 knockout LN229 cells on day 35. (B) Harvested xenografts from nude mice. (C) Tumors injected with NC and SLC3A2 knockout LN229 cells were measured every 7 days from 1 week after injection and the tumor growth curves were analyzed over the course of 35 days. (D) Tumor weight of the NC and knockout SLC3A2 of LN229 groups was evaluated. (E, F) Paraffin sections of tumor samples were used for immunohistochemical staining with antibodies against SLC3A2 or Ki-67. *p < 0.05, **p < 0.01; n=3. (G, H) Paraffin sections of tumor samples were used for immunohistochemical staining with antibodies against SLC7A11, p-AKT and NRF2. (I) A schematic illustration of the proposed model, showing that downregulation of SLC3A2 inhibited the malignant proliferation and enhanced ferroptosis susceptibility through the AKT/NRF2/GPX4 axis. EMT, epithelial-mesenchymal transition; GPX4, glutathione peroxidase 4; GSH, reduced glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species; NRF2, NF-E2-related factor 2; SLC7A11, solute carrier family 7 member 11.

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      Elevated SLC3A2 Expression Promotes the Progression of Gliomas and Enhances Ferroptosis Resistance through the AKT/NRF2/GPX4 Axis
      Characteristic Low expression of SLC3A2 (n=349) High expression of SLC3A2 (n=350) p-value
      Sex
       Female 150 (21.5) 148 (21.2) 0.853
       Male 199 (28.5) 202 (28.9)
      Age (yr)
       ≤ 60 289 (41.3) 267 (38.2) 0.033
       > 60 60 (8.6) 83 (11.9)
      WHO grade
       G2 133 (20.9) 91 (14.3) < 0.001
       G3 124 (19.5) 121 (19)
       G4 59 (9.3) 109 (17.1)
      IDH status
       WT 81 (11.8) 165 (23.9) < 0.001
       Mut 261 (37.9) 182 (26.4)
      1p/19q co-deletion
       Non-codel 273 (39.5) 247 (35.7) 0.022
       Codel 73 (10.5) 99 (14.3)
      Characteristic Total Univariate analysis
      		 Multivariate analysis
      HR (95% CI) p-value HR (95% CI) p-value
      SLC3A2 698 1.846 (1.445-2.358) < 0.001 1.533 (1.001-2.347) 0.050
      WHO grade 636
       G2 223 Reference Reference
       G3 245 2.967 (1.986-4.433) < 0.001 1.950 (1.238-3.071) 0.004
       G4 168 18.600 (12.448-27.794) < 0.001 5.946 (1.897-18.641) 0.002
      IDH status 688
       WT 246 Reference Reference
       Mut 442 0.116 (0.089-0.151) < 0.001 0.555 (0.327-0.941) 0.029
      1p/19q co-deletion 691
       Non-codel 520 Reference Reference
       Codel 171 0.225 (0.147-0.346) < 0.001 0.611 (0.353-1.056) 0.078
      Age (yr) 698
       ≤ 60 555 Reference Reference
       > 60 143 4.696 (3.620-6.093) < 0.001 4.253 (2.582-7.003) < 0.001
      Sex 698
       Female 297 Reference Reference
       Male 401 1.250 (0.979-1.595) 0.073 1.829 (1.163-2.878) 0.009
      Primary therapy outcome 464
       PD 112 Reference Reference
       SD 148 0.440 (0.294-0.658) < 0.001 0.363 (0.217-0.607) < 0.001
       PR 65 0.167 (0.073-0.385) < 0.001 0.186 (0.067-0.520) 0.001
       CR 139 0.131 (0.063-0.273) < 0.001 0.175 (0.081-0.377) < 0.001
      Table 1. Correlations between SLC3A2 expression and clinicopathological characteristics in GBM patients

      Values are presented as number (%). The dataset contains unaddressed missing values, resulting in discrepancies between the total counts of some clinical variables and the overall sample size. GBM, glioblastoma; IDH, isocitrate dehydrogenase; SLC3A2, solute carrier family 3 member 2; WHO, World Health Organization.

      Table 2. Univariate analysis and multivariate analysis for prognostic factors affecting overall survival

      CI, confidence interval; CR, complete response; HR, hazard ratio; IDH, isocitrate dehydrogenase; PD, progressive disease; PR, partial response; SD, stable disease; SLC3A2, solute carrier family 3 member 2; WHO, World Health Organization.

      Table 1.

      Table 2.

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