Data Sheet 3_IFNβ drives ferroptosis through elevating TRIM22 and promotes the cytotoxicity of RSL3.pdf

BackgroundCyclic GMP-AMP synthase (cGAS)-stimulator-of-interferon genes (STING) pathway is a cytosolic DNA sensor system. The production of this pathway, interferon-β (IFNβ), could suppress the growth of tumor cells, yet it is unclear whether ferroptosis is involved in IFNβ-induced cell death.
MethodsThe effects of IFNβ on ferroptosis were analyzed in HT1080, 4T1, HCT116 and 786-O cells. HT1080 and 4T1 cells treated with IFNβ were subjected to RNA-Seq analysis. STAT1, STAT3, TRIM21, and TRIM22 were silenced by siRNAs to examine their effects on IFNβ-induced ferroptosis. The cGAS-STING signaling pathway-activated mice were used to evaluate the effects of IFNβ on ferroptosis in vivo. HT1080 cells, three-dimensional (3D) spheroids, and the xenograft mouse models were treated with IFNβ, RSL3, or IFNβ combination with RSL3 to analyze whether IFNβ enhances RSL3-induced ferroptosis.
ResultsHere, we found that IFNβ could promote intracellular Fe2+ and lipid peroxidation levels, and decrease GSH levels in tumor cells. RNA sequencing data revealed that IFNβ induced a transcriptomic disturbance in ferroptosis-related genes. Knockdown of tripartite motif-containing 22 (TRIM22) suppressed the levels of intracellular Fe2+ and lipid ROS. It also reduced heme oxygenase (HMOX1) protein levels and increased ferroptosis suppressor protein 1 (FSP1) levels in HT1080 cells treated with IFNβ. Furthermore, our results illustrated that IFNβ enhanced the RAS-selective lethal 3 (RSL3)-induced ferroptosis and the inhibitory effect of RSL3 on GPX4. Meanwhile, compared to the groups treated with either IFNβ or RSL3 alone, the combination treatment of IFNβ and RSL3 significantly inhibited the growth of HT1080 three-dimensional (3D) spheroids and tumor in a mouse xenograft model.
ConclusionsOur work reveals a role for IFNβ in promoting ferroptosis and provides evidence that IFNβ could be used with RSL3 to increase cytotoxic effects in tumor cells.

研究背景
环GMP-AMP合酶（cyclic GMP-AMP synthase, cGAS）-干扰素基因刺激因子（stimulator of interferon genes, STING）信号通路是一种胞质DNA感知系统。该通路的产物干扰素-β（interferon-β, IFNβ）可抑制肿瘤细胞增殖，但目前尚不明确铁死亡（ferroptosis）是否参与IFNβ诱导的细胞死亡过程。

研究方法
本研究在HT1080、4T1、HCT116及786-O细胞中分析了IFNβ对铁死亡的影响。对经IFNβ处理的HT1080与4T1细胞进行RNA测序（RNA-Seq）分析。通过小干扰RNA（small interfering RNA, siRNAs）沉默信号转导与转录激活因子1（STAT1）、信号转导与转录激活因子3（STAT3）、三重基序蛋白21（tripartite motif-containing 21, TRIM21）及三重基序蛋白22（tripartite motif-containing 22, TRIM22）的表达，以探究其对IFNβ诱导的铁死亡的影响。本研究使用经cGAS-STING信号通路激活的小鼠，在体内评估IFNβ对铁死亡的影响。本研究用IFNβ、RAS选择性致死化合物3（RAS-selective lethal 3, RSL3）或IFNβ与RSL3联合处理HT1080细胞、三维（3D）球体及异种移植小鼠模型，以分析IFNβ是否可增强RSL3诱导的铁死亡。

研究结果
本研究发现，IFNβ可促进肿瘤细胞内的亚铁离子（Fe²+）与脂质过氧化水平，并降低细胞内谷胱甘肽（glutathione, GSH）的含量。RNA测序数据显示，IFNβ可引发铁死亡相关基因的转录组紊乱。沉默三重基序蛋白22（TRIM22）的表达可抑制经IFNβ处理的HT1080细胞内的亚铁离子与脂质活性氧（reactive oxygen species, ROS）水平。同时，该操作还可降低经IFNβ处理的HT1080细胞内的血红素氧合酶1（heme oxygenase 1, HMOX1）蛋白水平，并提升铁死亡抑制蛋白1（ferroptosis suppressor protein 1, FSP1）的表达量。此外，本研究结果表明，IFNβ可增强RAS选择性致死化合物3（RSL3）诱导的铁死亡，以及RSL3对谷胱甘肽过氧化物酶4（glutathione peroxidase 4, GPX4）的抑制作用。与此同时，与单独使用IFNβ或RSL3处理的组别相比，IFNβ与RSL3联合处理可显著抑制HT1080三维（3D）球体的生长及异种移植小鼠模型中的肿瘤增殖。

研究结论
本研究揭示了IFNβ在促进铁死亡中的作用，并提供了证据表明IFNβ可与RSL3联合使用，以增强其对肿瘤细胞的细胞毒性作用。