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: 9752256
• 负责人: Sydney Campbell
• 金额: $ 4.5万
• 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/9752256
• 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; 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; 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; pancreatic cancer model; 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.

项目摘要 超过90％的胰腺导管腺癌（PDAC）表达突变体Kras。突变体的表达 KRAS导致许多代谢变化。首先，细胞大大增加了葡萄糖摄取和 通过己糖胺生物合成途径（HBP）增加通量。 HBP产生尿苷二磷酸N- 乙酰葡萄糖（UDP-GLCNAC），这是N连接糖基化的主要底物。 n-聚糖组装 晚期内质网和高尔基体部分由N-乙酰基葡萄糖氨基转移酶（MGAT）酶，该酶，该酶，该酶 顺序修改糖结构，通过MGAT5修改MGAT1。具体而言，MGAT5的修改为 负责膜表面蛋白与半乳肠蛋白晶格的相互作用；更多的 与半肠晶格的相互作用，蛋白质的可能性较小，从而允许保留蛋白质 细胞膜的蛋白质。因此，GlcNAC的可用性，MGAT酶表达和推定的数量 给定蛋白上的N-糖基化位点确定了哪些蛋白在膜上呈现，因此可以 有助于细胞内的下游信号传导。而HBP通量和MGAT5表达却是 在PDAC中上调，其中任何一个对癌症生长和进展的功能影响都没有 经过深入研究。我假设增加的HBP通量和聚糖分支可以增加 膜上的特定蛋白质，MGAT5的表达是PDAC生长和 发展。为了检验这一假设，我提出了两个目标。在第一个目标中，我将确定增加的作用 通过操纵KRAS信号传导，GFAT1表达，HBP通量将蛋白质定位到细胞膜上 或MGAT5表达并准确确定哪些蛋白质或类别的蛋白质正在发生变化 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 表达突变体KRAS与表达WT KRA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 检查MGAT5在体内PDAC开发上的表达，以确定MGAT5之间的关系 表达和肿瘤等级。然后，我将使用CRISPR在鼠标PDAC细胞系中敲出MGAT5并使用 它们建立原位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。这些实验将对增加的功能影响有所了解 PDAC中的HBP通量和N-聚糖分支，并深入了解该疾病的发展 分子水平，可能识别这种致命疾病的新型治疗靶标。