Expression of xCT and activity of system xc(-) 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 xc(-), 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 xc(-), is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) 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 xc(-). 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.

Keywords: Hydrogen peroxide; KEAP1; NRF2; Oxidative stress; System x(c)(−); xCT.

Graphical abstract

Fig. 1

H₂O₂ increased NRF2 nuclear localization, xCT expression, and the activity of system x_c⁻ in MCF-7 cells. Cells treated with H₂O₂ were fractioned for analysis of cytosolic (C) and nuclear (N) protein levels of (A) NRF2 and (B) KEAP1 by Western blotting, with normalized fold changes relative to control obtained by densitometric analysis showing an increase in nuclear NRF2 in response to oxidative stress. (C) Real Time RT-PCR revealed that xCT mRNA levels increased significantly by 2-fold in response to treatment with H₂O₂, which also increased (D) xCT protein levels and (E) the release of glutamate by MCF-7 cells.

Fig. 2

Overexpressing NRF2 up-regulated xCT expression and the activity of system x_c⁻ in MCF-7 cells. (A) Luciferase assays were carried out on lysates isolated from MCF-7 cells transiently transfected with the human xCT promoter construct together with either an empty pcDNA3.1 vector (Vector) or an NRF2 overexpression vector (NRF2), demonstrating that NRF2 significantly up-regulated xCT at the level of transcription. (B) A representative Western blot of whole cell lysates prepared from MCF-7 cells transiently transfected with empty vector (V), or NRF2 (N) or ETS-1 (E) overexpression vectors, with densitometrically determined fold changes showing an up-regulation of xCT protein levels when NRF2 was overexpressed. (D) NRF2 overexpression also increased the release of glutamate by transiently transfected MCF-7 cells relative to Vector.

Fig. 3

Altering levels of KEAP1 in MCF-7 cells affected xCT expression and of the activity of system x_c⁻. (A) Luciferase assays carried out on lysates isolated from MCF-7 cells transiently transfected with the human xCT promoter construct together with either empty pcDNA3.1 vector (Vector) or a KEAP1 overexpression vector (KEAP1) demonstrated that KEAP1 significantly up-regulated xCT at the level of transcription. (B) A representative Western blot of whole cell lysates prepared from MCF-7 cells transiently transfected with empty pcDNA 3.1 vector (V) or KEAP1 (K) overexpression vector, showing decreased xCT protein levels upon KEAP1 overexpression. (C) Knock-down of KEAP1 using four different siRNAs (K1–K4) corresponded with a concomitant increase in xCT protein levels relative to a non-specific siRNA (NS). (D) Knock-down of KEAP1 also increased glutamate release by transiently transfected MCF-7 cells relative to NS.

Fig. 4

A summary of the KEAP1/NRF2 pathway regulating xCT expression and system x_c⁻ activity in MCF-7 breast cancer cells. In the current investigation, NRF2 upregulates xCT transcription, as well as mRNA and protein levels, in human MCF-7 breast cancer cells. Under basal conditions, KEAP1 facilitates the ubiquitination (UB) and proteasomal breakdown of NRF2, while in response to oxidative stress (ROS), NRF2 dissociates from KEAP1, enabling NRF2 to translocate to the nucleus. Nuclear NRF2 binds to the antioxidant response element (ARE) in the xCT promoter, activating transcription. xCT protein is then transported to the membrane, where it couples to 4F2hc, which together comprise system x_c⁻. The antiporter functions to export glutamate (GLU) and import cystine. Cystine is then reduced to cysteine, which is utilized in the production of glutathione (GSH), thereby protecting MCF-7 cells from oxidative stress.