*3.3. BCSCs with High ALDH<sup>+</sup> Activity Display Radioresistance upon Exposure to Fractionated Irradiation*

Although controversial, previous findings suggest that BCSCs might be less sensitive to irradiation than cancer cells in in vitro assays [4,5]. We used a clonogenic cell survival assay to analyze the relative radioresistance of BCSCs. A single-cell suspension of MCF-7 cells was plated and irradiated with an acute dose (6 Gy) and fractionated doses (2 Gy × 3 days) of γ-rays. Our clonogenic survival assay demonstrated significantly higher radioresistance in MCF-7 and MDA-MB-231 cells and their corresponding mammospheres upon exposure to fractionated doses of radiation compared to controls (Figure 3A,B). Not only did the number of the colonies formed increase significantly after fractionated irradiation but also proliferative capacity, as indicated by Ki67 staining, was higher in these cells (Figure 3C). Ionizing radiation significantly increased the proportion of these CSCs and also showed enhanced proliferation shortly after treatment, further resulting in rapid tumor repopulation [25]. As there was an increase in the proliferation in cancer cells and mammospheres after fractionated irradiation, we further assessed apoptosis and the expression of antiand proapoptotic genes, BCL2 and BAX. Although there was no significant change in the Annexin V<sup>+</sup> apoptotic population in MCF-7 cells and mammospheres after fractionated irradiation compared to their respective controls (Figure 3D), a significant increase in the BCL2/BAX ratio was observed at the protein levels, further supporting radioresistance in these cells (Figure 3E). *Cells* **2021**, *10*, x FOR PEER REVIEW 9 of 23 change in the Annexin V+ apoptotic population in MCF-7 cells and mammospheres after fractionated irradiation compared to their respective controls (Figure 3D), a significant increase in the BCL2/BAX ratio was observed at the protein levels, further supporting radioresistance in these cells (Figure 3E).

**Figure 3.** *Cont*.

**Figure 3.** Fractionated doses of radiation enhance radiation resistance and reduce apoptosis in BCSCs. (**A**) Clonogenic assay was carried out for up to 14 days. The representative images show an increase in the colony formation of MCF-7 cells. (**B**) MDA-MB-231 and their corresponding mammospheres after irradiation with fractionated doses. (**C**) Cell proliferation was measured by analyzing the expression of Ki67 using flow cytometry. (**D**) The dot plots depict Annexin V-FITC and PI staining by flow cytometry. The horizontal (*x*) axis represents Annexin V-FITC and the vertical (*y*) axis represents PI staining. The bar graph represents the percentage of apoptotic cells as Annexin-V-FITC-positive cells (early apoptotic cells) and the percentage of Annexin-V-FITC- and PI-positive cells (late apoptotic cells). (**E**) BCL2 and BAX levels were analyzed by Western blotting. GAPDH was used as loading control. The representative bar graph shows the ratio of BCL2 and BAX. All values are given mean ± SE; \* *p* < 0.05,; fractionated dose irradiation vs. acute irradiation. *3.4. The Emergence of Radioresistance Is Associated with High Migratory Potential and Tumor-***Figure 3.** Fractionated doses of radiation enhance radiation resistance and reduce apoptosis in BCSCs. (**A**) Clonogenic assay was carried out for up to 14 days. The representative images show an increase in the colony formation of MCF-7 cells. (**B**) MDA-MB-231 and their corresponding mammospheres after irradiation with fractionated doses. (**C**) Cell proliferation was measured by analyzing the expression of Ki67 using flow cytometry. (**D**) The dot plots depict Annexin V-FITC and PI staining by flow cytometry. The horizontal (*x*) axis represents Annexin V-FITC and the vertical (*y*) axis represents PI staining. The bar graph represents the percentage of apoptotic cells as Annexin-V-FITC-positive cells (early apoptotic cells) and the percentage of Annexin-V-FITC- and PI-positive cells (late apoptotic cells). (**E**) BCL2 and BAX levels were analyzed by Western blotting. GAPDH was used as loading control. The representative bar graph shows the ratio of BCL2 and BAX. All values are given mean ± SE; \* *p* < 0.05,; fractionated dose irradiation vs. acute irradiation.

