目的：谷胱甘肽过氧化物酶4 (GPX4) 作为铁死亡的一种关键内源性负调节因子，可以通过多种翻译后修饰途径调节其抗氧化功能。然而，在肝细胞癌（HCC）中，GPX4的磷酸化/去磷酸化状态对诱导性铁死亡的调控效应尚不清楚。方法：为了研究GPX4磷酸化/去磷酸化修饰对HCC细胞中铁死亡的影响和分子机制，我们使用索拉非尼（Sora）在体外建立HCC细胞中的铁死亡模型。通过定点突变方法，我们在GPX4的特定丝氨酸位点上构建了模拟GPX4磷酸化或去磷酸化的HCC细胞系。检测了GPX4磷酸化/去磷酸化修饰对HCC细胞中铁死亡的影响，并探讨了GPX4、p53和蛋白磷酸酶2A-B55β亚单位（PP2A-B55β）之间的相互关系。为了探索PP2A在索拉治疗的HCC中的协同抗肿瘤作用，我们在裸鼠模型中建立了PP2A-B55β过表达的异种移植瘤。结果：在体外HCC索拉诱导的铁死亡模型中，细胞质和线粒体GPX4水平降低、线粒体功能障碍和增强的p53逆向信号发生在索拉处理下。此外，我们发现线粒体p53明显向细胞核退化，并加重了索拉诱导的铁死亡。GPX4丝氨酸2位点（GPX4Ser2）的磷酸化状态显示，线粒体p-GPX4Ser2去磷酸化与铁死亡呈正相关，机制可能与线粒体p53退化入细胞核有关。在过表达PP2A-B55β的HCC细胞中发现，PP2A-B55β直接与线粒体GPX4作用，并促进HCC中索拉诱导的铁死亡。此外，PP2A-B55β减少了线粒体GPX4与p53的相互作用，导致线粒体p53退化到细胞核。此外，确证了PP2A-B55β在索拉处理的HCC异种移植瘤中增强了铁死亡介导的肿瘤生长抑制和线粒体p53逆向信号。结论：我们的数据揭示了PP2A-B55β/p-GPX4Ser2/p53轴是索拉诱导的铁死亡的一条新的调控途径。通过诱导线粒体p-GPX4Ser2的去磷酸化，触发了铁死亡，使线粒体p53退化入细胞核，PP2A-B55β是线粒体p-GPX4Ser2的去磷酸化的上游信号调节因子。我们的发现可能成为通过通过PP2A-B55β激活以靶向干预p-GPX4去磷酸化来增强索拉在HCC治疗中的疗效的一种潜在治疗策略。© The author(s).

Rationale: As a key endogenous negative regulator of ferroptosis, glutathione peroxidase 4 (GPX4) can regulate its antioxidant function through multiple post-translational modification pathways. However, the effects of the phosphorylation/dephosphorylation status of GPX4 on the regulation of inducible ferroptosis in hepatocellular carcinoma (HCC) remain unclear. Methods: To investigate the effects and molecular mechanism of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells. Sorafenib (Sora) was used to establish the ferroptosis model in HCC cells in vitro. Using the site-directed mutagenesis method, we generated the mimic GPX4 phosphorylation or dephosphorylation HCC cell lines at specific serine sites of GPX4. The effects of GPX4 phosphorylation/dephosphorylation modification on ferroptosis in HCC cells were examined. The interrelationships among GPX4, p53, and protein phosphatase 2A-B55β subunit (PP2A-B55β) were also explored. To explore the synergistic anti-tumor effects of PP2A activation on Sora-administered HCC, we established PP2A-B55β overexpression xenograft tumors in a nude mice model in vivo. Results: In the Sora-induced ferroptosis model of HCC in vitro, decreased levels of cytoplasmic and mitochondrial GPX4, mitochondrial dysfunction, and enhanced p53 retrograde signaling occurred under Sora treatment. Further, we found that mitochondrial p53 retrograded remarkably into the nucleus and aggravated Sora-induced ferroptosis. The phosphorylation status of GPX4 at the serine 2 site (GPX4Ser2) revealed that mitochondrial p-GPX4Ser2 dephosphorylation was positively associated with ferroptosis, and the mechanism might be related to mitochondrial p53 retrograding into the nucleus. In HCC cells overexpressing PP2A-B55β, it was found that PP2A-B55β directly interacted with mitochondrial GPX4 and promoted Sora-induced ferroptosis in HCC. Further, PP2A-B55β reduced the interaction between mitochondrial GPX4 and p53, leading to mitochondrial p53 retrograding into the nucleus. Moreover, it was confirmed that PP2A-B55β enhanced the ferroptosis-mediated tumor growth inhibition and mitochondrial p53 retrograde signaling in the Sora-treated HCC xenograft tumors. Conclusion: Our data uncovered that the PP2A-B55β/p-GPX4Ser2/p53 axis was a novel regulatory pathway of Sora-induced ferroptosis. Mitochondrial p-GPX4Ser2 dephosphorylation triggered ferroptosis via inducing mitochondrial p53 retrograding into the nucleus, and PP2A-B55β was an upstream signal modulator responsible for mitochondrial p-GPX4Ser2 dephosphorylation. Our findings might serve as a potential theranostic strategy to enhance the efficacy of Sora in HCC treatment through the targeted intervention of p-GPX4 dephosphorylation via PP2A-B55β activation.© The author(s).