Abstract

   Radiotherapy is the primary treatment for nasopharyngeal carcinoma
   (NPC); nonetheless, radioresistance remains the leading cause of
   localized recurrence. Our study demonstrates a significant increase in
   the N6-methyladenosine (m6A) methylase METTL3 in NPC and other tumors.
   Mechanistically, METTL3 acts as an m6A methylase, enhancing the m6A
   modification of the solute carrier family 7 member 11 (SLC7A11)
   transcript, which increases its stability and expression, thereby
   inhibiting radiation-induced ferroptosis and ultimately inducing
   radioresistance in NPC. Furthermore, silencing SLC7A11 or employing the
   ferroptosis inducer Erastin negated the promoting effect of METTL3 on
   NPC cell radioresistance. These findings suggest that METTL3 could be a
   novel therapeutic target for overcoming radiotherapy resistance in NPC.

   Keywords: nasopharyngeal carcinoma, m^6A modification, SLC7A11,
   radiosensitivity, ferroptosis

1. Introduction

   Nasopharyngeal carcinoma (NPC), a malignancy originating from the
   epithelium of the nasopharyngeal mucosa, can be highly locally invasive
   and prone to distant metastases. NPC is highly prevalent among people
   from Southeast Asian regions, especially Southern China [41]^1^,
   [42]^2. With the application of advanced radiotherapeutic techniques
   and concurrent chemoradiotherapy, the 5-year overall survival rate for
   NPC patients has surged from 17-35% to over 75% [43]^3. Nevertheless,
   approximately 10-20% of cases exhibit intrinsic resistance to
   radiotherapy, leading to local recurrence and distant metastasis
   [44]^4^, [45]^5. Therefore, it is significant to clarify the molecular
   mechanisms underlying NPC radioresistance to develop novel therapeutic
   strategies.

   Epigenetic alterations have been identified as key genetic features in
   tumorigenesis and progression, and their reversible nature makes them
   potential targets for overcoming treatment resistance [46]^6^, [47]^7.
   N6-methyladenosine (m6A) is the most prevalent dynamic modification of
   RNA methylation, involved in crucial cellular processes such as RNA
   metabolism, splicing, translation, nuclear export, and transport
   [48]^8^-[49]^11. We previously employed pyrophosphate sequencing to
   confirm that 70% of genes in NPC are hypermethylated [50]^12. METTL3 is
   significantly overexpressed in head and neck tumors and is linked to
   poor patient prognosis. Through screening m6A-related proteins, we
   discovered that the m6A methyltransferase METTL3 mediated the reduced
   expression of ZNF750, which activated the FGF14 signaling pathway and
   promoted the proliferation and invasion of NPC cells [51]^13.
   Simultaneously, we confirmed that METTL3 stabilized the non-coding
   lncRNA FAM225A, which functions as a ceRNA to upregulate ITGB3 and
   promotes the proliferation of NPC. This process promotes the
   proliferation, migration, and invasion of NPC [52]^14. Recently,
   several studies have clarified that N6-methyladenosine (m6A) methylase
   (METTL3) plays oncogenic roles in radiosensitivity in glioblastoma
   [53]^15, gastric cancer [54]^16, and hypopharyngeal squamous cell
   carcinoma [55]^17, representing an opportunity for the development of
   METTL3-targeted therapies for NPC.

   Ferroptosis, a new type of programmed cell death, is characterized by
   iron accumulation and lipid peroxidation during the cell death process
   [56]^18. Recent studies have identified that ferroptosis is closely
   related to the pathophysiological processes of many diseases, including
   cancers [57]^18^-[58]^20. Targeting ferroptosis-associated proteins for
   direct induction or secondary regulation has emerged as an effective
   therapeutic approach to triggering tumor cell demise, particularly in
   malignant tumors resistant to radiation [59]^21^, [60]^22. However, the
   underlying role of ferroptosis in NPC radiosensitivity remains unclear.

   In this study, we found an upregulation of METTL3 in various cancers,
   including NPC, which is associated with poor survival. Further in vitro
   and in vivo investigations indicate that METTL3-mediated m6A
   modification of SLC7A11, dependent on IGF2BP2, maintains its stability,
   inhibits NPC ferroptosis, and promotes radioresistance. Hence, METTL3
   may serve as a predictive marker and therapeutic target for
   radioresistance in NPC.

2. Materials and methods

2.1 Clinical specimen collection

   NPC specimens (n=78) were collected from the Affiliated Cancer Hospital
   of Guangzhou Medical University (Guangzhou, China) and prepared for
   immunohistochemical staining experiments. Fresh frozen biopsy specimens
   of NPC (n=8) and nasopharyngeal epithelial specimens (n=8) were
   collected for protein extraction. None of the patients had received any
   anti-tumor therapy prior to specimen collection. This study was
   approved by the Ethics Review Committee of the Affiliated Cancer
   Hospital of Guangzhou Medical University and adhered to the guidelines
   of the Helsinki Declaration.

2.2 Cell culture

   The normal human nasopharyngeal epithelial cell line NP69 was cultured
   in KSFM keratinocyte growth medium (Elgbio) supplemented with KSFGS
   cell growth factor. HEK-293T cells provided by the American Type
   Culture Collection (ATCC) were cultured in DMEM medium (Gibco)
   supplemented with 10% fetal bovine serum (FBS). Human NPC cell lines
   (HONE1, SUNE1) were cultured in RPMI-1640 medium (Gibco) supplemented
   with 10% FBS. All normal nasopharyngeal epithelial cell lines and NPC
   cell lines were generously provided by Professor Musheng Zeng (Sun
   Yat-sen University Cancer Center). HEK-293T cells were obtained from
   ATCC and cultured in DMEM medium (Invitrogen). Additionally, all cells
   were cultured at 37°C with 5% CO2. Negative results were obtained for
   all cells in mycoplasma testing, and cell lines were not passaged for
   more than three months.