*igenicity in Cancer Cells*

#### To analyze whether breast cancer cells irradiated with fractionated doses of radiation have functional characteristics of BCSCs, we examined their cell migration potential in *3.4. The Emergence of Radioresistance Is Associated with High Migratory Potential and Tumorigenicity in Cancer Cells*

vitro and tumorigenic properties in vivo. Compared to the controls and an acute dose, a To analyze whether breast cancer cells irradiated with fractionated doses of radiation have functional characteristics of BCSCs, we examined their cell migration potential in vitro and tumorigenic properties in vivo. Compared to the controls and an acute dose, a significant increase in migration efficiency was observed in cells irradiated with the fractionated doses of radiation in the scratch wound assay (Figure 4A). Further, tumors in mice derived from MCF-7 cells irradiated with fractionated doses of radiation weighed significantly more than tumors derived from nonirradiated or acute-dose-irradiated MCF-7 cells (Figure 4B,C). Consistent with the in vitro results, analysis of the xenograft tumors derived from tumor cells irradiated with fractionated doses also showed enhanced ALDH activity (Figure 4D). Overall, these data demonstrate that fractionated dose exposure enhances migration potential in vitro and increases tumorigenicity by elevating the ALDH<sup>+</sup> population in vivo.

significant increase in migration efficiency was observed in cells irradiated with the fractionated doses of radiation in the scratch wound assay (Figure 4A). Further, tumors in mice derived from MCF-7 cells irradiated with fractionated doses of radiation weighed significantly more than tumors derived from nonirradiated or acute-dose-irradiated MCF-7 cells (Figure 4B,C). Consistent with the in vitro results, analysis of the xenograft tumors derived from tumor cells irradiated with fractionated doses also showed enhanced ALDH activity (Figure 4D). Overall, these data demonstrate that fractionated dose exposure enhances migration potential in vitro and increases tumorigenicity by elevating the ALDH<sup>+</sup>

population in vivo.

**Figure 4.** *Cont*.

**D**

tion.

**Figure 4.** Fractionated doses of radiation enhance cell migration in vitro and tumor xenograft volume in vivo by increasing BCSC population. (**A**) Migration capacity was analyzed by scratch wound assay in confluent monolayers of irradiated MCF-7 cells and was expressed as % of gap closure of irradiated wells. (**B**) The flow diagram illustrates irradiated MCF-7 cells subcutaneously injected in SCID mice (n = 5). Tumors were dissected and dissociated in single cells, and ALDH activity was analyzed. (**C**) The image demonstrates isolated tumors. The bar graph represents tumor weight and volume of the xenograft tumors derived from MCF-7 control and irradiated cells. (**D**) ALDH activity was determined in isolated tumors using flow cytometry. All values are represented as mean ± SE. \* *p* < 0.05; \*\* *p* < 0.01; vs. fractionated dose irradiation.

**Figure 4.** Fractionated doses of radiation enhance cell migration in vitro and tumor xenograft volume in vivo by increasing

#### BCSC population*.* (**A**) Migration capacity was analyzed by scratch wound assay in confluent monolayers of irradiated MCF-7 cells and was expressed as % of gap closure of irradiated wells. (**B**) The flow diagram illustrates irradiated MCF-7 *3.5. Keap1-Nrf2 and not Bach1-Nrf2 Signaling Plays a Role in the Maintenance of Radioresistant ALDH<sup>+</sup> BCSCs*

