Abstract 5141: Stathmin mediates neuroblastoma migration and invasion via regulation of the actin network
Authors: Byrne Frances L. , McCarroll Joshua , Kavallaris Maria 
Published: February 2011

Aggressive metastatic neuroblastoma confers a poor clinical prognosis. Stathmin is a microtubule destabilizing protein highly expressed in advanced stage (metastatic) neuroblastoma (1) that promotes metastasis of non-neuronal cancers (2). However, stathmin's role in neuroblastoma metastasis is unclear. The aims of this study were to determine the functional and mechanistic role of stathmin in neuroblastoma metastasis.

During metastasis, restructuring of the cell cytoskeleton, in particular the actin network is necessary to drive cell migration/invasion. The Rho-GTPase/LIM kinase/cofilin pathway regulates actin remodeling. Activation of LIM kinases (LIMK1/2) by Rho-GTPases induces actin polymerization via phosphorylation (inactivation) of cofilin. Interestingly, stathmin is also regulated downstream of the Rho-GTPases. We therefore investigated this pathway as a potential mechanism underlying stathmin's role in neuroblastoma cell migration/invasion.

Methods: Studies were performed using siRNA to knockdown stathmin in 2 independent neuroblastoma cell lines, BE(2)-C and SY5Y. Stathmin knockdown was confirmed by qPCR and Western blot. SiRNA-treated cell lines were subjected to in vitro migration and invasion assays using fetal calf serum as a chemo-attractant. The expression of actin-regulatory proteins LIMK1/2 and the actin-severing protein cofilin were analyzed by Western blot. Actin filaments within neuroblastoma cells were visualized by phalloidin staining.

Results: Stathmin knockdown significantly reduced the migration of BE(2)C (47% reduction p&lt;0.001) and SY5Y (44% reduction p&lt;0.05) cells. Moreover, suppressing stathmin significantly reduced the ability of BE(2)C (62% reduction p&lt;0.05) and SY5Y (55% reduction p&lt;0.05) cells to invade an extra-cellular matrix. Stathmin knockdown BE(2)C cells displayed altered phosphorylation of both LIMK1/2 and cofilin upon serum-activation, compared to control cells. Cofilin phosphorylation was induced 5 minutes post serum activation in both control and stathmin knockdown cells. After 5 minutes, cofilin phosphorylation declined in control cells but was maintained in stathmin knockdown BE(2)C cells up to 60 minutes post serum-activation. The most notable difference was observed 15 minutes post serum-activation, a time point at which dense actin structures were visible at the cell periphery in phalloidin-stained stathmin knockdown cells but not controls cells. These data show that stathmin is a necessary intermediate for actin restructuring required for the migration and invasion of neuroblastoma cells.

Conclusion: Stathmin plays a pro-migratory and invasive role via regulation of the actin network in neuroblastoma. These identify stathmin as a promising new therapeutic target to treat metastatic neuroblastoma.

1. N. Hailat et al. J Clin Invest 1991; 88: 341

2. B. Belletti et al. Mol Biol Cell. 2008: 19: 2003

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5141.