Disrupting glutathione dependency in pancreatic cancer

破坏胰腺癌的谷胱甘肽依赖性

• 批准号: 10661797
• 负责人: Zeribe Chike Nwosu
• 金额: $ 13.18万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-07 至 2023-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10661797
• 关键词: Amino Acids; Antioxidants; Arginine; Automobile Driving; Bioinformatics; Biological Assay; CRISPR/Cas technology; Cancer Etiology; Cell Culture Techniques; Cell Line; Cell surface; Cells; Cessation of life; Coculture Techniques; Colorectal Cancer; Consumption; Coupled; Cysteine; Cytometry; Data; Data Set; Dependence; Epigenetic Process; Fibroblasts; Flow Cytometry; Future; G6PD gene; Gene Expression; Gene Expression Regulation; Genes; Genetic; Glutamates; Glutathione; Glycine; Goals; Growth; HDAC1 gene; Immunohistochemistry; Immunotherapy; In Vitro; Infiltration; KRAS oncogenesis; Macrophage; Malignant Neoplasms; Malignant neoplasm of liver; Malignant neoplasm of pancreas; Mentorship; Metabolic; Metabolic Pathway; Metabolism; Methods; Molecular; Nutrient; Nutrient availability; Nutritional Requirements; Pancreatic Ductal Adenocarcinoma; Pancreatic Ductal Carcinoma; Pathway interactions; Patients; Pentosephosphate Pathway; Persons; Phase; Regulation; Research; Resistance; Role; Sampling; Skin Cancer; Small Interfering RNA; Sorting; Source; Starvation; Stress; Survival Rate; Techniques; Testing; Training; Tumor-associated macrophages; United States; Vascular blood supply; Withdrawal; amino acid metabolism; cancer cell; chemotherapy; deprivation; dietary; epigenetic regulation; epigenomics; experimental study; extracellular; glutathione peroxidase; glutathione synthase; glutathione transporter; improved; in vivo; inhibitor; insight; knockout gene; metabolomics; mouse model; pancreatic cancer cells; pharmacologic; prevent; promoter; response; skills; small hairpin RNA; stable isotope; survival outcome; synergism; targeted treatment; therapy outcome; therapy resistant; transcriptome sequencing; tumor; tumor growth; tumor immunology; tumor metabolism; tumor microenvironment

PROJECT SUMMARY/ABSTRACT Pancreatic ductal carcinoma (PDAC) is the most common form of pancreatic cancer and is highly lethal and resistant to therapy. There is a need to explore new, effective, strategies to treat PDAC, given that only ~10% of the patients survive beyond five years. PDAC overutilize extracellular nutrients to sustain their growth. This nutrient dependency, coupled with a low blood supply, limits nutrient availability in the PDAC microenvironment. To achieve therapy and improve patient survival outcome, it is important to understand how PDAC survive in the nutrient-limited condition and the tumor-intrinsic or microenvironmental factors that sustain their survival. In this proposal, we show that PDAC cells rely on cysteine at a far greater extent than other amino acids. Metabolomics profiling revealed that the PDAC cells almost exclusively use cysteine to sustain intracellular glutathione (GSH). While some PDAC cells rapidly generate GSH when starved of cysteine, others maintain their GSH pool when starved of both cysteine and arginine, indicating the use of various mechanisms to sustain GSH and survival in PDAC cells. In addition, we found that under the same cysteine starvation, macrophages produce GSH, which is an important discovery given the high abundance of macrophages in PDAC microenvironment, their arginine catabolic function, and that the macrophage-derived GSH could sustain PDAC. In multiple gene expression datasets of patient tumors, we observed that PDAC express a high level of GSH pathway genes. Based on these data, we hypothesize that GSH is a core nutrient required for PDAC growth, is potentially sustained by tumor-associated macrophages, and that disrupting GSH utilization could improve therapy in PDAC. The aims of this study are 1). to determine the molecular mechanisms driving the dependency of PDAC on GSH – including the epigenetic regulation of GSH pathway, and 2). to determine the role of tumor-associated macrophages as a source and modulator of GSH in PDAC. The overarching goal is to explore whether blocking GSH utilization alone or alongside macrophage activities could be a way to improve PDAC therapy. Aim 1 will be pursued at the K99 phase, while most of Aim 2 will be pursued at the R00 phase. Methods will include gene interference (e.g., CRISPR/Cas9, shRNA, siRNA), pharmacological inhibitors of GSH pathways (including the pentose phosphate pathway), cell culture assays, metabolomics (including stable isotope tracing), dietary mouse models, bioinformatics, promoter analysis/epigenetic methods, RNA sequencing (single cell and bulk), immunohistochemistry, flow cytometry and mass cytometry. The project will receive input from a 5-person mentorship team that have expertise in tumor immunology, metabolism, bioinformatics, and epigenetics. The expected results could a) offer new insights on disrupting GSH pathway to suppress PDAC growth, b) reveal new microenvironmental mechanisms that enable tumor adaptation in nutrient-limited state, and c) reveal new opportunities to overcome resistance to chemotherapy or immunotherapy in PDAC.