cells subcutaneously injected in SCID mice (n = 5). Tumors were dissected and dissociated in single cells, and ALDH activity was analyzed. (**C**) The image demonstrates isolated tumors. The bar graph represents tumor weight and volume of the xenograft tumors derived from MCF-7 control and irradiated cells. (**D**) ALDH activity was determined in isolated tumors using flow cytometry. All values are represented as mean ± SE. \* *p* < 0.05; \*\* *p* < 0.01; vs. fractionated dose irradia-3.5. Keap1-Nrf2 and not Bach1-Nrf2 Signaling Plays a Role in the Maintenance of Radioresistant ALDH<sup>+</sup> BCSCs Diehn et al. [5] showed that CSCs in breast tumors contain low ROS levels and enhanced ROS defenses compared to their nontumorigenic progeny, and these differences appear to be critical for maintaining stem cell function, which could contribute to tumor radioresistance. Previous studies have shown the involvement of Nrf2 in chemoresistance in BCSCs [12,13], hence we hypothesized that Nrf2 could also play a significant role in the radioresistance of BCSCs. We first determined the levels of ROS in MCF-7 cells and mam-Diehn et al. [5] showed that CSCs in breast tumors contain low ROS levels and enhanced ROS defenses compared to their nontumorigenic progeny, and these differences appear to be critical for maintaining stem cell function, which could contribute to tumor radioresistance. Previous studies have shown the involvement of Nrf2 in chemoresistance in BCSCs [12,13], hence we hypothesized that Nrf2 could also play a significant role in the radioresistance of BCSCs. We first determined the levels of ROS in MCF-7 cells and mammospheres irradiated with fractionated doses of radiation. We did not see any change in the ROS levels in these cells compared to their respective controls. However, an acute dose of radiation increased the levels of ROS in MCF-7 cells as well as in mammospheres (Figure 5A). Western blot and qRT-PCR analysis revealed that Nrf2 expression (Figure 5B,C), activity (Figure 5D), as well as its targets HO1 and NQO1 (Figure 5E,F), increased significantly when treated with fractionated doses of radiation. We observed a significant decrease in the expression of Keap1, and there was no change in the expression of Bach1, MCF-7 and MDA-MB-231 cells and their corresponding mammospheres irradiated with fractionated doses (Figure 5G,H), indicating that Keap1-mediated Nrf2 degradation is impaired, leading to the stabilization of Nrf2 and its nuclear accumulation [10,12]. The reduced level of ROS in our study could therefore be attributed to the activation of the antioxidant defense mechanism.

> mospheres irradiated with fractionated doses of radiation. We did not see any change in the ROS levels in these cells compared to their respective controls. However, an acute dose of radiation increased the levels of ROS in MCF-7 cells as well as in mammospheres (Figure 5A). Western blot and qRT-PCR analysis revealed that Nrf2 expression (Figure 5B,C), activity (Figure 5D), as well as its targets HO1 and NQO1 (Figure 5E,F), increased significantly when treated with fractionated doses of radiation. We observed a significant decrease in the expression of Keap1, and there was no change in the expression of Bach1, MCF-7 and MDA-MB-231 cells and their corresponding mammospheres irradiated with

tioxidant defense mechanism.

fractionated doses (Figure 5G,H), indicating that Keap1-mediated Nrf2 degradation is impaired, leading to the stabilization of Nrf2 and its nuclear accumulation [10,12]. The reduced level of ROS in our study could therefore be attributed to the activation of the an-

62

**Figure 5.** *Cont*.

ated dose irradiation.

**Figure 5.** Fractionated doses of radiation generate low ROS and upregulate Nrf2 in BCSCs**.** (**A**) The bar graph represents ROS generation, assessed by DCF-DA staining using flow cytometry in MCF-7 cells and the corresponding CSC-enriched spheroids in control and irradiated cells (mean ± SE. \* *p* < 0.05, fractionated-dose-irradiated MCF-7 cells vs. mammospheres). (**B**) Western blot analysis and (**C**) qRT-PCR illustrating the expression of Nrf2 in MCF-7 cell and MDA-MB-231 and their mammospheres. GAPDH served as loading control. (**D**) The bar graph represents the quantification of Nrf2 activity in irradiated MCF-7 cells and mammospheres. (**E**) The blots depict the Nrf2 targets HO1 and NQO1 by Western blotting. (**F**) The bar graph depicts the transcript levels of HO1 and NQO1 in irradiated MCF-7 cells and mammospheres by qRT-PCR. (**G**) Keap1 and Bach1 expression in irradiated MCF-7 cells (upper) and MDA-MB-231 (lower) and their mammospheres using Western blot analysis. (**H**) Transcript levels of Keap1 by qRT-PCR. GAPDH served as loading control. Mean from three independent experiments. All values are given mean ± SE. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001; vs. fraction-**Figure 5.** Fractionated doses of radiation generate low ROS and upregulate Nrf2 in BCSCs. (**A**) The bar graph represents ROS generation, assessed by DCF-DA staining using flow cytometry in MCF-7 cells and the corresponding CSC-enriched spheroids in control and irradiated cells (mean ± SE. \* *p* < 0.05, fractionated-dose-irradiated MCF-7 cells vs. mammospheres). (**B**) Western blot analysis and (**C**) qRT-PCR illustrating the expression of Nrf2 in MCF-7 cell and MDA-MB-231 and their mammospheres. GAPDH served as loading control. (**D**) The bar graph represents the quantification of Nrf2 activity in irradiated MCF-7 cells and mammospheres. (**E**) The blots depict the Nrf2 targets HO1 and NQO1 by Western blotting. (**F**) The bar graph depicts the transcript levels of HO1 and NQO1 in irradiated MCF-7 cells and mammospheres by qRT-PCR. (**G**) Keap1 and Bach1 expression in irradiated MCF-7 cells (upper) and MDA-MB-231 (lower) and their mammospheres using Western blot analysis. (**H**) Transcript levels of Keap1 by qRT-PCR. GAPDH served as loading control. Mean from three independent experiments. All values are given mean ± SE. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001; vs. fractionated dose irradiation.

