JNK Signaling Promotes Bladder Cancer Immune Escape by Regulating METTL3-Mediated m6A Modification of PD-L1 mRNA.

The RNA N6-methyladenosine (m6A) writer methyltransferase-like 3 (METTL3) is upregulated in many types of cancer and promotes cancer progression by increasing expression of several oncogenes. Therefore, a better understanding of the mechanisms regulating METTL3 expression and the key targets of METTL3 in cancer cells could provide new therapeutic targets. In this study, we found that activated JNK signaling is associated with increased METTL3 expression in bladder cancer. Knockdown of JNK1 or administration of a JNK inhibitor impaired the binding of c-Jun with the METTL3 promoter, thereby decreasing the expression of METTL3 and global RNA m6A levels. Moreover, RNA m6A sequencing indicated enrichment of m6A in the 3'-UTR of immune checkpoint PD-L1 mRNA, which could be recognized by the m6A reader IGF2BP1 to mediate RNA stability and expression levels of PD-L1. Inhibition of JNK signaling suppressed m6A abundance in PD-L1 mRNA, leading to decreased PD-L1 expression. Functionally, METTL3 was essential for bladder cancer cells to resist the cytotoxicity of CD8+ T cells by regulating PD-L1 expression. Additionally, JNK signaling contributed to tumor immune escape in a METTL3-dependent manner both in vitro and in vivo. These data reveal the JNK/METTL3 axis as a mechanism of aberrant m6A modification and immune regulation in bladder cancer. SIGNIFICANCE: The identification of a novel m6A-dependent mechanism underlying immune system evasion by bladder cancer cells reveals JNK signaling as a potential target for bladder cancer immunotherapy.

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

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 METTL3 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 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 m6A modification in self-renewal and tumorigenicity of BCSCs through a novel signaling axis of METTL3-AFF4-SOX2/MYC.