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：胰腺腺癌（PDAC）是一种特别致命的癌症，可杀死40,000多人每年的美国人。 PDAC最常在疾病疾病时被诊断出，显而易见经常导致死亡的转移。患者的结果进一步受到通常对当前可用治疗的反应差。因此，建立更强大的了解导致PDAC发展和转移的过程，以及确定新颖，更有效的疗法靶标。我们假设线粒体线粒体膜离子通道钙Uniporter（MCU）可能有助于癌症发育，转移和肿瘤维持，因此可能提出可行的抗癌目标。该蛋白需要从内质中Ca2+的低水平，本构转移癌细胞似乎取决于生存的线粒体的网状。以前的缺乏适当的选择性，可渗透的药理学，阻碍了研究阻止这一路径的代理。因此，我们建议检查MCU在PDAC中的作用通过使用遗传方法的发展，转移和肿瘤维持新型动物和细胞培养模型。我们将使用遗传编辑技术，例如CRE/LOX 和CRISPR/CAS9系统，以观察MCU在这些过程中的作用。我们会淘汰 MCU在PDAC的鼠遗传模型（KPCY小鼠）的早期发育过程中观察该蛋白在肿瘤发育中的作用。我们还将测定增殖，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分析）在该模型产生的细胞系和3D培养物中，AS 以及患者来源的细胞系和已建立的人PDAC细胞系Panc-1。我们将重新在敲除单元中表达MCU，我们将使用CRISPR/CAS9在MCU中淘汰MCU 野生型细胞以确保效应是MCU依赖性的。观察MCU在转移性中的作用定植，我们将使用带有panc-1细胞表达荧光素酶的尾静脉转移模型点头/SCID小鼠，以及Transwell入侵，凝胶降解测定和蛋白质印迹上皮与间充质转变的转移相关标记，例如钙粘蛋白和基质金属蛋白酶。我们还将使用YFP从KPCY模型量化转移记者基因。观察MCU在肿瘤维持中的作用，从而建立其治疗性在更晚期疾病中的潜力，我们将使用诱导的CRISPR/CAS9敲除细胞培养体外鼠PDAC模型和原位模型，以观察急性MCU的影响在已经生长的肿瘤和细胞中消融是一种模拟药理抑制作用的方法。这些研究将阐明MCU在PDAC开发，转移和维持中的作用。值得注意的是，这项工作所产生的发现可能会为筛查的未来发展提供信息 PDAC中的方法或治疗方式，有可能挽救生命。