Lysosomal-mitochondrial signaling in non-apoptotic cancer cell death

项目总结 绝大多数临床使用或以下的常规和靶向化疗药物 发育依赖于参与凋亡途径来诱导肿瘤细胞死亡。然而，对细胞凋亡的抵抗-- 诱导剂是一个特别棘手的临床问题。靶向治疗耐药细胞的一种新方法是 利用细胞死亡机制而不是细胞凋亡来根除这些恶性亚群。这个 拟议研究的总体目标是定义溶酶体-线粒体细胞器间信号转导 肿瘤细胞特异性和程序性坏死溶酶体细胞死亡(LCD)过程的机制 由多种药物引起的。我们的先导化合物六亚甲基阿米洛利(HMA)，是一种药物的衍生品 已在临床上应用于血压管理超过45年，死亡分化 和干细胞，独立于肿瘤类型、亚型或种类，但不能有效地杀死正常 分化细胞或干细胞。此外，HMA杀死癌细胞不依赖于细胞周期、自噬 参与和caspase依赖的细胞凋亡；事实上，细胞死亡似乎是药物诱导的结果。 溶酶体限膜和随后的组织蛋白酶介导的质膜的通透性 破裂。我们的观察表明，有效的HMA诱导的细胞死亡需要产生和作用于 线粒体产生的活性氧(ROS)。我们的观察还表明，HMA诱导 鞘磷脂沉积症溶酶体储存疾病的一些特征，包括各种积聚 通常被溶酶体分解的脂类。值得注意的是，诸如乳糖基神经酰胺和 溶血磷脂酰胆碱已被证明在生产中充当信号第二信使 在HMA处理后，线粒体ROS特异性地聚集在肿瘤细胞中，而不是正常细胞中。我们的 观察指向一种模型，其中药物诱导异常脂质堆积和ROS介导的溶酶体 膜脂氧化破坏溶酶体膜的完整性，允许组织蛋白酶的释放和诱导 坏死性细胞死亡。为了测试这个模型，我们将使用生化、细胞生物学和代谢组学方法。 在目标1中，我们将评估双(单甘油)磷酸(BMP)，一种溶酶体驻留的脂质的贡献。 在肿瘤相对正常的细胞中被抑制，并被HMA治疗进一步抑制，在调节 溶酶体膜的稳定性和细胞活力通过其激活溶酶体酶的能力 鞘磷脂的分解途径。对这些研究的补充将是对脂肪组学和 与细胞转化和LCD诱导剂相关的代谢变化。在目标2中，我们将 研究溶酶体-线粒体信号事件将线粒体ROS的产生与失调联系在一起 溶酶体脂代谢。这些研究将揭示溶酶体靶标，使未来能够发展 更有效地诱导癌细胞特异性程序性坏死细胞死亡的新型治疗剂。

PROJECT SUMMARY The overwhelming majority of conventional and targeted chemotherapeutics in clinical use or under development rely on engaging apoptotic pathways to elicit tumor cell death. However, resistance to apoptosis- inducing agents is a particularly thorny clinical problem. A novel approach to targeting therapy-resistant cells is to engage cell death mechanisms other than apoptosis to eradicate these malignant subpopulations. The overall goal of the proposed studies is to define the lysosomal-mitochondrial inter-organelle signaling mechanisms underlying tumor cell-specific and programmed necrotic lysosomal cell death (LCD) process induced by a number of drugs. Our lead compound hexamethylene amiloride (HMA), a derivative of a drug that has been employed clinically in the management of blood pressure for over forty-five years, kills differentiated and stem cancer cells independent of tumor type, subtype, or species, but does not efficiently kill normal differentiated cells or stem cells. Moreover, HMA kills cancer cells independent of cell cycle, autophagy engagement, and caspase-dependent apoptosis; indeed, cell death appears to result from drug-induced permeabilization of the lysosomal limiting membrane and subsequent cathepsin-mediated plasma membrane rupture. Our observations indicate that efficient HMA-induced cell death requires the production and action of mitochondrially-produced reactive oxygen species (ROS). Our observations also indicate that HMA induces hallmarks of some of the sphingolipidosis lysosomal storage diseases, including the accumulation of a variety of lipid species that are normally broken down by the lysosome. Notably, lipids such as lactosylceramide and lysophosphatidylcholine that have been demonstrated to act as signaling second messengers in the production of mitochondrial ROS accumulate specifically in tumor cells but not normal cells upon HMA treatment. Our observations point to a model where drug-induced aberrant lipid accumulation and ROS-mediated lysosomal membrane lipid oxidation disrupt lysosomal membrane integrity, allowing cathepsin release and induction of necrotic cell death. To test this model, we will use biochemical, cell biological and metabolomics approaches. In Aim 1 we will assess the contribution of bis(monoacylglycerol)phosphate (BMP), a lysosome resident lipid that is suppressed in tumor relative normal cells and is further suppressed with HMA treatment, in regulating lysosomal membrane stability and cell viability via its ability to activate lysosomal enzymes of the sphingomyelin breakdown pathway. Complementing these studies will be an in-depth analysis of lipidomic and metabolomic changes associated with cellular transformation and LCD-inducing agents. In Aim 2, we will examine lysosomal-mitochondrial signaling events that couple mitochondrial ROS production to dysregulated lysosomal lipid metabolism. These studies will uncover lysosomal targets that will allow future development of novel therapeutic agents that more effectively elicit cancer cell-specific programmed necrotic cell death.