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

总结： 胰腺癌（PDAC）是一种特别致命的癌症， 每年都有美国人。PDAC最常在疾病进展时被诊断， 转移往往导致死亡。患者结局进一步受到以下因素的负面影响： 通常对目前可用的治疗反应不佳。因此，至关重要的是， 了解导致PDAC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 钙单向转运体（MCU）是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，因此可能是一种可行的抗癌药物。 目标这种蛋白质是必需的低水平，组成性转移钙+从内质网 癌细胞似乎依赖于线粒体来生存。先前 由于缺乏合适的选择性、细胞渗透性的药理学药物， 代理人来阻止这条途径。因此，我们建议研究MCU在PDAC中的作用 发展，转移和肿瘤的维持，通过使用遗传方法， 新的动物和细胞培养模型。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。我们将淘汰 在PDAC小鼠遗传模型KPCY小鼠的早期发育过程中， 观察该蛋白在肿瘤发生中的作用。我们还将检测增殖，细胞 代谢、氧消耗率和线粒体钙通量（通过电生理学和 线粒体钙摄取测定），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- 在敲除细胞中表达MCU，我们将使用CRISPR/Cas9敲除MCU中的MCU 野生型细胞，以确保效应是MCU依赖性的。观察MCU在肿瘤转移中的作用， 定植，我们将使用尾静脉转移模型与Panc-1细胞表达荧光素酶， NOD/SCID小鼠，以及transwell侵袭，凝胶降解试验和Western印迹法， 上皮向间质转化的转移相关标志物，如钙粘蛋白和 基质金属蛋白酶我们还将使用YFP定量KPCY模型的转移 报告基因观察MCU在肿瘤维持中的作用，从而确定其治疗作用。 在更晚期的疾病中，我们将使用诱导型CRISPR/Cas9敲除细胞培养物， 建立小鼠PDAC体外模型和原位模型，观察急性MCU的影响 在已经生长的肿瘤和细胞中进行消融，作为模拟药理学抑制的方法。 这些研究将阐明MCU在PDAC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告知未来的发展筛选 PDAC的方法或治疗方式，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.