Disrupting glutathione dependency in pancreatic cancer

破坏胰腺癌的谷胱甘肽依赖性

基本信息

批准号：
10526093
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
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-07 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10526093
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 GPX2 gene Gene Expression Gene Expression Regulation Genes Genetic Glutamates Glutathione Glycine Goals Growth HDAC1 gene Immunohistochemistry Immunotherapy In Vitro KRAS oncogenesis Malignant Neoplasms Malignant neoplasm of liver Malignant neoplasm of pancreas Mentorship Metabolic Metabolic Pathway Metabolism Methods Molecular Nutrient Pancreatic Ductal Adenocarcinoma Pancreatic Ductal Carcinoma Pathway interactions Patients Pentosephosphate Pathway Persons Pharmacology Phase Regulation Research Resistance Role Sampling Skin Cancer Small Interfering RNA Source Starvation Stress Survival Rate Techniques Testing Training Tumor-associated macrophages United States Vascular blood supply Withdrawal amino acid metabolism base cancer cell chemotherapy deprivation dietary epigenetic regulation epigenomics experimental study extracellular glutathione peroxidase glutathione synthase glutathione transporter improved in vivo inhibitor insight knockout gene macrophage metabolomics mouse model pancreatic cancer cells prevent promoter response skills small hairpin RNA stable isotope survival outcome targeted treatment therapy outcome therapy resistant transcriptome sequencing tumor tumor growth tumor immunology tumor metabolism tumor microenvironment

项目摘要

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，具有高度致死性和鉴于只有约 10% 的患者对治疗有抵抗力，因此需要探索新的、有效的策略来治疗 PDAC。 PDAC 患者会过度利用细胞外营养来维持生长。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。提案中，我们表明 PDAC 细胞对半胱氨酸的依赖程度远高于其他代谢组学。分析显示，PDAC 细胞几乎仅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 和生存另外，我们发现在同样的半胱氨酸饥饿下，巨噬细胞会产生GSH，即GSH。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，并且巨噬细胞衍生的 GSH 可以维持 PDAC 的多个基因表达。基于这些患者肿瘤数据集，我们观察到 PDAC 表达高水平的 GSH 通路基因。数据显示，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。本研究的目的是 1) 确定 PDAC 对 GSH 依赖的分子机制 – 包括GSH通路的表观遗传调控，以及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断。单独使用 GSH 或与巨噬细胞活动一起使用可能是改善 PDAC 治疗的一种方法。将在 K99 阶段进行，而目标 2 的大部分将在 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）、该项目将接受 5 人的输入。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。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。