nutlin-3a and Sarcoma--Ewing

Translocations involving ETS-transcription factors, most commonly leading to the EWSR1-FLI1 fusion protein, are the hallmark of Ewing sarcoma. Despite knowledge of this driving molecular event, an effective therapeutic strategy is lacking. To test potential treatment regimes, we established a novel Ewing sarcoma zebrafish engraftment model allowing time-effective, dynamic quantification of Ewing sarcoma progression and tumour burden in vivo, applicable for screening of single and combined compounds. In Ewing sarcoma the tumour-suppressor gene TP53 is commonly found to be wild-type, thus providing an attractive target for treatment. Here, we study TP53 wild-type (EW7, CADO-ES1 and TC32) and TP53-deleted (SK-N-MC) Ewing sarcoma cell lines to investigate the potentiating effect of p53 reactivation by Nutlin-3 on treatment with YK-4-279 to block transcriptional activity of EWSR1-FLI1 protein. Blocking EWSR1-FLI1 transcriptional activity reduced Ewing sarcoma tumour cell burden irrespective of TP53 status. We show that simultaneous YK-4-279 treatment with Nutlin-3 to stabilize p53 resulted in an additive inhibition of TP53 wild-type Ewing sarcoma cell burden, whilst not affecting TP53-deleted Ewing sarcoma cells. Improved inhibition of proliferation and migration by combinatorial treatment was confirmed in vivo by zebrafish engraftments. Mechanistically, both compounds together additively induced apoptosis of tumour cells in vivo by engaging distinct pathways. We propose reactivation of the p53 pathway in combination with complementary targeted therapy by EWSR1-FLI1 transcriptional activity disruption as a valuable strategy against p53 wild-type Ewing sarcoma.

Topics: Animals; Antineoplastic Agents; Bone Neoplasms; Cell Line, Tumor; Cells, Cultured; Disease Models, Animal; Drug Synergism; Heterografts; Humans; Imidazoles; Indoles; Piperazines; RNA-Binding Protein EWS; RNA-Binding Proteins; Sarcoma, Ewing; Signal Transduction; Transcription, Genetic; Tumor Suppressor Protein p53; Zebrafish; Zebrafish Proteins

Identification of factors to detect chemotherapy-resistant tumours at diagnosis is a first priority for risk-adapted therapy in the oncology of children and young adults, where more individualized, effective, and less toxic treatments are highly desirable. In this study, we analysed the miRNAs discriminating Ewing's sarcoma (EWS) patients with different clinical outcomes in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWSs by using the Agilent human miRNA microarray v.2 and/or qRT-PCR. Statistical power of the samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of five miRNAs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) as an independent predictor of risk for disease progression and survival. Validation analysis in the extended sample set indicated that both miR-34a and miR-490-3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR-34a, which appeared associated with either event-free or overall survival and emerged as a 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 2 years. High expression of miR34a can be detected also in paraffin-embedded tissues by in situ hybridization, thus contributing to an easy routine 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.

Topics: Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bone Neoplasms; Cell Line, Tumor; Disease-Free Survival; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Humans; Imidazoles; In Situ Hybridization; Inhibitory Concentration 50; Kaplan-Meier Estimate; MicroRNAs; Multivariate Analysis; Oligonucleotide Array Sequence Analysis; Paraffin Embedding; Piperazines; Proportional Hazards Models; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Retrospective Studies; Risk Assessment; Risk Factors; Sarcoma, Ewing; Time Factors; Transfection; Treatment Outcome; Tumor Suppressor Protein p53; Vincristine

Although mutations in the TP53 gene occur in half of all cancers, approximately 90% of Ewing sarcomas retain a functional wild-type p53. The low frequency of TP53 alterations in Ewing sarcoma makes this tumor type an ideal candidate for p53-targeted therapies. In this study, we have examined the molecular and cellular responses of cultured Ewing sarcoma cell lines following exposure to Nutlin-3a, a recently developed MDM2 antagonist.. The ability of Nutlin-3a to impart apoptosis or cell cycle arrest in a p53-dependent manner was determined in a comprehensive panel of Ewing sarcoma cell lines. The capacity of Nutlin-3a to augment the antitumor activity of MDM4 antagonists and cytotoxic agents currently used in the clinical treatment of Ewing sarcoma was also investigated.. Apoptosis was the primary response of wild-type p53 expressing Ewing sarcoma cell lines. The cytotoxicity of Nultin-3a was also synergistic with the chemotherapeutic agents, vincristine, actinomycin D, doxorubicin, and etoposide in a concentration-dependent manner. Significant MDM4 protein overexpression was observed in Ewing sarcoma cell lines of wild-type p53 status, providing a mechanism through which Ewing sarcomas can develop in the absence of TP53 alterations. This study provides the first evidence of synergism between targeted inhibition of MDM2 and MDM4.. Our findings suggest that p53-dependent apoptosis is the primary cellular response of Ewing sarcoma cell lines following exposure to Nutlin-3a. Furthermore, Nutlin-3a can synergize with the current Ewing sarcoma chemotherapy protocols, suggesting p53 activation as a novel systemic therapeutic approach for this disease.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Drug Synergism; Genes, p53; Humans; Imidazoles; Molecular Targeted Therapy; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Sarcoma, Ewing

Mutation of p53 is rare in Ewing's sarcoma (ES), suggesting that targeting and activation of wild-type p53 may be an effective therapeutic strategy for ES. The recently developed small-molecule MDM2 inhibitor nutlin-3 restores wild-type p53 function, resulting in the inhibition of cancer cell growth and the induction of apoptosis. In the present study, we explored the responsiveness of ES cell lines with wild-type or mutated p53 to nutlin-3. We found that treatment with nutlin-3 increased p53 level and induced p53 target gene expression (MDM2, p21, PUMA) in ES cells with wild-type p53, but not in ES cells with mutated p53. Consistently, nutlin-3 elicited apoptosis only in wild-type p53 cells, as assessed by caspase-3 activity assay and flow cytometric analyses of mitochondrial depolarisation and DNA fragmentation. In addition, we found nutlin-3 to evoke cellular senescence, indicating that nutlin-3 induces pleiotropic anticancer effects in ES. Furthermore, combined treatment with nutlin-3 and an inhibitor of NF-κB produced synergistic antineoplastic activity in ES cells. Our findings suggest that the direct activation of p53 by nutlin-3 treatment may be a useful new therapeutic approach for patients with ES.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cellular Senescence; Flow Cytometry; Genes, p53; Humans; Imidazoles; Membrane Potential, Mitochondrial; Membrane Potentials; Mice; Mutation; NF-kappa B; Piperazines; Sarcoma, Ewing; Tumor Suppressor Protein p53