Nrf2 promotes breast cancer cell migration via up‐regulation of G6PD/HIF‐1α/Notch1 axis

Abstract Abnormal metabolism of tumour cells is closely related to the occurrence and development of breast cancer, during which the expression of NF‐E2‐related factor 2 (Nrf2) is of great significance. Metastatic breast cancer is one of the most common causes of cancer death worldwide; however, the molecular mechanism underlying breast cancer metastasis remains unknown. In this study, we found that the overexpression of Nrf2 promoted proliferation and migration of breast cancers cells. Inhibition of Nrf2 and overexpression of Kelch‐like ECH‐associated protein 1 (Keap1) reduced the expression of glucose‐6‐phosphate dehydrogenase (G6PD) and transketolase of pentose phosphate pathway, and overexpression of Nrf2 and knockdown of Keap1 had opposite effects. Our results further showed that the overexpression of Nrf2 promoted the expression of G6PD and Hypoxia‐inducing factor 1α (HIF‐1α) in MCF‐7 and MDA‐MB‐231 cells. Overexpression of Nrf2 up‐regulated the expression of Notch1 via G6PD/HIF‐1α pathway. Notch signalling pathway affected the proliferation of breast cancer by affecting its downstream gene HES‐1, and regulated the migration of breast cancer cells by affecting the expression of EMT pathway. The results suggest that Nrf2 is a potential molecular target for the treatment of breast cancer and targeting Notch1 signalling pathway may provide a promising strategy for the treatment of Nrf2‐driven breast cancer metastasis.

antioxidant and cytoprotective systems. 4 The antioxidant response is principally mediated by the transcription factor Nrf2, which induces the transcriptional activation of several genes involved in glutathione (GSH) synthesis, chemoresistance and cytoprotection. 5 In recent years, accumulating evidence implies the importance of Nrf2 deregulation in tumourigenesis. 5 Despite the recent progress in the characterization of Nrf2 transcription factors, biological functions of Nrf2 remain to be explored. Several types of cancer cells display a large amount of reactive oxygen species (ROS), due to an aberrant metabolism, mitochondrial dysfunction or activation of oncogenes. 6 Under physiological conditions, Nrf2 localizes in the cytoplasm where it is bound by Kelch-like ECH-associated protein 1 (Keap1).
Kelch-like ECH-associated protein 1 forms a complex with Cul3 and Rbx1, and this E3 ubiquitin ligase complex can bind and ubiquitinate Nrf2, resulting in Nrf2 proteasomal degradation. 7 The stabilized Nrf2 accumulates in nuclei, heterodimerizes with small Maf proteins and activates target genes for cytoprotection through the antioxidant response element (ARE)/electrophile response element (EpRE). 8 The function of Nrf2 in chemoresistance has been demonstrated in diverse types of cancers, including cisplatin resistant bladder cancers. 9 The migration and invasion of tumour cells are crucial in cancer metastasis. 10 Warburg effect is an important expression of tumour cell metabolic reprogramming. However, tumour metabolic reprogramming occurs in many metabolic pathways, including glycolysis, the pentose phosphate pathway (PPP) and the Krebs cycle process. 11 The PPP is irreplaceable in the rapid proliferation of tumour cells regarding the provision of raw materials for macromolecular biosynthesis and maintenance of cellular redox status. 12 Recent studies have suggested that PPP is raised in many tumour cells, but maintenance of high hyperplastic in tumour cells through the PPP remains unanswered. Notch signalling pathway is a classical pathway. Recent studies show that Notch pathway was involved in cell proliferation, differentiation, migration and invasion. 13 It should be noted that Nrf2 is related to Notch pathway. 14 The synergy of Nrf2 and Notch pathway promotes survival rate of tumour cells, differentiation, invasion and metastasis in the condition of abnormal expression of Nrf2 and Notch pathway. 15 It is possible that Nrf2 can adjust the Notch pathway through affecting the PPP and leads to a change in breast cancer cell proliferation and migration, and need to explore its mechanism.
In this study, we demonstrate that Nrf2 leads to increased proliferation, migration, invasion in breast cancer cells. We found, for the first time, a novel function of Nrf2 in the Notch1 signalling pathway via PPP. Modulation of glucose-6-phosphate dehydrogenase (G6PD)/ Hypoxia inducing factor 1α (HIF-1α) expression by Nrf2 is therefore involved in the Notch1 pathway-mediated regulation proliferation, migration, invasion of breast cancer.

