Background Enhanced glucose metabolism is a feature of most tumors, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. Metabolic diseases including obesity and diabetes have a hyperglycemia component and are correlated with elevated pre-menopausal cancer risk for triple-negative breast cancer (TNBC). However, determining pathways for hyperglycemic disease-coupled cancer risk remains a major unmet need. One aspect of cellular sugar utilization is the addition of the glucose-derived protein modification O-GlcNAc (O-linked N-acetylglucosamine) via the single human enzyme that catalyzes this process, O-GlcNAc transferase (OGT). The data in this report implicate roles of OGT and O-GlcNAc within a pathway leading to cancer stem-like cell (CSC) expansion. CSCs are the minor fraction of tumor cells recognized as a source of tumors as well as fueling metastatic recurrence. The objective of this study was to identify a novel pathway for glucose-driven expansion of CSC as a potential molecular link between hyperglycemic conditions and CSC tumor risk factors. Methods We used chemical biology tools to track how a metabolite of glucose, GlcNAc, became linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-GlcNAc post-translational modification in three TNBC cell lines. Using biochemical approaches, genetic models, diet-induced obese animals, and chemical biology labeling, we evaluated the impact of hyperglycemia on CSC pathways driven by OGT in TNBC model systems. Results We showed that OGT levels were higher in TNBC cell lines compared to non-tumor breast cells, matching patient data. Our data identified that hyperglycemia drove O-GlcNAcylation of the protein TET1 via OGT-catalyzed activity. Suppression of pathway proteins by inhibition, RNA silencing, and overexpression confirmed a mechanism for glucose-driven CSC expansion via TET1-O-GlcNAc. Furthermore, activation of the pathway led to higher levels of OGT production via feed-forward regulation in hyperglycemic conditions. We showed that diet-induced obesity led to elevated tumor OGT expression and O-GlcNAc levels in mice compared to lean littermates, suggesting relevance of this pathway in an animal model of the hyperglycemic TNBC microenvironment. Conclusions Taken together, our data revealed a mechanism whereby hyperglycemic conditions activated a CSC pathway in TNBC models. This pathway can be potentially targeted to reduce hyperglycemia-driven breast cancer risk, for instance in metabolic diseases. Because pre-menopausal TNBC risk and mortality are correlated with metabolic diseases, our results could lead to new directions including OGT inhibition for mitigating hyperglycemia as a risk factor for TNBC tumorigenesis and progression.

Enhanced glucose metabolism is a feature of almost all cancers, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. The objective of this study is to characterize a mechanism by which elevated glucose level drives a tumorigenic pathway in triple negative breast cancer (TNBC). We used chemical biology methods to track how a metabolite of glucose, N-acetylglucosamine (GlcNAc), is linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-linked GlcNAc post translational modification (O-GlcNAc). In this work, we revealed that intracellular protein glycosylation by O-GlcNAc is driven by high glucose levels in TNBC models, including on TET1. A single enzyme, O-GlcNAc transferase (OGT), is responsible for catalyzing protein modification of O-GlcNAc. We showed that OGT activity is higher in TNBC cell lines compared to non-tumor breast cell lines and is associated with hyperglycemia. Furthermore, enhanced OGT activity activated a pathway for cancer stem-like cell (CSC) reprogramming in TNBC cells. In our model, O-GlcNAcylated TET1 upregulated expression of splicing factor TAR-DNA binding protein (TARDBP), which drives CSC induction as well as higher OGT levels. We show that this OGT-TET1-TARDBP axis “feeds-forward” in hyperglycemic conditions both in cell lines and diet-induced obese mice, which displayed higher blood glucose levels and tumor O-GlcNAc levels than lean littermates. This data converges on a novel pathway whereby hyperglycemia drives aberrant OGT activity, activating a pathway for CSC induction in TNBC. Our findings partially explain a key aspect of how obesity is associated with TNBC risk and negative outcomes. Statement of Implication This work presents a novel mechanism to explain how obesity is a risk factor for triple-negative breast cancer via elevated sugar-transfer activity by O-GlcNAc transferase in hyperglycemia, leading to the induction of a cancer stem-like cells and revealing a targetable pathway in obesity-associated tumors.