#### *3.6. Inhibition of Nrf2 Concealed Radioresistance, Tumorigenesis and Induced Apoptosis Via 3.6. Inhibition of Nrf2 Concealed Radioresistance, Tumorigenesis and Induced Apoptosis via Reducing BCSC Population*

*Reducing BCSC Population* To further investigate the role of Nrf2 in radioresistance, Nrf2 was knocked down in MCF-7 cells (shNrf2). These cells showed a 55% reduction in Nrf2 transcripts levels (Figure S2). A 50% reduction in the population of ALDH<sup>+</sup> cells was observed in Nrf2-knockdown mammospheres and MCF-7 cells after fractionated irradiation (Figure 6A). As a phenotypic effect, stable silencing of Nrf2 also resulted in the inhibition of mammosphere formation efficiency by two-fold in MCF-7 cells (Figure 6B). A reduction in the levels of SOX2, KLF4 and NANOG in these knockdown cells after irradiation indicated the role of Nrf2 in the suppression of BCSC population (Figure 6C). Tumorigenicity in SCID mice To further investigate the role of Nrf2 in radioresistance, Nrf2 was knocked down in MCF-7 cells (shNrf2). These cells showed a 55% reduction in Nrf2 transcripts levels (Figure S2). A 50% reduction in the population of ALDH<sup>+</sup> cells was observed in Nrf2 knockdown mammospheres and MCF-7 cells after fractionated irradiation (Figure 6A). As a phenotypic effect, stable silencing of Nrf2 also resulted in the inhibition of mammosphere formation efficiency by two-fold in MCF-7 cells (Figure 6B). A reduction in the levels of SOX2, KLF4 and NANOG in these knockdown cells after irradiation indicated the role of Nrf2 in the suppression of BCSC population (Figure 6C). Tumorigenicity in SCID mice was decreased after injection of the irradiated Nrf2 knockdown cells. A significant decrease in tumor size (Figure 6D) as well as the percentage of ALDH<sup>+</sup> population was observed

in these tumors compared to the corresponding control (Figure 6E). Further, a reduction in clonogenicity (Figure 6F) and a significantly higher number of Annexin-V-/PI-positive cells were observed compared to their respective controls in shNrf2 mammospheres and MCF-7 cells irradiated with fractionated doses (Figure 6G). Thus, these results suggest that Nrf2 plays a crucial role in the acquisition of radiation resistance in BCSCs. positive cells were observed compared to their respective controls in shNrf2 mammospheres and MCF-7 cells irradiated with fractionated doses (Figure 6G). Thus, these results suggest that Nrf2 plays a crucial role in the acquisition of radiation resistance in BCSCs.

was decreased after injection of the irradiated Nrf2 knockdown cells. A significant decrease in tumor size (Figure 6D) as well as the percentage of ALDH<sup>+</sup> population was observed in these tumors compared to the corresponding control (Figure 6E). Further, a reduction in clonogenicity (Figure 6F) and a significantly higher number of Annexin-V-/PI-

