**1. Introduction**

Radiotherapy (RT) is a critical factor of primary, adjuvant and palliative treatment for almost all kinds of cancers, including breast cancer. It alone is capable of lowering the 10-year risk of relapse by one half and reducing the 15-year risk of breast-cancer-related death [1]. Although profound benefits are achieved with RT due to its localized treatment, especially for ductal carcinoma and early invasive cancer, local control of the disease fails by 8–15% in radiotherapy-treated patients with advanced invasive tumors due to resistance and relapse of the tumor [2]. The reason for RT failure and the locoregional recurrence of breast cancer is the presence of a subset of radioresistant tumor cells, termed breast cancer stem cells (BCSCs), which show a difference in sensitivities to radiation [3–5]. Standard fractionated doses of radiation are sublethal for BCSCs as they typically evade radiation to develop innate or acquired resistance and establish tumor recurrence and metastasis, leading to the majority of cancer-related deaths. The molecular mechanisms that govern the emergence of aggressive radioresistance in BCSCs are yet unknown.

Low levels of reactive oxygen species (ROS) and enhanced ROS defenses appear to partially contribute to the adaptive tumor radioresistance in BCSCs [5–7]. Thus, the identification of underlying mechanisms and overcoming low ROS levels within BCSCs may be a useful method for improving radiation therapy. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the master regulator of antioxidant defense mechanisms, is a critical regulator of the redox balance. In the cytosol, Nrf2 activity is tightly regulated by two main inhibitors, Keap1 and GSK-3. The Neh2 and Neh6 domains of Nrf2 are the degron.

**Citation:** Kamble, D.; Mahajan, M.; Dhat, R.; Sitasawad, S. Keap1-Nrf2 Pathway Regulates ALDH and Contributes to Radioresistance in Breast Cancer Stem Cells. *Cells* **2021**, *10*, 83. https://doi.org/10.3390/ cells10010083

Received: 11 November 2020 Accepted: 28 December 2020 Published: 6 January 2021

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While the Neh2 domain binds the E3 ligase adapter Keap1 that presents Nrf2 for ubiquitination to a CUL3/RBX1 complex, the Neh6 domain requires previous phosphorylation by GSK-3 to bind the E3 ligase adapter b-TrCP and subsequent ubiquitination by a CUL1/RBX1 complex [8]. Inactivation of either of these regulators due to oxidative or electrophilic stress stabilizes Nrf2, which then translocates to the nucleus and binds to antioxidant response elements (ARE) in the promoter region of target genes by the formation of a heterodimer with small Maf proteins. In the nucleus, Bach1 negatively regulates nuclear Nrf2 activity by competitive-binding with small Maf proteins [9] and thereby inactivates HO1 [10,11]. Previous studies have shown elevated levels of Nrf2 as a critical regulator of chemoresistance in CSC-enriched breast tumors [12,13] and the activation of Nrf2-associated antioxidant genes, such as HO1, NQO1, Prx1, etc., that contribute to radioresistance in other cancer cells [14]. Since BCSCs contain low levels of ROS and enhanced antioxidant defense [5], the role of the Nrf2 pathway in the radioresistance of BCSCs deserves further investigation.

In this study, we observed an increase in ALDH activity, indicative of BCSCs with increased radioresistance, tumorigenesis, reduced apoptosis and the activation of signaling pathways, which promote mesenchymal–epithelial transition (MET) and migration. Additionally, enhanced tumorigenicity was observed after fractionated irradiation. Further investigation of the role of Nrf2 in radioresistance showed that Nrf2 and its associated genes HO1 and NQO1 were significantly increased after irradiation. The shRNA-mediated knockdown of Nrf2 expression led to a decrease in all of the above processes of radioresistance in BCSCs. The mechanism of Nrf2 activation was found to be regulated via Keap1 silencing, as we did not see any change in GSK-3β, as well as in Bach1, the negative regulator of Nrf2. We also did not find any change in the methylation status of the Keap1 promoter; however, a significant increase in the expression of miR200a was observed. This indicates that miR200a could be a possible mechanism of Keap1 silencing. This study provides evidence for the role of Nrf2 and its downstream genes and suggests mechanisms by which the Nrf2/Keap1 pathway induces radioresistance in BCSCs. Overall, the data indicate the contribution of ALDH<sup>+</sup> cell population to radioresistance via the Nrf2-Keap1 axis, suggesting that targeting ALDH<sup>+</sup> BCSC cell population with miR200a could be beneficial but warrants detailed studies.

#### **2. Materials and Methods**
