*Article* **Keap1-Nrf2 Pathway Regulates ALDH and Contributes to Radioresistance in Breast Cancer Stem Cells**

**Dinisha Kamble, Megharani Mahajan, Rohini Dhat and Sandhya Sitasawad \***

Redox Biology Lab, National Centre for Cell Science (NCCS), Pune 411007, India; dinisha.kamble19@gmail.com (D.K.); meghanandgaon@gmail.com (M.M.); rohini.dhat@nccs.res.in (R.D.) **\*** Correspondence: ssitaswad@nccs.res.in; Tel.: +20-2570-8109

**Abstract:** Tumor recurrence after radiotherapy due to the presence of breast cancer stem cells (BC-SCs) 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<sup>+</sup> 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<sup>+</sup> 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.

**Keywords:** BCSC; ALDH activity; fractionated dose of γ radiation; radioresistance; ROS; Nrf2; Keap1; miR200a; epithelial–mesenchymal transition (EMT)
