The m⁶A Methylation-Regulated AFF4 Promotes Self-Renewal of Bladder Cancer Stem Cells

Abstract

The dynamic N⁶-methyladenosine (m⁶A) modification of mRNA plays a role in regulating gene expression and determining cell fate. However, the functions of m⁶A mRNA modification in bladder cancer stem cells (BCSCs) have not been described. Here, we show that global RNA m⁶A abundance and the expression of m⁶A-forming enzyme METTL3 are higher in BCSCs than those in non-CSCs of bladder cancer (BCa) cells. The depletion of the <i>METTL3</i> inhibited the self-renewal of BCSCs, as evidenced by decreased ALDH activity and sphere-forming ability. Mechanistically, METTL3 regulates the m⁶A modification and thereby the expression of AF4/FMR2 family member 4 (AFF4), knockdown of which phenocopies the <i>METTL3</i> ablation and diminishes the tumor-initiating capability of BCSCs <i>in vivo</i>. AFF4 binds to the promoter regions and sustains the transcription of <i>SOX2</i> and <i>MYC</i> which have critical biological functions in BCSCs. Collectively, our results demonstrate the critical roles of m⁶A modification in self-renewal and tumorigenicity of BCSCs through a novel signaling axis of METTL3-AFF4-SOX2/MYC.