2.3 Transient transfection and establishment of stable cell lines

   The plasmids pEnter-IGF2BP1-Flag, pEnter-IGF2BP2-Flag,
   pEnter-IGF2BP3-Flag, pEnter-YTHDF1-Flag, pEnter-YTHDF2-Flag,
   pEnter-YTHDF3-Flag, pEnter-SLC7A11-Flag, and the corresponding
   pEnter-vector plasmids were purchased from WZ Biosciences. Plasmid DNA
   was purified using a plasmid extraction kit (TIANGEN). Transient
   transfection experiments were performed using the Lipofectamine 3000
   reagent kit (Invitrogen) according to the manufacturer's instructions.

   The plasmids Plko-u6-METTL3-gfp-puro, Plko-u6-METTL3-gfp-puro-vector,
   pCDH-METTL3-puro, and pCDH-METTL3-puro-vector were purchased from
   Getein Biotech. The catalytic mutant METTL3 (aa395-398, from DPPW to
   APPA) was generated by site-directed mutagenesis. To produce cells
   capable of stably expressing METTL3 or the vector, HEK-293T cells were
   co-transfected with lentiviral packaging plasmids and either
   pCDH-METTL3-puro or pCDH-METTL3-puro-vector. The collected lentiviruses
   were then used to infect NPC cells. Stable cells were selected using 2
   μg/ml puromycin (Solarbio) and verified by qRT-PCR and Western
   blotting.

2.4 Western blot assay

   Total cellular proteins were extracted using RIPA lysis buffer (CWBIO)
   supplemented with phosphatase and protease inhibitors (Beyotime). The
   protein concentration was measured using a protein quantification kit
   (CWBIO), and then the proteins were denatured. Equal amounts of protein
   (30 μg/sample) were separated by 5-15% sodium dodecyl
   sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred
   to a PVDF membrane (Millipore). The membrane was blocked with 5% bovine
   serum albumin (BSA) in TBST (1% Tween-20 in Tris-buffered saline) at
   room temperature for 1 hour. The membrane was then incubated with the
   primary antibodies against the target proteins overnight at 4°C
   ([61]Table S2). The membrane was incubated with the corresponding
   HRP-conjugated secondary antibodies for 1-2 hours at room temperature.
   The immunocomplexes were detected using a chemiluminescence reagent
   (EpiZyme), with GAPDH as the loading control.

2.5 RNA extraction and quantitative Real-Time PCR (qRT-PCR)

   Total RNA was extracted using RNAiso Plus (Takara) according to the
   manufacturer's instructions. The concentration of total RNA was
   measured using a NanoDrop spectrophotometer. Total RNA was
   reverse-transcribed into cDNA using Evo M-MLV reverse transcriptase
   pre-mixture (Accurate Biology). qRT-PCR samples were prepared using a
   SYBR Green reagent kit (Accurate Biology) and then quantitatively
   detected using a real-time fluorescence quantitative PCR instrument
   (Roche). Human GAPDH was used as an internal control for normalization.
   Primer sequences are provided in [62]Table S1.

2.6 Cell viability assay

   The CCK-8 cell counting kit (GLPBIO) was used to evaluate cell
   viability. NPC cells (5 × 10^3 cells/well) were seeded in 96-well
   plates and exposed to 2 Gy, 4 Gy, 6 Gy, and 8 Gy of radiation. After 24
   hours of incubation, 100 μl of RPMI-1640 medium containing 10% CCK-8
   solution was added to each well, and the cells were further incubated
   for 2 hours. The absorbance was then measured at 450 nm using a
   spectrophotometer (Tecan).

2.7 Colony formation assay

   Cells in the logarithmic growth phase were trypsinized and resuspended
   into a single-cell suspension. Cells (400-1000 cells/well) were seeded
   in 6-well plates. After 24 hours, the cells were exposed to different
   doses of X-ray radiation as required by the experiment. The cells were
   then cultured for 14 days, washed once with PBS, fixed with 4%
   paraformaldehyde, and stained with crystal violet.

2.8 RNA stability assay

   Cells were seeded in 12-well plates and treated with actinomycin D (5
   μg/mL, CST) for 0, 0.5, 1, 1.5, and 2 hours. RNA was extracted at the
   specified time points and analyzed by quantitative PCR. The half-life
   of the mRNA was calculated using linear regression analysis.

2.9 Glutathione (GSH) Quantification and Malondialdehyde (MDA) Determination

   The total cellular glutathione level was detected using GSH and GSSG
   Assay Kit (Beyotime) in accordance with the manufacturer's
   instructions, and the absorbance at 412 nm was measured using a
   spectrophotometer. For MDA detection, the cells were lysed and treated
   with Lipid Peroxidation MDA Assay Kit (Beyotime). The absorbance at 532
   nm was measured.

2.10 Quantification of intracellular ferrous ions (Fe^2+)

   According to the instructions, the total cellular Fe2+ level was
   detected using Cell Ferrous Iron Colorimetric Assay Kit (Elabscience).
   The collected cells were lysed by adding 0.2 mL of Reagent I per 1×10^6
   cells, mixed well, and incubated on ice for 10 minutes. After
   centrifugation at 15,000×g for 10 minutes, the supernatant was used for
   subsequent measurements. Reagent II was added to the standard wells,
   test wells, and control wells, mixed well, and incubated at 37°C for 10
   minutes. The absorbance was measured at 593 nm using a
   spectrophotometer.

