Abstract 1270: Polysialyltransferase ST8SiaII as a target for neuroblastoma dissemination

Polysialic acid (polySia) is expressed on the surface of NCAM (neuronal cell adhesion molecule) on neuroendocrine tumours, notably neuroblastoma and small cell lung cancer, where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis. PolySia expression is strongly associated with poor prognosis and aggressive disease in neuroblastoma patients in the clinic[1]. SiRNA knockdown of polysialyltransferase (polyST) ST8SiaII, the enzyme primarily responsible for polySia synthesis in tumours, abrogates tumour cell migration and invasion. Besides brain regions with persistent neuronal plasticity, polySia is essentially absent from the body post-embryogenesis. PolyST is thus a selective and largely unexplored therapeutic target for neuroblastoma dissemination [1]. We have established a highly sensitive HPLC-based polyST inhibition assay, amenable to high-throughput screening. We report our efforts to further optimise this cell-free assay, and include details of our novel methodology to quantify cell-surface polySia expression. Having demonstrated in vitro that inhibition of polyST by a small molecule leads to a reduction in tumour cell migration [2], we designed and synthesised ST8SiaII inhibitors. Using isogenic cell lines (C6-STX: polySia+/polyST+ and C6-WT: polySia-/polyST-) and naturally polySia expressing human neuroblastoma cells (SH-SY5Y, IMR-32) these compounds were evaluated for their ability to reduce polySia expression, to modulate tumour cell migration and invasion in vitro. We have identified novel agents which significantly reduce polySia expression, tumour cell migration and invasion. These effects were only found in cell lines expressing ST8SiaII and polySia. Specificity of agents for polySTs over other members of the sialyltransferase (ST) family (i.e. α-2,3-ST and α-2,6-ST) was subsequently investigated using lectin differential labelling probes. Agents did not inhibit sialyltransferase activity. We have investigated effects of agents on key intracellular signalling pathways. We demonstrated the effects of polyST inhibition on the dynamics of FAK and on ERK1/2, AKT, CREB and VEGFR-3 signalling. Furthemore, we have explored the behaviour of polySia-expressing cells under hypoxic conditions. Our data suggest that polySia is associated with a resistant phenotype, with C6-STX polySia-expressing cells demonstrating a survival advantage and additionally maintaining their migratory capacity under hypoxia (compared to WT cells