Effects of ROCK kinases inhibition on the invasive potential of Ewing sarcoma cell lines

Ewing\'s sarcoma (ES) is characterized by undifferentiated cells of neuroepithelial origin. It is the second most common malignant bone tumor in children and adolescents. Standard treatment consists of preoperative chemotherapy, followed by surgery and postoperative chemotherapy and/or radiotherapy. However, the spread of the tumor is very high and even in patients where the metastasis has not occurred, there may be circulating tumor cells. Furthermore, the 5-year survival which about 70% of patients without metastases at diagnosis achieve, falls to 25% in patients with metastases. Migration and invasion into adjacent tissues and blood vessels, cell adhesion and proliferation are essential for tumor metastasis to occur. Among the regulators of the processes are the Rho family of GTPases, which have as their effector kinases, ROCK1 and ROCK2. Thus, ROCK induce numerous cellular responses involving the regulation of many proteins associated with the cytoskeleton. Studies show that responses regulated by ROCK aggravate phenotypes associated with cancer and other diseases. This study aimed to verify the expression of ROCK1 and ROCK2 genes in ES tumor samples and to evaluate the effects of their pharmacological inhibition in vitro. Gene expression analysis showed lower expression of ROCK in both, patient samples (n=18) and the SK-ES- 1 and RD-ES cell lines. There was no relation of gene expression with clinical data. Nonetheless, the presence of EWS-FLI1 fusion appears to be associated with lower expression of ROCK1. Cell proliferation, clonogenic capacity and the cell cycle were not affected after different treatments with GSK429286 (ROCK1 inhibitor), SR3677 (ROCK2 inhibitor) and Hydroxyfasudil (pan-inhibitor). However, although not significant, there was a tenuous increase in cell migration and invasion following treatment with the drugs. Our data suggest that ROCK1 and ROCK2 might have a role in ES tumorigenesis and may be related im part to migration and invasion cell mechanisms, important processes for tumor metastasis. (AU)