Oxymatrine suppresses stiffness-induced epithelial-mesenchymal transition in breast cancer by targeting the PKN3-GRAF1 signaling axis.

PURPOSE: Increased extracellular matrix stiffness is a critical driver of epithelial-mesenchymal transition (EMT) and metastatic progression in breast cancer; however, effective pharmacological strategies targeting intracellular mechanotransduction remain limited. This study investigated whether oxymatrine, a bioactive alkaloid derived from Sophora flavescens, suppresses stiffness-induced EMT by modulating intracellular mechanical signaling. METHODS AND RESULTS: Using hydrogel substrates with tunable stiffness, we demonstrated that a stiff matrix promoted EMT, cell migration, and invasion in breast cancer cells. Oxymatrine treatment markedly attenuated these effects without cytotoxicity. A fluorescence resonance energy transfer-based tension biosensor revealed that matrix stiffening reduced the intramolecular tension and mobility of the membrane curvature protein GTPase regulator associated with focal adhesion kinase-1 (GRAF1), whereas oxymatrine restored GRAF1 mechanical activity, particularly at cell-cell junctions. Oxymatrine binds to PKN3 with nanomolar affinity and promotes its junctional localization, correlating with GRAF1 phosphorylation and tension. PKN3 phosphorylated GRAF1 at serine 588, enhancing relative intramolecular GRAF1 tension and suppressing EMT. Phosphorylation-deficient GRAF1 mutants abolished the anti-EMT and anti-metastatic effects of oxymatrine in vitro and in vivo. Clinical data analyses further showed that low expression of GRAF1 and PKN3 was associated with poor prognosis in breast cancer patients. CONCLUSIONS: These findings identify PKN3-dependent regulation of GRAF1 tension as a critical mechanical determinant of stiffness-induced EMT, and pharmacological restoration of this tension by oxymatrine effectively suppressed EMT progression, highlighting intracellular mechanoregulation as a promising therapeutic strategy for breast cancer.