The Mitochondrial Calcium Uniporter in Pancreatic Cancer Development, Metastasis, and Treatment

摘要： 胰腺癌(PDAC)是一种特别致命的癌症，导致超过4万人死亡 每年都是美国人。PDAC最常见的诊断是在疾病晚期，明显的 通常会导致死亡的转移。患者的预后进一步受到 通常对目前可用的治疗反应较差。因此，开发一种更强大的 了解导致动脉导管未闭发展和转移的过程，以及 确定新的、更有效的治疗靶点。我们假设线粒体 钙单转运体(MCU)，一种线粒体膜离子通道，可能与癌症有关 发展、转移和肿瘤维持，因此可能提供一种有效的抗癌药物 目标。这种蛋白是内质网钙离子低水平、结构性转移所必需的 癌细胞赖以生存的线粒体的网状结构。上一首 由于缺乏适当的选择性、细胞渗透性的药物，研究一直受到阻碍。 阻止这条通路的药物。因此，我们建议研究MCU在PDAC中的作用 发展、转移和肿瘤维持通过使用遗传方法和 新的动物和细胞培养模式。我们将使用基因编辑技术，例如Cre/lox 和CRISPR/CAS9系统，观察MCU在这些过程中的作用。我们将击倒对手 在PDAC小鼠遗传模型KPCY小鼠的早期发育过程中，MCU 观察该蛋白在肿瘤发生中的作用。我们还将分析细胞增殖、细胞 代谢、耗氧率和线粒体钙通量(通过电生理和 线粒体钙摄取分析)在细胞系和3D培养产生的该模型，如 以及患者来源的细胞系和已建立的人PDAC细胞系PANC-1。我们会重新- 在剔除单元中表示MCU，并在MCU中使用CRISPR/CAS9来剔除MCU 野生型细胞，以确保效果是MCU依赖的。MCU在肿瘤转移中的作用观察 定植后，我们将使用PANC-1细胞表达荧光素酶的尾静脉转移模型 NOD/SCID小鼠，以及跨孔侵袭，凝胶降解分析和Western blotting 上皮细胞向间充质细胞转化的转移相关标志物，如钙粘附素和 基质金属蛋白酶。我们还将使用YFP来量化KPCY模型中的转移 记者基恩。观察MCU在肿瘤维持中的作用，从而确定其治疗方法 在更严重的疾病中，我们将使用可诱导的CRISPR/Cas9基因敲除细胞培养 小鼠PDAC体外模型及观察急性MCU作用的原位模型 在已经生长的肿瘤和细胞中进行消融，作为一种模拟药物抑制的方法。 这些研究将阐明MCU在PDAC的发展、转移和维持中的作用。 值得注意的是，这项工作的结果可能会为筛查的未来发展提供参考 PDAC的方法或治疗方式，有可能挽救生命。

Summary: Pancreatic adenocarcinoma (PDAC) is a particularly lethal form of cancer which kills over 40,000 Americans every year. PDAC is most often diagnosed when disease is advanced, with apparent metastases that often lead to death. Patient outcomes are further negatively impacted by a typically poor response to currently available treatments. It is thus critical to develop a stronger understanding of the processes which lead to PDAC development and metastasis, as well as to determine novel, more efficacious targets for therapies. We hypothesize that the mitochondrial calcium uniporter (MCU), a mitochondrial membrane ion channel, may contribute to cancer development, metastasis, and tumor maintenance, and may therefore present a viable anticancer target. This protein is required for the low-level, constitutive transfer of Ca2+ from the endoplasmic reticulum to the mitochondria that cancer cells appear to be dependent upon for survival. Previous studies have been hampered by the lack of suitably selective, cell-permeable pharmacological agents to block this pathway. We thereby propose to examine the role of MCU in PDAC development, metastasis, and tumor maintenance through the use of genetic approaches and novel animal and cell culture models. We will use genetic editing techniques, such as the Cre/lox and CRISPR/Cas9 systems, to observe the role of MCU in these processes. We will knock out MCU during early development in the murine genetic model of PDAC, the KPCY mouse, to observe the role of this protein in tumor development. We will also assay proliferation, cellular metabolism, oxygen consumption rates, and mitochondrial calcium flux (via electrophysiology and mitochondrial calcium uptake assays) in cell lines and 3D cultures generated from this model, as well as patient-derived cell lines and the established human PDAC cell line, Panc-1. We will re- express MCU in the knockout cells, and we will use CRISPR/Cas9 to knock out MCU in MCU wild-type cells to ensure effects are MCU-dependent. To observe the role of MCU in metastatic colonization, we will use a tail-vein metastasis model with Panc-1 cells expressing luciferase in NOD/SCID mice, as well as transwell invasion, gel degradation assays, and Western blotting for metastasis-associated markers of epithelial to mesenchymal transition, such as cadherins and matrix metalloproteases. We will also quantify metastasis from the KPCY model using the YFP reporter gene. To observe the role of MCU in tumor maintenance and thus establish its therapeutic potential in more advanced disease, we will use an inducible CRISPR/Cas9 knockout cell culture model of murine PDAC in vitro and an orthotopic model to observe the effects of acute MCU ablation in already growing tumors and cells as a method to simulate pharmacological inhibition. These studies will elucidate the role of MCU in PDAC development, metastasis, and maintenance. Significantly, the findings resultant from this work may inform future development of screening methods or treatment modalities in PDAC, potentially saving lives.