NRF2 activation by cysteine as a mechanism of survival for Triple Negative Breast Cancer cells

Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous group of cancers, and there is an urgent need to identify the subtype-specific molecular mechanisms underlying TNBC progression and resistance to chemotherapy in order to develop effective therapeutic strategies. TNBC cells are highly dependent on exogenous cystine to proliferate and survive, and overexpress the cystine/glutamate antiporter SLC7A11/xCT to fuel glutathione synthesis and promote an oxidative stress response required by high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like (MSL) subtype utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism of oxidative stress defense. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1 at sensor cysteine residues 226 and 613. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in the response to oxidative stress (GCLM, TXNRD1, SRXN1, AKR1B10), but also in the epithelial-to-mesenchymal transition and stem-like phenotype (IGF2BP3, TGFBR3, POLR3G). Remarkably, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are poor prognostic markers for TNBC, and the expression of OSGIN1 correlates positively with the expression of SLC7A11 and negatively with claudin 3 and 7 as well as the expression of E-cadherin in the MSL subtype. Furthermore, exogenous expression of OSGIN1, similar to NRF2, is able to prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs. Hs 578T cells over expressing GFP (Control) or NRF2 were used for this experiment. For cysteine (Cys) depletion, cells were washed with phosphate-buffered saline solution (PBS), pH 7.4, and incubated for the indicated times in DMEM, high glucose, no glutamine, no methionine, no cystine (#21013024, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 0.2 mM L-Methionine, 100 μg/ml streptomycin, 100 U/ml penicillin, and 2 mM L-glutamine. 0.8 mM L-Cystine was added in control cells. Total RNA was extracted from Hs 578T cells and used for RNAseq library prepration.