Pharmacological evaluation of novel polysialyltransferase inhibitors as anti-metastatic agents and development of analytical methods for assessment of polysialylation inhibition. In vitro assessment of the effects of novel polysialyltransferase inhibitors on tumour cell function and development of quantitative HPLC-based methods for evaluation of novel polysialyltransferase inhibitors

Abstract

Polysialic acid (polySia) is a carbohydrate polymer highly expressed during embryonic development but rarely expressed during postnatal development. Two polysialyltransferase (polyST) enzymes are responsible for the synthesis of polySia: ST8SiaII and ST8SiaIV. During oncogenesis polySia is re-expressed and it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in neuroblastoma and many other tumours. PolyST inhibition thus presents a novel, selective and largely unexplored therapeutic opportunity to reduce tumour dissemination. Progress towards development of polyST inhibitors has been limited by lack of an efficient technique for quantitative assessment of enzyme activity. We have validated a highly sensitive cell-based and cell-free high throughput HPLC-based inhibition assays. Using isogenic cell lines (C6-STX: polySia+/ST8SiaII+ and C6-WT: polySia-/ST8SiaII-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y), a set of ST8SiaII inhibitors designed and synthesised in house were evaluated for their ability to reduce polySia expression and to modulate cell migration in vitro. We have identified CMP-sialic acid precursors, including ICT-3176, which reduced polySia expression and tumour cell migration by up to 70%. These effects were only found in cell lines expressing ST8SiaII and polySia. Furthermore, we have investigated the possible additive anti-migratory effect of combining polyST inhibition with the inhibition of certain signalling pathways that have been previously suggested to be modulated by polySia expression. Out of these combinations it was found that combining ST8SiaII and C-MET/ALK inhibition had a synergistic effect on inhibiting cancer cell migration. Additionally, the effect of polySia expression on cancer cell behaviour under hypoxic conditions was examined, where it was found that polySia expression enhanced cell migration and survival and inhibits cell adhesion. In summary, polyST inhibitors which dramatically decrease cell migration in vitro through modulation of polySia assembly were identified, using optimised cell-free and cell-based assays. Initial investigations into the role of polySia in hypoxia were also accomplished. This work paves the way for development of a novel therapeutic for the treatment of neuroblastoma.