ER Stress Reduces Angiogenesis and the Breast Tumor Progression

N-acetylglucosaminyl 1-phosphate transferase (GPT) catalyzes the first step of asparagine-linked (N-linked) protein glycosylation process. Angiogenesis is the hallmark for solid tumor growth. Microvascular capillary endothelial cells when treated with a competitive inhibitor of GPT tunicamycin, angiogenesis is halted by arresting cells in late G1. The treatment also inhibited the Matrigel™ invasion and chemotaxis. Genomic analysis by cDNA microarray indicated that a large number of upregulated genes correspond to transcription and translation. Exogenous addition of pro-angiogenic VEGF could not reverse the anti-angiogenic signaling of tunicamycin suggesting derangement of cell survival. Significant reduction of cell surface N-glycan expression supported the development of ER stress and induction of unfolded protein response (upr)– mediated apoptotic cell death. Down regulation of protein tyrosine kinase activity as well as the phosphorylation of VEGF receptors R1 and R2 within 3 hours of tunicamycin treatment strongly suggested that the action of tunicamycin starts much early.

Translating the in vitro knowledge into an in vivo model of athymic (Blab/c) nude mice (nu/nu) indicated that tunicamycin blocks neo-vascularization almost quantitatively in Matrigel™ implants. H & E staining of the Matrigel™ sections supported reduced vascularization. Immunohistochemical analysis of the sections also indicated reduced expression of CD34 and CD144. Intravenous injection of tunicamycin once a week slowed down a double negative grade-III breast adenocarcinoma growth by ∼50-60% in three weeks. The effect was dose-dependent. Histolopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel corroborated with the vessel size. High expression of GRP-78 in CD144 positive cells supported ER stress in tumor microvasculature. Supported in part by NIH U54-CA096297, Susan G. Komen for the Cure BCTR0600582 (DKB), and NIH/NCRR/RCMI G12-RR03035 (KB) grants.