| Western blotting
Cells were collected with RIPA lysis buffer that comprised protease inhibitor cocktail PMSF at a final concentration of 0.1%. Cells were placed for 30 minutes on the ice and collected at 12 000 g for 20 minutes at 4°C. Bicinchoninic acid protein assay kit to detect the contents of protein. Proteins were loaded onto a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane; immunoblotting was performed with primary and secondary antibodies. Proteins were visualized with the Canon Chemiluminescence imaging system. 17

| Cell proliferation assay
Cell proliferation assay was completed by CellTiter 96™ AQueous One Solution Cell Proliferation Assay (MTS) Kit. 18 Cells were inoculated at 5 × 10 3 per well into 96-well plates at 37°C, 5% CO 2 . Appropriate concentration of drug was added into the wells. After 12, 24, 36, 48, 60 and 72 hours, cells were added with 10 μL MTS Reagent and 90 μL medium for 1 hour at 37°C. Then absorbance of each well was tested at 490 nm using a microplate spectrophotometer.

| Wound healing assay
Cells were cultured in six-well plates and incubated in culture medium at 37°C in a 5% CO 2 incubator. A wound area was made with a 200 μL pipette tip following formation of a confluent monolayer.
Cells were then incubated in the same condition for 24 hours. Images were captured at 0 and 24 hours. All experiments were performed in triplicates. 16

| Transwell cell invasion assays
Transwell cell invasion assays were performed as previously described. 10 In brief, an appropriate number of cells was seeded in the upper insert chamber with serum-free medium. DMEM (500 μL) containing 10% FBS was plated at the bottom chamber. After 24 hours incubation, cells were rinsed and stained with crystal violet solution (Sigma-Aldrich). All the results of transwell assay have been quantified by counting the invasive cell number.

| Luciferase assay
The Notch1 promoter was cloned by PCR from genomic DNA of

| Statistical analysis
ANOVA was used for three-group comparisons; consequence was displayed as mean ± standard error of mean (mean ± SEM). A P-value less than 0.05 was considered statistically significant.

| Nrf2 regulated Notch1 signalling in breast cancer cells
The main functions of Notch pathway are regulating cells proliferation and migration. 20 In order to explore the influence of Nrf2 on Notch pathway in breast cancer cells, the expression of ligand  Figure 4A,B). The expressions of Notch2-4, Dll1, Dll3, Dll4 and Jagged 1-2 were not affected by Nrf2 overexpression or Nrf2 knockdown in MCF-7 and MDA-MB-231 cells ( Figure 4A,B).
It is suggested that Nrf2 is positively correlated with Notch1 in breast cancer.

| Nrf2 regulated Notch1 signalling via G6PD/ HIF-1α in breast cancer cells
In order to determine whether Nrf2 directly activated Notch1 signalling pathway, Notch1-promoter luciferase activity was assayed in MCF-7 and MDA-MB-231 cells. The results show that Notch1-promoter luciferase activity was not affected by Nrf2 overexpression or Nrf2 knockdown in MCF-7 and MDA-MB-231 cells ( Figure 5A).