*Cells* **2021**, *10*, x FOR PEER REVIEW 16 of 23

**Figure 6.** *Cont*.

**Figure 6.** Inhibition of Nrf2 radiosensitizes breast cancer cells by inducing apoptosis and suppressing BCSC population after radiation treatment. (**A**) BCSC population measured by ALDH activity using flow cytometry in shNrf2 MCF-7 cells and mammospheres. (**B**) Phase-contrast images depict the effect of fractionated and acute doses of radiation on sphere formation in shNrf2 MCF-7 cells. The bar graph represents mammosphere formation efficiency for the same. (**C**) Expression of stem cell markers, i.e., SOX2, KLF4 and NANOG, was analyzed in shNrf2 MCF-7 cells and mammospheres by Western blotting, GAPDH is used as loading control. All values are given as the mean ± SE, \*\*\* *p* < 0.001 vs. fractionateddose-irradiated shNrf2 cells. (**D**) The image demonstrates isolated tumors of the xenograft derived from shNrf2 MCF-7 control and irradiated cells. (**E**) ALDH activity was measured in shNrf2-derived tumors. (**F**) The representative images show a decrease in the colony formation of shNrf2 MCF-7 cells and mammospheres upon fractionated dose radiation treatment. (**G**) The bar graphs depict the percentage of apoptotic cells in shNrf2 MCF-7 cells and mammospheres. All values are given as the mean ± SE, \* *p* < 0.05, \*\* *p* < 0.01; vs. fractionated dose irradiation shNrf2 cells. All images are representative of three independent experiments. **Figure 6.** Inhibition of Nrf2 radiosensitizes breast cancer cells by inducing apoptosis and suppressing BCSC population after radiation treatment. (**A**) BCSC population measured by ALDH activity using flow cytometry in shNrf2 MCF-7 cells and mammospheres. (**B**) Phase-contrast images depict the effect of fractionated and acute doses of radiation on sphere formation in shNrf2 MCF-7 cells. The bar graph represents mammosphere formation efficiency for the same. (**C**) Expression of stem cell markers, i.e., SOX2, KLF4 and NANOG, was analyzed in shNrf2 MCF-7 cells and mammospheres by Western blotting, GAPDH is used as loading control. All values are given as the mean ± SE, \*\*\* *p* < 0.001 vs. fractionated-dose-irradiated shNrf2 cells. (**D**) The image demonstrates isolated tumors of the xenograft derived from shNrf2 MCF-7 control and irradiated cells. (**E**) ALDH activity was measured in shNrf2-derived tumors. (**F**) The representative images show a decrease in the colony formation of shNrf2 MCF-7 cells and mammospheres upon fractionated dose radiation treatment. (**G**) The bar graphs depict the percentage of apoptotic cells in shNrf2 MCF-7 cells and mammospheres. All values are given as the mean ± SE, \* *p* < 0.05, \*\* *p* < 0.01; vs. fractionated dose irradiation shNrf2 cells. All images are representative of three independent experiments.

#### *3.7. miR200a and not Promoter Methylation of Keap1 is Involved in Radioresistance of BCSC* Since we observed a significant decrease in the expression of Keap1 at mRNA and *3.7. miR200a and not Promoter Methylation of Keap1 is Involved in Radioresistance of BCSC*

protein levels, we further investigated its regulation at the epigenetic level, especially the methylation status of the Keap1 promoter by bisulfite sequencing [26]. We did not observe any change in the methylation status of the CpGs region in the Keap1 promoter, indicating that Keap1 promoter methylation may not be the key event in Nrf2 stabilization (Figure 7A,B). We next examined the role of the miR-200 family as it targets a conserved region in the Keap1 3′-UTR [27]. We observed no change in the expression of miR-141 but a significant increase in the expression of miR-200a, 1.4-fold in mammospheres and 1.85-fold in MCF-7 cells irradiated with the fractionated dose of radiation by RT-PCR (Figure 7C,D). Collectively, these results indicate that Keap1 downregulation could be due to increased miR200a; however, more studies are required to confirm the role of miR200a in this context. Since we observed a significant decrease in the expression of Keap1 at mRNA and protein levels, we further investigated its regulation at the epigenetic level, especially the methylation status of the Keap1 promoter by bisulfite sequencing [26]. We did not observe any change in the methylation status of the CpGs region in the Keap1 promoter, indicating that Keap1 promoter methylation may not be the key event in Nrf2 stabilization (Figure 7A,B). We next examined the role of the miR-200 family as it targets a conserved region in the Keap1 3 0 -UTR [27]. We observed no change in the expression of miR-141 but a significant increase in the expression of miR-200a, 1.4-fold in mammospheres and 1.85-fold in MCF-7 cells irradiated with the fractionated dose of radiation by RT-PCR (Figure 7C,D). Collectively, these results indicate that Keap1 downregulation could be due to increased miR200a; however, more studies are required to confirm the role of miR200a in this context.

