Abstract 481: Dualistic regulation of NF-kB signaling by CD99 and EWS-FLI1 in Ewing sarcoma: impact on cell differentiation

Abstract

Abstract Introduction: Ewing sarcoma (ES) is a high-grade rare malignant bone tumor in children and adolescents. This disease is primarily characterized by: i) Chromosomal translocations that fuse EWS gene and a gene of the ETS family of transcription factors (around 85% EWS-FLI1 and 10% EWS-ERG, respectively) which are the oncogenic drivers of this tumor; ii) High expression of CD99, a cell surface glycoprotein virtually present in all of ES cells. EWS-FLI1 and CD99 were reported to have divergent roles in modulating ES malignancy and neural differentiation In this study we investigated the possible role of nuclear factor-kappa B (NF-kB) signaling pathway on the neural differentiation of ES cells and its relationship with EWS-FLI1 and CD99. Materials and Methods: Transcriptional activity of several transcription factors (AP1, SRE, CREB, E2F and NF-kB) were evaluated by gene reporter assay. Using a knockdown approach in stable and transient conditions we silenced CD99, EWS-FLI1 and NF-kB itself, alone or in combination, in three ES cell lines (TC-71, IOR/CAR and ASP14). NF-kBp65 was also transiently overexpressed in TCshCD99 and CARshCD99 clones with pCMVp65 construct. Neural differentiation was detected evaluating β-III tubulin by immunofluorescence microscopy. Results: NF-kB was found heterogeneously expressed in a panel of ES cell lines. Stable CD99 silencing was found to decrease NF-kB activity (pValue <0.05) without any differences at RNA and protein levels. On the contrary, EWS-FLI1 knockdown increased NF-kB transactivation (pValue <0.01). CD99 affects NF-kBp65 activity and its effect are dominant with respect to EWS-FLI1. Accordingly, EWS-FLI1 silencing was not able to revert the reduction of NF-kB activity observed after CD99 deprivation. In addition cells silenced for CD99 but not for EWS-FLI1 (TCshCD99 and CARshCD99 clones) showed a neural differentiated phenotype, which was lost when cells were induced to re-express NF-kB. Conclusion: Our findings support the concept that inhibition of NF-kB is able to induce terminal neural differentiation of ES cells. This identifying NF-kB as a potential therapeutic target against Ewing sarcoma progression. (Supported by AIRC_2013_IG 14049 and Liddy Shriver Foundation) Citation Format: Selena Ventura, Chiara Manzalini, Dave Aryee, Piero Picci, Heinrich Kovar, Katia Scotlandi. Dualistic regulation of NF-kB signaling by CD99 and EWS-FLI1 in Ewing sarcoma: impact on cell differentiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 481. doi:10.1158/1538-7445.AM2014-481