Elucidating the role of microRNA-497 and WEE1 in neuroblastoma disease pathogenesis.

Neuroblastoma is a pediatric cancer that arises from precursor cells of the sympathetic nervous system and is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the 5 year overall survival rate remains poor in high-risk disease (20-30%). MiRNA profiling has emerged as a valuable tool in the diagnostic and prognostic evaluation of many cancers including neuroblastoma. Previously our laboratory identified two miRNA expression profiles; the first a 15 miRNA expression signature associated with survival and the second a miRNA expression profile associated with MYCN amplification. Based on this work, a number of individual miRNAs were chosen for further investigation. An evaluation of 4 selected miRNAs (miR-572, miR-30a*, miR-30e* and miR-497) revealed each was individually significantly associated with survival in neuroblastoma. The subsequent initial functional characterisation of these miRNAs revealed that only miR-497 significantly impacted cell proliferation and apoptosis levels in the MYCN amplified cell lines examined. MiR-497 had previously been identified as a member of the 15 miRNA expression profile associated with survival and in the present study it is demonstrated that miR-497 is individually significantly associated with both event free and overall survival in neuroblastoma. Low miR-497 expression was also significantly associated with MYCN amplification and the over-expression of miR- 497 resulted in the inhibition of cell proliferation and caspase mediated apoptosis in MYCN amplified high-risk neuroblastoma cells. A novel target for miR-497, WEE1, was also discovered in neuroblastoma and it was determined that miR-497 exerts a tumour suppressive function through the efficient down-regulation of WEE1 protein. WEE1 is a tyrosine kinase regulator of the cell cycle that functions by negatively regulating CDC2 mediated apoptosis. The over-expression of WEE1 has been demonstrated in a variety of cancer types and has been associated with a poor clinical prognosis in these cancers. This study demonstrated that high WEE1 expression was associated with poor clinical outcome in neuroblastoma and that high WEE1 expression was significantly associated with MYCN amplification. Treatment of high-risk neuroblastoma cells with MK-1775, a small molecule inhibitor of WEE1 currently undergoing clinical trials, resulted in the same phenotypic effect of increased apoptosis. A comparative study between the downstream effects of MK-1775 treatment and miR-497 over-expression revealed that both methods of WEE1 inhibition resulted in decreased CDC2 phosphorylation, increased cleaved PARP and increased levels of gamma-H2AX, demonstrating the similarities between with small molecule inhibitor and the natural antisense inhibitor of WEE1. Additionally this study identified a second novel target for miR-497 in neuroblastoma, CHK1. The over-expression of both WEE1 and CHK1 has been previously demonstrated in neuroblastoma. CHK1 functions as a key cell cycle checkpoint regulator by positively modulating WEE1 activity and inhibiting CDC25a. CDC25a functions to dephosphorylate and subsequently reactivate CDC2, therefore promoting apoptosis induction. This study revealed that high expression of CHK1 is significantly associated with both poor event free and overall survival in neuroblastoma and that CHK1 expression is significantly associated with MYCN amplification. The identification of both WEE1 and CHK1 as novel targets of miR-497 in neuroblastoma has further emphasised the tumour suppressive role of miR-497 and highlights the therapeutic potential for miR-497 mediated therapy in high-risk neuroblastoma