Investigating the Roles of Phospholipase A2s and Diacylglycerol Signaling in Ferroptosis

项目摘要 尽管癌症治疗发展迅速，但获得性耐药性仍在继续， 威胁现有化疗药物的长期疗效，需要扩大治疗 靶向诱导肿瘤细胞死亡的新机制的选择。为此，诱导铁凋亡，一种非- 细胞凋亡的调节性细胞死亡模式已被证明是一种有前途的抗癌策略。到目前为止， 铁凋亡易感性已在许多癌细胞类型中得到证实，包括肾透明细胞癌 和肝细胞癌25.铁凋亡也已被证明参与治疗机制 包括索拉非尼（一种多激酶抑制剂）和免疫检查点阻断在内的现有疗法 治疗26，27，30.此外，多项研究报道，治疗后持续存在的耐药癌细胞 常规的一线药物治疗或经历了上皮-间质转化的患者表现出 对可药用磷脂氢过氧化物酶（即谷胱甘肽过氧化物酶）的依赖性增加 4（GPX 4）22-24，铁凋亡细胞死亡途径的中心组分和主要抑制剂。因此，我们认为， 确定控制铁凋亡的分子机制将可能揭示开发的潜在目标， 新型癌症治疗方法。 亚铁凋亡的特征是对生物膜的广泛脂质过氧化损伤， 破坏GPX 4解毒这些脂质活性氧的能力，最终导致细胞死亡。 虽然已经阐明了这种受调节的细胞死亡途径的中心机制，但是， 铁凋亡的易感性还没有完全表征，最终导致铁凋亡的分子信号和事件 在细胞死亡中仍然是未知的。我们的实验室和其他实验室已经证明， 细胞脂质组在铁凋亡期间发生，其特征在于溶血磷脂的显著上调 和某些含多不饱和脂肪酸的二酰基甘油（PUFA-DAG）2-7。的机制 介导这些变化和功能，他们在ferroptosis是未知的。在这项研究中，我将使用 小分子剂量反应，遗传调节技术和脂质组学分析，以研究 磷脂酶A2（PLA 2，产生溶血磷脂的脂质重塑酶）和PUFA-DAG 铁凋亡的信号通路。我建议，特定的磷脂酶A2酶（具体来说，ABHD 12， PLA 2G 15和PLA 2G 10）在从膜去除关键致敏脂质物质中是有活性的， 在铁凋亡过程中可能修复氧化损伤的膜（目的1）。我并建议 特异性PUFA-DAG通过激活某些蛋白激酶C协调下游信号级联 亚型，它们参与氧化应激途径15，指导细胞走向细胞死亡（Aim 2）。这些对立过程之间平衡的倾斜可能决定细胞的命运。

PROJECT SUMMARY Despite rapid advances in cancer therapeutic development, acquired drug resistance continues to threaten the long-term efficacy of existing chemotherapeutic agents, necessitating the expansion of treatment options targeting novel mechanisms to induce tumor cell death. To that end, induction of ferroptosis, a non- apoptotic, regulated cell death modality, has been shown to be a promising anti-cancer strategy. To date, ferroptosis susceptibility has been demonstrated in many cancer cell types, including clear-cell renal carcinoma and hepatocellular carcinoma25. Ferroptosis has also been shown to be involved in the therapeutic mechanism of existing therapies including sorafenib, a multi-kinase inhibitor, and immune checkpoint blockade therapy26,27,30. Furthermore, multiple studies have reported that therapy-resistant cancer cells that persist after conventional first-line drug treatments or that have undergone epithelial-to-mesenchymal transition exhibit an increased dependency on a druggable phospholipid hydroperoxidase enzyme, namely glutathione peroxidase 4 (GPX4)22-24, the central component and major inhibitor of the ferroptotic cell death pathway. Therefore, defining the molecular mechanisms governing ferroptosis will likely reveal potential targets for the development of novel cancer therapeutics. Ferroptosis is characterized by extensive lipid peroxidative damage to biological membranes that overwhelms the ability of GPX4 to detoxify these lipid reactive oxygen species, eventually leading to cell death. While the central mechanisms of this regulated cell death pathway have been elucidated, the mediators of ferroptosis susceptibility are not fully characterized and the molecular signals and events that ultimately result in cellular demise remain unknown. Our lab and others have demonstrated that widespread changes in the cellular lipidome occur during ferroptosis, characterized by the significant upregulation of lysophospholipids and certain polyunsaturated fatty acid-containing diacylglycerols (PUFA-DAGs)2-7. The mechanisms that mediate these changes and the functions they play in ferroptosis are as yet unknown. In this study, I will use small molecule dose response, genetic modulation techniques, and lipidomic profiling to investigate the role of phospholipase A2s (PLA2s, lipid remodeling enzymes that generate lysophospholipids), and PUFA-DAG signaling pathways in ferroptosis. I propose that specific phospholipase A2 enzymes (specifically, ABHD12, PLA2G15, and PLA2G10) are active in the removal of key sensitizing lipid species from the membrane and potentially in the repair of oxidatively damaged membranes during ferroptosis (Aim 1). I further propose that specific PUFA-DAGs coordinate downstream signaling cascades through activation of certain protein kinase C subtypes, which have been implicated in oxidative stress pathways15, directing the cell toward cell death (Aim 2). A tipping of the balance between these opposing processes may determine cell fate.