Abstract 5134: Mechanistic dissection of EWS-FLI impact on Ewing's sarcoma cell behavior

Abstract

Abstract Ewing's sarcoma is caused by the oncogenic fusion of the EWS and FLI genes to encode a chimeric EWS-FLI protein product. The disease is often metastatic at presentation. This study aims to define the molecular mechanism by which the EWS-FLI oncoprotein influences metastatic potential by testing the impact of EWS-FLI expression on cytoarchitecture, adhesion, and migration of Ewing's sarcoma cells. We compared the behavior of A673 Ewing's sarcoma cells with A673 cells in which we knocked down expression of EWS-FLI by RNA-interference. Knock-down of EWS-FLI results in enhanced cell spreading and the establishment of robust actin stress fibers and focal adhesions. Consistent with these morphological attributes, a quantitative cell adhesion assay revealed that parental A673 cells are less adhesive than those in which EWS-FLI expression was reduced by RNA interference. Knockdown of EWS-FLI in A673 cells is also associated with increased cell motility and loss of anchorage-independent cell growth. Gene expression profiling revealed that expression of EWS-FLI causes downregulation of transcripts encoding several focal adhesion proteins and integrin adhesion receptors by 2-4 fold. By Western immunoblot analysis, we demonstrated that knock-down of EWS/FLI oncoprotein expression in human A673 cells leads to increased expression of focal adhesion constituents such as the actin regulator, zyxin. Introduction of a zyxin expression construct into A673 Ewing's sarcoma cells was sufficient to restore the integrity of the actin cytoskeleton and focal adhesions, and to enhance cell adhesion. In contrast, restoration of zyxin expression in A673 sarcoma cells did not affect cell motility, matrigel invasion, or anchorage-independent cell growth. Thus, oncogenic transformation by EWS-FLI is associated with changes in cytoskeletal architecture, cell adhesion, cell motility/invastion, and cell growth control. Re-expression of a single focal adhesion protein, zyxin, is sufficient to restore cytoskeletal integrity and partially rescue the adhesion deficits seen in A673 sarcoma cells. These studies provide evidence for a zyxin-dependent pathway downstream of EWS-FLI that controls cell morphology and adhesion, as well as zyxin-independent pathways that influence motility/invasion and anchorage-independent cell growth. Loss of adhesion coupled to reduced zyxin expression may be an early step in the establishment of metastatic potential of Ewing's sarcoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5134.