Role of the hexosamine biosynthesis pathway in pancreatic cancer.

项目摘要 超过90%的胰腺导管腺癌（PDAC）表达突变型KRAS。表达突变型 KRAS导致许多代谢变化;例如，细胞显著增加葡萄糖摄取， 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- 乙酰葡糖胺（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通过N-乙酰葡糖胺转移酶（MGAT）酶， 依次修饰糖结构，MGAT 1至MGAT 5。具体地，通过MGAT 5的修饰是 负责膜表面蛋白与半乳糖凝集素晶格的相互作用; 与半乳糖凝集素晶格的相互作用，蛋白质将被内吞的可能性越小，允许保留半乳糖凝集素。 细胞膜上的蛋白质。因此，GlcNAc的可用性、MGAT酶表达和推定的GLA-C的数量是不确定的。 给定蛋白质上的N-糖基化位点确定了哪些蛋白质存在于膜上，从而可以 有助于细胞内的下游信号传导。虽然HBP通量和MGAT 5表达都是 在PDAC中上调，这些对癌症生长和进展的功能影响中的任何一个都没有 被好好研究过了。我假设增加的HBP通量和聚糖分支允许增加的保留。 MGAT 5的表达是PDAC生长所需的， 发展为了验证这个假设，我提出了两个目标。在第一个目标中，我将建立一个更大的作用， HBP通量通过操纵KRAS信号传导、GFAT 1表达， 或MGAT 5表达，并准确确定哪些蛋白质或蛋白质类别在细胞周期中发生变化。 通过N-糖蛋白质组学。我还将确定营养环境对膜蛋白的影响 在表达突变型KRAS的PDAC细胞与表达WT KRAS的PDAC细胞中，KRAS蛋白的表达与表达WT KRAS的那些相比有显著差异。在第二个目标中，我将 测试PDAC肿瘤生长和转移是否需要MGAT 5表达。为此，我将首先 检查体内PDAC发育过程中Mgat 5的表达，以确定Mgat 5与PDAC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Mgat 5， 通过建立原位PDAC模型，监测Mgat 5基因敲除对肿瘤的影响 生长和转移。这些实验将提供一个了解的功能影响，增加 HBP通量和PDAC中的N-聚糖分支，并提供了对这种疾病在 在分子水平上，有可能为这种致命的疾病确定新的治疗靶点。

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.