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. 2023 Jan:27:101576.
doi: 10.1016/j.tranon.2022.101576. Epub 2022 Nov 4.

m6A demethylase FTO renders radioresistance of nasopharyngeal carcinoma via promoting OTUB1-mediated anti-ferroptosis

Wei-Mei Huang  1 , Zhi-Xun Li  1 , Ying-Hui Wu  2 , Zhi-Ling Shi  1 , Jing-Lin Mi  1 , Kai Hu  3 , Ren-Sheng Wang  4
Affiliations

m6A demethylase FTO renders radioresistance of nasopharyngeal carcinoma via promoting OTUB1-mediated anti-ferroptosis

Wei-Mei Huang et al. Transl Oncol. 2023 Jan.

Abstract

Radiotherapy is a valid treatment for nasopharyngeal carcinoma (NPC), and radioresistance is the main cause of local NPC treatment failure. However, the underlying mechanisms and valuable markers of radioresistance for NPC remain have not been established. In this study, we observed that the m6A mRNA demethylase fat mass and obesity-associated protein (FTO) was significantly upregulated in radioresistant NPC tissues and cells relative to parental radiosensitive NPC tissues and cells. FTO enhances radioresistance by repressing radiation-induced ferroptosis in NPC. Mechanistically, FTO acts as an m6A demethylase to erase the m6A modification of the OTUB1 transcript and promote the expression of OTUB1, thereby inhibiting the ferroptosis of cells induced by radiation and finally triggering the radiotherapy resistance of NPC. Furthermore, our in vivo experiment results showed that the FTO inhibitor, FB23-2, and the ferroptosis activator, erastin, altered tumor responsiveness to radiotherapy in NPC cell lines and patient-derived xenografts. Our findings reveal, for the first time, that FTO enhances NPC radiotherapy resistance by withstanding radiation-induced ferroptosis, suggesting that FTO may serve as a potential therapeutic target and valuable prognostic biomarker in patients with NPC.

Keywords: Nasopharyngeal carcinoma (NPC), FTO, Ferroptosis; OTUB1; m6A.

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Conflict of interest statement

Declaration of Competing Interest The authors declare that they have no competing interests.

