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EWS-FLI1 Suppresses NOTCH-Activated p53 in Ewing's Sarcoma

¹ Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria and ² Institute of Pathology, Heinrich-Heine University, Duesseldorf, Germany

Requests for reprints: Heinrich Kovar, Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Kinderspitalgasse 6, A-1090 Vienna, Austria. Phone: 43-1-40470-4090; Fax: 43-1-40470-7150; E-mail: heinrich.kovar{at}ccri.at.

Key Words: EWS-FLI1 • Ewing's sarcoma • NOTCH • p53 • tumor suppression

Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21^WAF1/CIP1 levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21^WAF1/CIP1 induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21^WAF1/CIP1. These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53. [Cancer Res 2008;68(17):7100–9]