铁死亡是一种非凋亡的细胞死亡模式，其特征是铁依赖性脂质过氧化的积累。虽然谷胱甘肽过氧化物酶 4 (GPX4) 催化的脂质自由基消除反应是一种主要的抗铁死亡机制，但从药物角度抑制该途径有望成为一种抗肿瘤策略。然而，某些肿瘤细胞在脂质自由基消除途径中表现出冗余，导致它们对 GPX4 抑制剂没有反应。在这项研究中，我们对不同的癌细胞系和 FDA 批准的药物进行了筛选，最终确定替西罗莫司与 GPX4 抑制剂 RSL3 组合作为肝癌细胞铁死亡的有效诱导剂。从机制上讲，替西罗莫司通过直接结合并抑制铁死亡抑制蛋白 1 (FSP1) 酶，使肝癌细胞对铁死亡敏感。值得注意的是，虽然替西罗莫司被认为是一种有效的 mTOR 抑制剂，但其诱导铁死亡的作用主要归因于其对 FSP1 的抑制，而不是 mTOR 活性。通过进行体外集落形成测定和体内肿瘤异种移植模型，我们证明替西罗莫司和RSL3的组合可有效抑制肝肿瘤进展。这种杀肿瘤作用与脂质过氧化的增加和铁死亡的诱导有关。总之，我们的研究结果强调了结合多靶点铁死亡诱导剂来规避肝癌细胞对铁死亡的抗性的潜力，并强调替西罗莫司作为一种有前途的 FSP1 抑制剂和铁死亡诱导剂，这也值得在转化医学中进一步研究。© 作者(s) 2024。牛津大学出版社代表中国科学院上海生命科学研究院生物化学与细胞生物学研究所出版。

Ferroptosis is a non-apoptotic mode of cell death characterized by iron-dependent accumulation of lipid peroxidation. While lipid radical elimination reaction catalyzed by glutathione peroxidase 4 (GPX4) is a major anti-ferroptosis mechanism, inhibiting this pathway pharmaceutically shows promise as an anti-tumor strategy. However, certain tumor cells exhibit redundancy in lipid radical elimination pathways, rendering them unresponsive to GPX4 inhibitors. In this study, we conducted screens across different cancer cell lines and FDA-approved drugs, leading to the identification of temsirolimus in combination with the GPX4 inhibitor RSL3 as a potent inducer of ferroptosis in liver cancer cells. Mechanistically, temsirolimus sensitized liver cancer cells to ferroptosis by directly binding to and inhibiting ferroptosis suppressor protein 1 (FSP1) enzyme. Notably, while temsirolimus is recognized as a potent mTOR inhibitor, its ferroptosis-inducing effect is primarily attributed to its inhibition of FSP1 rather than mTOR activity. By performing in vitro colony formation assays and in vivo tumor xenograft models, we demonstrated that the combination of temsirolimus and RSL3 effectively suppressed liver tumor progression. This tumoricidal effect was associated with increased lipid peroxidation and induction of ferroptosis. In conclusion, our findings underscore the potential of combining multi-target ferroptosis-inducing agents to circumvent resistance to ferroptosis in liver cancer cells and highlight temsirolimus as a promising FSP1 inhibitor and ferroptosis inducer, which also deserves further investigation in translational medicine.© The Author(s) 2024. Published by Oxford University Press on behalf of Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.