Neuroblastoma is the most common extra-cranial solid tumour in childhood and overall 5-year survival rates are dismal, primarily due to high prevalence of metastatic disease1,2. Metastasis occurs in a series of discrete steps, modelled into a "metastatic cascade"3. Recently, our laboratory has shown that targeting the microtubule destabilizing protein, stathmin, decreased lung metastasis in a neuroblastoma xenograft orthotopic model4. However stathmin's role in neuroblastoma metastasis is yet to be determined. This study aims to understand how stathmin mediates key events in the "metastatic cascade" by examining the role of stathmin in migration, invasion, anoikis and transendothelial migration.
Methods: In SK-N-BE(2) neuroblastoma cells, stathmin was transiently suppressed using two separate siRNA sequences (siRNAa/siRNAb). Migration and invasion were assessed in stathmin suppressed SK-N-BE(2) using transwell chemotactic migration and invasion assays. To examine anoikis, stathmin suppressed SK-N-BE(2) were cultured separately as adherent and in suspension, and apoptosis measured. To examine transendothelial migration, stathmin suppressed SK-N-BE(2) were assessed for their ability to cross an endothelial cell monolayer and matrigel barrier.
Results: Stathmin siRNA treated SK-N-BE(2) had decreased stathmin expression (>80%) compared to controls; this signficantly decreased migration index (Control: 48.87%; siRNAa: 29.87%; siRNAb: 31.23%; p<0.05, n=3) and invasion index (Control: 21.73%; siRNAa: 8.00%; siRNAb: 9.57%, p<0.0005, n=3). Stathmin suppression did not influence anoikis (Control: 25.14%; siRNAa: 26.63%; siRNAb: 23.82%; n=4). In contrast, stathmin suppression reduced transendothelial migration (siRNAa: 1.5-fold; siRNAb: 2.6-fold) compared to controls.
Conclusions: Stathmin suppression interferes with multiple key steps in the metastatic cascade, including migration, invasion and transendothelial migration.
Implications: Examining stathmin suppression may improve the understanding of the neuroblastoma metastatic cascade. Furthermore, targeting stathmin may provide a novel appraoch in the treatment of metastatic neuroblastoma.