Tumor recurrence after radiotherapy due to the presence of breast cancer stem cells (BCSCs) is a clinical challenge, and the mechanism remains unclear. Low levels of ROS and enhanced antioxidant defenses are shown to contribute to increasing radioresistance. However, the role of Nrf2-Keap1-Bach1 signaling in the radioresistance of BCSCs remains elusive. Fractionated radiation increased the percentage of the ALDH-expressing subpopulation and their sphere formation ability, promoted mesenchymal-to-epithelial transition and enhanced radioresistance in BCSCs. Radiation activated Nrf2 via Keap1 silencing and enhanced the tumor-initiating capability of BCSCs. Furthermore, knockdown of Nrf2 suppressed ALDH+ population and stem cell markers, reduced radioresistance by decreasing clonogenicity and blocked the tumorigenic ability in immunocompromised mice. An underlying mechanism of Keap1 silencing could be via miR200a, as we observed a significant increase in its expression, and the promoter methylation of Keap1 or GSK-3β did not change. Our data demonstrate that ALDH+ BCSC population contributes to breast tumor radioresistance via the Nrf2-Keap1 pathway, and targeting this cell population with miR200a could be beneficial but warrants detailed studies. Our results support the notion that Nrf2-Keap1 signaling controls mesenchymal–epithelial plasticity, regulates tumor-initiating ability and promotes the radioresistance of BCSCs.

Hypoxia and oxidative stress significantly contribute to breast cancer (BC) progression. Although hypoxia-inducible factor 1α (Hif-1α) is considered a key effector of the cellular response to hypoxia, nuclear factor erythroid 2–related factor 2 (Nrf2), a master antioxidant transcription factor, is a crucial factor essential for Hif-1α-mediated hypoxic responses. Hence, targeting Nrf2 could provide new treatment strategies for cancer therapy. miRNAs are potential regulators of hypoxia-responsive genes. In a quest to identify novel hypoxia-regulated miRNAs involved in the regulation of Nrf2, we found that miR-140-5p significantly affects the expression of Nrf2 under hypoxia. In our study, miR-140-5p expression is downregulated in BC cells under hypoxic conditions. We have identified Nrf2 as a direct target of miR-140-5p, as confirmed by the luciferase assay. Knockdown of miR-140-5p under normoxic conditions significantly enhanced Nrf2/HO-1 signaling and tumor growth, angiogenesis, migration, and invasion in BC. In contrast, overexpression of miR-140-5p under hypoxic conditions revealed opposite results. Further silencing Nrf2 expression mimicked the miR-140-5p-induced anti-tumor effects. Consistent with the knockdown of miR-140-5p in vitro, mice injected with miR-140-5p-KD cells exhibited dramatically reduced miR-140-5p levels, increased Nrf2 levels, and increased tumor growth. In contrast, tumor growth is potently suppressed in mice injected with miR-140-5p-OE cells. Collectively, the above results demonstrate the importance of the Nrf2/HO-1 axis in cancer progression and, thus, targeting Nrf2 by miR-140-5p could be a better strategy for the treatment of Nrf2-driven breast cancer progression.