项目概要/摘要 胰腺导管癌 (PDAC) 是胰腺癌最常见的形式，具有高度致死性和 对治疗有抵抗力。鉴于只有约 10% 的患者患有 PDAC，因此需要探索新的、有效的策略来治疗 PDAC。 患者存活超过五年。 PDAC 过度利用细胞外营养物质来维持其生长。这 营养依赖性加上血液供应不足，限制了 PDAC 微环境中营养的可用性。 为了实现治疗并改善患者生存结果，了解 PDAC 如何在环境中生存非常重要 营养有限的条件和维持其生存的肿瘤内在或微环境因素。在这个 提案中，我们发现 PDAC 细胞对半胱氨酸的依赖程度远高于其他氨基酸。代谢组学 分析显示，PDAC 细胞几乎完全使用半胱氨酸来维持细胞内谷胱甘肽 (GSH)。 虽然一些 PDAC 细胞在缺乏半胱氨酸时会快速生成 GSH，但其他细胞在缺乏半胱氨酸时仍会维持其 GSH 库。 缺乏半胱氨酸和精氨酸，表明使用各种机制来维持 GSH 和生存 PDAC 细胞。此外，我们发现在同样的半胱氨酸饥饿下，巨噬细胞会产生GSH，这 鉴于 PDAC 微环境中巨噬细胞的丰富性，它们的精氨酸是一个重要的发现 分解代谢功能，巨噬细胞衍生的 GSH 可以维持 PDAC。在多基因表达中 在患者肿瘤数据集中，我们观察到 PDAC 表达高水平的 GSH 通路基因。基于这些 根据数据，我们假设 GSH 是 PDAC 生长所需的核心营养素，可能通过以下方式维持： 肿瘤相关巨噬细胞，并且破坏 GSH 的利用可以改善 PDAC 的治疗。这 本研究的目的是1)。确定驱动 PDAC 对 GSH 依赖性的分子机制 – 包括 GSH 途径的表观遗传调控，2)。确定肿瘤相关的作用 巨噬细胞作为 PDAC 中 GSH 的来源和调节剂。首要目标是探索是否会阻塞 单独利用 GS​​H 或与巨噬细胞活动一起利用可能是改善 PDAC 治疗的一种方法。目标1将 目标2的大部分内容将在R00阶段进行。方法将包括基因 干扰（例如 CRISPR/Cas9、shRNA、siRNA）、GSH 途径的药理学抑制剂（包括 磷酸戊糖途径）、细胞培养测定、代谢组学（包括稳定同位素示踪）、饮食小鼠 模型、生物信息学、启动子分析/表观遗传学方法、RNA 测序（单细胞和批量）、 免疫组织化学、流式细胞术和质量细胞术。该项目将收到 5 人的意见 导师团队拥有肿瘤免疫学、代谢、生物信息学和表观遗传学方面的专业知识。这 预期结果可以 a) 提供关于破坏 GSH 通路以抑制 PDAC 生长的新见解，b) 揭示 新的微环境机制使肿瘤能够适应营养有限的状态，c）揭示了新的 克服 PDAC 化疗或免疫治疗耐药性的机会。