Dual inhibition of PSME2 and STAT3 induces ferroptosis in esophageal squamous cell carcinoma cells by regulating the SLC7A11/GPX4 axis

doi:10.12122/j.issn.1673-4254.2026.09.21

Abstract

Abstract:

Objective To investigate the form of cell death induced by PSME2 silencing combined with the STAT3 inhibitor WP1066 in esophageal squamous cell carcinoma (ESCC) cells and explore the underlying mechanism. Methods ESCC cell lines KYSE30 and NE6-T with PSME2 silencing were treated with WP1066. Phenotypic rescue experiments were performed to determine the predominant form of cell death using different cell death-related inhibitors. Western blotting was used to detect the expressions of the protein markers of ferroptosis, apoptosis, pyroptosis and necroptosis. Intracellular reactive oxygen species (ROS) levels and lipid peroxidation were assessed using DCFH-DA and C11-BODIPY fluorescent probes, respectively. Mitochondrial ultrastructural changes were observed by transmission electron microscopy (TEM). mRNA transcriptomic analysis was performed to identify differentially expressed genes in cells treated with PR171 or siPSME2 combinedwith WP1066. Gene set enrichment analysis to analyze the enriched biological functions. Results Dual inhibition of PSME2 and STAT3 induced obvious ESCC cell death. This lethal effect was markedly reversed by the ferroptosis inhibitor ferrostatin-1 and attenuated by the apoptosis inhibitor Z-VAD-FMK, whereas 2-bromohexadecanoic acid or necrosulfonamide did not show significant inhibitory effects. The combined treatment with siPSME2 and WP1066 significantly downregulated SLC7A11 and GPX4 protein expressions and increased intracellular ROS levels and lipid peroxide accumulation in ESCC cells. The ESCC cells with dual inhibition showed obvious mitochondrial shrinkage, reduced cristae, and increased membrane density. mRNA transcriptomic analysis identified 45 overlapping differentially expressed genes between the PR171 plus WP1066 group and the siPSME2 plus WP1066 group, enriched in pathways related to stress response, redox imbalance, protein translation/ribosomal function, metabolism, and cell cycle regulation. Conclusion Dual inhibition of PSME2 and STAT3 promotes lipid peroxide accumulation and induces ferroptosis in ESCC cells possibly by regulating the SLC7A11/GPX4 antioxidant axis, indicating a therapeutic strategy targeting both PSME2 and STAT3 for ESCC.

Key words: proteasome activator complex subunit 2, STAT3, esophageal squamous cell carcinoma, ferroptosis, SLC7A11, GPX4

Chunhui QI, Heqian ZHANG, Xiaoshuang WU, Yongan KANG, Jingyi YANG, Di ZHAO, Shegan GAO, Yijun QI. Dual inhibition of PSME2 and STAT3 induces ferroptosis in esophageal squamous cell carcinoma cells by regulating the SLC7A11/GPX4 axis[J]. Journal of Southern Medical University, 2026, 46(9): 2230-2241.

Figures/Tables 5

Fig.1 Ferroptosis inhibition markedly reverses cell death induced by dual inhibition of PSME2 and STAT3 in esophageal squamous cell carcinoma (ESCC) cells. A, B: Released LDH activity (mU/mL) in ESCC cells after different treatments (A: KYSE30; B: NE6-T). C: Immunofluorescence staining of Calcein-AM/PI and quantitative assessment across different groups (green indicates live cells, and red indicates dead cells; scale bar=275 μm). D, E: Quantitative analyses of the percentage of cell death (%) in ESCC cells after different treatments (D: KYSE30; E: NE6-T). Data are presented as Mean±SD (n=3). **P<0.01,***P<0.001, ****P<0.0001 vs siPSME2+WP1066.

Fig.2 Dual inhibition of PSME2 and STAT3 induces downregulation of ferroptosis-related molecules GPX4 and SLC7A11. A, B: Western blotting of the protein levels of PSME2, PARP, cleaved PARP, caspase-3 and cleaved caspase-3 in KYSE30 and NE6-T cells. C, D: Results of Western blotting of GSDME and RIP in KYSE30 and NE6-T cells. E, F: Results of Western blotting of GPX4 and SLC7A11 in KYSE30 and NE6-T cells. G: GPX4 and SLC7A11 mRNA expressions in KYSE30 cells. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs siControl.

