Role of O-GlcNAc-ylation on tumor progression

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Abstract O-linked ß-N-acetylglucosamine (O-GlcNAc) is a sugar attachment to the side chain hydroxyl of a serine or threonine residue on proteins. O-GlcNAcylation controls key signaling and biological processes such as signal transduction, transcription, cell cycle progression, and metabolism. Perturbations in O-GlcNAc homeostasis have been linked with diabetes, cancer, and neurodegenerative diseases. Increased glucose levels channel flux through the Hexoseamine Biosynthetic Pathway (HBP), culminating in increased O-GlcNAc levels. The activity of HBP and consequently cellular O-GlcNAc-ylation are elevated in several cancer types, including breast cancer. We recently reported that inhibiting HBP activity significantly decreased the invasive phenotype of breast tumor cells. We surmised that abundance of glucose, a readily-metabolizable carbohydrate, will drive flux through HBP, resulting in enrichment of a portfolio of proteins that are modified by O-GlcNAc-ylation. Using unbiased proteomics analysis, we identified that elevated glucose culture conditions enrich for O-GlcNAc- modified GLI proteins, transcription factors of the Hedgehog (Hh) pathway. Importantly, we identified that in elevated glucose conditions, O-GlcNAc-modification of GLI exacerbates Hh/GLI activity; and inhibiting HBP mitigated this effect. We hypothesize that HBP-directed O-GlcNAc-ylation fundamentally programs invasive and chemoresistant attributes in tumor cells through activating Hh/GLI signaling. In Aim 1 we will determine the molecular underpinnings of HBP-directed O-GlcNAc-ylation of GLI. We will determine the causes and consequences of GLI O-GlcNAc-ylation. We will first identify engagement of the HBP in O-GlcNAc-modification of GLI proteins. Next, we will undertake investigations to identify establish the mechanistic basis of how HBP signaling engages O-GlcNAc-modified GLI to program invasive and chemoresistant attributes in tumor cells. In Aim 2 we will evaluate the impact of an elevated O-GlcNAc landscape on molecular and cellular attributes of the mammary tumor and the associated immune microclimate using two distinct and complementary syngeneic mouse models of mammary cancer. To enrich the relevance, we will also evaluate human TNBC and PDX model systems. We will test if inhibiting GLI activity, in the context of elevated O-GlcNAc, uncouples the influence of O- GlcNAc-ylation on invasive and chemoresistant attributes of mammary tumor cells. Relevance: Our proposed studies are structured to systematically investigate how O-GlcNAc-driven metabolic reprogramming in cancer cells connects at the molecular level to aberrantly activate Hh/GLI signaling. The cumulative outcomes will create mechanistic understanding of how O-GlcNAc-ylation programs tumor invasion, progression and response to anti-neoplastics.