Loss of connexin43 expression in Ewing's sarcoma cells favors the development of the primary tumor and the associated bone osteolysis

Abstract

Ewing's sarcoma (ES) is a primary bone tumor characterized by a chromosomic translocation between the EWS gene and a member of the ETS gene family, mainly FLI1, which leads to an aberrant transcription factor EWS-FLI1 that promotes tumorigenicity. Gap junctions are intercellular channels composed of transmembrane proteins (connexin: Cx), that allow direct intercellular communication between adjacent cells. Numerous studies have shown that tumorigenesis may be associated with a loss of gap junctional intercellular communication (GJIC). Loss of Cx43 expression was observed at the protein and mRNA levels in ES cell lines compared to those measured in human mesenchymal stem cells. A673 ES cells stably transfected with an shRNA targeting EWS-FLI1 showed an increase in Cx43 expression (at the mRNA, protein and transcriptional levels) and GJIC. In an osteolytic murine model of ES, the overexpression of Cx43 in ES cells dramatically reduced tumor growth, leading to a significant increase in animal survival. In vitro assays showed that Cx43 overexpression increases the p27 level with an associated marked decrease of Rb phosphorylation, consistent with the observed blockade of the cell cycle in G0/G1 phase. In addition, the bone microarchitectural parameters, assessed by micro-CT analysis, showed an increased bone volume when Cx43 expression was enhanced. Histological analysis demonstrated that the overexpression of Cx43 in ES tumor cells inhibits osteoclast activity and therefore bone resorption. Our study demonstrated that the loss of Cx43 expression in ES cells plays a crucial role in the development of the primary tumor and the associated bone osteolysis.