Fig.3 Dual inhibition of PSME2 and STAT3 induces lipid peroxidation, Fe²⁺ accumulation, ROS accumulation, and mitochondrial ultrastructural changes in ESCC cells. A, B: Lipid peroxidation levels measured by C11-BODIPY fluorescence staining in ESCC cells after different treatments (green for oxidation state, and red for reduction state; A: KYSE30; B: NE6-T; Scale bars=10 μm ). C, D: MDA levels (umol/ug) in ESCC cells after indicated treatments (C: KYSE30; D: NE6-T). E: Transmission electron microscopy of ESCC cells after different treatments (Scale bar=5 μm ). F, G: Intracellular ROS detected using DCFH-DA staining in KYSE30 and NE6-T cells (Scale bar=125 μm). H, I: Intracellular Fe²⁺ levels detected using FerroOrange staining in KYSE30 and NE6-T cells (Scale bar=125 μm). Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs siControl.

Fig.4 Dual inhibition of PSME2 and STAT3 reduces glycolytic activity and mitochondrial respiration in ESCC cells. A: ECAR curves showing glycolysis rates of indicated cells after different treatments. B: OCR curves reflecting mitochondrial respiration capacity. C, D: Quantification of basal respiration, ATP production, proton leak, spare respiration capacity, non-glycolytic acidification, glycolysis, glycolysis capacity and reserve. Data are presented as Mean±SD (n=3). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs siControl.

Fig.5 Identification of 45 differentially expressed genes (DEGs) and 9 key genes associated with dual inhibition of PSME2 and STAT3 and their Hallmark/KEGG enrichment analyses. A: Venn diagram showing 45 overlapping DEGs between PR171+WP1066 and siPSME2+WP1066 groups. B: Hallmark enrichment analysis of the overlapping DEGs. C: KEGG pathway enrichment of common DEGs. D: Intersection analysis of DEGs among PR171+WP1066, siPSME2+WP1066, siPSME2, WP1066, and PR171 groups. Nine candidate DEGs related to combined treatment are retained (Red indicates upregulation and blue indicates downregulation; log2FC > 0 upregulated genes, log2FC < 0 downregulated genes).

