Abstract 1382: 2-Deoxy-glucose downregulates endothelial Akt and Erk via interference with N-linked glycosylation, induction of endoplasmic reticulum stress, and GSK-3β activation

BACKGROUND: Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analog, 2-deoxy-D-Glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity (PloS One2010;5(10):e13699), and down regulates endothelial Akt and Erk pathways (Kovacs et al. 2012 AACR annual meeting; abstract no 2232). The aims of this study are to characterize the mechanisms of Akt and Erk down regulation by 2-DG. METHODS: Differences in 2-DG uptake between endothelial and tumor cells were assessed by determination of radioactive labeled 2-DG uptake at different time points. The effects of 2-DG on VEGFR2 status were determined by immunocytochemistry (ICC), N-glycan digestions and cell surface biotinylation experiments. 2-DG9s effects on ER stress pathways, apoptosis, and differential effects of other ER stress inducers were assessed by Western blot (WB). The in vivo effects of 2-DG were further evaluated in the LH BETA T AG retinoblastoma model by immunofluorescence staining (IF) for microvessel density and markers of unfolded protein response (UPR). In vivo UPR induction was further determined in Laser capture microdissection (LCM) isolated tumor cells using immunoblots for GRP 78. RESULTS/CONCLUSIONS: No significant differences in 2-DG uptake were found between endothelial cells (HUVECs/HMVECs) or tumor cells (MDA-MB231, HT-29). 2-DG mediated ERK and AKT down regulation were associated with changes in VEGFR2 migration pattern and downstream signaling, as evidenced by decreased phosphorylation of the downstream effector PLC-γ1 pTyr 783 . Changes in VEGFR2 migration pattern were due to receptor hypoglycosylation, as supported by mannose reversal of these effects, and N-glycan digestion showing that Endo H and PNGase F digestion of 2-DG treated samples resulted in less number of cleaved bands than ctrl. Biotinylation experiments showed that in 2-DG treated cells, VEGFR2 was not present on the cell surface. 2-DG induced endoplasmic reticulum (ER) stress (increased PERK and eIF2-α phosphorylation), leading to GSK-3β activation and apoptosis (increased cleaved caspase-3 and PARP) in a mannose reversible manner. Chemical inhibition of GSK-3β prevented 2-DG induced apoptosis. In vivo, periocular administration of 2-DG in LH BETA T AG mice was associated with significant reduction of newly formed (CD 105 +) tumor capillaries and induction of ER stress (GRP 78 expression). These findings uniquely link N-linked glycosylation inhibition, ER stress and endothelial Erk/Akt down regulation, and provide a novel strategy for angiogenesis inhibition, which may have the potential to overcome resistance to standard antiangiogenic agents. Citation Format: Krisztina Kovacs, Christina Decatur, Dien G. Pham, Huaping Liu, Yuqi Jing, Timothy G. Murray, Theodore J. Lampidis, Jaime R. Merchan. 2-Deoxy-glucose downregulates endothelial Akt and Erk via interference with N-linked glycosylation, induction of endoplasmic reticulum stress, and GSK-3β activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1382. doi:10.1158/1538-7445.AM2015-1382