2.11 Quantification of Reactive Oxygen Species (ROS)

   According to the instructions, ROS Assay Kit (Beyotime) was used to
   detect intracellular ROS levels. DCFH-DA was diluted 1:1000 in
   serum-free culture medium to achieve a final concentration of 10 μM.
   Cells were seeded at a density of 2 × 10^5 cells per well in a 6-cm
   dish and cultured for 12 hours. The cell culture medium was then
   replaced with either drug-free medium (control group), drug-containing
   medium, or medium treated with 4 Gy X-ray irradiation, and incubated
   for an additional 24 hours. The cells were trypsinized, collected, and
   resuspended in the diluted DCFH-DA solution at a concentration of
   10^6-10^7 cells/mL, then incubated at 37°C in a cell culture incubator
   for 20 minutes.

2.12 Immunohistochemistry (IHC) staining and hematoxylin and eosin (H&E)
Staining

   Paraffin-embedded NPC tissues were sectioned at a thickness of 4 μm.
   After incubation at 65°C for 2 hours, the tissue sections were
   deparaffinized in xylene and rehydrated through a graded ethanol
   series. Endogenous peroxidase activity was blocked using 4% hydrogen
   peroxide. Normal goat serum was used to block the tissue. The tissue
   was incubated with the primary antibody overnight at 4°C ([63]Table
   S2). The tissue was incubated with the secondary antibody for 20
   minutes and stained with 3,3'-diaminobenzidine (DAB) substrate
   (ZSGB-BIO). The percentage of positive tumor cells and their staining
   intensity were evaluated. According to a semi-quantitative system, the
   staining intensity was scored as 0 (no staining), 1 (weak), 2
   (moderate), or 3 (strong). The percentage of positive tumor cells was
   scored as 1 (<25%), 2 (26-50%), 3 (51-75%), or 4 (>75%). The percentage
   of positive cells was determined from five different random areas. The
   staining intensity score was multiplied by the positive cell percentage
   score to quantify the expression and obtain the final score. For H&E
   staining, the sections were immersed in hematoxylin for 5 minutes,
   washed in water for 5 minutes, and then stained with eosin for 5-10
   minutes. After dehydration in different concentrations of ethanol, the
   sections were mounted with coverslips. The stained tissue sections were
   imaged using a slide scanner microscope (3DHISTECH).

2.13 Immunofluorescence (IF) staining

   Cell suspensions (1-2 × 10^3 cells/mL) were seeded onto cell slides,
   fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.5%
   Triton X-100 for 10 minutes, and blocked with 5% BSA for 30 minutes.
   The cells were then incubated with the primary antibodies overnight at
   4°C ([64]Table S2). The following day, the cells were incubated with
   the corresponding fluorescent-labeled secondary antibodies for 1 hour
   at room temperature. The cells were then counterstained with an
   anti-fluorescence quenching mounting medium containing DAPI for nuclear
   staining. The slides were observed using a laser scanning confocal
   microscope (ZEISS).

2.14 Transmission electron microscopy (TEM)

   The cells were subjected to routine trypsin digestion, which was
   terminated using complete culture medium. The cell suspension was then
   centrifuged at 1,000 rpm for 5-10 minutes to collect the cell pellet.
   The supernatant was discarded, and the compact cell pellet was gently
   resuspended in 1 mL of 2.5% glutaraldehyde fixative at room
   temperature. The cells were fixed in the dark for 15 minutes at room
   temperature. After post-fixation with 1% osmium tetroxide and
   dehydration, the samples were embedded in EPON resin. The embedded
   samples were sectioned and stained with uranyl acetate. The stained
   sections were then examined under a transmission electron microscope.

2.15 M6A immunoprecipitation sequencing (MeRIP-seq) and mRNA sequencing
(mRNA-seq)

   MeRIP-seq and mRNA-seq experiments were conducted by Gene Denovo
   Biology, utilizing the Illumina NovaSeq 6000 sequencing system. The
   same company also assisted with subsequent bioinformatics analyses. The
   m6A peaks were visualized using the UCSC genome browser or IGV
   software. Motif discovery, both de novo and known, was performed with
   HOMER, followed by motif localization using Perl scripts. FPKM
   (fragments per kilobase of transcript per million) values for all mRNAs
   in the input libraries were calculated using StringTie. Gene Set
   Enrichment Analysis (GSEA) was conducted using the R package
   clusterProfiler.

2.16 RNA immunoprecipitation (RIP)

   RIP detection was conducted using the RIP reagent kit according to the
   manufacturer's protocol (Boxin Biotech, China). Cells were lysed to
   remove DNA, and the resulting lysate was divided into input, IgG, and
   IP groups. In the IgG and IP groups, 5 μg of IgG and primary antibody
   were added ([65]Table S2), and the samples were incubated overnight at
   4°C. The following day, 20 μL of Protein A/G magnetic beads were added
   to the IgG and IP groups, and the samples were incubated for 1 hour at
   4°C. RNA-protein complexes bound to the magnetic beads were then
   eluted. RNA was extracted and purified using a
   phenol-chloroform-isoamyl alcohol solution. RT-qPCR was performed to
   detect the expression levels of the target gene.

2.17 Animal experiments

   Five-week-old female BALB/c nude mice were purchased from the Guangdong
   Medical Laboratory Animal Center. HONE1 cells (1×10^7 cells) were mixed
   with Matrigel (1:1, Corning) and subcutaneously injected into the fat
   pad of the nude mice. After palpable tumors formed, the mice were
   randomly divided into six groups: Ctrl group, IR group (4 Gy), and IR
   (4 Gy) combined with the ferroptosis inducer Erastin (10 mg/kg; ip)
   group. Tumor volumes were measured every three days and calculated as
   volume = length × width^2/2. The primary tumor cells were stained with
   primary antibody ([66]Table S2).

