Unknown,Transcriptomics,Genomics,Proteomics

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CD99 triggering induces a novel processes of death in Ewing's sarcoma cells involving reactivation of p53 functions and RAS upregulation.

ABSTRACT: CD99 is a transmembrane protein, whose expression is constantly associated to EwingM-bM-^@M-^Ys Sarcoma (EWS), a class of pediatric bone tumors with particular poor prognosis. We previously reported that engagement of CD99 leads to massive and rapid EWS cell death through a non-canonical path. Here we report that death occurs through a novel mechanism starting from classical apoptotic features, such as phosphatydilserine exposure on cell surface and mitochondrial depolarization and ending with massive cytoplasmic hypervacuolization (autophagosomes and micropinosomes). Mechanistically, CD99 induces upregulation of IGFI-R and RAS and rapid MDM2 degradation, which leads to p53 reactivation. We propose that upon CD99 engagement and subsequent p53 reactivation, the EWS-ets oncogene becomes insufficient to sustain EWS transformation; in this context, up-regulated RAS, deprived of a cooperating oncogenic stimulus, might contribute to the delivery of fatal rather than pro-survival signal. The most CD99-responsive EWS cells have either wild type or transcriptional active P53, though mutated, and greatly benefit from MDM2 degradation. Due to the low rate of P53 inactivating mutations in EWS patients, these findings sustain CD99-targeting with specific MAbs either to directly kill EWS cells or to increase sensitivity to chemotherapy. By recruiting also RAS, CD99 delivers a signal which exceeds that of drugs designed exclusive to reactivate p53 functions, besides being easily druggable Ewing Sarcoma cell lines 6647 control or treated with the anti-CD99 0662 MAb were profiled to discover mechanisms related to CD99 targeting. Treated cells were compared to control cells after 30, 60 and 120 minutes of exposure to 0662 (10M-BM-5g/ml); all treatments are in duplicate.

ORGANISM(S): Homo sapiens

SUBMITTER: Andrea Grilli

PROVIDER: E-GEOD-36097 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The t(11;22)(p13;q12) translocation is pathognomonic for the highly aggressive desmoplastic small round cell tumour (DSRCT). The translocation fuses exon 7 of the EWS gene to exon 8 of the WT1 gene (EWS/WT1). Two splice variants of EWS/WT1 exist, arising from the presence (+KTS) or absence (-KTS) of three amino acids: lysine, threonine and serine between zinc fingers 3 and 4. To investigate the oncogenic properties of both isoforms of EWS/WT1, we over-expressed EWS/WT1 in untransformed murine embryonic fibroblasts (MEFs). We demonstrate that neither isoform of EWS/WT1 is sufficient to transform wild type MEFs, however the oncogenic potential of both isoforms is unmasked by the loss of p53. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 resulted in enhanced cell proliferation, clonogenic survival and anchorage-independent growth. In addition EWS/WT1 expression in wild type MEFs attenuated several p53-dependent responses, including cell cycle arrest after irradiation and daunorubicin induced apoptosis. We show that DSRCT commonly have copy number amplification of MDM2 and MDMX, suggesting loss of p53 function in the tumours. Expression of either isoform of EWS/WT1 in MEFs induced characteristic mRNA expression profiles, including up-regulation of canonical Wnt pathway signaling. This was validated in cell lines and in a series of DSCRT, which confirmed canonical Wnt pathway activation in the tumours. We show for the first time that both isoforms of EWS/WT1 have oncogenic potential and that in addition to co-operating with loss of p53 function can also further attenuate p53-mediated responses. In addition we provide the first link between EWS/WT1 and Wnt pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterizes DSCRT. Four independently generated pools of wild type MEFs were infected with tetracycline repressible EWS/WT1+KTS, EWS/WT1-KTS or GFP, selected with hygromycin and protein expression confirmed. Four GFP, four KTS+ and four KTS- samples were hybridized to the Ilumina BeadChip.