**Figure 7.** Promoter methylation and the role of miRNA200 in Keap1 regulation. (**A**) Primers' design for bisulfite sequencing. The original genomic sequence of the Keap1 promoter region is shown. The Keap1 promoter contains 13 CpGs sites. (**B**) Keap1 promoter methylation by Quantification Tool for Methylation Analysis (QUMA) analysis: ○, unmethylated CpGs; ●, methylated CpGs. (**C**) Predicted binding sites between miR200a and Keap1 at 3′ UTR. (**D**) miR200a expression **Figure 7.** Promoter methylation and the role of miRNA200 in Keap1 regulation. (**A**) Primers' design for bisulfite sequencing. The original genomic sequence of the Keap1 promoter region is shown. The Keap1 promoter contains 13 CpGs sites. (**B**) Keap1 promoter methylation by Quantification Tool for Methylation Analysis (QUMA) analysis: #, unmethylated CpGs; •, methylated CpGs. (**C**) Predicted binding sites between miR200a and Keap1 at 3<sup>0</sup> UTR. (**D**) miR200a expression level in control and fractionated-dose-irradiated MCF-7 cells and mammospheres. All images are representative of three independent experiments. All values are given mean ± SE; \* *p* < 0.05; vs. fractionated dose irradiation.

level in control and fractionated-dose-irradiated MCF-7 cells and mammospheres. All images are representative of three

#### independent experiments. All values are given mean ± SE; \* *p* < 0.05; vs. fractionated dose irradiation. **4. Discussion**

**4. Discussion** Radiation can induce cancer cell death by generating ROS and DNA damage; however, it is inefficient in targeting CSCs, which are largely responsible for therapy resistance, tumorigenesis and tumor recurrence [28–30]. Our study demonstrates that fractionated doses of radiation enhanced the E-BCSC marker ALDH<sup>+</sup> and transcription factors of embryonic stem cells in BCSC-enriched mammospheres, indicating the E-BCSC phenotype, which is proliferative in nature. BCSC plasticity plays a crucial role in therapy resistance. BCSCs exhibit plasticity, which transitions between quiescent mesenchymal- (M-BCSCs) and proliferative epithelial-like (E-BCSCs) states [31]. An increase in E-BCSCs such as ALDH+ population and E-cadherin, indicative of MET, and a decrease in M-BCSCs such as CD44<sup>+</sup> /24<sup>−</sup> population, the mesenchymal markers Vimentin, SNAIL and SLUG, demonstrated that fractionated doses of radiation increase the epithelial type of BCSCs [24,31,32]. Thus, these results support the notion that BCSC markers are not restricted to a particular population but change according to their plasticity based on the therapy. Hence, plasticity from M- BCSCs to E-BCSCs contributes to radioresistance. Since NANOG, SOX2 and KLF4 are essential for converting tumor cells into aggressive stem-Radiation can induce cancer cell death by generating ROS and DNA damage; however, it is inefficient in targeting CSCs, which are largely responsible for therapy resistance, tumorigenesis and tumor recurrence [28–30]. Our study demonstrates that fractionated doses of radiation enhanced the E-BCSC marker ALDH<sup>+</sup> and transcription factors of embryonic stem cells in BCSC-enriched mammospheres, indicating the E-BCSC phenotype, which is proliferative in nature. BCSC plasticity plays a crucial role in therapy resistance. BCSCs exhibit plasticity, which transitions between quiescent mesenchymal- (M-BCSCs) and proliferative epithelial-like (E-BCSCs) states [31]. An increase in E-BCSCs such as ALDH+ population and E-cadherin, indicative of MET, and a decrease in M-BCSCs such as CD44+/24<sup>−</sup> population, the mesenchymal markers Vimentin, SNAIL and SLUG, demonstrated that fractionated doses of radiation increase the epithelial type of BCSCs [24,31,32]. Thus, these results support the notion that BCSC markers are not restricted to a particular population but change according to their plasticity based on the therapy. Hence, plasticity from M- BCSCs to E-BCSCs contributes to radioresistance. Since NANOG, SOX2 and KLF4 are essential for converting tumor cells into aggressive stem-like cells, an increase in the expression of these markers in our study after irradiation further supports the increased cancer stem cell population.