2.18 Database analysis

   The METTL3 transcriptome data were downloaded from the TCGA database
   ([67]https://tcga-data.nci.nih.gov/tcga/) and the Gene Expression
   Omnibus (GEO) database ([68]https://www.ncbi.nlm.nih.gov/geo/).
   Patients with incomplete clinicopathological information were excluded
   from data curation and analysis. The gene expression data from the
   microarray were normalized using Transcripts Per Million (TPM).

2.19 Statistical analysis

   All experiments were performed at least three times. Data were
   statistically analyzed using Prism 8.0 software. An unpaired t-test was
   used to analyze the differences between two groups. One-way ANOVA was
   used for comparisons among multiple groups. Kaplan-Meier survival
   curves were plotted, and the log-rank test was used for comparison. The
   results are presented as mean ± standard error of the mean (mean ±
   SEM). p < 0.05 was considered statistically significant.

3. Results

3.1 METTL3 is upregulated and correlates with poor prognosis in NPC

   To elucidate the role of METTL3 in NPC, we first assessed METTL3
   expression in eight paired clinical samples. The results revealed a
   marked upregulation of METTL3 in NPC tissues (Figure [69]1A). However,
   we found no significant difference in METTL3 expression in the fifth
   tissue pair. This finding suggests potential tissue-specific variation
   and aligns with the broader biological context we are investigating.
   Compared to the nasopharyngeal epithelial cell line NP69, METTL3 was
   also upregulated in NPC cell lines (Figure [70]1B-C). Meanwhile, GEO
   ([71]GSE12452) showed that the mRNA levels of METTL3 were markedly
   upregulated in NPC (Figure [72]1D). TCGA database reveals differential
   expression of METTL3 mRNA across 27 solid tumor types, with notably
   higher expression in HNSC (Figure [73]1E). Survival analysis indicated
   that METTL3 was associated with poor survival (Figure [74]1F and G).
   Subgroup analysis indicated that higher METTL3 expression was
   significantly associated with clinical stage and T stage (p<0.05,
   Figure [75]1H-I, [76]Table S3). Collectively, our data indicated that
   METTL3 may act as an oncogenic factor in the progression of NPC.

Figure 1.

   [77]Figure 1
   [78]Open in a new tab

   Elevated expression of METTL3 associated with poor prognosis in NPC
   patients. (A) Differential expression of METTL3 in normal
   nasopharyngeal epithelial tissues and NPC tissues (N: normal; T:
   tumor). The right panel displays quantification data from three
   replicates of WB assay. (B) Protein expression of METTL3 in normal and
   NPC cell lines. The right panel shows quantification data from three
   replicates of Western blot analysis. (C) Expression of METTL3 mRNA in
   nasopharyngeal normal cell line (NP69) and NPC cell lines. (D)
   Differential expression of METTL3 mRNA in the [79]GSE12452 dataset
   (N=10, T=31). (E) Expression of METTL3 in various cancer types in TCGA
   data. (F) Representative IHC staining images of METTL3 expression in
   NPC tissues. (G) Kaplan-Meier analysis of overall survival rates of NPC
   patients with high/low expression of METTL3. (H-I) Distribution of
   patients with high and low expression of METTL3 in tumor grade (H) and
   comparison of T stage (I). Data are presented as mean ± SEM of three
   independent experiments, * p < 0.05; ** p < 0.01; *** p < 0.001.

3.2 METTL3 suppresses NPC radiosensitivity in vitro and in vivo

   To identify potential targets regulated by METTL3, we conducted RNA
   sequencing (RNA-seq) on HONE1 cells with overexpressed METTL3.
   Overexpression of METTL3 caused dysregulated gene expression, resulting
   in 943 genes being upregulated and 2201 genes being downregulated
   (Figure [80]2A). Subsequently, using m6A-seq technology, we mapped the
   m6A methylome in NPC cells overexpressing METTL3, identifying 25,070
   m6A peaks across 20,332 genes. These m6A modifications were mainly
   enriched in protein-coding transcripts (27%), stop codons (25%), and
   3'-UTRs (20%) (Figure [81]2B). In line with previous studies, GGAC
   [U/A] was identified as the consensus sequence for m6A in HONE1 cells
   (Figure [82]2C). By integrating m6A-seq and RNA-seq data, we identified
   430 overlapping differentially expressed genes (DEGs) with m6A
   modifications (Figure [83]2D). KEGG and GO analyses revealed that the
   overlapping DEGs were significantly enriched in pathways and processes
   related to radiation damage and ferroptosis, such as DNA damage repair,
   homologous and non-homologous recombination, biosynthesis of aromatic
   compounds, and metal ion binding (Figure [84]2E-F).

Figure 2.

