The m(6)A Methylation-Regulated AFF4 Promotes Self-Renewal of Bladder Cancer Stem Cells

The dynamic N(6)-methyladenosine (m(6)A) modification of mRNA plays a role in regulating gene expression and determining cell fate. However, the functions of m(6)A mRNA modification in bladder cancer stem cells (BCSCs) have not been described. Here, we show that global RNA m(6)A abundance and the expression of m(6)A-forming enzyme METTL3 are higher in BCSCs than those in non-CSCs of bladder cancer (BCa) cells. The depletion of the METTL3 inhibited the self-renewal of BCSCs, as evidenced by decreased ALDH activity and sphere-forming ability. Mechanistically, METTL3 regulates the m(6)A modification and thereby the expression of AF4/FMR2 family member 4 (AFF4), knockdown of which phenocopies the METTL3 ablation and diminishes the tumor-initiating capability of BCSCs in vivo. AFF4 binds to the promoter regions and sustains the transcription of SOX2 and MYC which have critical biological functions in BCSCs. Collectively, our results demonstrate the critical roles of m(6)A modification in self-renewal and tumorigenicity of BCSCs through a novel signaling axis of METTL3-AFF4-SOX2/MYC.