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Cited by 277Ferroptosis and cancer prognosisEsophageal Cancer Research and TreatmentGenomics, phytochemicals, and oxidative stress

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SLC7A11 regulated by NRF2 modulates esophageal squamous cell carcinoma radiosensitivity by inhibiting ferroptosis

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Feng, Lei, Zhao, Kaikai, Sun, Liangchao, Yin, Xiaoyang, Zhang, Junpeng, Liu, Conghe, Li, Baosheng·2021·Journal of Translational Medicine
DOI 10.1186/s12967-021-03042-7PMC PMC8393811
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Abstract

Background: Solute carrier family 7 member 11(SLC7A11) is a component of cysteine/glutamate transporter, which plays a key role in tumor growth; however, its underlying effect on radiosensitivity in esophageal squamous cell carcinoma (ESCC) remains unclear. This study aimed to clarify SLC7A11’s expression and correlation with nuclear expression of nuclear factor erythroid-2 ( NRF2)-associated radioresistance in ESCC. Methods: We included 127 ESCC patients who received radical chemoradiotherapy. Immunohistochemical staining was used to detect SLC7A11 and NRF2 nuclear expression, and the relationship between clinicopathological characteristics and survival rates or therapy response were evaluated. Western blot, dual-reporter assays and Chromatin immunoprecipitation (ChIP)-sequencing were used to analyze their relationship in vitro. Their roles in radioresistance were then investigated through multiple validation steps. Results: NRF2 nuclear expression and SLC7A11 expression were overexpressed in ESCC tissues and were positively correlated with one another. NRF2 nuclear expression was significantly associated with tumor length, lymph node metastasis, and TNM stage, while SLC7A11 expression was associated with lymph node metastasis. Patients with high NRF2 nuclear expression and SLC7A11 expression had significantly shorter overall and progression-free survival, and poor treatment response. The multivariate model showed that NRF2 nuclear expression and SLC7A11 expression, sex and tumor location are independent prognostic factors. In vitro analysis confirmed that hyperactivation of NRF2 induced SLC7A11 expression by directly binding to its promoter region, promoting radioresistance, reducing radiotherapy-induced lipid peroxidation levels, PTGS2 expression, and radiotherapy-related ferroptosis morphologic features. Conclusion: Our study reveals a connection between high SLC7A11 expression and NRF2 nuclear expression in patients with ESCC that was related to worse survival and poorer therapy outcomes. SLC7A11-mediated ferroptosis inhibition induced NRF2-associated radioresistance, highlighting potential of NRF2/SLC7A11/ferroptosis axis as future therapeutic targets against therapy resistance biomarker.

