Abstract 483: Functional validation of CEBPB as an oncogenic target of EWS-FLI1 in Ewing sarcoma

Abstract

Abstract We previously identified increased copies of CEBPB in a subset of Ewing sarcoma (ES) tumors, and this gain was associated with worse clinical outcome. Due to CEBPB's role in cell growth and differentiation, as well as the gene's ability to transform normal mammary epithelial cells, we hypothesized that CEBPB acts as an oncogene in ES by increasing cell proliferation and transformation. To address this hypothesis, we altered gene expression using viral gene delivery systems in ES cell lines. First, we tested if CEBPB is a target of the EWS-FLI1 fusion protein using a FLI1 shRNA to knockdown EWS-FLI1 in an ES cell line (A673), followed by re-expression of the fusion gene. We then measured expression of the three C/EBPβ (protein) isoforms by western blot. Knockdown of EWS-FLI1 expression led to decreased protein expression of all three C/EBPβ isoforms. Re-expression of EWS-FLI1 rescued C/EBPβ protein expression, suggesting that C/EBPβ is a target of EWS-FLI1. To explore the functional consequence of altered expression of CEBPB, we transduced ES cell lines to knockdown, overexpress, and rescue C/EBPβ. Changes in protein expression were confirmed by western blot. Following transduction and antibiotic selection, cell proliferation and colony formation were measured by quantification of cellular ATP (Cell Titer Glo, Promega). Overexpression of C/EBPβ-1, the largest of the three C/EBPβ isoforms, led to a significant (p<0.005) increase in colony formation when cells were grown in soft agar compared to empty vector transduced cells. In addition, knockdown of C/EBPβ decreased colony formation (p<0.05), and re-expression of either C/EBPβ-1 (p<0.0001) or C/EBPβ-2 (p<0.005) rescued the phenotype. To identify downstream targets of C/EBPβ we measured changes in protein expression by western blot of potential CEBPB targets in cells with overexpression of each C/EBPβ isoform. Overexpression of C/EBPβ-1 and CEBPβ-2 led to increased protein expression of ALDH1A1. In addition, overexpression of C/EBPβ-1 and C/EBPβ-2 led to resistance to chemotherapeutic agents similar to previous reports of chemoresistance for ES cells that overexpressed ALDH. In conclusion, the increased transformation potential of ES cells that overexpress either C/EBPβ-1 or C/EBPβ-2 indicates that CEBPB is an oncogenic target of EWS-FLI1. Poor outcome for patients with CEBPB amplifications may result from chemoresistance mediated by increased expression of ALDH1A1. In addition, increased expression of ALDH may suggest that overexpression of C/EBPβ in ES tumors induces a stem cell like state. Patients with CEBPB amplifications may benefit from pretreatment with ALDH inhibitors prior to chemotherapy. Citation Format: Lisa M. Abegglen, Jamie D. Gardiner, Clinton C. Mason, Bryce E. Carter, Elizabeth A. Schackmann, Marcus Stucki, Angelica R. Putnam, R Lor Randall, Heinrich Kovar, Stephen L. Lessnick, Joshua D. Schiffman. Functional validation of CEBPB as an oncogenic target of EWS-FLI1 in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 483. doi:10.1158/1538-7445.AM2015-483