Unfolded Protein Response Is Required in nu/nu Mice Microvasculature for Treating Breast Tumor with Tunicamycin

Aditi Banerjee,Jing-Yu Lang,Mien-Chie Hung,Krishanu Sengupta,Sushanta K Banerjee,Krishna Baksi,Dipak K Banerjee

doi:10.1074/jbc.m110.169771

Abstract

Up-regulation of the dolichol pathway, a "hallmark" of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF(165). Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF(165)-specific phosphotyrosine kinase activity; and (iii) Matrigel(TM) invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in Matrigel(TM) implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in Matrigel(TM) implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ∼50-60% in 3 weeks. Histopathological 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 was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ∼65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycin's potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.

Highlights

Up-regulation of the dolichol pathway, a “hallmark” of asparagine-linked protein glycosylation, enhances angiogenesis in vitro
The present study demonstrates that tunicamycin (i) inhibits angiogenesis in vivo and reduces a double (ERϪ/PRϪ/EGFRϩ) and a triplenegative (ERϪ/PRϪ/EGFRϪ) breast tumor growth in nude mice; and (ii) the inhibition of angiogenesis is stable under tumor microenvironment as it is irreversible by exogenous VEGF165
To evaluate the stability of the anti-angiogenic effect of tunicamycin in tumor microenvironment, a time course was set up where VEGF165-stimulated capillary endothelial cells were treated with tunicamycin (1 ␮g/ml) and monitored every 24 h for 144 h

Summary

Introduction

Up-regulation of the dolichol pathway, a “hallmark” of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. To verify the status of the N-glycans on the breast tumor microvascular endothelial cell surface, the tissue sections were stained with Texas-Redconjugated WGA and examined under a fluorescence microscope.

Results

Conclusion