引起铁死亡（ferroptosis）的药物在癌症治疗中具有潜在的作用。在探讨这些药物对乳腺癌（BC）的影响时，我们发现一种诱导铁死亡的药物铁死亡诱导剂（erastin）在小鼠人BC细胞的肿瘤生长中起到了显著的抑制作用，但是当在已知能有效杀死其他细胞类型的浓度下，在二维单层培养中，对BC细胞的生长只有轻微的减少。BC在体内以三维聚集物的形式生长，我们发现，诱导铁死亡的药物铁死亡诱导剂和磺胺嘧啶在三维培养中对多种人类乳腺癌细胞株的生长抑制效果明显强于二维培养中。为了理解这种差异效应的机制，我们发现，铁死亡诱导剂上调了编码多种直接和间接自噬刺激因子的mRNA，如ATG16L2，ATG9A，ATG4D，GABARAP，SQSTM/p62，SEC23A 和BAX等，在2D培养中的肿瘤细胞中，而在3D培养中没有上调。此外，这些药物促进了2D培养的肿瘤细胞的自噬，但并不促进3D培养的肿瘤细胞的自噬。我们观察到，药物抑制自噬促动蛋白激酶ULK1或通过RNA干扰介导的自噬介质ATG12的敲低明显增强了2D培养中对铁死亡诱导剂的BT细胞对其敏感性。我们还发现促进铁死亡的药物上调了BC细胞的血红素氧化酶1（HO-1），HO-1增加了细胞游离铁池的量，可能促进了铁死亡。实际上，我们观察到通过RNA干扰HO-1逆转了铁死亡诱导剂对BC细胞3D生长的影响。因此，这些药物对于这种生长的作用是由HO-1介导的。总结起来，诱导铁死亡的药物引发的自噬会降低它们在2D生长环境中杀死BC的能力。这种保护机制在BC细胞以3D聚集物形式生长时被抑制，诱导铁死亡的药物更有效地杀死这些细胞。而且，这种细胞死亡是由HO-1介导的。因此，诱导铁死亡是阻断3D BC生长的一个有前景的策略。© 2023. 作者

Drugs causing ferroptosis, iron-mediated cell death, represent promising tools for cancer treatment. While exploring the effect of these drugs on breast cancer (BC), we found that a ferroptosis-inducing drug erastin dramatically inhibits tumorigenicity of human BC cells in mice but when used at a concentration known to effectively kill other cell types only modestly reduces such growth in 2D monolayer culture. BCs grow in vivo as 3D masses, and we found that ferroptosis inducers erastin and sulfasalazine inhibit growth of multiple human BC cell lines in 3D culture significantly stronger than in 2D culture. To understand the mechanism of this differential effect, we found that ferroptosis inducers upregulate mRNAs encoding multiple direct and indirect autophagy stimulators, such as ATG16L2, ATG9A, ATG4D, GABARAP, SQSTM/p62, SEC23A and BAX, in tumor cells growing in 2D but not in 3D culture. Furthermore, these drugs promoted autophagy of tumor cells growing in a 2D but not in a 3D manner. We observed that pharmacological inhibition of autophagy-stimulating protein kinase ULK1 or RNA interference-mediated knockdown of autophagy mediator ATG12 significantly sensitized tumor cells to erastin treatment in 2D culture. We also found that ferroptosis-promoting treatments upregulate heme oxygenase-1 (HO-1) in BC cells. HO-1 increases cellular free iron pool and can potentially promote ferroptosis. Indeed, we observed that HO-1 knockdown by RNA interference reversed the effect of ferroptosis inducers on BC cell 3D growth. Hence, the effect of these drugs on such growth is mediated by HO-1. In summary, autophagy triggered by ferroptosis-promoting drugs reduces their ability to kill BC growing in a 2D manner. This protection mechanism is inhibited in BC cells growing as a 3D mass, and ferroptosis-promoting drugs kill such cells more effectively. Moreover, this death is mediated by HO-1. Thus, ferroptosis induction represents a promising strategy for blocking 3D BC growth.© 2023. The Author(s).