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.

项目摘要/摘要胰腺导管癌（PDAC）是胰腺癌最常见的形式，高度致命，并且对治疗的抗药性。鉴于只有约10％患者的生存超过五年。 PDAC过度利用细胞外营养以维持其生长。这养分依赖性，再加上血液供应低，限制了PDAC微环境中的养分利用率。为了实现治疗并改善患者的生存结果，重要的是要了解PDAC如何生存营养限制的状况以及维持其存活率的肿瘤内部或微环境因素。在这个提案，我们表明PDAC细胞比其他氨基酸更大程度地依赖半胱氨酸。代谢组学分析表明，PDAC细胞几乎完全使用半胱氨酸来维持细胞内谷胱甘肽（GSH）。虽然某些PDAC细胞在半胱氨酸饿死时会迅速产生GSH，而其他PDAC细胞则保持其GSH池同时饿死的半胱氨酸和精氨酸，表明使用各种机制来维持GSH和生存 PDAC细胞。此外，我们发现在相同的半胱氨酸饥饿下，巨噬细胞产生了GSH，这是GSH的，考虑到PDAC微环境中巨噬细胞的高抽象，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。在多基因表达中患者肿瘤的数据集，我们观察到PDAC表达了高水平的GSH途径基因。基于这些数据，我们假设GSH是PDAC生长所需的核心营养素，可能会受到与肿瘤相关的巨噬细胞以及破坏GSH利用的巨噬细胞可以改善PDAC的治疗。这项研究的目的是1）。确定驱动PDAC依赖性GSH的分子机制包括GSH途径的表观遗传调节和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。总体目标是探索是否阻止单独或与巨噬细胞活动一起使用GSH可以是改善PDAC治疗的一种方法。目标1意志在K99阶段进行追踪，而AIM 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途径的药物抑制剂（包括戊糖磷酸盐途径），细胞培养测定，代谢组学（包括稳定的同位素追踪），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（单细胞和大量），免疫组织化学，流式细胞仪和质量细胞术。该项目将收到5人的意见具有肿瘤免疫学，代谢，生物信息学和表观遗传学方面的专业知识的资术团队。这预期结果a）关于破坏抑制PDAC增长的GSH途径的新见解，b）揭示新的微环境机制，可以在营养限制状态下适应肿瘤，并且c）揭示了新的在PDAC中克服对化学疗法或免疫疗法的抗性的机会。