Abstract
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.
Highlights
The p53 tumor suppressor plays a pivotal role in the prevention of oncogenic transformation by inducing cell cycle arrest or apoptosis in response to various cellular stresses [1]
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
Our findings suggest that p53-dependent apoptosis is the primary cellular response of Ewing sarcoma cell lines following exposure to Nutlin-3a
Summary
Nutlin-3a induces cell death in Ewing sarcoma cell lines in a p53-dependent manner. The ability of Nutlin-3a to induce p53-dependent cell death was determined in vitro using a comprehensive panel of 9 Ewing sarcoma cells lines with varying p53 status. The viability of all Ewing sarcoma cell lines expressing wild-type p53 was markedly reduced following exposure to Nutlin-3a in a dose-dependent manner (Supplementary Fig. 1). Neither stabilization of p53 protein levels nor transactivation of p53 target genes was observed following Nutlin-3a treatment of Ewing sarcoma cell lines (SK-ES1 and RD-ES) expressing mutant p53 (Fig. 2C and Supplementary Fig. 3). The observed synergism required p53 pathway activation, as Nutlin-3a did not influence the cytotoxic activity of these drugs in the mutant p53 expressing cell line TC-71 (Supplementary Fig. 8) or in NHB (Supplementary Fig. 9). Pharmacologic activation of p53 by Nutlin-3a has been shown to cause a growth arrest in normal dividing cells, thereby protecting them from the cytotoxic effects of chemotherapeutic agents that target rapidly dividing cells—a concept termed "cyclotherapy" [31, 32]. Further investigations into the key genetic and molecular events that allow these sarcomas to develop in the presence of a wild-type p53 were required
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