Abstract A56: Expression of CXCR4 is highly dynamic in Ewing sarcoma and its upregulation in response to microenvironmental stress promotes cell migration and invasion

Background and Objective: Ewing sarcoma (ES) is the second most common malignant bone tumor in pediatric patients. Although the primary cause of tumor-related death in ES is relapse at distant sites, the mechanisms underlying metastatic relapse remain to be fully elucidated. In the current study we are investigating the role of the CXCR4/CXCL12 chemokine axis as a mediator of ES metastasis. Methods: Expression of CXCR4 transcript and surface protein were quantified using qRT-PCR and flow cytometry. Label-retaining, putative cancer stem cells were isolated 10-12 days following PKH-dye incorporation into cell membranes. Chemotactic migration and invasion to SDF-1α were evaluated in unmodified cells and in cells following CXCR4 knockdown or exposure to the CXCR4 antagonist AMD3100. Small molecule inhibitors of Rho-GTPases were used to assess the contribution of these proteins to CXCR4-dependent migration. Results: Baseline expression of CXCR4 transcript and surface protein varied widely among ES cell lines. Immunohistochemical staining of primary tumors also revealed marked variability of CXCR4, ranging from completely undetectable to high-level expression. Interestingly, CXCR4 transcript was increased in slowly proliferating (PKH-dye-retaining) TC71 and A673 cells implicating CXCR4 as a potential marker of ES cancer stem cells. To determine if CXCR4 expression is static or responsive to changes in the microenvironment we measured transcript levels and the frequency of CXCR4+ cells under different experimental conditions. Significantly, serum deprivation, hypoxia and growth constraint (cell confluence) all resulted in upregulation of CXCR4 mRNA and CXCR4+ cell frequency in each of 4 different ES cell lines. This upregulation of CXCR4 was rapidly reversed upon removal of each of the stressors indicating the plastic and dynamic nature of CXCR4 regulation. In addition, within 3 weeks of FACS sorting, TC-32 ES cells that had been sorted into CXCR4+ and CXCR4- populations reverted to their baseline state, demonstrating that CXCR4+ cells can change into CXCR4- cells and vice versa. Functionally, CXCR4+ ES cells migrated and invaded towards SDF-1α, the ligand for CXCR4 and this chemotactic migration and invasion were impeded pharmacologically by AMD3100, and genetically by CXCR4 knockdown. Interestingly, we observed complete abrogation of invasion through Matrigel in CXCR4 knockdown and AMD3100 treated cells suggesting a critical role for CXCR4 in mediating ES cell invasion through basement membranes. To begin to define the mechanism of CXCR4-mediated invasion we exposed ES cells to small molecule inhibitors of the Rho-GTPases CDC42 and RAC1. Importantly, sub-cytotoxic doses of both compounds suppressed ES cell migration towards SDF-1α. Conclusions: We have demonstrated the highly dynamic nature of CXCR4 expression in ES cells and have shown that this molecular plasticity allows ES to switch back and forth between CXCR4- and CXCR4+ states in response to changes in the microenvironment. Significantly, ES cells that express high levels of CXCR4 are highly migratory and invasive in response to chemotactic signaling from SDF-1α and preliminary data suggest that Rho-GTPases CDC42 and/or RAC1 mediate this aggressive phenotype. We propose a model in which stressors in the microenvironment induce ES cells to upregulate CXCR4 thus contributing to cellular migration and invasion. Ongoing work is testing whether CXCR4+ ES cells are more metastatic than CXCR4- cells in vivo. The CXCR4/CXCL12 axis may thus be a novel therapeutic target for the prevention of ES metastasis. Citation Format: Melanie A. Krook, Lauren A. Nicholls, Christopher A. Scannell, Rashmi Chugh, Dafydd G. Thomas, Elizabeth R. Lawlor. Expression of CXCR4 is highly dynamic in Ewing sarcoma and its upregulation in response to microenvironmental stress promotes cell migration and invasion. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A56.