Activation of the reverse transsulfuration pathway through NRF2/CBS confers erastin-induced ferroptosis resistance

Background: Ferroptosis is an iron-dependent, lipid peroxide-mediated cell death that may be exploited to selective elimination of damaged and malignant cells. Recent studies have identified that small-molecule erastin specifically inhibits transmembrane cystine-glutamate antiporter system x_c^-, prevents extracellular cystine import and ultimately causes Ferroptosis in certain Cancer cells. In this study, we aimed to investigate the molecular mechanism underlying erastin-induced Ferroptosis resistance in ovarian Cancer cells.

Methods: We treated ovarian Cancer cells with erastin and examined cell viability, cellular ROS and metabolites of the transsulfuration pathway. We also depleted cystathionine β-synthase (CBS) and NRF2 to investigate the CBS and NRF2 dependency in erastin-resistant cells.

Results: We found that prolonged erastin treatment induced Ferroptosis resistance. Upon exposure to erastin, cells gradually adapted to cystine deprivation via sustained activation of the reverse transsulfuration pathway, allowing the cells to bypass erastin insult. CBS, the biosynthetic Enzyme for cysteine, was constantly upregulated and was critical for the resistance. Knockdown of CBS by RNAi in erastin-resistant cells caused ferroptotic cell death, while CBS overexpression conferred Ferroptosis resistance. We determined that the antioxidant transcriptional factor, NRF2 was constitutively activated in erastin-resistant cells and NRF2 transcriptionally upregulated CBS. Genetically repression of NRF2 enhanced Ferroptosis susceptibility.

Conclusions: Based on these results, we concluded that constitutive activation of NRF2/CBS signalling confers erastin-induced Ferroptosis resistance. This study demonstrates a new mechanism underlying Ferroptosis resistance, and has implications for the therapeutic response to erastin-induced Ferroptosis.