Targeting neuropilin-1 attenuates 4-hydroxytamoxifen-induced stemness and sensitizes ERα-re-expressing TNBC to tamoxifen.

Reactivating functional estrogen receptor alpha (ERα) expression represents a promising strategy for tamoxifen (TAM)-based endocrine therapy in triple-negative breast cancer (TNBC). However, TAM and its metabolites may exert potential oncogenic effects, which could compromise TAM efficacy in TNBC. Here, we report that low-dose 4-hydroxytamoxifen (4-OHT), an active metabolite of TAM, enhances stemness in TNBC cells via the Neuropilin-1 (NRP-1)/Calpain-2 (CAPN2)/β-catenin axis. Mechanistically, 4-OHT stimulates CAPN2 activation by promoting its membrane localization, increasing substrate cleavage, and inducing degradation of its endogenous inhibitor calpastatin. Activated CAPN2 suppresses ubiquitin-mediated degradation of β-catenin, leading to elevated β-catenin stability and intracellular accumulation. This enhances β-catenin transcriptional activity and ultimately promotes stemness in TNBC cells. Kaplan-Meier survival analysis showed that high NRP1 expression correlates with poorer overall survival in breast cancer patients undergoing endocrine therapy. Moreover, 4-OHT triggers Ca2+ release and ERK phosphorylation in TNBC cells through the NRP-1/PLC-γ1 pathway, thereby activating CAPN2. Finally, we demonstrate that the NRP-1 inhibitor EG00229 sensitizes ERα-re-expressing TNBC cells to TAM treatment. Our study uncovers a molecular mechanism by which 4-OHT promotes TNBC stemness via the NRP-1/CAPN2/β-catenin signaling cascade. Targeting NRP-1 may serve as a valuable strategy to improve TAM efficacy in ERα-re-expressing TNBC.