FBXW7通过抑制c-Myc/SOX2/SLC7A11促进头颈鳞癌细胞铁死亡

doi:10.13591/j.cnki.kqyx.2024.06.005

摘要/Abstract

摘要：

目的探究FBXW7对头颈鳞状细胞癌铁死亡的影响。方法体外培养头颈鳞癌细胞系HN4和HN6,构建FBXW7,SOX2过表达质粒,将质粒稳定转染细胞系培养,采用qRT-PCR和Western blot实验分别在转录水平和蛋白水平验证过表达转染效率。通过检测丙二醛(MDA)、谷胱甘肽(GSH)和活性氧(ROS)水平来验证过表达FBXW7后头颈鳞癌细胞的脂质过氧化水平。用铁死亡抑制剂Fer-1处理细胞后进一步检测细胞活力变化验证FBXW7对铁死亡的影响。通过 Western blot检测转染过表达质粒后对细胞通路的影响。结果 HN4和HN6细胞系在过表达FBXW7后表现出脂质过氧化水平增加,铁死亡抑制剂Fer-1能够有效逆转过表达FBXW7引起的细胞铁死亡。Western blot实验结果显示过表达FBXW7降低了c-Myc、SOX2和SLC7A11的表达。结论 FBXW7通过降解c-Myc来调控SOX2-SLC7A11的表达,从而有效调控头颈鳞状细胞癌铁死亡。

关键词: 头颈鳞状细胞癌, FBXW7, 铁死亡, SOX2

Abstract:

Objective To explore the effect of FBXW7 on ferroptosis in head and neck squamous cell carcinoma. Methods Head and neck squamous cell lines HN4 and HN6 were cultured in vitro. FBXW7 and SOX2 overexpression plasmids were constructed, and the plasmids were stably transfected into cell lines. The overexpression transfection efficiency was verified at the transcription level and protein level by qRT-PCR and Western blot experiments, respectively. The lipid peroxidation levels of head and neck squamous cell carcinoma cells with overexpressing FBXW7 were verified by measuring malondialdehyde(MDA), glutathione(GSH), and reactive oxygen species(ROS) levels. After treating cells with ferroptosis inhibitor Fer-1, the changes in cell viability were further detected to verify the effect of FBXW7 on ferroptosis. The effect of transfection of the overexpressed plasmid on cellular pathways was detected by Western blot. Results HN4 and HN6 cell lines showed increased levels of lipid peroxidation after overexpression of FBXW7, and the ferroptosis inhibitor Fer-1 was able to effectively reverse the ferroptosis induced by overexpression of FBXW7. Western blot assay results showed that overexpression of FBXW7 reduced the expression of c-Myc, SOX2 and SLC7A11. Conclusion FBXW7 regulates the expression of SOX2-SLC7A11 by degrading c-Myc, thereby effectively regulating ferroptosis in HNSCC.

Key words: head and neck squamous cell carcinoma, FBXW7, ferroptosis, SOX2

中图分类号:

R739.85

陈以任, 赵振远, 郑阳玉, 张玮, 宋晓萌. FBXW7通过抑制c-Myc/SOX2/SLC7A11促进头颈鳞癌细胞铁死亡[J]. 口腔医学, 2024, 44(6): 426-432.

CHEN Yiren, ZHAO Zhenyuan, ZHENG Yangyu, ZHANG Wei, SONG Xiaomeng. FBXW7 promotes ferroptosis in head and neck squamous cell carcinoma cells through inhibiting c-Myc/SOX2/SLC7A11[J]. Stomatology, 2024, 44(6): 426-432.

