Prognostic significance of miR-34a in Ewing sarcoma is associated with cyclin D1 and ki-67 expression.

BACKGROUND: At diagnosis, identification of reliable biological indicators of prognosis to allow stratification of patients according to different risks is an important but still unresolved aspect in the treatment of Ewing sarcoma (EWS) patients. This study aimed to explore the role of miR-34A expression on prognosis of EWS patients. PATIENTS AND METHODS: Specimens from 109 patients with non-metastatic EWS treated at the Rizzoli Institute with neoadjuvant chemotherapy (protocols ISG/SSGIII, EW-1, EW-2, EW-REN2, EW-REN3, EW-PILOT) and 17 metastases were studied. Sixty-eight patients (62%) remained disease-free and 41 (38%) relapsed (median follow-up: 67 months, range 9-241 months). Expression of miR-34a and of some of its targets (cyclin D1, bcl-2, SIRT1 and YY1) was evaluated by qRT-PCR using TaqMan MicroRNA Assays and/or by immunohistochemistry on tissue microarrays from the same patients. RESULTS: High expression of miR-34a in localized tumors was significantly related to better event-free and overall survival (P = 0.004). Relevance of miR-34a was confirmed by using different calibrators (normal mesenchymal stem cells and different normal tissues). By multivariate Cox regression analysis, low miR-34a expression as well as nontotal necrosis and high levels of lactate dehydrogenase were all confirmed as independent risk factors associated with poor outcome. Expression of miR-34a was lower in metastases than in primary tumors. It inversely correlated with expression of cyclin D1 and Ki-67. CONCLUSIONS: By demonstrating its relationship with clinical outcome, we propose evaluation of miR-34a at diagnosis of EWS patients to allow early risk stratification. Validation of these results would nonetheless ultimately need a prospective assessment.

CD99 suppresses osteosarcoma cell migration through inhibition of ROCK2 activity.

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and ß-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and ß-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.