Breast Cancer‐Derived AZU1 Educates Neutrophils to Promote Tumor Progression and Metastasis Through PAD4‐Dependent Extracellular Trap Formation

ABSTRACT

Neutrophil extracellular traps (NETs) play crucial roles in cancer progression, but their regulatory mechanisms in breast cancer remain poorly understood. We developed a NETs‐related prognostic risk model using TCGA breast cancer data and identified key biomarkers through bioinformatics analysis. AZU1 expression was validated in clinical samples using qRT‐PCR and immunohistochemistry. In vitro experiments investigated AZU1's effects on neutrophil activation and NET formation using recombinant protein treatment, co‐culture assays, and flow cytometry. Mechanistic studies employed phospholipase C (PLC) inhibition and PAD4 knockdown approaches. An orthotopic mouse model validated in vivo findings. Four NETs‐related genes (F2RL2, AZU1, IL33, ELANE) constituted a robust prognostic model with good predictive performance. AZU1 showed significant upregulation in breast cancer tissues and correlated with advanced tumor stages. AZU1 overexpression in breast cancer cells enhanced neutrophil recruitment and NET formation through PLC signaling activation. Recombinant AZU1 dose‐dependently activated neutrophils, promoted NET formation, and enhanced cancer cell invasion via epithelial‐mesenchymal transition induction. PLC inhibition and PAD4 knockdown effectively blocked AZU1‐induced neutrophil activation. In vivo experiments confirmed that AZU1 overexpression accelerated tumor growth and metastasis, while PAD4 inhibition reversed these effects. AZU1 promotes breast cancer progression through PAD4‐dependent NET formation, representing a potential therapeutic target for breast cancer treatment.