Systems Biology and Experimental Validation Enable Discovery of MMP9-Centered Networks, Anticancer Activity, and...

Matrix metalloproteinase-9 (MMP9) is involved in extracellular matrix remodeling, inflammation, and metastasis, and its overexpression is associated with poor prognosis in lung cancer. However, the systems-level effects of MMP9 inhibition remain incompletely understood. We combined systems biology with experimental validation to characterize MMP9-centered signaling in non-small cell lung cancer (NSCLC) and identify downstream pharmacodynamic biomarkers. Network and pathway analyses revealed extracellular matrix- and inflammation-related interaction modules and prioritized an MMP9-associated gene panel. Synthesized compounds were evaluated using purified enzyme assays and A549 NSCLC cells. At 50 μM, compounds M34 and M33 showed the strongest inhibition of MMP9 activity (65.01% and 61.32%, respectively). Seven compounds (M1, M2, M8, M9, M10, M27, and M34) demonstrated antiproliferative activity at 72 h (IC₅₀ = 40-115 μM), suppressed migration and colony formation, induced apoptosis, and reduced MMP9 protein expression. Quantitative PCR confirmed coordinated downregulation of MMP9, VEGFA, APP, and ETV4, with upregulation of CDH1, COL5A1, COL6A2, CCL2, and CCL17. Enrichment analysis linked these changes to inflammatory signaling, immune activation, and extracellular matrix remodeling. These findings establish an experimentally supported eight-gene pharmacodynamic biomarker signature and support biomarker-guided development of MMP9-targeted strategies in NSCLC.