Nuclear PD-L1 drives IFN-γ-promoted lung metastasis of triple-negative breast cancer via POLR2A-mediated transcriptional activation of LY6E.

BACKGROUND: ​​Triple-negative breast cancer (TNBC) patients exhibiting high PD-L1 expression demonstrate poor responses to anti-PD-L1 therapy and aggressive lung metastasis. The paradoxical role of PD-L1 beyond its immune checkpoint function and the impact of interferon-γ-secreted during immunotherapy-on metastasis remain poorly understood. METHODS: Integrated reanalysis of single-cell RNA sequencing (scRNA-seq) data from TNBC lung metastases identified enriched signaling pathways. IFN-γ function was assessed using murine and human TNBC cell lines, employing in vitro assays and in vivo modeling in both immunocompetent and immunodeficient mice. CRISPR/Cas9-mediated PD-L1 ablation, pharmacological inhibitors, RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), co-immunoprecipitation (Co-IP), bioinformatics analyses, and in vivo metastasis assays were utilized to dissect underlying mechanisms. RESULTS: scRNA-seq revealed significant enrichment of IFN-γ signaling within a distinct metastatic TNBC cluster. IFN-γ pretreatment potently enhanced the lung metastatic capacity of TNBC cells in both immunocompetent and immunodeficient murine models. CRISPR/Cas9-mediated PD-L1 ablation abolished IFN-γ-driven metastasis without affecting proliferation, indicating an immune checkpoint-independent mechanism. Mechanistically, IFN-γ facilitated HDAC2-mediated deacetylation of PD-L1, promoting its nuclear translocation. RNA-seq identified lymphocyte antigen 6 complex locus E (LY6E) as a key downstream effector, with expression correlating with PD-L1 in TNBC patient samples. Nuclear PD-L1 bound to the RNA polymerase II subunit POLR2A to form a transcriptional complex that directly activated LY6E expression, thereby driving metastatic dissemination. CONCLUSION: Our findings unveil a novel IFN-γ-nuclear PD-L1/POLR2A-LY6E signaling axis critical for TNBC lung metastasis. This immune-independent mechanism, driven by nuclear PD-L1 transcriptional activity, provides a mechanistic basis for the limited efficacy of anti-PD-L1 antibodies against metastasis and nominates nuclear PD-L1 complexes and LY6E as potential therapeutic targets to overcome metastatic resistance in TNBC.