2-Deoxy-Glucose Downregulates Endothelial AKT and ERK via Interference with N-Linked Glycosylation, Induction of Endoplasmic Reticulum Stress, and GSK3ß Activation.

Interference with endothelial cell metabolism is a promising, yet unexploited strategy for angiogenesis inhibition. We reported that the glucose analogue 2-deoxy-D-glucose (2-DG) inhibits angiogenesis at significantly lower concentrations than those required for tumor cytotoxicity. Here, we found that hypersensitivity to 2-DG in endothelial cells is not associated with enhanced drug uptake compared with tumor cells, but with time-dependent, endothelial-selective inhibition of AKT and ERK phosphorylation. Downregulation of these critical survival pathways is shown to be due to 2-DG's interference with N-linked glycosylation, leading to alterations in VEGFR2 (and downstream signaling) as well as induction of endoplasmic reticulum (ER) stress, GSK3ß activation, and apoptosis. In vivo, periocular administration of 2-DG in LHBETATAG mice was associated with significant reduction of newly formed (CD105(+)) tumor capillaries, ER stress (GRP 78 expression), and endothelial apoptosis (TUNEL). These findings uniquely link N-linked glycosylation inhibition, ER stress, and ERK/AKT downregulation in endothelial cells, and provide a novel drug development strategy to overcome resistance mechanisms to currently available antiangiogenic agents.

Adoptively transferred tumor-specific T cells stimulated ex vivo using herpes simplex virus amplicons encoding 4-1BBL persist in the host and show antitumor activity in vivo.

4-1BB is a T-cell costimulatory receptor which binds its ligand 4-1BBL, resulting in prolonged T cell survival. We studied the antitumor effects of adoptively transferred tumor-specific T cells expanded ex vivo using tumors transduced with herpes simplex virus (HSV) amplicons expressing 4-1BBL as a direct source of antigen and costimulation. We constructed HSV amplicons encoding either the 4-1BBL (HSV.4-1BBL) or B7.1 (HSV.B7.1) costimulatory ligands. Lewis lung carcinoma cells expressing ovalbumin (LLC/OVA) were transduced with HSV.4-1BBL, HSV.B7.1, or control HSV amplicons and used to stimulate GFP+ OVA-specific CD8+ T cells (OT-1/GFP) ex vivo. Naive or ex vivo stimulated OT-1/GFP cells were adoptively transferred into LLC/OVA tumor-bearing mice. Higher percentages of OT-1/GFP cells were seen in the peripheral blood, spleen, and tumor bed of the HSV.4-1BBL-stimulated OT-1/GFP group compared with all other experimental groups. OT-1 cells identified within the tumor bed and draining lymph nodes of the HSV.4-1BBL-stimulated OT-1 group showed enhanced bromodeoxyuridine (BrdUrd) incorporation, suggesting ongoing expansion in vivo. Mice receiving HSV.4-1BBL-stimulated OT-1/GFP had significantly decreased tumor volumes compared with untreated mice (P<0.001) or to mice receiving naive OT-1/GFP (P<0.001). Transfer of HSV.B7.1-stimulated OT-1/GFP did not protect mice from tumor. Mice that received HSV.4-1BBL-stimulated OT-1/GFP exhibited increased cytolytic activity against LLC/OVA and higher percentages of Ly-6C+ OT-1/GFP in the spleen and tumor bed compared with controls. Tumor-specific T cells stimulated ex vivo using tumor transduced with HSV.4-1BBL expand in vivo following adoptive transfer, resulting in tumor eradication and the generation of tumor-specific CD44+Ly-6C+CD62L- effector memory T cells.