Emerging evidence indicates that Nrf2 plays a crucial role in CSC survival and resistance [33]. It is shown to be involved in chemotherapeutic drug resistance due to enhanced antioxidant capacity and detoxification of anticancer agents [14,34,35]. However, the involvement of the Nrf2-Keap1 axis in radioresistance of BCSCs is poorly understood. A strong association between low levels of ROS and enhanced antioxidant defense in BCSC radioresistance reported by Diehn et al. [5] prompted us to further investigate the role of Nrf2. Enhanced expression of Nrf2 and its downstream genes HO1 and NQO1 after irradiation in breast cancer cells and their corresponding mammospheres ascertains the involvement of Nrf2 in radioresistance. A recent report has shown that Nrf2 enhances ALDH<sup>+</sup> E-BCSCs [24]. This supports our results, as we have observed a decrease in the ALDH<sup>+</sup> E-BCSCs after Nrf2 inhibition. A decrease in embryonic stem cell markers, colony and sphere formation ability and reduced tumorigenicity after Nrf2 knockdown further indicate that Nrf2 is involved in the reprogramming process, and Nrf2 signaling is an important target for radiation resistance of BCSCs.

In the current study, Nrf2 appears to be regulated by Keap1 as we observed a decrease in the Keap1 levels with no change in the expression of either GSK-3β (Figure S3) [36] or Bach1. Additionally, as Bach1 binds to HO1 [10,11], an increase in the levels of HO1 in our study further confirms that Bach1 does not play a role in the regulation of Nrf2. Loss of Keap1 function is shown to mediate Nrf2 stabilization and is often associated with reduced drug sensitivity in several cancers [37–39]. A reduction in Keap1 expression with a concomitant increase in the expression of Nrf2 and its downstream targets HO1 and NQO1 clearly demonstrates the role played by Keap1 in Nrf2 regulation in the facilitation of acquired radioresistance. Hence, we tried to understand the mechanism of Keap1 regulation in this study.

Besides mutations through cysteine residues, epigenetic mechanisms, particularly the promoter hypermethylation [26], and miRNAs are the main regulators of Keap1. We did not see any change in the promoter methylation status of Keap1 after irradiation, which suggested that irradiation may regulate Keap1 post-transcriptionally rather than epigenetically. Hence, we further studied the role of the miR200 family as it is known to be involved in the regulation of Keap1. A significant increase in the transcript levels of miR200a indicates its role in the regulation of Keap1 in the radioresistance of BCSCs. Furthermore, reports from other studies have shown that miR200a suppresses the expression of transcriptional factors ZEB1/2 and inhibits the transition from the epithelial-to-mesenchymal phenotype [40]. This further strengthens and supports our studies where miR200a could be responsible for the inhibition of Keap1 as well as EMT in BCSC-enriched mammospheres.

In conclusion, the current study provides interesting insights into the mechanism by which fractionated doses of radiation increases radioresistance in the BCSC population. Our results indicate the enrichment of the E-BCSC phenotype. The regulation of Nrf2 in irradiated conditions occurs via the downregulation of Keap1 and not by GSK3β or Bach1. We provide mechanistic insight into the regulation of Keap1, possibly via posttranscriptional modification through miR200a and not via promoter methylation. Although the current study is limited to only the higher expression of miR200a, and given its potential for therapeutic purposes, additional mechanistic studies regarding its role in Keap1 inhibition and thus radioresistance is highly warranted. Nevertheless, alteration in the Nrf2-Keap1 pathway establishes relationships between radioresistance and BCSCs.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2073-4 409/10/1/83/s1, Figure S1: Characterization of mammospheres. Figure S2: Nrf2 expression in Nrf2 knockdown cells. Figure S3: p-GSK3β levels in fractionated-dose-irradiated MCF-7 cells and mammospheres. Table S1: List of primers.

**Author Contributions:** D.K.: conceptualization, methodology, resources, formal analysis, validation and original draft preparation; M.M.: contribution in shNrf2 cell line generation; R.D.: contribution in promoter methylation studies; S.S.: investigation, conceptualization, formal analysis, visualization, validation, writing—original draft preparation and writing, review and editing, supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by grants from: BRNS, D.A.E. (Department of Atomic Energy). M.M. and R.D. are SRF of Council of Scientific and Industrial Research (CSIR), Govt. of India.

**Institutional Review Board Statement:** All animal experiments were carried out in accordance with the procedures and guidelines of the Institutional Animal Ethics Committee (NCCS) for animal experiments and approved by the Institute Animal Ethical Committee (IAEC) for Animal Experiments at the National Centre for Cell Science, S.P Pune University, Pune, India (IAEC/2016/B-264).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We sincerely acknowledge B.S Patro, Bio-Organic Division, BARC, Mumbai, India for his technical support. We wish to thank Sarojini Singh for proof reading the manuscript. We also acknowledged Director (NCCS) for providing all institutional facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.
