Loss of Ferroptosis-suppressors Accelerates Breast Cancer Recurrence by Promoting Dormancy Exit

Breast cancer mortality is principally due to recurrent metastatic disease that may commonly emerge from a state of dormant minimal residual disease following definitive treatment of a primary tumor. Our genomic analysis of paired primary and metastatic breast cancers arising in the same patients resulted in the identification of the ferroptosis pathway as being preferentially mutated in metastases. An in vivo CRISPR-Cas9 screen of known ferroptosis regulators in mice identified guides targeting PNPLA2, GOT1, and GPX4 as being selected for in late dormancy and recurrence, which was surprising given the ferroptosis-suppressing functions ascribed to these proteins. However, recurrence-free survival assays in mice confirmed that deletion of PNPLA2, GOT1, or GPX4 accelerated tumor recurrence in mice and provided further evidence that this acceleration was attributable to an increased probability that dormant tumor cells deleted for these genes would reenter the cell cycle. Using a model of therapy-associated breast cancer dormancy, we also found that dormant cancer cells were markedly resistant to ferroptosis-inducing stimuli, including erastin and RSL3. Moreover, dormant tumor cells lacking PNPLA2, GOT1, or GPX4 displayed enhanced ferroptosis resistance compared to wildtype cells. Probing this ferroptosis-resistant state revealed that the ratio of reduced to oxidized glutathione was elevated in dormant cells. Moreover, deletion of PNPLA2, GOT1, or GPX4 further increased this ratio in dormant cells, suggesting that an increased ratio of reduced to oxidized glutathione may contribute to ferroptosis resistance in dormant tumor cells, especially those lacking these genes, potentially by rendering them more capable of handling lipid oxidative stress. Consistent with our findings in mice, analysis of human breast cancer patient data revealed that patients with copy number loss of PNPLA2, GOT1, or GPX4 exhibited worse recurrence-free and overall survival. Together, our study implicates three genes in the process of recurrence from dormant tumor cells and identifies a unique metabolic feature of the dormant tumor cell state. The surprising resistance to ferroptosis inducers exhibited by dormant tumor cells, as well as counterintuitive effects of deleting ferroptosis-suppressing genes on tumor recurrence following dormancy, raise questions regarding the utility of targeting ferroptosis in cancer that merit further study.