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: 10679078
• 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/10679078
• 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; Life; 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; Play; Process; Prognosis; Proliferating; Property; Proteins; Regulation; Renal carcinoma; Reporter Genes; Role; 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; pharmacologic; 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4万美国人死亡 每年都有。PDAC最常见的诊断是在疾病晚期，有导致死亡的转移。 患者的结果进一步受到对现有治疗的典型不良反应的负面影响。 因此，对导致PDAC开发和开发的流程有更深入的了解是至关重要的 研究转移，以及确定新的、更有效的治疗靶点。低层次的，结构性的 内质网(ER)到线粒体的钙转移是最佳生物能量学和 癌细胞存活。我们假设这一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一转运体离子通道(MCU)调节钙离子 在膜接触部位的两个细胞器之间传递。然而，不可能针对这一点 体内途径，因为缺乏针对这些离子的选择性、细胞渗透性的药理学试剂 频道。因此，我们建议研究内质网到线粒体的钙转运在PDAC中的作用 通过使用新的动物和细胞培养模型来发展、转移和维持肿瘤。我们 将在PDAC(KPCY小鼠)的小鼠遗传模型的早期发育中从基因上删除MCU，以 观察该蛋白在肿瘤发生中的作用。此外，我们将使用肿瘤细胞以及基因- 使用CRE/LOX和CRISPR/Cas9系统的修饰细胞，以及患者来源的细胞系和 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，使用生化、细胞生物学和生物物理学 包括电生理学、活细胞成像和荧光测定法在内的方法，以确定内质网到细胞外信号通路的作用。 线粒体在这些过程中的钙转移。为了确定MCU在转移中的作用，我们将量化 使用敏感的YFP报告基因的KPCY模型中的转移，我们将使用体内尾静脉 表达荧光素酶基因修饰的PANC-1细胞在NOD/SCID小鼠体内的转移模型 体外跨井侵袭和凝胶降解试验，以及生化和形态评价 上皮向间充质转化的转移相关标记物。观察ER-to-ER的作用 线粒体钙转移在肿瘤维持中的作用及其治疗潜能的进一步提高 疾病，我们将使用可诱导的小鼠PDAC的CRISPR/Cas9细胞培养模型在体外和体内 可诱导原位模型观察急性MCU消融对已经生长的肿瘤和AS细胞的影响 一种模拟深层药物抑制的方法。这些研究将阐明内质网到内质网的作用。 线粒体钙转运在PDAC发生、转移和维持中的作用及其可能的信息 PDAC新治疗靶点的未来发展，有可能挽救生命。