P10.07 Sialate O-acetylesterase (SIAE) 
over-expression leads to a collapse of the mitochondrial membrane potential and predisposes cells to etoposide treatment in RES256 medulloblastoma cells

Medulloblastoma is the most common malignant paediatric brain tumour and is the leading cause of childhood cancer related mortality. Current treatment includes maximal safe surgical resection, radiotherapy if the patient is over 3 years of age, and high-dose chemotherapy that usually includes methotrexate, cisplatin, etoposide, vincristine, lomustine and cyclophosphamide. Survivors often suffer significant treatment-related sequelae including endocrine disorders and neurocognitive deficits that negatively impact quality of life. GD3, an oncofoetal ganglioside is re-expressed in medulloblastoma and is thought to be associated with mitochondrially-mediated apoptosis. 9-O-acetyl GD3 (GD3A), its acetylated form, has been shown to protect cells from GD3-mediated apoptosis. Sialate-O-acetylesterase (SIAE) is responsible for cleaving the acetyl group from GD3A. A genome-wide transcriptional analysis of 103 MB patients revealed that this enzyme is significantly down-regulated in MB tissue compared to non-neoplastic foetal and adult brain (p>0.02). To determine if this pathway may serve as a potential therapeutic target we used a well described tet-responsive inducible expression system. SIAE and a mutant of that has been shown to have very little catalytic activity (SIAE-S127A) were expressed in the RES256 cell line. SIAE and SIAE-S127A expression was verified by Western blot analysis after 48 hours of induction with low levels of doxycycline. SIAE expression was also confirmed by SIAE-S127A expressing RES256 cells in conditioned media, but was not secreted by wild-type expressing cells. Over-expression of SIAE showed a significant increase in GD3 expression (p