N-acetylglucosaminyl 1-phosphate transferase: an excellent target for developing new generation breast cancer therapeutic

Abstract

Studies from our laboratory have explained that breast tumor progression can be attenuated by targeting the N-linked glycoproteins of the tumor microvasculature and that of tumor cells alike with a protein N-glycosylation inhibitor, tunicamycin. Absence of N-glycosylation leads to an accumulation of un- or mis-folded proteins in the ER and the cell develops “ER stress”. The result is cell cycle arrest, and induction of apoptosis mediated by unfolded protein response (upr) signaling. Tunicamycin inhibited in vitro and in vivo (Matrigel™ implants in athymic nude mice) angiogenesis in a dose dependent manner. The action is irreversible and survived under tumor microenvironment, i.e., in the presence of FGF-2 or VEGF or higher serum concentration. Importantly, tunicamycin prevented the progression of double negative (ER^-/PR^-/Her2⁺) and triple negative (ER^-/PR^-/Her2^-) breast tumors by ∼55% - 65% in three weeks in athymic nude mice [Balb/c(nu/nu)]. Analyses of paraffin sections exhibited “ER stress” in both microvasculature and in tumor tissue.

Figure 1

Proliferation of capillary endothelial cells as a function of tunicamycin concentration and the time of treatment (a). Morphological changes associated with apoptotic death of microvascular endothelial cells following tunicamycin treatment (b)

Figure 2

The capillary endothelial cells do not regain their virulence upon withdrawal of tunicamycin. (a) Monitoring cell number after 24 hour, 48 hour and 72 hours. (b) Clonogenic assay.

Figure 3

Nuclear fragmentation of capillary endothelial cells following tunicamycin treatment.

Figure 4

Tunicamycin survives under tumor microenvironment. (a) in the presence of FGF-2; and (b) in the presence of VEGF₁₆₅.

Figure 5

Tunicamycin treatment down regulates VEGF receptors I & II expression (a), phosphorylation of VEGFRI & II (b) and VEGF₁₆₅ –specific protein tyrosine kinase (c).

Figure 6

Tunicamycin inhibits (a) migration and chemotaxis of capillary endothelial through Matrigel^™ matrices in a wound healing assay; (b) angiogenesis in Matrigel^™ plug assay.

Figure 7

Tunicamycin inhibits double (orthotopic tumor) and triple negative (xenograft) breast tumor progression. Staining of paraffin sections exhibits reduced microvessel density, reduced mitotic index as well as reduced Ki-67 and VEGF expression.

Figure 8

Tunicamycin treatment develops “Er stress” in tumor microvasculature as well as in tumor cells (a &b). Analysis of cellular proteome by Raman Spectroscopy supports protein denaturation following tunicamycin treatment (c & d).

Figure 9

Tunicamycin gold nanoparticles are three times more potent than the native compound. Tunicamycin nanoparticles reduces cellular proliferation by down regulating the expression of cyclin D1 and/or CDK4n (a, b, & c). The cells experience “ER stress” (d) but does not undergo apoptosis.