Abstract 506: H2O2 upregulates xCT in human breast cancer cells via NRF-2

Abstract Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate, making the transcription factors involved in antioxidant defense regulation targets of interest. The transcription factor NF E2 Related Factor 2 (NRF-2) regulates cellular defense genes including those encoding intracellular redox-balancing proteins such as enzymes involved in glutathione metabolism. Glutathione in particular is an important intracellular antioxidant molecule. NRF-2 binds to the Antioxidant Response Element (ARE) in the promoter of its target genes. Under basal conditions, KEAP1 acts as an inhibitor that targets NRF-2 for ubiquitination. During oxidative stress, NRF-2 dissociates from KEAP1 and enters the nucleus to bind to the ARE sequence. It is hypothesized that the elevated Reactive Oxygen Species may be depleting the glutathione levels within the cancer cell. System xc- is a cysteine/glutamate antiporter that exports glutamate while importing cysteine to synthesize glutathione. In response to oxidative stress the cells increase system xc- activity in order to provide cysteine for glutathione synthesis. There is evidence that expression of xCT the specific subunit of System X is regulated by NRF-2. However this has not yet been demonstrated in human cancer cells, which is the focus of this project. Basal expression of NRF-2, Keap1 and xCT was characterized in two breast cancer cell lines (MDA MB 231 and MCF-7) compared to normal human breast epithelial 184B5 cells. Compared to 184B5 cells, Keap-1, NRF-2 and xCT were upregulated in MDA MB 231 cells, but down regulated in MCF-7 cells. MCF-7 cells were treated with H202 resulting in NRF-2 protein accumulation in the nucleus and decreased levels in the cytosol. Under the same treatment conditions, Keap-1 protein decreased in the nucleus and increased in the cytosol. With H202 treatment, xCT mRNA levels increased in MCF-7 cells. We have cloned approximately 2.6 kilobase (-2329 to +278 bp) of the human xCT promoter region from genomic DNA isolated from MDA MB 231 breast cancer cells. We found that the xCT promoter was responsive to treatment with H2O2, which increased transcriptional activity. This is consistent with results described for the mouse promoter, which is known to contain an antioxidant response element that can be bound by NRF-2 during oxidative stress. Co transfection of MDA MB 231 with the xCT promoter and an NRF-2 expression vector also increased promoter activity. This data suggests that under oxidative stress, NRF-2 is localized to the nucleus and transcriptionally upregulates xCT. This is the first study in which the regulation of xCT has been linked to oxidative stress via NRF-2 in human breast cancer cells. This work was supported by the Canadian Institutes for Health Research. Citation Format: Eric Habib, Katja Linher-Melville, Gurmit Singh. H2O2 upregulates xCT in human breast cancer cells via NRF-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2014-506