Abstract
Abstract Neuroblastoma is a highly metastatic childhood cancer of the sympathetic nervous system. Stathmin is a microtubule destabilizing protein highly expressed in neuroblastoma though studies addressing its functional role in this malignancy have been limited. The Rho- Rho-associated coiled-coil forming kinase (Rho-ROCK) signaling pathway has been implicated in metastasis through its regulation of the cell cytoskeleton namely the actin remodeling proteins, cofilin and myosin light chain (MLC), and microtubule dynamics. The aims of this study were to determine whether stathmin influenced the Rho-ROCK signaling pathway and metastasis in neuroblastoma. Methods: SiRNA-mediated stathmin suppression was confirmed in 2 independent neuroblastoma cell lines, BE(2)-C and SY5Y, by qPCR and western blot. The cell cytoskeleton of siRNA-transfected cells was visualized by staining with phalloidin (actin filaments) and β-tubulin (microtubules) and tubulin polymer levels were examined by western blot. The expression of cofilin and MLC in siRNA-transfected cells, following treatment with the ROCK inhibitor Y-27632, were analyzed by western blot. SiRNA-transfected cells were subjected to chemotaxis transwell migration and invasion assays. To assess stathmin's role in neuroblastoma metastasis, two million control or stathmin shRNA/luciferase-expressing neuroblastoma cells [SK-N-BE(2)/TGL] were injected into the left adrenal fat pad of 8 week old SCID-Beige mice. Tumor growth was monitored weekly using the Xenogen IVIS System. Mice were sacrificed when primary tumors reached 1500mm3, 38 days post-injection or upon health decline. Ex vivo imaging and IHC confirmed neuroblastoma cells in the lungs of mice. Results: SiRNA-mediated stathmin suppression significantly reduced neuroblastoma cell migration by 44-47% [SY5Y cells 44% reduction (p<0.05) and BE(2)C cells 47% reduction (p<0.001)] and invasion through an extra-cellular matrix by 55-62% [SY5Y cells 55% reduction (p<0.05) and BE(2)C cells 62% reduction (p<0.05)]. Stathmin suppression also altered neuroblastoma cell morphology and this was associated with changes in the cytoskeleton including increased tubulin polymer levels and the phosphorylation of cofilin and MLC. Treatment of stathmin-suppressed neuroblastoma cells with Y-27632 ablated MLC phosphorylation, and returned the level of cofilin phosphorylation and cell invasion back to that of untreated control cells. This highlights a novel link between stathmin expression and ROCK signaling. In vivo, shRNA-mediated stathmin suppression did not influence neuroblastoma tumor growth. In contrast, stathmin suppression significantly reduced metastatic neuroblastoma tumor burden in the lungs by 71% (p<0.01) compared to controls. Conclusion: Stathmin is a novel mediator of metastasis in neuroblastoma and potential therapeutic target for this deadly disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1444. doi:1538-7445.AM2012-1444
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