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

• Published in: Cancer research (Chicago, Ill.) Vol. 68; no. 17; pp. 7100 - 7109
• Main Authors: BAN, Jozef, BENNANI-BAITI, Idriss M, KOVAR, Heinrich, KAUER, Max, SCHAEFER, Karl-Ludwig, POREMBA, Christopher, JUG, Gunhild, SCHWENTNER, Raphaela, SMRZKA, Oskar, MUEHLBACHER, Karin, ARYEE, Dave N. T
• Format: Journal Article
• Language: English
• Published: Philadelphia, PA American Association for Cancer Research 01.09.2008
• Subjects: Antineoplastic agents; Base Sequence; Basic Helix-Loop-Helix Transcription Factors - genetics; Biological and medical sciences; Blotting, Western; Cell Cycle Proteins - genetics; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21 - genetics; Diseases of the osteoarticular system; DNA Primers; Fluorescent Antibody Technique; Gene Silencing; Genes, p53; Humans; Medical sciences; Oligonucleotide Array Sequence Analysis; Oncogene Proteins, Fusion - genetics; Oncogene Proteins, Fusion - physiology; Pharmacology. Drug treatments; Proto-Oncogene Protein c-fli-1; Receptors, Notch - physiology; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Protein EWS; Sarcoma, Ewing - metabolism; Transcription Factors - genetics; Transcription Factors - physiology; Tumors; Tumors of striated muscle and skeleton; Ewing sarcoma; Notch receptor; TP53 Gene; Transcription factor Fli1; C-Onc gene; Diseases of the osteoarticular system; Malignant tumor; Protooncogene; Cell signaling; Cancer; Tumor suppressor gene
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-07-6145

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.