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

• 批准号: 10443604
• 负责人: James Kevin FOSKETT
• 金额: $ 51.61万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 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/10443604
• 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

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，伴有导致死亡的转移。 患者结局进一步受到当前可用治疗的典型不良反应的负面影响。 因此，至关重要的是要更好地理解导致PDAC发展的过程， 转移，以及确定新的，更有效的治疗靶点。低水平，组成型 钙从内质网（ER）到线粒体的转移是最佳生物能量学所必需的， 癌细胞存活率我们假设这一途径有助于胰腺癌的发展， 转移和肿瘤维持，并且因此可以呈现可行的抗癌靶点。ER定位 IP 3R钙释放离子通道和线粒体钙单向转运体离子通道MCU介导钙 两个细胞器在膜接触部位之间的转移。然而，一直无法以此为目标 由于缺乏针对这些离子的选择性的、细胞可渗透的药理学试剂， 渠道因此，我们建议研究ER向线粒体的钙转移在PDAC中的作用。 发展，转移和肿瘤的维持，通过使用新的动物和细胞培养模型。我们 将在PDAC小鼠遗传模型KPCY小鼠的早期发育过程中遗传删除MCU， 观察该蛋白在肿瘤发生中的作用。另外，我们会用肿瘤细胞和基因- 使用Cre/lox和CRISPR/Cas9系统修饰的细胞，以及患者来源的细胞系， 建立人PDAC细胞系Panc-1。我们将检测细胞增殖细胞生物能量学氧气 消耗率和线粒体钙稳态，使用生物化学，细胞生物学和生物物理学 方法，包括电生理学，活细胞成像和荧光测定法，以确定ER的作用， 线粒体的钙离子转运。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模型中观察转移，并且我们将使用体内尾静脉 在NOD/SCID小鼠中用表达荧光素酶的基因修饰的Panc-1细胞转移模型，以及在 体外transwell侵袭和凝胶降解试验，以及生物化学和形态学评估 上皮细胞向间质细胞转化的转移相关标志物。观察ER对 线粒体钙转移在肿瘤维持中的作用，因此它对更先进的治疗潜力 疾病，我们将在体外和体内使用鼠PDAC的诱导型CRISPR/Cas9细胞培养模型， 诱导原位模型，以观察急性MCU消融在已经生长的肿瘤和细胞中的作用， 一种模拟深度药理学抑制的方法。这些研究将阐明ER对 线粒体钙转运在PDAC的发展、转移和维持中起重要作用， 在PDAC中的新的治疗靶点的未来发展，可能挽救生命。