Lysosomal-mitochondrial signaling in non-apoptotic cancer cell death

项目摘要 绝大多数常规和靶向化疗药物在临床使用或 发展依赖于参与凋亡途径以引起肿瘤细胞死亡。然而，抗凋亡- 诱导剂是一个特别棘手的临床问题。一种靶向治疗抗性细胞的新方法是 参与细胞凋亡以外的细胞死亡机制来根除这些恶性亚群。的 所提出的研究的总体目标是确定溶酶体-线粒体细胞器间信号传导 肿瘤细胞特异性和程序性坏死性溶酶体细胞死亡（LCD）过程的潜在机制 由多种药物引起。我们的主要化合物阿替米洛利（HMA），一种药物的衍生物， 已经在临床上用于血压管理超过45年， 和干细胞癌细胞独立于肿瘤类型，亚型，或物种，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，和半胱天冬酶依赖性凋亡;事实上，细胞死亡似乎是由于药物诱导的细胞凋亡。 溶酶体限制膜的透化和随后的组织蛋白酶介导的质膜 破裂我们的观察表明，高效的HMA诱导的细胞死亡需要以下物质的产生和作用： 体内产生的活性氧（ROS）。我们的观察还表明，HMA诱导 一些鞘脂病溶酶体贮积病的标志，包括各种 通常被溶酶体分解的脂质种类。值得注意的是，脂质如乳糖神经酰胺和 溶血磷脂酰胆碱已被证明作为信号第二信使在生产 线粒体活性氧的积累，特别是在肿瘤细胞，而不是正常细胞后，HMA治疗。我们 观察结果指向一个模型，其中药物诱导的异常脂质蓄积和ROS介导的溶酶体 膜脂质氧化破坏溶酶体膜的完整性，使组织蛋白酶释放和诱导 坏死性细胞死亡为了测试这个模型，我们将使用生物化学，细胞生物学和代谢组学方法。 在目标1中，我们将评估双（单酰基甘油）磷酸（BMP），溶酶体驻留脂质的贡献 其在肿瘤相对正常细胞中被抑制，并且在HMA治疗中被进一步抑制， 溶酶体膜稳定性和细胞活力，通过其激活溶酶体酶的能力， 鞘磷脂分解途径。作为对这些研究的补充，将对脂质组学和 与细胞转化和LCD诱导剂相关的代谢组学变化。在目标2中，我们 研究溶酶体-线粒体信号传导事件，其将线粒体ROS产生与失调 溶酶体脂质代谢这些研究将揭示溶酶体靶点，这将使未来的发展， 更有效地引发癌细胞特异性程序性坏死细胞死亡的新治疗剂。

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.