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.

Public Health Relevance

The hexosamine biosynthesis pathway and complex glycan branching are upregulated in pancreatic cancer, but the functional impacts of these alterations and their roles in pancreatic cancer progression have not been established. Understanding the roles of hexosamine flux and glycan branching in tumor onset, growth, and metastasis in a pancreatic cancer model will provide crucial insight into disease development and could point toward therapeutic targets for pancreatic cancer.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Schmidt, Michael K
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University of Pennsylvania
Schools of Medicine
United States
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