   [85]Figure 2
   [86]Open in a new tab

   METTL3 facilitates radiotherapy resistance both in vitro and in vivo.
   (A) Volcano plot illustrates the RNA-seq results of differentially
   expressed genes in control and METTL3-overexpressing HONE1 cells. Blue
   dots represent significantly upregulated genes, red dots indicate
   significantly downregulated genes, and gray dots denote genes without
   significant differential expression (|log2FC| ≥ 2, P-value ≤ 0.05). (B)
   Pie chart shows the distribution of m6A peaks in different gene regions
   in METTL3-overexpressing HONE1 cells, including CDS (27%), stop codon
   (25%), 3' UTR (20%), start codon (15%), unmapped (9%), and stop codon
   (3%). (C) The sequence logo displays the enriched consensus sequence
   around the m6A peaks. (D) Venn diagram shows the overlap between
   differentially expressed genes identified by RNA-seq and m6A-modified
   genes identified by MeRIP-seq. (E-F) KEGG pathway enrichment analysis
   (E) and Gene Ontology (GO) term enrichment analysis (F) of overlapping
   differentially expressed genes (DEGs). (G-J) Cell viability (G-H) and
   clonogenic capability (I-J) of NPC cells with METTL3 knockdown after
   exposure to different doses of irradiation (0, 2, 4, 6, 8 Gy). (K-L)
   Temporal dynamics of γ-H2AX levels in NPC cells following treatment
   with varying doses of irradiation, as assessed by WB. (M-N) Alterations
   in γ-H2AX levels after irradiation (4 Gy) for 24 hours in control and
   METTL3 knockdown cells. (O) Enhanced growth of subcutaneous xenografts
   in nude mice upon METTL3 overexpression (n=4). (P) Tumors weight. (Q)
   The growth curve of tumors. (R) Histological examination of tumor
   sections stained with H&E, along with IHC staining using anti-METTL3
   and anti-γ-H2AX antibodies (scale bar = 20 µm). Data represent the mean
   ± SEM of three independent experiments, * p < 0.05; ** p < 0.01; *** p
   < 0.001.

   To investigate the impact of METTL3 on NPC, we downregulated METTL3
   expression in NPC cells using shRNA and validated it through Western
   blot (WB) and quantitative PCR (qPCR) assays ([87]Figure S1A-B). CCK-8
   assays revealed that METTL3-KD significantly decreased the survival
   ability of NPC cells after IR treatment (Figure [88]2G-H). METTL3
   knockdown reduced the proliferative capacity of the cells ([89]Figure
   S1C). METTL3-KD decreased the clonogenic capacity of NPC cells (Figure
   [90]2I-J). To determine the optimal time and dosage for radiation, NPC
   cells were treated with different doses and durations. The results
   demonstrated that γ-H2AX expression was dose-dependent and
   time-dependent, particularly at 4 Gy for 24 hours (Figure [91]2K-L).
   METTL3-KD contributed to an upregulation of γ-H2AX expression, which
   was significantly enhanced when combined with IR treatment (Figure
   [92]2M-N). Likewise, IF staining revealed a pronounced increase in the
   number of γ-H2AX foci in METTL3-KD cells 24 hours post-exposure to 4 Gy
   irradiation ([93]Figure S1D-E). Xenograft tumor models indicated that
   METTL3 promoted NPC growth in vivo (Figure [94]2O-Q). IHC analysis
   revealed that METTL3 could decrease γ-H2AX expression (Figure [95]2R).
   These findings suggest that METTL3 is associated with NPC malignant
   features, such as radioresistance.

3.3 METTL3 inhibits NPC radiation-induced ferroptosis

   Radiotherapy uses high-energy ionizing radiation (IR) to produce DNA
   double-strand breaks, inducing cell cycle arrest, senescence, and
   various forms of cell death, including apoptosis, necrosis, and
   autophagy. In addition to directly damaging DNA, IR induces indirect
   cellular effects by generating reactive oxygen species (ROS) such as
   hydroxyl radicals and hydrogen peroxide, which further contribute to
   DNA damage [96]^23.

   This suggests a potential link between radiotherapy and ferroptosis, a
   regulatory form of cell death [97]^24. Ferroptosis is an iron-dependent
   regulated cell death driven by lipid peroxidation and plays a
   significant role in radiation-induced cell death and tumor suppression
   [98]^25. We investigated the role of METTL3 by conducting WB analysis
   on a stable METTL3 knockdown cell line. The results indicated that the
   expression levels of ferritin ferroptosis-related genes SLC7A11 and
   GPX4 were significantly reduced in METTL3 KD cells compared to the
   control group, with this difference becoming more pronounced
   post-combined irradiation (Figure [99]3A). SLC7A11 and GPX4 are key
   molecules closely linked to ferroptosis (iron-dependent cell death).
   Studies have shown that METTL3 regulates the mRNA methylation of
   SLC7A11, which indirectly influences the expression and function of
   GPX4 [100]^20^, [101]^26^, [102]^27. Transmission electron microscopy
   unveiled distinctive morphological features of ferroptosis in METTL3-KD
   cells, characterized by smaller mitochondria and increased membrane
   density (Figure [103]3B). A series of experiments were conducted in
   METTL3-KD cells to evaluate the levels of ferroptosis. Subsequent to
   METTL3 suppression, GSH/GSSH levels declined (Figure [104]3C), while
   MDA, Fe2+, and ROS levels escalated, with this effect becoming more
   pronounced post-irradiation (Figure [105]3D-F). Treatment of NPC cells
   with the ferroptosis inducer Erastin resulted in decreased cell
   viability, whereas cell viability increased following treatment with
   the ferroptosis inhibitor Fer-1 at different radiation doses (Figure
   [106]3G-H and [107]Figure S1F-G). Colony formation assays demonstrated
   that Erastin treatment reversed METTL3 inhibition-induced cell death
   and post-irradiation growth suppression, whereas Fer-1 treatment
   yielded opposite outcomes in METTL3-overexpressing NPC cells (Figure
   [108]3I-J). These findings suggest that METTL3 promotes radioresistance
   by inhibiting NPC ferroptosis.

Figure 3.

