title: EWS-FLI1 suppresses NOTCH-activated p53 in Ewing's sarcoma.
abstract: 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.
authors.affiliation: Children's Cancer Research Institute, St. Anna Kinderkrebsforschung, Vienna, Austria.
authors.forename: Jozef
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authors.lastname: Ban
authors.forename: Idriss M
authors.initials: IM
authors.lastname: Bennani-Baiti
authors.forename: Max
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authors.lastname: Kauer
authors.forename: Karl-Ludwig
authors.initials: KL
authors.lastname: Schaefer
authors.forename: Christopher
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authors.lastname: Poremba
authors.forename: Gunhild
authors.initials: G
authors.lastname: Jug
authors.forename: Raphaela
authors.initials: R
authors.lastname: Schwentner
authors.forename: Oskar
authors.initials: O
authors.lastname: Smrzka
authors.forename: Karin
authors.initials: K
authors.lastname: Muehlbacher
authors.forename: Dave N T
authors.initials: DN
authors.lastname: Aryee
authors.forename: Heinrich
authors.initials: H
authors.lastname: Kovar