Figures

Fig 1
Fig. 1
High FTO levels are closely associated with the radioresistance of NPC. (A) Kaplan–Meier survival curves show the correlations between FTO expression and overall survival (OS, p = 0.021), as well as recurrence-free survival (RFS, no significance), in HNSC patients. (B-C) RT-qPCR assay and IHC staining were used to detect the level of FTO mRNA (Mean ± SEM, n = 3, two-sided t test) and protein in NPC tissues. (D) The CCK8 assay was used to detect the proliferation activity of indicated NPC cells after radiation (single-hit multitarget model was used to compare the sensitivity of different groups of cells to radiation). (E) Flow cytometry assay was used to detect the cell apoptosis after receiving 4 Gy radiation (mean± SEM, n = 3, two-sided t test). (F) Colony formation assay was used to test the proliferation activity of indicated cells after 4Gy radiation (mean ± SEM, n = 3, two-sided t test). (G) The mRNA and protein levels of FTO in indicated cells were detected by RT-qPCR assay and western blot assay (mean ± SEM, n = 3, two-sided t test).
Fig 2
Fig. 2
FTO promotes the radiation resistance of NPC cells. (A) Dose responses of survival factions of C666-1R and HONE1R cells treated with or without FB23-2. (B) The proliferation of NPC cell lines was evaluated by colony formation assay under treatment with or without FB23-2 (mean ± SEM, n = 3, two-sided t test). (C) Treatment with an FTO inhibitor FB23-2 promoted the apoptosis of NPC cells after irradiation, confirmed by flow cytometry (mean ± SEM, n = 3, two-sided t test). (D) RT-qPCR assay and western blot assay were used to detect the expression of FTO and Gapdh in C666-1 and HONE1 cells after transfecting with Flag-FTO plasmid (FTO) or corresponding empty plasmid (Con). (E) Dose responses of surviving factions of C666-1 and HONE1 cells with or without overexpressing FTO. (F) Overexpression of FTO promotes the proliferation of NPC cells under radiation (4 Gy), which was confirmed by colony formation assay (mean ± SEM, n = 3, two-sided t test). (G) Overexpression of FTO inhibited the apoptosis of NPC cells under radiation (4 Gy), which was confirmed by flow cytometry assay (mean ± SEM, n = 3, two-sided t test). (H) Comet assay was used to evaluate the DNA damage of indicated cells after radiation (4 Gy).
Fig 3
Fig. 3
FB23-2 suppresses NPC radiotherapy resistance in vivo. (A) Outline of the experimental approach. (B) Images of subcutaneous tumors comprising HONE1R cells after treating with FB23-2 or DMSO (Vehicle) (n = 5). (C) (Left) The growth curves of subcutaneous tumors derived from HONE1 cells with or without FB23-2 combination with radiotherapy are shown. (Right) Tumor weight (means, n = 5, two-sided t test) was measured at the endpoint. (D) Representative images of immunohistochemical staining of pH2A.X and Ki67 in tissue sections taken from tumors of the mice treated as indicated in (B). pH2A.X is a marker of DNA damage. Scale bars, 100 μm.
Fig 4
Fig. 4
Induction of ferroptosis contributes to the radiosensitivity of NPC cells. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that multiple deregulated genes were annotated in lipid metabolism pathways when knock down FTO in HEK293T cells. (B) Basal lipid peroxidation levels increased with cells exposure to radiation. Indicated cells were stained with C11-BODIPY 581/591. Reduced-BODIPY was measured by flow cytometry using a 488 nm laser and oxidized-BODIPY was measured with a 561 nm laser. A significant shift of oxidized-BODIPY occurred on exposure to radiation. (C) Cellular lipid peroxidation levels were measured by the malondialdehyde (MDA) kit. (D) Intracellular reduced glutathione (GSH) levels of NPC cells were significantly decreased with exposure to radiation. (E) Electron micrographs showing mitochondria in cells with or without radiation. (F) Cell apoptosis rate analyses of NPC cells that received the indicated treatments are shown.
Fig 5
Fig. 5
FTO promotes radiotherapy resistance by antagonizing radiation-induced ferroptosis in NPC cells. (A–C) BODIPY 581/591C11, MDA, and intracellular GSH were detected in NPC cells when treating cells with FB23-2 or overexpressed FTO combination with radiation (mean ± SEM, n = 3, two-sided t test). (D) Electron micrographs showing mitochondria in NPC cells treated with FB23-2 or overexpressed FTO, as well as corresponding control NPC cells. Red arrows showed the shrunken mitochondria. Blue arrows showed lipid droplets. (E) Dose responses of surviving factions of C666-1 and HONE1 cells that received the indicated treatments are shown. (F) Representative images of colony formation assay in NPC cells that received the indicated treatments are shown.
Fig 6
Fig. 6
FTO suppresses radiation-induced ferroptosis in an OTUB1 and SLC7A11-dependent manner. (A) The m6A RNA methylation status in NPC cells were evaluated using an RNA Methylation Quantification Kit (mean ± SEM, n = 3, two-sided t test). (B) The transcription and protein levels of OTUB1 were evaluated by RT-qPCR and Western blot assay. (C) The transcription and protein levels of OTUB1 were detected when treating NPC cells with FB23-2 or overexpressing FTO. (D) Kaplan–Meier survival curves show the correlations between OTUB1 expression and overall survival (OS, p0.05) in patients with HNSC. (E) The transcription levels of OTUB1 were positively correlated with those of FTO in HNSC tissue samples. (F, G) RIP experiments were performed using antibodies against m6A and against FTO with extracts from NPC cells. (H) Western blot analysis of SLC7A11 and OTUB1 after immunoprecipitation of Anti-OTUB1 in NPC cells treated with FB23-2 or transfected with Flag-FTO; 2.5% of the sample was loaded as the input. (I) Dose responses of surviving factions of C666-1 and HONE1 cells that received the indicated treatments are shown. (J) Cell apoptosis of indicated NPC cells was quantified using Flow Cytometry assay.
Fig 7
Fig. 7
Accelerating ferroptosis overcomes radioresistance of NPC patient-derived xenografts. (A) Outline of the experimental approach. (B) Images of subcutaneous tumors comprising HONE1R cells after treating with erastin or DMSO (vehicle) (n = 5). (C) (Left) The growth curves of subcutaneous tumors derived from HONE1R cells with or without erastin in combination with radiotherapy are shown; (Right) Tumor weight was measured at the endpoint. (D) Outline of the experimental approach, beginning with the primary sample. (E) Histological comparison of the founder (P0) PDX tumor and primary patient-derived specimens. (F) (a) Images of tumor-bearing mice and subcutaneous tumors derived from mice; (b) the growth curves of xenografted tumor derived from mice treated with erastin or DMSO (vehicle) and under 20 Gy radiotherapy; (c) tumor weight was measured at the endpoint (mean ± SEM, two-sided t test).
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