Abstract
BACKGROUND: Disruption of circadian rhythm (DCR) has been connected with breast cancer (BC) susceptibility; whereas it is unclear whether status of key clock genes could be used in predicting BC prognosis, tumor immune microenvironment, and immunotherapy responses. RESULTS: Circadian clock genes demonstrate significant dysregulation in BC, where elevated CLOCK expression emerges as an independent prognostic factor strongly correlated with adverse clinical outcomes. CLOCK-overexpressing BC cells exhibit enhanced proliferative ability and strong resistance to chemotherapy drugs doxorubicin and gemcitabine. High CLOCK expression correlates with reduced CD8(+) T cell infiltration and increased M2 macrophage polarization, consistent with increased immune checkpoint molecule PD-L1 expression in the TCGA BC dataset. Additionally, patients with high CLOCK expression display lower Tumor Immune Dysfunction and Exclusion (TIDE) score. Mechanistically, RNA-sequencing identified suppressed NF-κB, TNF, MAPK pathways, and PD-L1 expression in sh-CLOCK MCF-7 cells. Subsequent in vitro validation demonstrated that CLOCK mediates NF-κB p65 acetylation at K56 site, potentiating its transcriptional activation of PD-L1, thereby facilitating immune evasion in BC. CONCLUSIONS: CLOCK functions as a critical prognostic biomarker in BC by promoting tumor proliferation, chemoresistance, and immune evasion. Mechanistically, CLOCK mediates NF-κB p65 acetylation to enhance PD-L1 transcription, promoting immune evasion in BC.