Expression of xCT and activity of system x _c ⁻ are regulated by NRF2 in human breast cancer cells in response to oxidative stress

Abstract

Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System x _c ⁻, an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system x _c ⁻, is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H ₂O ₂) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system x _c ⁻. We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.

Graphical abstract

Highlights

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  Acute H ₂O ₂ treatment of MCF-7 human breast cancer cells increases NRF2 nuclear translocation.

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  H ₂O ₂ also increases levels of xCT mRNA and protein, and extracellular glutamate release.

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  NRF2 overexpression in MCF-7 cells up-regulates the activity of the human xCT promoter, which contains a proximal ARE.

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  KEAP1 overexpression in MCF-7 cells represses promoter activity, correlating with decreased xCT protein levels.

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  siRNA-mediated knockdown of KEAP1 up-regulates xCT protein levels and glutamate release.