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

线粒体钙单向转运蛋白在胰腺癌发生、转移和治疗中的作用

基本信息

批准号：
10434660
负责人：
Jillian Weissenrieder
金额：
$ 6.76万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-03-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10434660
关键词：
3-Dimensional Ablation Acute Adenocarcinoma Cell American Animals Biological Biological Assay Biological Markers CRISPR/Cas technology Cadherins Calcium Cancer Patient Cell Culture Techniques Cell Line Cell Survival Cells Cessation of life Citric Acid Cycle DNA biosynthesis Data Development Diagnosis Disease Early Diagnosis Electrophysiology (science)Endoplasmic Reticulum Ensure Enzymes Extravasation Flow Cytometry Future Gel Genetic Genetic Models Growth Homeostasis Human Image Immune response In Vitro Injections Ion Channel KRAS2 gene Knock-out Lead Luciferases Maintenance Malignant Neoplasms Malignant neoplasm of pancreas Measures Metabolic Metabolism Metalloproteases Methods Mitochondria Mitochondrial Membrane Protein Mitoxantrone Modality Modeling Molecular Morphology Mus Mutation NOD/SCID mouse Neoplasm Metastasis Normal Cell Organoids Outcome Oxygen Consumption Pancreas Pancreatic Adenocarcinoma Pancreatic Ductal Adenocarcinoma Pathway interactions Patient-Focused Outcomes Patients Permeability Pharmacology Process Prognosis Property Proteins Reporter Genes Research Respiratory Chain Role Ruthenium Savings Survival Rate System Tail Techniques Testing Therapeutic Time Veins Weight Western Blotting Work Xenograft Model Xenograft procedure advanced disease anti-cancer anti-cancer therapeutic base calcium uniporter cancer cell cell motility cell type epithelial to mesenchymal transition falls genetic approach improved in vivo inhibitor innovation knock-down migration mitochondrial membrane mouse model neoplastic cell novel pancreas development release of sequestered calcium ion into cytoplasm response screening targeted treatment therapeutic target three dimensional cell culture transcriptome sequencing treatment response tumor tumorigenesis uptake

项目摘要

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.

概括：胰腺癌 (PDAC) 是一种特别致命的癌症，导致超过 40,000 人死亡美国人每年。 PDAC 最常在疾病进展时诊断，有明显的症状转移常常导致死亡。患者的治疗效果进一步受到以下因素的负面影响：通常对当前可用的治疗反应不佳。因此，发展更强大的了解导致 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蛋白（MCU）是一种线粒体膜离子通道，可能导致癌症发展、转移和肿瘤维持，因此可能是一种可行的抗癌药物目标。该蛋白质是从内质中低水平、组成性转移 Ca2+ 所必需的网织到线粒体，癌细胞似乎依赖线粒体生存。以前的由于缺乏适当选择性、细胞渗透性的药理学药物，研究受到阻碍阻断该途径的药物。因此，我们建议研究 MCU 在 PDAC 中的作用通过使用遗传方法和方法来控制肿瘤的发展、转移和维持新颖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 MCU 在 PDAC 小鼠遗传模型（KPCY 小鼠）的早期发育过程中，观察该蛋白质在肿瘤发展中的作用。我们还将检测增殖、细胞新陈代谢、耗氧率和线粒体钙通量（通过电生理学和线粒体钙摄取测定）在从此模型生成的细胞系和 3D 培养物中，如以及患者来源的细胞系和已建立的人类 PDAC 细胞系 Panc-1。我们将重新在敲除细胞中表达MCU，我们将使用CRISPR/Cas9在MCU中敲除MCU 野生型细胞，以确保效果依赖于 MCU。观察MCU在转移中的作用定植后，我们将使用尾静脉转移模型，其中 Panc-1 细胞表达荧光素酶 NOD/SCID 小鼠，以及 Transwell 侵袭、凝胶降解测定和蛋白质印迹上皮间质转化的转移相关标志物，例如钙粘蛋白和基质金属蛋白酶。我们还将使用 YFP 量化 KPCY 模型的转移报告基因。观察MCU在肿瘤维持中的作用，从而建立其治疗方法在更晚期疾病中的潜力，我们将使用诱导型 CRISPR/Cas9 敲除细胞培养物小鼠 PDAC 体外模型和观察急性 MCU 影响的原位模型消融已经生长的肿瘤和细胞作为模拟药理抑制的方法。这些研究将阐明 MCU 在 PDAC 发育、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信息 PDAC 的方法或治疗方式有可能挽救生命。