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Figures

Fig. 1 NRF2 and SLC7A11 expression in ESCC tissues. Representative images for negative, low, moderate, and high NRF2 expression; negative, low, moderate, and high SLC7A11 expression (× 400 magnification). ESCC, esophageal squamous cell carcinoma
Fig. 1 NRF2 and SLC7A11 expression in ESCC tissues. Representative images for negative, low, moderate, and high NRF2 expression; negative, low, moderate, and high SLC7A11 expression (× 400 magnification). ESCC, esophageal squamous cell carcinomaCC BY · PubMed Central
Fig. 2 NRF2 nuclear and SLC7A11 expression in ESCC tumor and matched adjacent normal tissues. Representative images of nuclear NRF2 (a) and SLC7A11 (b) expression in tumor and matched adjacent normal tissues (×400 magnification)
Fig. 2 NRF2 nuclear and SLC7A11 expression in ESCC tumor and matched adjacent normal tissues. Representative images of nuclear NRF2 (a) and SLC7A11 (b) expression in tumor and matched adjacent normal tissues (×400 magnification)CC BY · PubMed Central
Fig. 3 Correlation between SLC7A11 and NRF2 expression. a SLC7A11 expression was positively correlated with nuclear NRF2 expression among all the patients (n = 127). b SLC7A11 and NRF2 protein expression were analyzed in six ESCC cell lines. Western blotting revealed that SLC7A11 expression positively correlated with NRF2 level. c Sequence of the SLC7A11 promoter showing the position of the most representative putative NRF2 binding sites of the promoter. Schematic of putative NRF2 binding sites in the proximal region of SLC7A11 promoter. d Dual-luciferase reporter assays showing NRF2 acting as a trans-activator of SLC7A11 promoter activity. Luciferase activity assays (n = 3) were performed after transfection of either firefly luciferase promoter plasmid, NRF2 overexpression plasmid (TF), or TF-nc plasmid into 293T cells. Data are presented as the mean ± SEM. *p < 0.05. e Integrative Genomics Viewer (IGV) snapshots of NRF2 binding sites at the promoter of SLC7A11 region
Fig. 3 Correlation between SLC7A11 and NRF2 expression. a SLC7A11 expression was positively correlated with nuclear NRF2 expression among all the patients (n = 127). b SLC7A11 and NRF2 protein expression were analyzed in six ESCC cell lines. Western blotting revealed that SLC7A11 expression positively correlated with NRF2 level. c Sequence of the SLC7A11 promoter showing the position of the most representative putative NRF2 binding sites of the promoter. Schematic of putative NRF2 binding sites in the proximal region of SLC7A11 promoter. d Dual-luciferase reporter assays showing NRF2 acting as a trans-activator of SLC7A11 promoter activity. Luciferase activity assays (n = 3) were performed after transfection of either firefly luciferase promoter plasmid, NRF2 overexpression plasmid (TF), or TF-nc plasmid into 293T cells. Data are presented as the mean ± SEM. *p < 0.05. e Integrative Genomics Viewer (IGV) snapshots of NRF2 binding sites at the promoter of SLC7A11 regionCC BY · PubMed Central
Fig. 4 Relationship between SLC7A11 or NRF2 nuclear expression and clinical response to CRT. SLC7A11 (a) and nuclear NRF2 (b) treatment responses of ESCC patients. CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease
Fig. 4 Relationship between SLC7A11 or NRF2 nuclear expression and clinical response to CRT. SLC7A11 (a) and nuclear NRF2 (b) treatment responses of ESCC patients. CR: complete response; PR: partial response; SD: stable disease; PD: progressive diseaseCC BY · PubMed Central
Fig. 5 Kaplan–Meier survival curves for NRF2 nuclear expression and SLC7A11 expression. OS survival curves for nuclear NRF2 (a), SLC7A11 (b), and their combined (c) expression. PFS survival curves for nuclear NRF2 (d), SLC7A11 (e), and their combined (f) expression
Fig. 5 Kaplan–Meier survival curves for NRF2 nuclear expression and SLC7A11 expression. OS survival curves for nuclear NRF2 (a), SLC7A11 (b), and their combined (c) expression. PFS survival curves for nuclear NRF2 (d), SLC7A11 (e), and their combined (f) expressionCC BY · PubMed Central
Fig. 6 Hyperactive NRF2 promotes radioresistance by inhibiting ferroptosis in ESCC cells. a Western blot analysis showing NRF2 levels in KYSE 30 and KYSE 150 cell lines with overexpression of NRF2 (NF-OE), and negative control (NC). b Lipid peroxidation assessment of NC and NF-OE 24 h after 6 Gy RT in KYSE 30 and KYSE 150 cells, respectively. Bar graph showing RT-induced relative fold changes of lipid peroxidation levels per 10,000 cells by C11-BODIPY staining in the indicated cells. c Quantitative RT-PCR analysis of PTGS2 expression in NC and NF-OE KYSE 30 or KYSE 150 cells 24 h after 6 Gy RT, respectively. d TEM images of NC and NF-OE KYSE 150 cells 24 h after exposure to 6 Gy of RT. Nu: nucleus; red arrows: mitochondria; yellow arrows: autophagosome; Scale bars: 5 µm. e Representative images of clonogenic survival assays in NC and NF-OE KYSE 30 or KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 2 Gy or 4 Gy of RT, respectively. f The quantified clonogenic survival assay in NC and NF-OE KYSE 30 or KYSE 150 cells that were pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h followed by 2 Gy or 4 Gy of RT. g Quantified cell proliferation assay curves analyzed by CCK-8 in NC and NF-OE KYSE 30 or KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 6 Gy RT. Each experiment was conducted independently triplicate. Student’s t-test (two-tailed) was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bar graphs: mean ± SEM, n = 3
Fig. 6 Hyperactive NRF2 promotes radioresistance by inhibiting ferroptosis in ESCC cells. a Western blot analysis showing NRF2 levels in KYSE 30 and KYSE 150 cell lines with overexpression of NRF2 (NF-OE), and negative control (NC). b Lipid peroxidation assessment of NC and NF-OE 24 h after 6 Gy RT in KYSE 30 and KYSE 150 cells, respectively. Bar graph showing RT-induced relative fold changes of lipid peroxidation levels per 10,000 cells by C11-BODIPY staining in the indicated cells. c Quantitative RT-PCR analysis of PTGS2 expression in NC and NF-OE KYSE 30 or KYSE 150 cells 24 h after 6 Gy RT, respectively. d TEM images of NC and NF-OE KYSE 150 cells 24 h after exposure to 6 Gy of RT. Nu: nucleus; red arrows: mitochondria; yellow arrows: autophagosome; Scale bars: 5 µm. e Representative images of clonogenic survival assays in NC and NF-OE KYSE 30 or KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 2 Gy or 4 Gy of RT, respectively. f The quantified clonogenic survival assay in NC and NF-OE KYSE 30 or KYSE 150 cells that were pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h followed by 2 Gy or 4 Gy of RT. g Quantified cell proliferation assay curves analyzed by CCK-8 in NC and NF-OE KYSE 30 or KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 6 Gy RT. Each experiment was conducted independently triplicate. Student’s t-test (two-tailed) was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bar graphs: mean ± SEM, n = 3CC BY · PubMed Central
Fig. 7 Hyperactive NRF2 inhibits RT-induced ferroptosis by regulating SLC7A11 in ESCC cells. a Western blotting and qRT-PCR analysis of NRF2 and SLC7A11 expression in NC and NF-OE KYSE 30 and KYSE 150 cells. b Western blot analysis of SLC7A11 levels in NF-OE KYSE 150 cells with SLC7A11 siRNA (NF-OE-SLC-Si), negative control (NF-OE-SLC-con), and NF-OE KYSE 150. c Lipid peroxidation assessment in NF-OE-SLC-Si and NF-OE-SLC-con KYSE 150 cells 24 h after exposure to 6 Gy of RT. Bar graph showing RT-induced relative fold changes of lipid peroxidation levels per 10,000 cells by C11-BODIPY staining in the indicated cells. d qRT-PCR analysis of PTGS2 expression in NF-OE-SLC-Si and NF-OE-SLC-con KYSE 150 cells 24 h after exposure to 6 Gy of RT. e Representative images of clonogenic survival assays in NF-OE-Con and NF-OE-SLC-Si KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 4 Gy of RT, respectively. f The quantified clonogenic survival assay in NF-OE-Con and NF-OE-SLC-Si KYSE 150 cells that were pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h followed by 4 Gy of RT, respectively. g Quantified cell proliferation assay curves analyzed by CCK-8 in NF-OE-SLC-con and NF-OE-SLC-Si KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 6 Gy RT. Each experiment was conducted independently triplicate. Student’s t-test (two-tailed) was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bar graphs: mean ± SEM, n = 3
Fig. 7 Hyperactive NRF2 inhibits RT-induced ferroptosis by regulating SLC7A11 in ESCC cells. a Western blotting and qRT-PCR analysis of NRF2 and SLC7A11 expression in NC and NF-OE KYSE 30 and KYSE 150 cells. b Western blot analysis of SLC7A11 levels in NF-OE KYSE 150 cells with SLC7A11 siRNA (NF-OE-SLC-Si), negative control (NF-OE-SLC-con), and NF-OE KYSE 150. c Lipid peroxidation assessment in NF-OE-SLC-Si and NF-OE-SLC-con KYSE 150 cells 24 h after exposure to 6 Gy of RT. Bar graph showing RT-induced relative fold changes of lipid peroxidation levels per 10,000 cells by C11-BODIPY staining in the indicated cells. d qRT-PCR analysis of PTGS2 expression in NF-OE-SLC-Si and NF-OE-SLC-con KYSE 150 cells 24 h after exposure to 6 Gy of RT. e Representative images of clonogenic survival assays in NF-OE-Con and NF-OE-SLC-Si KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 4 Gy of RT, respectively. f The quantified clonogenic survival assay in NF-OE-Con and NF-OE-SLC-Si KYSE 150 cells that were pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h followed by 4 Gy of RT, respectively. g Quantified cell proliferation assay curves analyzed by CCK-8 in NF-OE-SLC-con and NF-OE-SLC-Si KYSE 150 cells pre-treated with 5 μM ferrostatin-1 or DMSO for 24 h, followed by exposure to 6 Gy RT. Each experiment was conducted independently triplicate. Student’s t-test (two-tailed) was used for statistical analysis, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Bar graphs: mean ± SEM, n = 3CC BY · PubMed Central

