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.