Role of the hexosamine biosynthesis pathway in pancreatic cancer.

项目总结 90%以上的胰腺导管腺癌(PDAC)表达突变的KRAS。突变体的表达 KRAS导致了许多代谢变化；首先，细胞极大地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- 乙酰氨基葡萄糖(UDP-GlcNAc)，N-连接糖基化的主要底物。N-葡聚糖组装在 晚期内质网和高尔基体部分由N-乙酰氨基葡萄糖基转移酶(MGAT)酶作用，其中 按顺序修改糖结构，从MGAT1到MGAT5。具体地说，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； 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，允许保留 细胞膜上的蛋白质。因此，GlcNAc的可用性、MGAT酶的表达以及推测的 特定蛋白质上的N-糖基化位点决定了哪些蛋白质出现在膜上，从而可以 对细胞内的下行信号有贡献。而HBP通量和MGAT5表达均为 在PDAC中上调，其中任何一种对癌症生长和进展的功能影响都没有 已经做了很好的研究。我假设增加的HBP通量和多聚糖分支允许增加的保留 并且MGAT5的表达是PDAC生长和生长所必需的 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，我将确立增加的作用 HBP通过调节KRAS信号，GFAT1表达， 或MGAT5的表达，并准确地确定哪些蛋白质或蛋白质类别在 通过N-糖蛋白组学分析膜。我还将确定营养环境对膜蛋白的影响 在表达突变KRAS的PDAC细胞和表达WT KRAS的PDAC细胞中的表达。在第二个目标中，我将 检测PDAC肿瘤生长和转移是否需要MGAT5的表达。为此，我将首先 检测体内发育过程中mGat5的表达以确定mGat5与PDAC发育的关系 表达与肿瘤分级有关。然后我将用CRISPR敲除小鼠PDAC细胞系中的Mgat5，并使用 他们将建立原位PDAC模型，通过该模型我将监测Mgat5基因敲除对肿瘤的影响 生长和转移。这些实验将提供对增加的功能影响的理解 HBP通量和PDAC中的N-葡聚糖分支，并提供了对该疾病发展的洞察 分子水平，有可能确定这种致命疾病的新治疗靶点。

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.