Invited Speaker

Targeting N-Glycans for Anti-angiogenic Therapeutics Treating Breast Cancer
Dipak K. Banerjee
USA

Breast cancer is affecting the lives of million women worldwide every year and is becoming a major public health issue. The disease is multi-factorial but angiogenesis is crucial for tumor progression. Current choice of therapeutics targeting the tumor microvessels is limited. In addition, there are serious side effects and the treatment cost is also high. Therefore, new anti-angiogenic therapeutics is indeed required. Our approach focuses on glycotherapeutics.

Asparagine-linked glycoproteins are evolutionary conserved in which the glycan chains are attached N-glycosidically to the asparagine-residue of the protein core present in Asn-X-Ser/Thr motif giving either a "high mannose", or a "hybrid", or a "complex" type structure. Accumulated evidence from our and other laboratories indicates that N-glycans are crucial for cellular proliferation and differentiation. We have observed a dynamic relationship between dolichol-linked glycan (LLO, Glc₃Man₉GlcNAc₂-PP-Dol) biosynthesis and capillary endothelial cell proliferation. Cells when treated with cAMP-related stimuli LLO level is up-regulated so does the cellular proliferation and capillary lumen formation. Mannosylphospho dolichol synthase (DPMS; GDP-mannose + Dol-P = Dol-P-Man + GDP), a 31kDa phosphoprotein and a "key" step in LLO biosynthesis is activated. cDNA cloning and sequencing identifies a PKA motif in DPMS (Arg-Lys-Ileu-Ileu-Ser¹⁶⁵; GenBank #GQ367549). Disabling the PKA motif by site-directed mutagenesis results in loss of phosphorylation activation of DPMS and consequently inhibition of cellular proliferation. To establish the role of DPMS in angiogenesis further, a capillary endothelial cell clone overexpressing DPMS has been isolated. The clone exhibits high expression of complex glycans on the cell surface, increased proliferation rate and accelerated healing of the wound induced by a mechanical stress. All angiogenic activity however is down-regulated in a cell clone where the DPMS is silenced by siRNA.

In a subsequent study, we have used tunicamycin, a competitive inhibitor of N-acetylglucosaminyl 1-phosphate transferase (the first step of LLO biosynthesis). Mechanistic details indicate tunicamycin down regulates cell surface glycoproteins expression and arrests cells in G1. Down-regulation of Bcl-2 and CDK-4 as well as up-regulation of p53, p21^Cip/WAF1 is also observed. c-Jun and c-Myc up-regulation and c-Fos down-regulation further support the loss of survival potential. Increased DNA fragmentation, annexin V binding and caspase activation indicate apoptosis. High GRP-78/Bip, ATF6 and PERK supports induction of unfolded protein response (upr) and the apoptosis is mediated by transcriptional and translational attenuation. Tunicamycin is stable under tumor microenvironment and VEGF₁₆₅ is unable to overcome the inhibition in vitro as well as in Matrigel^TM implants in nude mice. Expression of phospho-VEGFR1 and VEGFR2 and phospho-tyrosine kinase activity are all down regulated. Western blotting, QRT-PCR and cDNA microarray results indicate inhibition of endogenous angiogenesis inhibitor, thrombospondin (TSP-1) expression. When MDA-MB-435 induced breast tumor in athymic nude mice is treated with tunicamycin exhibits reduced angiogenesis and consequently the tumor growth. The effect is time and dose dependent. For example, ~55% reduction in tumor growth is achieved in three weeks at 1.0mg/Kg body weight. H & E and immunohistochemical staining of paraffin sections of tumor tissue show down regulation of Ki-67 and VEGF expression and decreased mitotic index. It is therefore concluded that N-glycan is an excellent target for developing anti-angiogenic therapeutics treating breast cancer. Supported by grants from Susan G. Komen for the Cure BCTR0600582 and NIH U54-CA096297.