   [109]Figure 3
   [110]Open in a new tab

   METTL3 knockdown sensitizes NPC cells to radiotherapy by promoting
   ferroptosis. (A) WB images depicting key ferroptosis-associated
   proteins in cells with or without irradiation (IR) treatment upon
   METTL3 knockdown. (B) TEM revealing distinctive morphological features
   of ferroptosis in METTL3-suppressed cells. (C-F) Measurement of
   GSH/GSSH ratio (C), MDA levels (D), Fe^2+ levels (E), and ROS levels
   (F) in control and METTL3 knockdown cells with or without IR treatment.
   (G-H) CCK-8 assay assessing the viability of HONE1 cells treated with
   the ferroptosis inducer Erastin (G) or the ferroptosis inhibitor
   ferrostatin-1 (H) at different doses. (I-J) Cell survival rates
   following irradiation at various doses, with corresponding D0, Dq, and
   N values depicted on the right. Erastin: 30 μM, ferrostatin-1: 1 μM.
   Data represent the mean ± SEM of three independent experiments, * p <
   0.05; ** p < 0.01; *** p < 0.001.

3.4 METTL3-mediated m6A modification of SLC7A11 promotes its stability in an
IGF2BP2-dependent manner

   To clarify the potential mechanism of METTL3 in ferroptosis, Gene Set
   Enrichment Analysis (GSEA) was performed based on the [111]GSE12452
   dataset, which indicated that METTL3 expression was significantly
   associated with ferroptosis-related pathways, including the oxidative
   stress pathway (Figure [112]4A). We first established stable
   METTL3-overexpressing NPC cells (Figure [113]4B-C). Based on previous
   studies [114]^28^-[115]^33, nine oxidative stress-related genes were
   selected for validation. The results showed that the expression levels
   of NFE2L2, SLC7A11, GPX4, and phosphorylated AKT were upregulated in
   METTL3-overexpressing cells, with the most significant expression of
   SLC7A11 (Figure [116]4D-E and S1H). As shown in Figures [117]4F and
   S1I, the METTL3-specific antibody significantly enriched SLC7A11 mRNA
   in the RIP-qPCR experiment, compared to the IgG control antibody.
   Furthermore, IHC revealed that SLC7A11 expression was positively
   correlated with METTL3 expression (Figure [118]4G-H).

Figure 4.

   [119]Figure 4
   [120]Open in a new tab

   METTL3 enhances the stability and expression of SLC7A11 mRNA through
   m6A modification recognized by IGF2BP2. (A) GSEA of METTL3
   overexpression phenotypes showing high/low expression signatures. (B-C)
   Validation of METTL3 overexpression by WB (B) and qPCR (C). (D-E) mRNA
   and protein expression of ferroptosis-related genes after METTL3
   overexpression. (F) RIP-qPCR assay utilizes an METTL3-specific antibody
   and an IgG control antibody to detect the enrichment of METTL3 binding
   at the SLC7A11 m6A modification site. (G-H) Representative images (G)
   and scoring (H) of SLC7A11 expression in NPC tissues after IHC
   staining. (I) Confirmation of METTL3 overexpression in wild-type and
   mutant METTL3 cells. (J) qRT-PCR was used to assess the expression
   levels of SLC7A11 at specific time points after Act D (2 µg/mL)
   treatment in HONE1 wild-type METTL3, mutant METTL3, and their
   respective control cells. (K) Expression changes of ferroptosis-related
   genes in wild-type and mutant METTL3 cells confirmed by WB. (L)
   Expression of six m6A reader enzymes detected by qPCR in control and
   METTL3-overexpressing HONE1 cells. (M) RIP-qPCR assay utilizes an
   IGF2BP2-specific antibody and an IgG control antibody to detect the
   enrichment of IGF2BP2 binding at the SLC7A11 m6A modification site.
   (N-O) Representative images (N) and scoring (O) of SLC7A11 expression
   in NPC tissues after IHC staining. (P) Kaplan-Meier analysis of overall
   survival rates in NPC patients with high/low expression of IGF2BP2. (Q)
   qRT-PCR was used to assess the expression levels of SLC7A11 at specific
   time points after treatment with Act D (2 µg/mL) in HONE1 control
   cells, METTL3 knockdown cells, and METTL3 knockdown cells transfected
   with IGF2BP2. Data represent the mean ± SEM of three independent
   experiments, * p < 0.05; ** p < 0.01; *** p < 0.001.

   Given that m6A modification positively regulated the mRNA level of
   SLC7A11, we investigated whether m6A modification affected the
   stability of SLC7A11 mRNA. The NPC cells were treated with actinomycin
   D, a transcription inhibitor, for the indicated times. The level of
   SLC7A11 mRNA was shown to be highly stable with METTL3 overexpression,
   while the effect was reversed by METTL3 mutation (Figure [121]4I-K and
   S1J). A recent study reported that IGF2BPs and YTHDFs are distinct
   families of m6A readers that target thousands of mRNA transcripts
   through the recognition of the m6A motif [122]^9^, [123]^34. The
   effects of IGF2BPs and YTHDFs on SLC7A11 mRNA stabilization were
   further explored. We transiently transfected IGF2BPs and YTHDFs
   plasmids to detect SLC7A11 expression. Only overexpression of IGF2BP2
   markedly enhanced SLC7A11 mRNA expression (Figure [124]4L and S1K). The
   IGF2BP2-specific antibody notably enriched SLC7A11 mRNA in the RIP-qPCR
   assay, in contrast to the IgG control antibody (Figure [125]4M and
   S1L). Additionally, a significant correlation was observed between the
   expression of IGF2BP2 and SLC7A11 (r²=0.5919; p<0.0001, Figure
   [126]4N-O and S1M). Based on the median of IGF2BP2 immunohistochemistry
   scores, patients with expression levels higher than the median were
   classified as the "high expression" group, while those with levels
   below the median were classified as the "low expression" group.
   Kaplan-Meier analysis indicates that high IGF2BP2 expression correlates
   with poor prognosis in patients with NPC (Figure [127]4P and S1N). The
   reduction of SLC7A11 mRNA caused by METTL3 knockdown could be rescued
   by IGF2BP2 (Figure [128]4Q and S1O). Together, our findings suggest
   that METTL3-mediated m6A modification maintains the stability of
   SLC7A11 expression in an IGF2BP2-dependent manner.

