Abstract 4987: Stathmin regulates cell migration, invasion and transendothelial migration via RhoA activation in neuroblastoma

Abstract

Abstract Neuroblastoma is an aggressive childhood cancer with dismal 5-year survival rates, primarily due to metastatic disease. The microtubule protein stathmin is highly expressed in neuroblastoma, and silencing stathmin decreases metastatic disease in a neuroblastoma orthotopic mouse model1. However, stathmin's role in the metastatic cascade and mechanism of action in this process is unknown. Our aim was to investigate the role of stathmin in neuroblastoma metastasis. Methods: To study the role of stathmin (STMN) in neuroblastoma, siRNA gene knockdown (using two individual siRNAs STMNsiRNA1 and STMNsiRNA2) or a non-targeting control siRNA (ContsiRNA) was performed in both SK-N-BE(2) and SH-SY5Y cell lines. 3D spheroid invasion assessed by counting and measuring cellular protrusion length into collagen I matrix. Stathmin expression was assessed by RT-qPCR and western blotting. Migration and invasion assays were performed using Boyden-chambers. Anoikis was assessed using Annexin V staining. Transendothelial migration was assessed by measurement of lysed migrated cells using a fluorescent plate reader. RhoA activation was assessed by RhoA activity pulldown and western blotting. Results: Stathmin suppression significantly decreased SK-N-BE(2) and SH-SY5Y cell migration (p<0.05) and invasion (p<0.0005). Spheroid invasion was significantly decreased in SK-N-BE(2) as measured by the number and length of cellular protrusions (p<0.05). Stathmin did not influence anoikis in either of the neuroblastoma cell lines, however stathmin knockdown significantly reduced transendothelial migration in both cell lines compared to ContsiRNA cells (p<0.0001). To determine whether stathmin's effect on transendothelial migration was mediated via ROCK signalling SK-N-BE(2) cells were treated with two separate ROCK inhibitors. Importantly, ROCK inhibition restored transendothelial migration levels in stathmin knockdown cells to those of ContsiRNA cells (STMNsiRNA1:86.0±4.9%; STMNsiRNA2:66.2±4.5%; control:80.95±10.85%; p>0.25), indicating stathmin mediates transendothelial migration via inhibition of ROCK signalling. Importantly, stathmin suppression dramatically increased RhoA activation (upstream regulator of ROCK signalling) indicating that stathmin's effect on ROCK signalling is mediated via its effects on its regulator RhoA. Conclusions: Stathmin suppression interferes with key metastatic events via RhoA/ROCK signalling in neuroblastoma. Targeting stathmin may provide a novel treatment approach for metastatic neuroblastoma. 1 Byrne, FL, Yang, L, Phillips, PA, Hansford, LM, Fletcher, JI, Ormandy, CJ, McCarroll, JA & Kavallaris, M. (2013) Oncogene [epub Feb 11] Citation Format: Christopher M. Fife, Frances L. Byrne, Sharon M. Sagnella, Thomas P. Davis, Joshua A. McCarroll, Maria Kavallaris. Stathmin regulates cell migration, invasion and transendothelial migration via RhoA activation in neuroblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4987. doi:10.1158/1538-7445.AM2014-4987