图/表 7

表1

图1

图2

图3

图4

图5

图6

参考文献 29

[1]	Battaglia AM, Chirillo R, Aversa I, et al. Ferroptosis and cancer: Mitochondria meet the “iron maiden”cell death[J]. Cells, 2020, 9(6):1505.
[2]	Chen X, Kang R, Kroemer G, et al. Broadening horizons: The role of ferroptosis in cancer[J]. Nat Rev Clin Oncol, 2021, 18(5):280-296. doi: 10.1038/s41571-020-00462-0 pmid: 33514910
[3]	Chen X, Kang R, Kroemer G, et al. Organelle-specific regulation of ferroptosis[J]. Cell Death Differ, 2021, 28(10):2843-2856. doi: 10.1038/s41418-021-00859-z pmid: 34465893
[4]	Dixon SJ, Pratt DA. Ferroptosis: A flexible constellation of related biochemical mechanisms[J]. Mol Cell, 2023, 83(7):1030-1042.
[5]	Gao WT, Wang XY, Zhou Y, et al. Autophagy, ferroptosis, pyroptosis, and necroptosis in tumor immunotherapy[J]. Signal Transduct Target Ther, 2022, 7(1):196.
[6]	Jiang XJ, Stockwell BR, Conrad M. Ferroptosis: Mechanisms, biology and role in disease[J]. Nat Rev Mol Cell Biol, 2021, 22(4):266-282.
[7]	Lan T, Sun TT, Wei C, et al. Epigenetic regulation of ferroptosis in central nervous system diseases[J]. Mol Neurobiol, 2023, 60(7):3584-3599. doi: 10.1007/s12035-023-03267-1 pmid: 36847936
[8]	de Cova CC. The highs and lows of FBXW7: New insights into substrate affinity in disease and development[J]. Cells, 2023, 12(17):2141.
[9]	Di Fiore R, Suleiman S, Drago-Ferrante R, et al. The role of FBXW7 in gynecologic malignancies[J]. Cells, 2023, 12(10):1415.
[10]	Fan JY, Bellon M, Ju MY, et al. Clinical significance of FBXW7 loss of function in human cancers[J]. Mol Cancer, 2022, 21(1):87. doi: 10.1186/s12943-022-01548-2 pmid: 35346215
[11]	An HJ, Lee CJ, Lee GE, et al. FBXW7-mediated ERK3 degradation regulates the proliferation of lung cancer cells[J]. Exp Mol Med, 2022, 54(1):35-46.
[12]	Chen SY, Leng P, Guo JL, et al. FBXW7 in breast cancer: Mechanism of action and therapeutic potential[J]. J Exp Clin Cancer Res, 2023, 42(1):226. doi: 10.1186/s13046-023-02767-1 pmid: 37658431
[13]	Lan HY, Sun Y. FBXW7 E3 ubiquitin ligase: Degrading, not degrading, or being degraded[J]. Protein Cell, 2019, 10(12):861-863. doi: 10.1007/s13238-019-0652-x pmid: 31342282
[14]	Zheng Y, Song A, Wang CD, et al. Isoform specific FBXW7 mediates NOTCH1 Abruptex mutation C1133Y deregulation in oral squamous cell carcinoma[J]. Cell Death Dis, 2020, 11(8):615. doi: 10.1038/s41419-020-02873-4 pmid: 32792479
[15]	Gu FY, Lin YX, Wang Z, et al. Biological roles of LSD1 beyond its demethylase activity[J]. Cell Mol Life Sci, 2020, 77(17):3341-3350. doi: 10.1007/s00018-020-03489-9 pmid: 32193608
[16]	Jiménez-Izquierdo R, Morrugares R, Suanes-Cobos L, et al. FBXW7 tumor suppressor regulation by dual specificity tyrosine-regulated kinase 2[J]. Cell Death Dis, 2023, 14(3):202. doi: 10.1038/s41419-023-05724-0 pmid: 36934104
[17]	Minami JK, Morrow D, Bayley NA, et al. CDKN2A deletion remodels lipid metabolism to prime glioblastoma for ferroptosis[J]. Cancer Cell, 2023, 41(6):1048-1060.e9.
[18]	Swanda RV, Ji QQ, Wu XC, et al. Lysosomal cystine governs ferroptosis sensitivity in cancer via cysteine stress response[J]. Mol Cell, 2023, 83(18):3347-3359.e9.
[19]	Zeng FT, Nijiati S, Tang LG, et al. Ferroptosis detection: From approaches to applications[J]. Angew Chem Int Ed Engl, 2023, 62(35):e202300379.
[20]	Jehl A, Conrad O, Burgy M, et al. Blocking EREG/GPX4 sensitizes head and neck cancer to cetuximab through ferroptosis induction[J]. Cells, 2023, 12(5):733.
[21]	Sánchez-Ortega M, Carrera AC, Garrido A. Role of NRF2 in lung cancer[J]. Cells, 2021, 10(8):1879.
[22]	Farkhondeh T, Pourbagher-Shahri AM, Azimi-Nezhad M, et al. Roles of Nrf2 in gastric cancer: Targeting for therapeutic strategies[J]. Molecules, 2021, 26(11):3157.
[23]	Tossetta G, Fantone S, Montanari E, et al. Role of NRF2 in ovarian cancer[J]. Antioxidants (Basel), 2022, 11(4):663.
[24]	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 pmid: 34446045
[25]	Sun Y, Lian T, Huang QC, et al. Nanomedicine-mediated regulated cell death in cancer immunotherapy[J]. J Control Release, 2023, 364: 174-194.
[26]	Gong CD, Ji QK, Wu MJ, et al. Ferroptosis in tumor immunity and therapy[J]. J Cell Mol Med, 2022, 26(22):5565-5579. doi: 10.1111/jcmm.17529 pmid: 36317423
[27]	Zhong LJ, Zhang YY, Li MY, et al. E3 ligase FBXW7 restricts M2-like tumor-associated macrophage polarization by targeting c-Myc[J]. Aging (Albany NY), 2020, 12(23):24394-24423.
[28]	Bass AJ, Watanabe H, Mermel CH, et al. SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas[J]. Nat Genet, 2009, 41(11):1238-1242. doi: 10.1038/ng.465 pmid: 19801978
[29]	Kim R, Hashimoto A, Markosyan N, et al. Ferroptosis of tumour neutrophils causes immune suppression in cancer[J]. Nature, 2022, 612(7939):338-346.

基因名称	引物序列
si-c-Myc-1	F:5'-CCCAAGGUAGUUAUCCUUAAATT-3' R:5'-UUUAAGGAUAACUACCUUGGGTT-3'
si-c-Myc-2	F:5'-UACGGAACUCUUGUGCGUAAGTT-3' R:5'-CUUACGCACAAGAGUUCCGUATT-3'
FBXW7	F:5'-GGCCAAAATGATTCCCAGCAA-3' R:5'-ACTGGAGTTCGTGACACTGTTA-3'
SOX2	F:5'-GCCGAGTGGAAACTTTTGTCG-3' R:5'-GGCAGCGTGTACTTATCCTTCT-3'
c-Myc	F:5'-GGCTCCTGGCAAAAGGTCA-3' R:5'-CTGCGTAGTTGTGCTGATGT-3'
SLC7A11	F:5'-TCTCCAAAGGAGGTTACCTGC-3' R:5'-AGACTCCCCTCAGTAAAGTGAC-3'
GAPDH	F:5'-GAAGGTGAAGGTCGGAGTC-3' R:5'-GAGATGGTGATGGGATTTC-3'