3.5 The elevation of METTL3 accelerates NPC progression by upregulating
SLC7A11 expression

   Western blot assays revealed that SLC7A11 overexpression counteracted
   the reduction in GPX4, NFE2L2, and phosphorylated AKT levels induced by
   METTL3 knockdown (Figure [129]5A). CCK8 assays demonstrated that
   SLC7A11 overexpression rescued the cell viability of METTL3-KD cells
   (Figure [130]5B). Furthermore, SLC7A11 overexpression reversed the
   decreased GSH/GSSH ratio (Figure [131]5C) and the increased levels of
   MDA, Fe2+, and ROS induced by METTL3 inhibition (Figure [132]5D-F). Our
   findings indicate that METTL3 inhibits radiotherapy sensitivity by
   upregulating SLC7A11 expression and mitigating ferroptosis, thereby
   accelerating the malignant progression of NPC.

Figure 5.

   [133]Figure 5
   [134]Open in a new tab

   SLC7A11 overexpression reverses ferroptosis-related effects induced by
   METTL3 knockdown in NPC cells. (A) SLC7A11 overexpression in METTL3
   knockdown cells restores the protein expression of phosphorylated AKT,
   GPX4, and NFE2L2. (B) Enhanced growth capacity of nasopharyngeal
   carcinoma cells upon SLC7A11 expression in METTL3 knockdown cells.
   (C-F) Measurement of GSH/GSSH ratio (C), MDA (D), Fe²⁺ levels (E), and
   ROS levels (F) in METTL3 knockdown cells overexpressing SLC7A11 with or
   without IR. Data represent the mean ± SEM of three independent
   experiments, * p < 0.05; ** p < 0.01; *** p < 0.001.

3.6 METTL3 collaborates with ferroptosis inducers to regulate
radiosensitivity in vivo

   To further investigate whether METTL3 enhances the radioresistance of
   NPC in vivo, we established subcutaneous xenografts in nude mice, as
   depicted in Figure [135]6A. Compared to the control group, METTL3
   overexpression significantly increased the volume and weight of the
   tumors, regardless of the presence of IR and Erastin. Moreover, the
   combination treatment with IR and Erastin resulted in lower tumor
   volume and weight compared to the IR group. Importantly, the
   combination therapy of IR and Erastin demonstrated superior efficacy,
   overcoming METTL3-induced radiotherapy resistance (Figure [136]6B-D).
   Figure [137]6E illustrates that radiation combined with the ferroptosis
   inducer Erastin led to decreased expression of METTL3, IGF2BP2, and
   SLC7A11, while upregulating γ-H2AX expression. In summary, METTL3 in
   combination with ferroptosis inducers can promote radiotherapy
   sensitivity in vivo. These data suggest that the METTL3/IGF2BP2/SLC7A11
   axis promotes NPC radioresistance by inhibiting NPC ferroptosis.

Figure 6.

   [138]Figure 6
   [139]Open in a new tab

   METTL3 promotes radioresistance in NPC in vivo. (A) Schematic
   representation of the experimental design and workflow for subcutaneous
   tumor transplantation in nude mice. (B) Tumor growth status in each
   group after 18 days of treatment (n=4). (C) Tumor volume. (D) Tumor
   weight. (E) Representative images of H&E staining and IHC staining for
   METTL3, IGF2BP2, SLC7A11, and γ-H2AX protein expression in
   subcutaneously transplanted tumors. Scale bar: 50 µm. Quantitative data
   from three repeated immunohistochemical images are presented on the
   right. Data represent the mean ± SEM of three independent experiments,
   * p < 0.05; ** p < 0.01; *** p < 0.001.

4. Discussion

   Due to its special anatomical location and nonspecific symptoms, most
   NPC cases are diagnosed at advanced stages. For advanced-stage
   patients, radiotherapy combined with chemotherapy has become the
   primary treatment strategy [140]^35. Despite advancements in treatment
   and technology, some patients still experience residual tumors after
   radiotherapy or develop local recurrence, with complications and
   mortality being prominent. The inherent radiotherapy resistance of NPC
   cells may be a crucial factor contributing to treatment failure
   [141]^1^, [142]^36. Hence, there is an urgent need to elucidate the
   molecular mechanisms underlying radioresistance and to identify
   potential therapeutic targets. In this study, we identified that the
   m6A level is significantly increased due to the upregulation of
   methyltransferase METTL3 in NPC. This upregulation stimulates the m6A
   modification of SLC7A11 mRNA. The m6A reader IGF2BP2 then directly
   binds to the m6A site on SLC7A11 mRNA and maintains its stability,
   which inhibits ferroptosis in NPC. This process promotes the
   progression of NPC and leads to a worse clinical prognosis.

