Abstract B33: Plasticity of the Ewing sarcoma invasive program: How microenvironmental stresses alter the drivers and repressors of metastasis
Authors: Bailey Kelly M. , Airkik Merlin , Krook Melanie , Magnuson Brian , Ljungman Mats , Lawlor Elizabeth 
Published: May 2016

Purpose of the Study: Metastatic Ewing sarcoma is notoriously difficult to treat and patient survival rates are still dismal. Therefore, better understanding the modulators of Ewing Sarcoma metastasis is required. The purpose of this study was to define the contribution of tumor cell plasticity to dynamic changes in cell migration, invasion, and invadopodia formation, in vitro proxies of the metastatic phenotype in vivo. We hypothesized that microenvironmental stress (i.e. nutrient deprivation and hypoxia) may promote transition of Ewing sarcoma cells from less motile to more migratory and invasive states by repressing proteins known to inhibit invadopodia formation, such as caldesmon, while also activating drivers of invasion, such as Src kinase.

Experimental Procedures: The Ewing Sarcoma cell lines A673, CHLA10 and CHLA25 were stressed with either nutrient deprivation (serum withdrawal) or hypoxia (1% oxygen). Cell migration and invasion assays with transwells and x-Celligence real time cell monitoring assays were utilized. Genome-wide RNA synthesis was analyzed by Bru-Seq technology, where bromouridine labeled RNA is used to examine newly synthesized RNA in an unbiased manner. To specifically focus on invadopodia (actin based cell invasive structures) formation, we performed matrix degradation assays by plating cells on FITC-labeled substrate and analyzing matrix degradation via confocal microscopy. Protein expression and phosphorylation status was confirmed by western blot. The commercially available Src inhibitor dasatinib was used to disrupt stress induced Src activation.

Data Summary: Nutrient deprivation enhanced Ewing Sarcoma migration and invasion. Bru-Seq analysis was performed on cells deprived of serum for 24 hours to determine RNA that was newly synthesized under this specific stress. Geneset enrichment analysis of this data revealed significant overlap with gene signatures of hypoxic stress. Given this result, we examined cell motility and invasive behaviors under hypoxic stress. We found that hypoxia also significantly enhanced migration and invasion compared to normoxic controls. Invadopodia are actin-rich cellular structures that harbor matrix metalloproteinases and are “lead-points” to matrix degradation and cancer cell invasion. Src kinase is a driver of invadopodia formation, while the actin modulating protein caldesmon is a reported inhibitor of invadopodia formation. Western blot analysis of cells exposed to serum deprivation and/or hypoxic conditions revealed changes in Src phosphorylation and/or caldesmon expression in each of the cell lines examined. Using FITC-labeled gelatin based matrix assays, we examined invadopodia formation and matrix degradation in Ewing sarcoma cells under stress. While single stresses (i.e. nutrient withdrawal or hypoxia in isolation) led to increases in cell matrix degradation, combining these two stresses dramatically enhanced the ability of these cells to form invadopodia and degrade underlying matrix. Current studies are underway to modify stress-induced changes in Src activity with the commercially available drug dasatinib, a known potent inhibitor of Src at nanomolar concentrations.

Conclusion: The combination of nutrient deprivation and hypoxia commonly occur simultaneously in tumor microenvironments. We have shown that this stress combination promotes invadopodia formation and Ewing sarcoma cell invasion. Further, changes in expression and phosphorylation of key regulators of invadopodia formation (i.e. Src and caldesmon) were noted in stress conditions. Our current and future work aims to further dissect the role of Src and caldesmon in metastatic Ewing sarcoma. We will attempt to use drugs already in clinical use, such as dasatinib, to block invadopodia matrix degradation under conditions of stress.

Citation Format: Kelly M. Bailey, Merlin Airkik, Melanie Krook, Brian Magnuson, Mats Ljungman, Elizabeth Lawlor. Plasticity of the Ewing sarcoma invasive program: How microenvironmental stresses alter the drivers and repressors of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B33.