RNA demethylase ALKBH5 regulates hypopharyngeal squamous cell carcinoma ferroptosis by posttranscriptionally activating NFE2L2/NRF2 in an m6A‐IGF2BP2‐dependent manner

Background Having emerged as the most abundant posttranscriptional internal mRNA modification in eukaryotes, N6‐methyladenosine (m6A) has attracted tremendous scientific interest in recent years. However, the functional importance of the m6A methylation machinery in ferroptosis regulation in hypopharyngeal squamous cell carcinoma (HPSCC) remains unclear. Methods We herein performed bioinformatic analysis, cell biological analyses, transcriptome‐wide m6A sequencing (m6A‐seq, MeRIP‐seq), RNA sequencing (RNA‐seq), and RNA immunoprecipitation sequencing (RIP‐seq), followed by m6A dot blot, MeRIP‐qPCR, RIP‐qPCR, and dual‐luciferase reporter assays. Results The results revealed that ALKBH5‐mediated m6A demethylation led to the posttranscriptional inhibition of NFE2L2/NRF2, which is crucial for the regulation of antioxidant molecules in cells, at two m6A residues in the 3′‐UTR. Knocking down ALKBH5 subsequently increased the expression of NFE2L2/NRF2 and increased the resistance of HPSCC cells to ferroptosis. In addition, m6A‐mediated NFE2L2/NRF2 stabilization was dependent on the m6A reader IGF2BP2. We suggest that ALKBH5 dysregulates NFE2L2/NRF2 expression in HPSCC through an m6A‐IGF2BP2‐dependent mechanism. Conclusion Together, these results have revealed an association between the ALKBH5‐NFE2L2/NRF2 axis and ferroptosis, providing insight into the functional importance of reversible mRNA m6A methylation and its modulators in HPSCC. RSL3‐induced head and neck cancer cell ferroptosis is closely correlated with the global m6A abundance. (a, b) Cell viability (a) and LDH release assays (b) of HNSCC cell lines that had been exposed to different concentrations of RSL3 for 24 h. (c) Measurement of cellular lipid ROS levels by BODIPY C11 staining after exposure to RSL3 for 24 h. (d) Western blot analysis of Detroit 562 and SCC25 cells exposed to various concentrations of RSL3 for 24 h. β‐Actin was used as the loading control. (e, f) Representative images of the m6A dot blot assay (e) and quantitation of m6A (f) showing the global m6A abundance in HNSCC cells. MB, methylene blue staining (as a loading control). (g) Heat map of HNSCC cells with hierarchical clustering of ferroptosis‐sensitive and ferroptosis‐insensitive cells; each value was normalized to the corresponding mean value.