Antimicrobial peptide WK-13-3D promotes apoptosis, autophagy, and ubiquitination in triple-negative breast cancer via binding immunoglobulin protein (BiP)

Purpose

To elucidate the inhibitory mechanism of antimicrobial peptide WK-13-3D on triple-negative breast cancer (TNBC) by targeting the binding immunoglobulin protein (BiP), a key endoplasmic reticulum (ER) chaperone regulating unfolded protein response and tumor survival.

Methods

TNBC cell lines (MDA-MB-231 and MDA-MB-468) were treated with WK-13-3D to assess proliferation, migration, invasion, and apoptosis. Pull-down assays identified interacting proteins, and Western blotting (WB) analyzed alterations in BiP, PERK, eIF2α, p-eIF2α, Caspase3, Cleaved-Caspase3, Bax, LC3, P62, AKT, p-AKT, mTOR, and p-mTOR. Transmission electron microscopy examined intracellular structures, while qPCR measured BiP mRNA levels. The effects of WK-13-3D on BiP ubiquitination were explored via co-immunoprecipitation (Co-IP). Animal tumor models were used to confirm the inhibitory effects, with BiP and Ki67 (a nuclear proliferation marker indicating actively dividing tumor cells) expression analyzed by immunohistochemistry (IHC).

Results

WK-13-3D inhibited TNBC cell proliferation, migration, and invasion, while promoting apoptosis. Pull-down experiments identified 268 interacting proteins, with BiP being the most frequent. Databases (TIMER and TCGA) showed high BiP expression in breast cancer, associated with poor prognosis. WB assays revealed that WK-13-3D activated ER stress-induced apoptosis and autophagy via BiP. Co-IP demonstrated that WK-13-3D mediated BiP ubiquitination at sites 352 and 547 through K6 and K29 chains. IHC analysis further confirmed decreased Ki67 levels in WK-13-3D-treated tumors, reflecting suppressed proliferative activity. Animal experiments confirmed tumor growth inhibition.

Conclusion

WK-13-3D promotes apoptosis, autophagy and Ubiquitination in TNBC by modulating BiP.