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: 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
• 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

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.

胰腺腺癌（PDAC）是一种特别致命的癌症，杀死了40,000多名美国人 每年。 PDAC最常在疾病疾病时被诊断出，转移导致死亡。 对当前可用治疗的典型反应不佳，患者的结果进一步影响。 因此，至关重要的是要对过程进行更深入的了解，从而导致PDAC发展和 转移以及确定新颖，更有效的疗法靶标。低级，构成 最佳生物能和钙的钙转移是内质网（ER） - 最佳生物能量的转移 癌症生存。我们假设这种途径有助于胰腺癌的发展， 转移和肿瘤维持，因此可能提出一个可行的抗癌靶标。 ER定位 IP3R钙释放离子通道和线粒体钙 - 抹骨离子通道，MCU，介导钙 在膜 - 接触部位的两个细胞器之间转移。但是，不可能针对这个 由于缺乏针对这些离子的选择性，可渗透的药理剂，因此体内途径 频道。因此，我们建议检查ER到线粒体钙转移在PDAC中的作用 通过使用新型动物和细胞培养模型的发展，转移和肿瘤维持。我们 将在PDAC的鼠遗传模型（KPCY小鼠的鼠遗传模型中）在早期发育过程中遗传删除MCU， 观察该蛋白在肿瘤发育中的作用。此外，我们将使用肿瘤细胞以及遗传 - 使用CRE/LOX和CRISPR/CAS9系统以及患者衍生的细胞系以及 建立的人类PDAC细胞系，Panc-1。我们将测定增殖，细胞生物能，氧气 消耗率和线粒体钙稳态，使用生化，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，以定义ER到ER的作用 线粒体钙转移在这些过程中。为了确定MCU在转移中的作用，我们将量化 使用敏感的YFP-Reporter基因的KPCY模型中的转移，我们将使用体内尾静脉 具有遗传改性的panc-1细胞的转移模型，表达在NOD/SCID小鼠中以及在 体外移植和凝胶降解测定法，以及生化和形态评估 上皮到间质转变的转移相关标记。观察ER到ER的作用 线粒体钙的维持中的钙转移，因此其治疗潜力更先进 疾病，我们将在体外使用诱导的CRISPR/CAS9细胞培养模型和体内 可诱导的原位模型观察急性MCU消融在已经成长的肿瘤和细胞中的影响 一种模拟深刻药理抑制的方法。这些研究将阐明ER到达的作用 PDAC开发，转移和维护中的线粒体钙转移，它们可能会告知 PDAC中新型治疗靶标的未来开发，有可能挽救生命。