   Among m6A modification regulatory factors, METTL3 has been thoroughly
   and extensively studied in the progression of various cancer types,
   with multiple studies confirming METTL3 as an independent prognostic
   factor significantly associated with survival rates in NPC patients
   [143]^37^, [144]^38. Previous reports have highlighted the role of
   METTL3 in DNA damage [145]^39^, [146]^40. It is generally believed that
   ionizing radiation can induce DNA double-strand breaks (DSBs), leading
   to cell death in tumor cells. However, DNA DSBs also activate complex
   and highly regulated DNA damage response (DDR) signaling, resulting in
   radiotherapy resistance in cancer cells [147]^41. Currently, the
   relationship and underlying mechanisms between METTL3 and
   radioresistance in NPC remain largely unexplored. In this study, we
   initially analyzed the high expression of METTL3 in NPC tissues. TCGA
   database revealed that METTL3 was significantly upregulated in various
   cancer types. Furthermore, we found that METTL3 is associated with poor
   prognosis in NPC patients, correlating with clinical characteristics.
   In vitro and in vivo assays found that ectopic expression of METTL3
   regulated radioresistance through ferroptosis-related pathways.

   Ferroptosis is a crucial pathway through which radiation induces tumor
   cell death. Radiation-induced reactive oxygen species (ROS) can lead to
   lipid peroxidation of cell membranes, ultimately triggering ferroptosis
   [148]^42. Irradiation also induces adaptive responses involving SLC7A11
   or GPX4, inhibiting radiation-induced ferroptosis and promoting tumor
   cell survival, leading to radioresistance [149]^25. Previous studies
   have reported that METTL3 is associated with lipid peroxidation and
   ferroptosis [150]^27. However, there is no literature reporting the
   relationship among METTL3, radioresistance, and ferroptosis in NPC.
   Functional experiments indicated that inhibiting METTL3 can lead to
   increased lipid peroxidation and eventual ferroptosis in NPC cells,
   with or without irradiation. Overexpression of METTL3 followed by
   treatment with the ferroptosis inducer Erastin promoted the
   radiosensitivity of NPC cells. Thus, METTL3-induced ferroptosis in NPC
   cells may play a crucial role in radiotherapy resistance. We
   investigated the potential mechanisms by which METTL3 regulates
   radiotherapy resistance in NPC. By integrating RNA-seq and m6A-seq
   data, we identified SLC7A11 as a potential downstream target of METTL3
   in NPC. SLC7A11 has been validated as a potential biomarker closely
   associated with tumorigenesis [151]^43^, [152]^44. SLC7A11 promotes
   glutathione (GSH) synthesis and reduces the generation of lipid
   hydroperoxides (L-OOH) to protect tumor cells from oxidative stress and
   ferroptosis [153]^45. Our data revealed a significant positive
   correlation between the expression levels of SLC7A11 and METTL3, with
   high levels of SLC7A11 indicating poor prognosis.

   Moreover, we observed that overexpression of METTL3 leads to
   upregulation of the phosphorylation levels of NFE2L2, GPX4, and AKT.
   Nuclear factor erythroid 2-related factor 2 (NFE2L2) is considered the
   major transcription factor regulating stress-induced transcription of
   SLC7A11. NFE2L2 primarily mediates transcriptional programs in response
   to oxidative stress, regulating SLC7A11 expression and thereby altering
   cellular sensitivity to ferroptosis [154]^46. Glutathione peroxidase 4
   (GPX4) serves as a major oxidative-reduction enzyme utilizing GSH as a
   reducing agent to detoxify lipid peroxides [155]^47. Multiple studies
   have demonstrated that AKT signaling promotes radioresistance in tumor
   cells by modulating DNA damage repair processes [156]^48^-[157]^50. In
   this study, we confirmed a novel regulatory pathway whereby METTL3
   mediates m6A modification-dependent stability of SLC7A11 mRNA via
   IGF2BP2, sustaining SLC7A11 expression in NPC cells. The
   radioresistance and ferroptosis induced by METTL3 could be partially
   reversed by downregulation of SLC7A11. The combined application of the
   ferroptosis inducer Erastin and radiation may serve as a potential
   therapeutic avenue for NPC patients exhibiting METTL3 overexpression.

   In summary, this study elucidates the oncogenic role of METTL3 in the
   radioresistance and progression of NPC. Mechanistically, the
   METTL3/IGF2BP2/SLC7A11 axis fosters radioresistance and inhibits
   ferroptosis, thereby facilitating the progression of NPC.

   The relatively small sample size of the animal experiments in this
   study may limit the statistical power of the results. Some
   heterogeneity exists between groups, potentially due to individual
   biological differences and minor variations in experimental conditions.
   However, we used appropriate statistical methods to control these
   factors, and the results align with existing literature [158]^26^,
   [159]^27^, [160]^51^-[161]^54. Increasing the sample size in future
   studies will be essential to improve the robustness and
   generalizability of the findings. Furthermore, future research should
   emphasize combining the development and clinical application of METTL3
   inhibitors to facilitate their translation into radiosensitizers. In
   personalized treatment, integrating METTL3 and SLC7A11 expression
   levels for patient stratification may offer more precise treatment
   plans for nasopharyngeal carcinoma patients. Additionally, combining
   radiotherapy with ferroptosis inducers like Erastin could become a
   novel therapeutic approach, especially for patients with resistant or
   recurrent disease. Validating the therapeutic potential and prognostic
   value of the METTL3/IGF2BP2/SLC7A11 axis via clinical trials will be
   crucial for translating this research into clinical practice.

Supplementary Material

   Supplementary figure.
   [162]ijbsv21p1837s1.pdf^ (606KB, pdf)

   Supplementary tables.
   [163]ijbsv21p1837s2.xlsx^ (14.9KB, xlsx)

Acknowledgments