Identification of EEF1A1 as a therapeutic target in TNBC: Anticancer action of a novel Penicillide-derived inhibitor through ribosomal protein regulation.

Triple-negative breast cancer (TNBC) is one of the most prevalent and aggressive subtypes of breast cancer worldwide, contributing significantly to cancer-related mortality in women. Due to the absence of effective targeted therapies, there is an urgent need to identify novel molecular targets and bioactive compounds for TNBC treatment. Eukaryotic elongation factor 1A1 (EEF1A1), a key regulator of protein synthesis through its role in peptide chain elongation, has emerged as a potential therapeutic candidate; however, its functional role in TNBC remains poorly understood. In this study, we isolated a series of Penicillide-like compounds from the mycelium of Penicillium sp. NBU2256, designated as compounds 1-5. Using CCK-8 cytotoxicity assays, compound 2 was identified as the most potent inhibitor of TNBC cell viability. Subsequent mechanistic investigations revealed that compound 2 induced apoptosis, triggered cell cycle arrest, and modulated cancer stem cell properties. To elucidate its molecular target, we employed drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA), which identified EEF1A1 as a direct binding partner of compound 2. Network pharmacology analysis further predicted RPL27A and RPLP0 as downstream effectors of EEF1A1 signaling. Functional validation using actinomycin D and cycloheximide treatments demonstrated that compound 2 suppresses RPL27A and RPLP0 expression at the translational level, thereby inhibiting tumor cell invasion and migration and exerting robust antitumor effects. Collectively, these findings provide novel insights into the anticancer mechanisms of EEF1A1-targeting agents and highlight EEF1A1 as a promising therapeutic target for the treatment of TNBC.