| Nrf2 can affect breast cancer cell proliferation and migration by influencing the expression of Notch1
One of the main functions of the Notch pathway involves regulating breast cancer cell proliferation and migration. 21 We assumed that the effect of Nrf2 on breast cancer cell proliferation and migra-  (Figure 7D-F). Therefore, we suggest that Nrf2 controls

| D ISCUSS I ON
Metabolic reprogramming by transcription factor Nrf2 could contribute to the development of breast cancer. 11 However, the mechanisms that drive breast cancer progression need further research. NF-E2-related factor 2 is important in protection of oxidative stress. However, some studies have shown that the survival and development of cancer cells were promoted through excessive expression of Nrf2 and its target genes. 22 Recent data suggest that the there are different degrees of Keap1 mutations in patients with lung cancer. 23 Activation of Nrf2 can promote cell tumourigenicity, and knockdown of Nrf2 can inhibit tumour growth, metastasis and invasion. 24 The target genes of Nrf2 are vital in tumour metastasis. The main function of heme oxygenase 1 (HO-1), the main target gene of Nrf2, is to participate in maintaining cells redox equilibrium.
It is reported that the survival rate of lung cancer patients with high level of HO-1 is lower than patients with low expression of HO-1. 25 NAD(P)H quinone dehydrogenase 1 (NQO1) is another gene activated by Nrf2 and also the key gene of oxidative stress. Compared with no-metastatic tumours, NQO1 in metastatic tumours showed higher expression. 26 In this study, we observed that high levels of Nrf2 expression significantly correlated with higher proliferation and migration of breast cancers cells, all of which precludes shorter overall survival and a higher recurrence rate in breast cancer patients. These observations suggest that Nrf2 promotes breast cancer metastasis and may serve as a biomarker for poor prognosis.
In addition to its role in regulation of oxidative stress, Nrf2 also involves in the anabolic metabolism. NF-E2-related factor 2 directly activates six genes involved in PPP and NADPH production pathway, including G6PD, PGD, TKT, TALDO1 and malic enzyme 1 (ME1), through binding of this transcription factor to AREs of these gene promoters. 11 The metabolic reprogramming provides energy and metabolites to facilitate rapid growth and proliferation of cancer cells. The PPP is important in promoting cancer proliferation, differentiation and survival. 27 Overexpression of G6PD is a prognostic factor in multiple cancers and is associated with tumour progression, metastasis or recurrent disease. 28 The oxidative PPP converts G6P, a glycolytic intermediate, into ribulose-5-phosphate and generates NADPH, which is used for GSH production, detoxification and biosynthesis of lipids. Pentose phosphate pathway is up-regulated in many types of tumours. The activities of G6PD and TKT, key PPP enzymes, were increased in cancer cells. 29 In this work, we found that the two key PPP enzymes, G6PD and TKT, were up-regulated in Nrf2-overexpressed MCF-7 and MDA-MB-231 cells, whereas ectopic overexpression of Keap1 and Nrf2 knockdown counteracts the increase of G6PD and TKT expressions in breast cancer cells. These observations indicate that PPP is activated in Nrf2-overexpressed breast cancer cells. In addition, targeting Nrf2 as a combination therapy/chemically induced synthetic lethality target, an approach that has shown excellent results for breast cancer therapy. 30,31 NF-E2-related factor 2 can interact with other signalling pathways key genes. Notch pathway is one of them, Notch1 is crucial in cell survival, abnormal expression of Notch1 has been considered as a universal phenomenon in small cell lung cancer. 34 NF-E2-related factor 2 can activate Notch1 and regulate the proliferation of haematopoietic progenitor cells (HPCs). 35 The results are consistent with our findings that inhibition of Notch1 lowered the expression of its target genes HES-1 and inhibited the tumour proliferation. The Our conclusion confirmed the involvement of Nrf2 transcription factor in tumour metastasis. In summary, our findings suggest that Nrf2 contributes to metastatic ability of basal type breast cancer cells through G6PD/HIF-1α/Notch1 signalling axis. Nrf2-dependent G6PD/HIF-1α activation provokes Notch1 signalling by up-regulation of Jagged1 and Hes1, thereby promoting EMT in breast cancer cells. Further study on Nrf2/G6PD/HIF-1α/Notch1 signalling axis is demanded for realization of basal type breast cancer treatment.

ACK N OWLED G EM ENTS
This study was supported by Beijing Natural Science Foundation

CO N FLI C T O F I NTE R E S T
The authors declare that there are no conflicts of interest.