Integrative systems biology, transcriptomic profiling, and experimental validation reveal enterolactone as a...

Triple-negative breast cancer (TNBC) is characterized by aggressive metastatic behaviour and limited therapeutic options. Here, we present a multi-tiered systems biology framework to investigate the anti-metastatic potential of enterolactone (EL), integrating computational, transcriptomic, and experimental approaches in the MDA-MB-231 TNBC model. Network pharmacology identified 78 EL-metastatic TNBC (mTNBC) overlapping targets as potential therapeutic targets of EL against mTNBC, with PPI and GMFA network enrichment uncovering key metastasis-associated pathways including Notch, TGF-β, TNF, and ErbB signaling. Molecular docking and 100 ns molecular dynamics simulations revealed stable binding of EL to several core oncogenic proteins (e.g., EGFR, PARP1, AURKB, SMAD4, CDK4), suggesting poly-pharmacological engagement. Genome-wide transcriptomic profiling of EL-treated cells coupled with GSEA revealed coordinated downregulation of oncogenic programs including E2F, G2/M checkpoint, MYC targets, EMT, and metabolic plasticity, alongside induction of NRF2 signaling and ferroptosis. This study reports, for the first time, transcriptome-wide effects of EL in MDA-MB-231 cells, linking its activity to the repression of stemness, invasion, and immune-evasive traits. Experimental validation via qPCR confirmed EL-mediated suppression of key molecular targets of TGF-β and Notch signaling pathways. EL also impaired cortactin expression and disrupted cytoskeletal remodeling, validated by immunofluorescence and flow cytometry studies, indicating attenuation of invasive machinery. Furthermore, EL reduced metastatic dissemination in a zebrafish xenograft model, reinforcing its in vivo anti-metastatic potential. Together, our integrative study elucidates EL's multitarget mechanism against metastatic TNBC and highlights its translational promise as a systems-level modulator of oncogenic signaling. These findings warrant further in-depth mechanistic investigations to validate EL's therapeutic potential.