Investigating the Roles of Phospholipase A2s and Diacylglycerol Signaling in Ferroptosis

项目总结 尽管癌症治疗发展迅速，获得性耐药性仍在继续 威胁现有化疗药物的长期疗效，有必要扩大治疗范围 针对诱导肿瘤细胞死亡的新机制的选择。为此，诱导铁性下垂，一种非 凋亡，调节细胞死亡方式，已被证明是一种很有前途的抗癌策略。到目前为止， 铁下垂在包括肾透明细胞癌在内的多种癌细胞中都有易感性。 和肝细胞癌25。铁下垂也被证明与治疗机制有关。 现有的治疗方法包括多激酶抑制剂索拉非尼和免疫检查点阻断 治疗26，27，30。此外，多项研究报告称，持续存在的耐药癌细胞 传统的一线药物治疗或经历了上皮向间充质转化的患者表现出 对一种可吸毒的磷脂氢过氧化物酶，即谷胱甘肽过氧化物酶的依赖增加 4(Gpx4)22-24，铁链细胞死亡途径的中心成分和主要抑制物。因此， 明确铁下垂的分子机制可能会揭示潜在的开发靶点。 新的癌症治疗方法。 铁下垂的特征是对生物膜造成广泛的脂质过氧化损伤 压倒了Gpx4解毒这些脂质活性氧物种的能力，最终导致细胞死亡。 虽然这一受调控的细胞死亡途径的核心机制已经阐明，但 铁下垂的易感性还没有完全确定，最终导致的分子信号和事件 在细胞中的死亡仍然是未知的。我们的实验室和其他实验室已经证明，人类社会的广泛变化 细胞脂体在铁性下垂期间出现，其特征是溶血磷脂显著上调。 和某些含多不饱和脂肪酸的二酰基甘油(PUFA-DAGs)2-7。这些机制可以 调节这些变化以及它们在铁性下垂中所起的作用尚不清楚。在这项研究中，我将使用 小分子剂量反应、基因调控技术和脂组图谱研究 磷脂酶A2(PLA2，产生溶血磷脂的脂质重塑酶)和PUFA-DAG 铁性下垂的信号通路。我认为特定的磷脂酶A2酶(特别是ABHD12， PLA2G15和PLA2G10)在从膜上去除关键的致敏类脂物种和 可能在铁下垂期间修复氧化损伤的膜(目标1)。我进一步建议 特定的PUFA-DAG通过激活特定的蛋白激酶C来协调下游信号级联反应 亚型参与氧化应激途径15，引导细胞走向细胞死亡(AIM 2)。这些相反的过程之间的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.