Polyphyllin VI induces Forkhead box O3 nuclear liquid-liquid phase separation to promote mitophagy in breast cancer

Mitochondrial dysfunction and defective mitophagy are closely associated with cancer progression. Forkhead box O3 (FOXO3), a key transcription factor, promotes autophagy by activating autophagy-related genes. Polyphyllin VI (PPVI), a Paris polyphylla-derived steroidal saponin, displays potent antitumor activity.

To investigate the antitumor effect of PPVI in breast cancer (BC) and elucidate its underlying mechanism.

Differential gene expression and pathway enrichment were analyzed using RNA sequencing. Mitochondrial autophagic flux was assessed using the mKeima-Red-Mito-7 plasmid. Expression of mitophagy-related markers was evaluated by qPCR and Western blot. FOXO3 was identified as a direct PPVI-binding protein using a biotin-labeled PPVI and mass spectrometry. Their interaction was validated by Microscale thermophoresis. The role of FOXO3 in PPVI-induced mitophagy was studied using gene knockout, gene/protein expression analysis, and functional assays. FOXO3-driven liquid-liquid phase separation (LLPS) and PPVI's regulatory role were analyzed by live-cell imaging, fluorescence recovery after photobleaching, truncation mutants, and molecular dynamics simulations. The in vivo anticancer activity of PPVI was evaluated using zebrafish and xenograft mouse models. Drug toxicity was assessed in zebrafish, while pharmacokinetics and tissue distribution were evaluated in rats.

PPVI induced BNIP3/NIX-mediated mitophagy and mitochondrial dysfunction. It directly bound FOXO3, promoting nuclear translocation and transcriptional activation. FOXO3 underwent LLPS, mainly driven by its C2 region (151-673 aa), and PPVI enhanced the nuclear LLPS of FOXO3. PPVI suppressed tumor growth in vivo but exhibited cardiotoxicity in zebrafish. It primarily accumulated in the liver, had a short half-life, and showed extremely low oral bioavailability.

PPVI exerts its anti-BC activity by directly binding FOXO3, inducing its nuclear condensation with LLPS-like properties, and thereby predominantly engages BNIP3/NIX-mediated mitophagy. These findings reveal that the antitumor effects of PPVI rely on an unrecognized critical role of FOXO3 phase behavior, establishing PPVI as a unique pharmacological probe and lead compound that functions through direct modulation of FOXO3, offering a new direction for therapeutic development.