RNA demethylase ALKBH5 regulates hypopharyngeal squamous cell carcinoma ferroptosis by posttranscriptionally activating NFE2L2/NRF2 in an m A-IGF2BP2-dependent manner.

Background: Having emerged as the most abundant posttranscriptional internal mRNA modification in eukaryotes, N6-methyladenosine (m A) has attracted tremendous scientific interest in recent years. However, the functional importance of the m A methylation machinery in ferroptosis regulation in hypopharyngeal squamous cell carcinoma (HPSCC) remains unclear.

Methods: We herein performed bioinformatic analysis, cell biological analyses, transcriptome-wide m A sequencing (m A-seq, MeRIP-seq), RNA sequencing (RNA-seq), and RNA immunoprecipitation sequencing (RIP-seq), followed by m A dot blot, MeRIP-qPCR, RIP-qPCR, and dual-luciferase reporter assays.

Results: The results revealed that ALKBH5-mediated m A demethylation led to the posttranscriptional inhibition of NFE2L2/NRF2, which is crucial for the regulation of antioxidant molecules in cells, at two m A 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, m A-mediated NFE2L2/NRF2 stabilization was dependent on the m A reader IGF2BP2. We suggest that ALKBH5 dysregulates NFE2L2/NRF2 expression in HPSCC through an m A-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 m A methylation and its modulators in HPSCC.