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  1. Koppula P, Zhuang L, Gan B. Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell. 2020. https://doi.org/10.1007/s13238-020-00789-5. · DOI

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Metadata

Authors
Feng, L., Zhao, K., Sun, L., Yin, X., Zhang, J., Liu, C., & Li, B.
Affiliations
  • Shandong Tumor Hospital (CN)
  • Binzhou Medical University (CN)
Corresponding author
Li, Baosheng
ORCID
Venue
Journal of Translational Medicine
Publisher
Springer Science and Business Media LLC
Published
2021-08-26
Volume
19
Issue
1
Pages
367-367
ISSN
1479-5876 (ISSN-L)
DOI
10.1186/s12967-021-03042-7
PMC
PMC8393811
OpenAlex
W3194902068
Semantic Scholar
e55a74d417d2810a7e103ad2eaccdd036e12dc8c
Cited by
277
References
65
Citations by year
2026: 46 · 2025: 67 · 2024: 67 · 2023: 61 · 2022: 35 · 2021: 1
FWCI
26.79
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Translational Medicine (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (No. 81874224)
  • Academic promotion program of Shandong First Medical University (2019LJ004)
  • National Key Research and Development Program of China (2016YFC0105106)
  • Taishan Scholar Construction Project (ts20120505)
  • Tumor prevention and control Joint Fund of Shandong province natural science fund (ZR2019LZL008)
  • Yantai Science and Technology Innovation Development Program (2020YT06121070)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Ferroptosis and cancer prognosis
  • Health Sciences › Medicine › Surgery › Esophageal Cancer Research and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics, phytochemicals, and oxidative stress
Keywords
SLC7A11, Nrf2, esophageal squamous cell carcinoma, radioresistance (biology), ferroptosis, chemoradiotherapy, prognostic factors, lipid peroxidation, cyclooxygenase-2, promoter binding, treatment resistance
MeSH terms
Esophageal Squamous Cell Carcinoma, Ferroptosis, Esophageal Neoplasms, Head and Neck Neoplasms, Humans, Prognosis, Radiation Tolerance, Amino Acid Transport System y+, NF-E2-Related Factor 2

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