The effect of Wilms tumor 1-associated protein on ferroptosis and immune escape in non-small-cell lung cancer by mediating N6-methyladenosine modification of Twist1

Objective: Non-small-cell lung cancer (NSCLC) is one of the most frequently occurring and aggressive cancers globally, but its complex molecular mechanisms remain only partially understood. This study investigated the effects of Wilms tumor 1-associated protein (WTAP) on NSCLC cell ferroptosis and immune escape through N6-methyladenosine (m6A) modification of twist family BHLH transcription factor 1 (Twist1). Material and Methods: Western blot and reverse transcription-quantitative polymerase chain reaction were used to assess Twist1 expression in NSCLC cell lines and its circular structure. Small interfering RNAs targeting Twist1 and WTAP were transfected into H2170 cells, and their effects on cell viability, apoptosis, ferroptosis markers (reactive oxygen species [ROS], malondialdehyde [MDA], and Fe 2+ ), and immune escape were evaluated using cell counting kit-8, flow cytometry, terminal deoxynucleotidyl transferase dUTP Nick-End labeling, ROS staining, and enzyme-linked immunosorbent assay. Co-culture with CD8 + T cells was employed to assess cytotoxicity and exhaustion marker expression (Lymphocyte activation gene-3, Programmed cell death protein 1, and T-cell immunoreceptor with Ig and ITIM domains). Magna methylated RNA immunoprecipitation, photo-activatable ribonucleoside-enhanced cross-linking and immunoprecipitation, and treatment with the methylation inhibitor 3-Deazaneplanocin A (3-DAA) confirmed the m6A modification of Twist1 and WTAP’s regulatory role. Results: Twist1 was dramatically elevated in the NSCLC cell lines ( P < 0.05). Knockdown of Twist1 inhibited NSCLC cell viability; promoted apoptosis; increased ROS, MDA, and Fe 2+ levels; enhanced the cytotoxicity of CD8 + T cells; and reduced the expression of exhaustion markers ( P < 0.05). These effects were partially reversed by the ferroptosis inhibitor Fer-1. WTAP expression increased in the NSCLC cells, and its knockdown markedly reduced the m6A modification and expression of Twist1 ( P < 0.05). Furthermore, overexpression of Twist1 partially reversed the inhibitory effects of WTAP knockdown on NSCLC cell proliferation, ferroptosis, and immune escape ( P < 0.05). Conclusion: Twist1 is highly expressed in NSCLC. It promotes NSCLC progression by enhancing ferroptosis and immune escape. WTAP-mediated m6A methylation plays a critical role in regulating Twist1 expression. The WTAP/Twist1 axis may serve as a potential therapeutic target for NSCLC.