Antimicrobial peptide WK-13-3D inhibits triple-negative breast cancer by blocking autophagic flux through inhibiting the AKT/mTOR pathway and targeting binding immunoglobulin protein (BiP).

PURPOSE: To investigate the antitumor efficacy of a novel antimicrobial peptide WK-13-3D, against triple-negative breast cancer (TNBC) and elucidate its mechanism of action via targeting binding immunoglobulin protein BiP and modulating the AKT/mTOR pathway to disrupt autophagy-lysosome homeostasis.

METHODS

TNBC cell lines MDA-MB-231 and MDA-MB-468 were employed as in vitro models. Cell viability and clonogenic potential were assessed using CCK-8 and colony formation assays. Autophagy-related proteins (LC3-II/I, p62) and phosphorylation levels of key AKT/mTOR pathway components were analyzed by Western blotting. Autophagosome formation was visualized via transmission electron microscopy and immunofluorescence. Autophagic flux was monitored using mCherry-GFP-LC3 tandem fluorescent adenovirus. Lysosomal acidification was evaluated by LysoTracker Red and acridine orange (AO) staining. Functional rescue experiments were conducted using the AKT activator SC79. The in vivo antitumor activity of WK-13-3D was validated in a nude mouse xenograft model.

RESULTS

WK-13-3D significantly suppressed TNBC cell viability and clonogenicity. Mechanistically, WK-13-3D concurrently inhibited AKT/mTOR signaling and directly bound to BiP, triggering endoplasmic reticulum (ER) stress. Notably, WK-13-3D treatment led to autophagic flux blockade, evidenced by autophagosome accumulation (increased LC3-II/I ratio) and impaired degradation of the autophagic substrate p62, without compromising lysosomal acidification or hydrolytic function. Activation of the AKT/mTOR pathway partially reversed WK-13-3D-induced autophagy dysregulation, whereas BiP overexpression restored autophagic flux and enhanced lysosomal activity. These dual-targeting effects converged to exert a potent and enhanced antitumor response.

CONCLUSIONS

WK-13-3D disrupts autophagy-lysosome crosstalk in TNBC via coordinated inhibition of AKT/mTOR signaling and BiP-mediated ER stress, representing a promising lead candidate that provides mechanistic insights for TNBC treatment and warrants further preclinical investigation.