m(6)A mRNA Methylation Regulates Ferroptosis in HPSCC by Targeting NFE2L2/NRF2

Background: Emerging as the most abundant posttranscriptional internal mRNA modification in eukaryotes, N6-methyladenosine (m6A) modification has gathered tremendous scientific interest in recent years. However, no study addresses the role of m6A modification in ferroptosis. Here, we showed that m6A modifications are decreased in RSL3-induced ferroptosis in hypopharyngeal squamous cell carcinoma (HPSCC). We found that AlkB homolog 5 (ALKBH5), one of the m6A demethylases, is the primary factor involved in aberrant m6A modification. Methods: Bioinformatics analysis, sample analysis, cell biological analyses and transcriptome sequencing were performed to evaluate the correlation between m6A modification and ferroptosis as well as molecular mechanism of ALKBH5 function. Transcriptome-wide m6A-seq and RIP-seq data and following m6A dot blot, MeRIP-qPCR, RIP-qPCR and dual luciferase reporter assays were mapped to screen and validate the candidate targets of ALKBH5. Results: ALKBH5-knockdown impaired ferroptotic cell death in HPSCC. However, overexpression of ALKBH5 has an opposite effect, suggesting that ALKBH5 is a positive regulator of ferroptosis. Mechanistically, ALKBH5-mediated m6A demethylation led to a post-transcriptional inhibition of NFE2L2/NRF2, the central player in the regulation of antioxidant molecules in cells, at two m6A residues in the 3ʹ-UTR. Therefore, knocking down ALKBH5 subsequently increases the expression levels of NFE2L2/NRF2 and increased cell resistance to ferroptosis. In addition, m6A -mediated NFE2L2/NRF2 stabilization relied on the m6A reader IGF2BP2. Conclusion: ALKBH5 functions as a tumor suppresser through ferroptosis in HPSCC. ALKBH5 destabilizes NFE2L2/NRF2 expression in HPSCC through an m6A-IGF2BP2-dependent mechanism. Together, our work uncovers a critical link between ALKBH5-NFE2L2/NRF2 and ferroptosis, providing insight into the functional importance of the reversible mRNA m6A methylation and its modulators in HPSCC. Funding Information: This work was supported by the Natural Science Foundation of Zhejiang Province (No. LY21H160031), National Natural Science Foundation of China (No. 62071415, No.81903160), Medical Health Science and Technology Project of Zhejiang Provincial Health Commission Grants (No. 2019336033 and No. 2020367813). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: According to the protocol approved by the Ethical Review Committee of the Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University. Patient tissue samples were obtained with informed consent. The in vivo assay using nude mice was approved by the Ethics Review Committee of Zhejiang University college of Medicine. All animal studies were carried out in accordance with Institute of Laboratory Animal Resources guidelines and approved by the University Committee on the Use and Care of Animals at the Zhejiang University.