The presence of cancer stem cells (CSC), which possess the ability of self-renewal and cancer initiation, is correlated with poor prognosis and drug resistance of breast cancer patients. But the molecular regulatory networks for maintenance of CSC function still remain unclear. Here, we identified that an estrogen-inducible gene FXYD3, whose expression is significantly upregulated in ER⁺ breast CSCs, is a critical player for regulating ER⁺ breast CSC function. FXYD3 amplification is crucial in mediating tamoxifen resistance in ER⁺ breast cancer cells. Interestingly, we also find that stem cell–related transcription factor SOX9 directly promotes FXYD3 expression, and FXYD3 is indispensable for SOX9 nucleus localization, thus forming a positive regulatory feedback loop for FXYD3 amplification and function. In terms of mechanism, FXYD3 interacts with Src and ERα to form an activated complex and triggers Src to transduce nongenomic estrogen signaling for facilitating ER⁺ breast CSCs. Collectively, these results establish a critical role for SOX9/FXYD3/Src axis in boosting nongenomic estrogen signaling and SOX9 nucleus entry, which is required for maintenance of ER⁺ breast CSCs and endocrine resistance. Targeting FXYD3-mediated pathway might be a promising therapeutic strategy for hormone therapy–refractory ER⁺ breast cancer.

SOX9/FXYD3/Src axis is critical for promoting CSC function and tamoxifen resistance in ER⁺ breast cancer.