The Function and Regulation of CXCR4 in Ewing Sarcoma.

Abstract

Ewing sarcoma is an aggressive and deadly bone and soft tissue cancer predominantly effecting the pediatric population. The prognosis for patients with metastatic or relapsed disease is dismal, with fewer than 20% surviving. However, little is known about the biological mechanisms driving Ewing sarcoma metastasis and in order to improve outcomes for Ewing sarcoma patients, it is imperative that we understand these metastatic processes. The chemokine receptor, CXCR4, is the most commonly expressed chemokine receptor in human cancer. There is evidence demonstrating that the CXCL12/CXCR4 signaling axis contributes to tumor metastasis in over twenty human malignancies but its function in Ewing sarcoma is not yet known. Evaluation of CXCR4 transcript and surface protein in Ewing sarcoma cell lines and primary tumors revealed variability of CXCR4, ranging from undetectable expression to high-level expression. We have demonstrated the highly dynamic nature of CXCR4 expression in Ewing sarcoma cells and have shown that this molecular plasticity allows Ewing sarcoma to switch back and forth between CXCR4- and CXCR4+ states in response to changes in the microenvironment. Significantly, Ewing sarcoma cells that express high levels of CXCR4 are highly migratory and invasive toward a CXCL12 gradient. The CXCR4 antagonist, AMD3100, impeded the migratory and invasive properties of the CXCR4+ cells. Having demonstrated that CXCR4 is dynamic and that its expression is induced in response to microenvironmental cues we investigated the potential contribution of epigenetic regulation to these cell-state transitions. Chromatin immunoprecipitation studies revealed that the CXCR4 promoter exists in a bivalent state, as characterized by simultaneous enrichment of the H3K4me3 and H3K27me3 histone marks at the CXCR4 promoter. Exposure of Ewing sarcoma cells to microenvironmental cues resulted in loss of bivalency, as evidenced by loss of H3K27me3 and retention of the H3K4me3. Additionally, we demonstrated that an active enhancer exists upstream of CXCR4 and also contributes to CXCR4 regulation. In support of this, treatment of Ewing sarcoma cells with JQ1, resulted in marked down-regulation of CXCR4 expression in ambient conditions and blocked stress-dependent induction. These findings support the potential use of epigenetic modifiers as therapeutics that could be used to antagonize cell state transitions.