miRNA signature predicts survival of Ewing’s sarcoma patients and miR34a directly influences cell chemosensitivity and malignancy

Identification of factors to detect chemotherapy-resistant tumours at diagnosis is a first priority for risk-adapted therapy in children and young adults oncology where more individualized, effective and less toxic treatments are highly desirable. In this study, we analyzed the miRNAs discriminating Ewing's sarcoma (EWS) patients with different clinical outcome in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWS by using the Agilent Human miRNA microarray v.2 and/or qRT-PCR. Statistical power of samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of 5 miRNAs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) as an independent predictor of risk to disease progression and survival. Validation analysis in the extended samples set indicated that both miR-34a and miR490-3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR-34a, which appeared associated to either event-free or overall survival and emerged as significant predictor also after multivariate analysis. Patients with the highest expression of miR-34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within two years. High expression of miR34a can be detected also in paraffin-embedded tissues by in situ hybridization, thus contributing to an easy routinely evaluation of this miRNA. Functional analysis of miR-34a in EWS cell lines indicated that when miR-34a expression was enforced cells were less proliferative, less malignant and sensitized to doxorubicin and vincristine. Expression of miR-34a could be increased in p53wt cells by treatment with Nutlin-3a. Accordingly, Nutlin-3a synergizes with doxorubicin. Overall, our data indicate that miR-34a expression is a strong predictor of outcome in EWS. Restoration of miR-34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long-term toxicity to EWS patients