Role of the hexosamine biosynthesis pathway in pancreatic cancer.

己糖胺生物合成途径在胰腺癌中的作用。

• 批准号: 9327535
• 负责人: Sydney Campbell
• 金额: $ 4.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-31 至 2020-08-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9327535
• 关键词: 2-acylglycerol O-acyltransferase; Adenocarcinoma Cell; Adhesions; Anabolism; Cancer Model; Cell Culture Techniques; Cell Line; Cell membrane; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Development; Disease; Endoplasmic Reticulum; Enzymes; Fatty Acids; Galactose Binding Lectin; Generations; Glucosamine; Glucose; Glutamine; Glycoproteins; Golgi Apparatus; Growth; Growth Factor Receptors; Growth and Development function; Hexosamines; In Vitro; Injectable; KRAS2 gene; Knock-out; Lectin; Link; Maintenance; Malignant Neoplasms; Malignant neoplasm of pancreas; Mass Spectrum Analysis; Membrane; Membrane Proteins; Metabolic; Modeling; Modification; Molecular; Monitor; Mus; N-Acetylglucosaminyltransferases; N-Glycosylation Site; Neoplasm Metastasis; Nutrient; Nutritional; Oncogenic; Oxygen; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Polysaccharides; Proteins; Regulation; Role; Serum; Signal Transduction; Site; Staining method; Stains; Structure; Tail; Testing; Transaminases; Up-Regulation; Uridine Diphosphate N-Acetylglucosamine; Veins; cell motility; comparative; detection of nutrient; experimental study; fructose-6-phosphate; glucose uptake; glycoproteomics; glycosylation; in vivo; insight; migration; mouse model; mutant; neoplastic cell; new therapeutic target; nucleotide metabolism; protein folding; sugar; therapeutic target; tumor; tumor growth; tumor microenvironment; tumor progression

PROJECT SUMMARY Over 90% of pancreatic ductal adenocarcinomas (PDAC) express mutant KRAS. Expression of mutant KRAS leads to a number of metabolic changes; for one, cells dramatically increase glucose uptake and increase flux through the hexosamine biosynthesis pathway (HBP). The HBP produces uridine diphosphate N- acetylglucosamine (UDP-GlcNAc), the major substrate for N-linked glycosylation. N-glycans are assembled in the late endoplasmic reticulum and Golgi in part by N-acetylglucosaminyltransferase (MGAT) enzymes, which modify the sugar structure sequentially, MGAT1 through MGAT5. Specifically, modification by MGAT5 is responsible for the interaction of membrane surface proteins with the galectin lattice; the greater amount of interaction with the galectin lattice, the less likely the protein will be endocytosed, allowing for retention of the protein at the cell membrane. Thus, GlcNAc availability, MGAT enzyme expression, and the number of putative N-glycosylation sites on a given protein establish which proteins are presented at the membrane and can thus contribute to downstream signaling within the cell. While both HBP flux and MGAT5 expression are upregulated in PDAC, the functional impacts of either of these on cancer growth and progression have not been well studied. I hypothesize that increased HBP flux and glycan branching allows for increased retention of specific proteins at the membrane, and that expression of MGAT5 is required for PDAC growth and development. To test this hypothesis, I propose two aims. In the first aim, I will establish the role of increased HBP flux on localization of proteins to the cell membrane by manipulating KRAS signaling, GFAT1 expression, or MGAT5 expression and determining exactly what proteins or classes of proteins are changing at the membrane by N-glycoproteomics. I will also determine the impact of nutritional context on membrane protein presentation in PDAC cells expressing mutant KRAS vs those expressing WT KRAS. In the second aim, I will test whether MGAT5 expression is required for PDAC tumor growth and metastasis. To do this, I will first examine expression of Mgat5 over PDAC development in vivo to determine the relationship between Mgat5 expression and tumor grade. I will then knock out Mgat5 in mouse PDAC cell lines using CRISPR and use them to establish orthotopic PDAC models through which I will monitor the impact of Mgat5 knockout on tumor growth and metastasis. These experiments will provide an understanding of the functional impacts of increased HBP flux and N-glycan branching in PDAC, and provide insight into the development of this disease at the molecular level, potentially identifying novel therapeutic targets for this deadly disease.

项目概要 超过 90% 的胰腺导管腺癌 (PDAC) 表达突变型 KRAS。突变体的表达 KRAS 会导致许多代谢变化；其一，细胞显着增加葡萄糖摄取， 增加通过己糖胺生物合成途径（HBP）的通量。 HBP 产生尿苷二磷酸 N- 乙酰氨基葡萄糖 (UDP-GlcNAc)，N-连接糖基化的主要底物。 N-聚糖组装在 晚期内质网和高尔基体部分由 N-乙酰氨基葡萄糖转移酶 (MGAT) 酶作用， 按顺序修改糖结构，MGAT1 到 MGAT5。具体地，MGAT5的修改是 负责膜表面蛋白与半乳糖凝集素晶格的相互作用；的量越大 与半乳糖凝集素晶格的相互作用，蛋白质被内吞的可能性越小，从而允许保留 细胞膜上的蛋白质。因此，GlcNAc 可用性、MGAT 酶表达以及假定的数量 给定蛋白质上的 N-糖基化位点确定了哪些蛋白质出现在膜上，从而可以 有助于细胞内的下游信号传导。虽然 HBP 通量和 MGAT5 表达均 PDAC 中上调，但其中任何一个对癌症生长和进展的功能影响尚未 得到了很好的研究。我假设增加 HBP 通量和聚糖分支可以增加保留 膜上的特定蛋白质，并且 MGAT5 的表达是 PDAC 生长和 发展。为了检验这个假设，我提出了两个目标。在第一个目标中，我将确立增强的作用 通过操纵 KRAS 信号、GFAT1 表达，HBP 在蛋白质定位到细胞膜上的通量， 或 MGAT5 表达并准确确定哪些蛋白质或蛋白质类别在变化 N-糖蛋白质组学的膜。我还将确定营养环境对膜蛋白的影响 表达突变型 KRAS 的 PDAC 细胞与表达 WT KRAS 的 PDAC 细胞中的呈现。在第二个目标中，我会 测试 PDAC 肿瘤生长和转移是否需要 MGAT5 表达。为此，我首先要 检查 Mgat5 在体内 PDAC 发育中的表达以确定 Mgat5 之间的关系 表达和肿瘤分级。然后我将使用 CRISPR 敲除小鼠 PDAC 细胞系中的 Mgat5 并使用 他们建立了原位 PDAC 模型，通过该模型我将监测 Mgat5 敲除对肿瘤的影响 生长和转移。这些实验将有助于了解增加的功能影响 HBP 通量和 PDAC 中的 N-聚糖分支，并深入了解这种疾病的发展 分子水平，有可能确定这种致命疾病的新治疗靶点。