m6A-modified circFOXA1 promotes tumor immune evasion via enhancing PD-L1 deubiquitination in triple-negative breast cancer

Restoring CD8⁺T cell infiltration and potentiating anti-tumor immune responses in the tumor microenvironment (TME) is critical for developing effective immunotherapies against triple-negative breast cancer(TNBC), while the regulatory role of N⁶-methyladenosine (m⁶A)-modified circular RNAs (circRNAs) in TNBC immune escape remains largely unelucidated. Whole-transcriptome microarray analysis combined with bioinformatics mining was conducted on TNBC tissues to screen circRNAs associated with immune evasion. RNA immunoprecipitation (RIP), RNA pulldown, and methylated RNA immunoprecipitation (MeRIP) assays were performed to verify the m⁶A modification of circFOXA1 and its interaction with gasdermin C (GSDMC). In vitro T cell-mediated tumor cytotoxicity assays and in vivo xenograft models in C57BL/6 mice were used to investigate the functional roles of the circFOXA1/GSDMC axis in TNBC anti-tumor immunity. Luciferase reporter and actinomycin D assays were further applied to clarify the regulatory mechanism of GSDMC on OTUB1 and programmed cell death-ligand 1 (PD-L1) expression. CircFOXA1 was identified as an m⁶A-modified circRNA with high stability and upregulation in TNBC, and its expression was significantly negatively correlated with CD8⁺T cell infiltration in TNBC tissues. Functionally, circFOXA1 induced immunosuppression in a CD8⁺T cell-dependent manner both in vitro and in vivo. Mechanistically, the m⁶A writer METTL14 mediated the m⁶A modification of circFOXA1, and the m⁶A reader YTHDF2 promoted circFOXA1 circularization. CircFOXA1 impaired immune cell-dependent tumor killing by upregulating GSDMC expression, which further enhanced OTUB1 mRNA stability at the post-transcriptional level. OTUB1-mediated deubiquitination subsequently stabilized PD-L1 protein, ultimately inhibiting CD8⁺T cell infiltration and driving TNBC immune escape. This study identifies a novel regulatory axis of m⁶A-modified circFOXA1/GSDMC/OTUB1/PD-L1 in mediating TNBC immune escape, where GSDMC enhances PD-L1 protein stability via OTUB1-dependent deubiquitination. These findings reveal a new molecular mechanism underlying TNBC immune evasion and identify circFOXA1 as a potential therapeutic target to improve the efficacy of anti-PD-1/PD-L1 immunotherapy in TNBC.