Endoplasmic Reticulum-to-Mitochondria Calcium Transfer in Pancreatic Cancer Development, Metastasis, and Treatment

胰腺癌发生、转移和治疗中的内质网至线粒体钙转移

基本信息

批准号：
10208636
负责人：
James Kevin FOSKETT
金额：
$ 52.66万
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：
2021-08-15 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10208636
关键词：
Ablation Acute American Animal Model Animals Autophagocytosis Biochemical Bioenergetics Biological Biological Assay Biophysics CRISPR/Cas technology Calcium Cancer Patient Cancer cell line Cause of Death Cell Culture Techniques Cell Line Cell Survival Cells Cellular Metabolic Process Cessation of life Citric Acid Cycle Cytoplasm Dependence Development Diagnosis Disease Early Diagnosis Early identification Electrophysiology (science)Endoplasmic Reticulum Fibroblasts Future Gel Genetic Genetic Models Growth Homeostasis Human ITPR1 gene Immune response In Vitro Interruption Invaded Ion Channel Lead Luciferases Maintenance Malignant Neoplasms Malignant neoplasm of pancreas Malignant neoplasm of prostate Mediating Membrane Metabolic Metabolism Methods Mitochondria Modeling Molecular Morphology Mus NOD/SCID mouse Neoplasm Metastasis Normal Cell Organelles Oxidoreductase Oxygen Consumption Pancreatic Adenocarcinoma Pancreatic Ductal Adenocarcinoma Pathway interactions Patient-Focused Outcomes Patients Permeability Pharmaceutical Preparations Pharmacology Play Process Prognosis Property Proteins Regulation Renal carcinoma Reporter Genes Role Savings Signal Transduction Site Survival Rate System Tail Technology Therapeutic Veins Work advanced disease anti-cancer anti-cancer therapeutic biophysical techniques calcium uniporter cancer cell cancer therapy cell motility cell type designer receptors exclusively activated by designer drugs epithelial to mesenchymal transition falls genetic predictors genetically modified cells improved in vivo innovation live cell imaging malignant breast neoplasm migration mouse model neoplastic cell new therapeutic target novel novel therapeutics pancreatic cancer patients pancreatic cell line response targeted treatment therapeutic target three-dimensional modeling tumor tumor progression tumorigenesis tumorigenic uptake

项目摘要

Pancreatic adenocarcinoma (PDAC) is a particularly lethal form of cancer that kills over 40,000 Americans every year. PDAC is most often diagnosed when disease is advanced, with metastases that lead to death. Patient outcomes are further negatively-impacted by a typical 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. Low-level, constitutive endoplasmic reticulum (ER)-to-mitochondria transfer of calcium is required for optimal bioenergetics and cancer-cell survival. We hypothesize that this pathway contributes to pancreatic cancer development, metastasis, and tumor maintenance, and may therefore present a viable anticancer target. The ER-localized IP3R calcium-release ion channel and the mitochondrial calcium-uniporter ion channel, MCU, mediate calcium transfer between the two organelles at membrane-contact sites. However, it has been impossible to target this pathway in vivo because of the lack of selective, cell-permeable pharmacological agents against these ion channels. We therefore propose to examine the role of ER-to-mitochondria calcium transfer in PDAC development, metastasis, and tumor maintenance through the use of novel animal and cell-culture models. We will genetically delete MCU during early development in a murine genetic-model of PDAC, the KPCY mouse, to observe the role of this protein in tumor development. In addition, we will use tumor cells as well as genetically- modified cells using Cre/lox and CRISPR/Cas9 systems, as well as patient-derived cell lines and the established human PDAC cell line, Panc-1. We will assay proliferation, cellular bioenergetics, oxygen consumption rates, and mitochondrial calcium homeostasis, using biochemical, cell biological and biophysical approaches, including electrophysiology, live-cell imaging and fluorimetry, to define the role of ER-to- mitochondria calcium transfer in these processes. To determine the role of MCU in metastasis, we will quantify metastasis in the KPCY model using the sensitive YFP-reporter gene, and we will use an in vivo tail-vein metastasis model with genetically-modified Panc-1 cells expressing luciferase in NOD/SCID mice, as well as in vitro transwell-invasion and gel-degradation assays, and biochemical and morphological assessment of metastasis-associated markers of epithelial-to-mesenchymal transition. To observe the role of ER-to- mitochondria calcium transfer in tumor maintenance and thus its therapeutic potential for more advanced disease, we will use an inducible CRISPR/Cas9 cell-culture model of murine PDAC in vitro and an in vivo inducible orthotopic model to observe the effects of acute MCU ablation in already-growing tumors and cells as a method to simulate profound pharmacological inhibition. These studies will elucidate the role of ER-to- mitochondria calcium transfer in PDAC development, metastasis, and maintenance, and they may inform future development of novel therapeutic targets in PDAC, potentially saving lives.

胰腺癌 (PDAC) 是一种特别致命的癌症，导致超过 40,000 名美国人死亡每年。 PDAC 最常在疾病进展时被诊断出来，出现转移导致死亡。对当前可用治疗的典型反应不佳进一步对患者治疗结果产生负面影响。因此，对导致 PDAC 开发和实施的流程有更深入的了解至关重要。转移，以及确定新的、更有效的治疗靶点。低级、本构型内质网 (ER) 至线粒体的钙转移对于最佳生物能学和癌细胞的存活。我们假设该途径有助于胰腺癌的发展，转移和肿瘤维持，因此可能是一个可行的抗癌靶点。 ER 本地化 IP3R 钙释放离子通道和线粒体钙单向转运蛋白离子通道，MCU，介导钙两个细胞器在膜接触位点之间的转移。然而，却无法以此为目标由于缺乏针对这些离子的选择性、细胞渗透性药理学试剂，体内途径渠道。因此，我们建议研究 ER 至线粒体钙转移在 PDAC 中的作用通过使用新型动物和细胞培养模型来研究肿瘤的发展、转移和维持。我们将在 PDAC 小鼠遗传模型（KPCY 小鼠）的早期发育过程中从基因上删除 MCU，以观察该蛋白质在肿瘤发展中的作用。此外，我们将使用肿瘤细胞以及基因- 使用 Cre/lox 和 CRISPR/Cas9 系统修饰细胞，以及患者来源的细胞系和建立了人PDAC细胞系Panc-1。我们将检测增殖、细胞生物能量学、氧气使用生物化学、细胞生物学和生物物理学来测量消耗率和线粒体钙稳态方法，包括电生理学、活细胞成像和荧光测定法，来定义 ER 的作用这些过程中线粒体钙转移。为了确定 MCU 在转移中的作用，我们将量化使用敏感的 YFP 报告基因在 KPCY 模型中进行转移，我们将使用体内尾静脉使用表达荧光素酶的转基因 Panc-1 细胞在 NOD/SCID 小鼠以及体外 Transwell 侵袭和凝胶降解测定，以及生化和形态学评估上皮间质转化的转移相关标志物。观察 ER 的作用线粒体钙转移在肿瘤维持中的作用及其更先进的治疗潜力疾病，我们将使用小鼠 PDAC 的体外诱导型 CRISPR/Cas9 细胞培养模型和体内模型诱导原位模型观察急性 MCU 消融对已经生长的肿瘤和细胞的影响一种模拟深度药理抑制的方法。这些研究将阐明 ER 的作用 PDAC 发育、转移和维持中线粒体钙转移，它们可能会提供信息未来开发 PDAC 新型治疗靶点，有可能挽救生命。