References 32

[1]	Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-49. doi：10.3322/caac.21660
[2]	Pennathur A, Gibson MK, Jobe BA, et al. Oesophageal carcinoma[J]. Lancet, 2013, 381(9864): 400-12. doi：10.1016/s0140-6736(12)60643-6
[3]	孙魁, 陈攀, 刘永萱, 等. PSME2和STAT3双重抑制通过合成致死效应诱导食管鳞癌细胞死亡[J]. 中国生物化学与分子生物学报, 2024, 40(4): 534-43. doi：10.13865/j.cnki.cjbmb.2024.02.1465
[4]	Dixon SJ, Lemberg KM, Lamprecht MR, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death[J]. Cell, 2012, 149(5): 1060-72. doi：10.1016/j.cell.2012.03.042
[5]	Jiang XJ, Stockwell BR, Conrad M. Ferroptosis: mechanisms, biology and role in disease[J]. Nat Rev Mol Cell Biol, 2021, 22(4): 266-82. doi：10.1038/s41580-020-00324-8
[6]	Tang DL, Chen X, Kang R, et al. Ferroptosis: molecular mechanisms and health implications[J]. Cell Res, 2021, 31(2): 107-25. doi：10.1038/s41422-020-00441-1
[7]	Stockwell BR, Jiang XJ, Gu W. Emerging mechanisms and disease relevance of ferroptosis[J]. Trends Cell Biol, 2020, 30(6): 478-90. doi：10.1016/j.tcb.2020.02.009
[8]	Liu X, Chen C, Han D, et al. SLC7A11/GPX4 inactivation-mediated ferroptosis contributes to the pathogenesis of triptolide-induced cardiotoxicity[J]. Oxid Med Cell Longev, 2022, 2022: 3192607. doi：10.1155/2022/3192607
[9]	Chen X, Kang R, Kroemer G, et al. Ferroptosis in disease: mechanisms and therapeutic implications[J]. Nat Rev Clin Oncol, 2021, 18(5): 280-96. doi：10.1038/s41571-020-00462-0
[10]	Lei G, Zhang YL, Koppula P, et al. The role of ferroptosis in ionizing radiation-induced cell death and tumor suppression[J]. Cell Res, 2020, 30(2): 146-62. doi：10.1038/s41422-019-0263-3
[11]	Feng L, Zhao KK, Sun LC, et al. SLC7A11 regulated by NRF2 modulates esophageal squamous cell carcinoma radiosensitivity by inhibiting ferroptosis[J]. J Transl Med, 2021, 19(1): 367. doi：10.1186/s12967-021-03042-7
[12]	Liu CC, Li HH, Lin JH, et al. Esophageal cancer stem-like cells resist ferroptosis-induced cell death by active Hsp27-GPX4 pathway[J]. Biomolecules, 2022, 12(1): 48. doi：10.3390/biom12010048
[13]	Miyauchi W, Shishido Y, Matsumi Y, et al. Simultaneous regulation of ferroptosis suppressor protein 1 and glutathione peroxidase 4 as a new therapeutic strategy of ferroptosis for esophageal squamous cell carcinoma[J]. Esophagus, 2023, 20(3): 492-501. doi：10.1007/s10388-022-00982-x
[14]	Tao H, Song SJ, Fan ZW, et al. PKCiota inhibits the ferroptosis of esophageal cancer cells via suppressing USP14-mediated autophagic degradation of GPX4[J]. Antioxidants (Basel), 2024, 13(1): 114. doi：10.3390/antiox13010114
[15]	Yan L, Hu HD, Feng L, et al. ML385 promotes ferroptosis and radiotherapy sensitivity by inhibiting the NRF2-SLC7A11 pathway in esophageal squamous cell carcinoma[J]. Med Oncol, 2024, 41(12): 309. doi：10.1007/s12032-024-02483-6
[16]	Peng H, He YP, Hu YP, et al. Berbamine promotes ferroptosis of esophageal squamous cell carcinoma by facilitating USP51-mediated GPX4 ubiquitination and degradation[J]. Biomed Pharmacother, 2024, 179: 117309. doi：10.1016/j.biopha.2024.117309
[17]	Jiang B, Zhao YQ, Shi M, et al. DNAJB6 promotes ferroptosis in esophageal squamous cell carcinoma[J]. Dig Dis Sci, 2020, 65(7): 1999-2008. doi：10.1007/s10620-019-05929-4
[18]	Yao WJ, Wang JJ, Meng FR, et al. Circular RNA CircPVT1 inhibits 5-fluorouracil chemosensitivity by regulating ferroptosis through miR-30a-5p/FZD3 axis in esophageal cancer cells[J]. Front Oncol, 2021, 11: 780938. doi：10.3389/fonc.2021.780938
[19]	Yu H, Lee H, Herrmann A, et al. Revisiting STAT3 signalling in cancer: new and unexpected biological functions[J]. Nat Rev Cancer, 2014, 14(11): 736-46. doi：10.1038/nrc3818
[20]	Bersuker K, Hendricks JM, Li ZP, et al. The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis[J]. Nature, 2019, 575(7784): 688-92. doi：10.1038/s41586-019-1705-2
[21]	Vander Heiden MG, DeBerardinis RJ. Understanding the intersections between metabolism and cancer biology[J]. Cell, 2017, 168(4): 657-69. doi：10.1016/j.cell.2016.12.039
[22]	Pavlova NN, Thompson CB. The emerging hallmarks of cancer metabolism[J]. Cell Metab, 2016, 23(1): 27-47. doi：10.1016/j.cmet.2015.12.006
[23]	Gao MH, Monian P, Quadri N, et al. Glutaminolysis and transferrin regulate ferroptosis[J]. Mol Cell, 2015, 59(2): 298-308. doi：10.1016/j.molcel.2015.06.011
[24]	Zhang YL, Swanda RV, Nie LT, et al. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation[J]. Nat Commun, 2021, 12: 1589. doi：10.1038/s41467-021-21841-w
[25]	Gao MH, Yi JM, Zhu JJ, et al. Role of mitochondria in ferroptosis[J]. Mol Cell, 2019, 73(2): 354-63.e3. doi：10.1016/j.molcel.2018.10.042
[26]	Ye LL, Wen XQ, Qin JL, et al. Metabolism-regulated ferroptosis in cancer progression and therapy[J]. Cell Death Dis, 2024, 15(3): 196. doi：10.1038/s41419-024-06584-y
[27]	Ouyang S, Li HX, Lou LL, et al. Inhibition of STAT3-ferroptosis negative regulatory axis suppresses tumor growth and alleviates chemoresistance in gastric cancer[J]. Redox Biol, 2022, 52: 102317. doi：10.1016/j.redox.2022.102317
[28]	Badgley MA, Kremer DM, Maurer HC, et al. Cysteine depletion induces pancreatic tumor ferroptosis in mice[J]. Science, 2020, 368(6486): 85-9. doi：10.1126/science.aaw9872
[29]	Dang CV. MYC on the path to cancer[J]. Cell, 2012, 149(1): 22-35. doi：10.1016/j.cell.2012.03.003
[30]	Stine ZE, Walton ZE, Altman BJ, et al. MYC, metabolism, and cancer[J]. Cancer Discov, 2015, 5(10): 1024-39. doi：10.1158/2159-8290.cd-15-0507
[31]	朱权, 黄柏胜, 位磊艳, 等. 过表达LncRNA MEG3通过促进铁死亡增强肝癌细胞对顺铂的化疗敏感性[J]. 南方医科大学学报, 2024, 44(1): 17-24.
[32]	王元国, 张鹏. 铁死亡抑制基因在食管癌中的高表达分析[J]. 南方医科大学学报, 2024, 44(7): 1389-96.