PO.ET06.01 · 实验与分子治疗
用EC359抑制LIFR触发铁死亡以克服子宫内膜癌的化疗耐药
LIFR inhibition with EC359 triggers ferroptosis to overcome chemoresistance in endometrial cancer
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：子宫内膜癌（ECa）是女性生殖道最常见的恶性肿瘤，其发病率和死亡率不断上升，凸显了对更好治疗手段的需求。用EC359靶向白血病抑制因子受体（LIFR）显示出前景，可降低I型和II型ECa的活力和侵袭。由于LIFR信号与铁死亡（一种铁依赖性细胞死亡）相关联，本研究探讨EC359是否诱导铁死亡并增强化疗，从而为晚期ECa提供一种新型治疗策略。
方法：通过长期暴露于化疗药物建立化疗耐药ECa细胞系，并通过RNA测序验证以确认耐药相关的转录谱。通过集落形成测定、基于MTT的细胞活力测定和凋亡分析，在多个已建立的及患者来源的原代ECa细胞模型中评估EC359的抗肿瘤效应。利用Western blot、RT-qPCR、透射电子显微镜（TEM）和流式细胞术开展机制研究，以评估铁死亡相关标志物和细胞变化。为进行临床前验证，采用ECa的患者来源类器官（PDO）和异种移植（PDX）模型，以确定EC359体内联合治疗的疗效。
结果：taxol耐药ECa细胞的RNA-seq分析揭示了与细胞增殖、干性和LIFR信号相关通路的上调，以及凋亡通路的下调。EC359显著降低了taxol耐药晚期ECa模型的细胞活力。值得注意的是，当使用Ferrostatin-1药理学性抑制铁死亡时，EC359的细胞毒性效应明显减弱，表明铁死亡是EC359诱导细胞死亡的关键机制。机制研究表明，EC359抑制谷胱甘肽抗氧化防御系统，从而促进铁死亡。与此一致，补充细胞外胱氨酸可在EC359处理后恢复细胞活力。此外，EC359显著降低了关键抗铁死亡蛋白的表达。流式细胞术证实EC359处理的细胞中脂质过氧化增加，而TEM研究揭示了与铁死亡相关的特征性线粒体损伤。重要的是，EC359与化疗联合显著降低了PDO的活力，并在晚期ECa的PDX模型中强力抑制了肿瘤进展。
结论：我们的研究提示，EC359通过破坏抗氧化防御诱导铁死亡并与化疗协同，EC359可作为克服晚期ECa化疗耐药的一种有前景的策略。
查看英文原文 English abstract
Background: Endometrial cancer (ECa), the most common malignancy of the female reproductive tract, has rising incidence and mortality, highlighting the need for better treatments. Targeting leukemia inhibitory factor receptor (LIFR) with EC359 shows promise by reducing viability and invasion in both Type I and II ECa. Since LIFR signaling is linked to ferroptosis, an iron-dependent cell death, this study explores whether EC359 induces ferroptosis and enhances chemotherapy, offering a novel therapeutic strategy for advanced ECa.
Methods: Chemotherapy-resistant ECa cell lines were established through prolonged exposure to chemotherapeutic agents and validated by RNA sequencing to confirm resistance-associated transcriptional profiles. The antitumor effects of EC359 were assessed in multiple established and patient-derived primary ECa cell models through colony formation assays, MTT-based cell viability assays, and apoptosis analysis. Mechanistic studies were conducted using Western blotting, RT-qPCR, transmission electron microscopy (TEM), and flow cytometry to evaluate ferroptosis-related markers and cellular changes. For preclinical validation, patient-derived organoids (PDO) and xenograft (PDX) models of ECa were employed to determine the therapeutic efficacy of EC359 in combination in vivo .
Results: RNA-seq analysis of taxol-resistant ECa cells revealed upregulation of pathways associated with cell proliferation, stemness, and LIFR signaling, along with downregulation of apoptotic pathways. EC359 significantly reduced cell viability in taxol-resistant advanced ECa models. Notably, the cytotoxic effect of EC359 was markedly attenuated when ferroptosis was pharmacologically inhibited using Ferrostatin-1, indicating that ferroptosis is a key mechanism of EC359-induced cell death. Mechanistic studies demonstrated that EC359 suppresses the glutathione antioxidant defense system, thereby promoting ferroptosis. Consistent with this, supplementation with extracellular cystine restored cell viability following EC359 treatment. Moreover, EC359 markedly reduced the expression of key anti-ferroptosis proteins. Flow cytometry confirmed increased lipid peroxidation in EC359-treated cells, while TEM studies revealed characteristic mitochondrial damage associated with ferroptosis. Importantly, EC359 combined with chemotherapy significantly decreased the viability of PDOs and robustly inhibited tumor progression in PDX models of advanced ECa.
Conclusion: Our studies suggest that EC359 induces ferroptosis by disrupting antioxidant defenses and synergizes with chemotherapy and that EC359 serve as a promising strategy to overcome chemoresistance in advanced ECa.
利益披露 Disclosure
E. J. Aller, None..
B. Subramani, None..
X. Yang, None..
P. Ramirez, None.
B. Santhamma,
Evestra Employment.
H. B. Nair, None..
E. Kost, None..
R. K. Vadlamudi, None..
S. Viswanadhapalli, None.