Stathmin mediates neuroblastoma metastasis in a tubulin-independent manner via RhoA/ROCK signaling and enhanced transendothelial migration.

Neuroblastoma, the most common solid tumor of young children, frequently presents with aggressive metastatic disease and for these children the 5-year survival rates are dismal. Metastasis, the movement of cancer cells from one site to another, involves remodeling of the cytoskeleton including altered microtubule dynamics. The microtubule-destabilizing protein, stathmin, has recently been shown to mediate neuroblastoma metastasis although precise functions remain poorly defined. In this study we investigated stathmin's contribution to the metastatic process and potential mechanism(s) by which it exerts these effects. Stathmin suppression significantly reduced neuroblastoma cell invasion of 3D tumor spheroids into an extracellular matrix. Moreover, inhibiting stathmin expression significantly reduced transendothelial migration in two different neuroblastoma cell lines in vitro. Inhibition of ROCK, a key regulator of cell migration, in neuroblastoma cells highlighted that stathmin regulates transendothelial migration through ROCK signaling. Reduced stathmin expression in neuroblastoma cells significantly increased the activation of the RhoA small GTPase. Notably, re-expression of either wild type or a phospho-mimetic stathmin mutant (4E) made defective in tubulin binding returned cell migration and transendothelial migration back to control levels, indicating that stathmin may influence these processes in neuroblastoma cells independent of tubulin binding. Finally, stathmin suppression in neuroblastoma cells significantly reduced whole body, lung, kidney and liver metastases in an experimental metastases mouse model. In conclusion, stathmin suppression interferes with the metastatic process via RhoA/ROCK signaling in neuroblastoma cells. These findings highlight the importance of stathmin to the metastatic process and its potential as a therapeutic target for the treatment of neuroblastoma.

Shear-induced platelet activation and adhesion on human pulmonary artery endothelial cells seeded onto hydrophilic polymers.

We evaluated platelet activation and adhesion on two plasma polymerized surfaces, N-vinyl pyrrolidone (NVP) and gamma-butyro lactone (GBL), which have been shown previously to promote endothelial cell growth and adhesion as well as fibronectin-coated glass (1 microg/cm(2)) coverslips. Human pulmonary artery endothelial cells were seeded onto coverslips at a low density ( approximately 20,000 cells/cm(2)) and grown to confluence (3-5 days). The materials, both with and without ECs, were then exposed to a shear rate of 400 s(-1) in a closed loop recirculating flow system containing human platelet-rich plasma. Plasma samples were taken at 0, 5, 15, 30, and 60 min and analyzed for platelet and coagulation activation. The coverslips were examined for EC coverage and platelet adherence. EC retention over a 1-h period was approximately 75% for all three materials. All three materials without ECs were highly platelet activating having similar P-selectin expression, platelet factor 4 (PF4) release, mepacrine uptake, and microparticle production. Both microparticle production and platelet adhesion were significantly lower in EC-seeded materials. Dense granule and PF4 release were both slightly diminished in all three materials seeded with ECs. P-selectin expression was reduced slightly for GBL, but remained the same for the other two materials. The EC-seeded materials displayed favorable characteristics with respect to platelet activation and adhesion; however, they still demonstrated some thrombogenic tendencies due to EC loss and exposure of the underlying substrate. Therefore, both EC coverage and EC hemostatic function are important factors in determining the thromboresistance of an EC-seeded surface.