Abstract 5030: Serum deprivation induces Ewing Sarcoma cells to adopt a more invasive phenotype and to upregulate RHO-MKL transcriptional activity.
Authors: Airik Merlin , Krook Melanie , Scannell Christopher , Haak Andrew , Neubig Richard , Lawlor Elizabeth 
Published: August 2014

Background: Ewing sarcoma (ES) is an aggressive and metastatic tumor of bone and soft tissue that primarily affects children and adolescents. The primary cause of tumor-related death in patients with ES is metastatic disease. In epithelial-derived cancers metastasis is dependent on phenotypic transition of cells from epithelial- to mesenchymal-like states (EMT). In addition, Rho signaling pathways are primary mediators of cancer cell motility and can induce cell migration and invasion, in part through transcriptional activation of MKL/SRF target genes. The mechanisms of metastasis in non-epithelial tumors such as ES remain to be elucidated.

Objective: We sought to determine whether phenotypic plasticity between non-invasive and invasive states is also a feature of ES and to determine the molecular mechanism underlying this transition.

Methods: ES cell lines were subjected to serum deprivation and phenotypic differences measured using scratch assays, transwell studies, and the xCelligence system. qRT-PCR, Western blot and immunofluorescence were used to quantitate or visualize changes in gene and protein expression, respectively.

Results: Serum deprivation induced ES cells to rapidly adopt a more aggressive phenotype, reflected by changes in cell shape and increased motility, while proliferation was inhibited. Serum replenishment rapidly reverted these phenotypic changes. Mechanistically, markers of EMT were largely unaffected indicating that the observed phenotypic plasticity in ES cells was not mediated by classic EMT-MET transitions. Instead, activation of Rho-MKL signaling was apparent as evidenced by nuclear localization of MKL and upregulation of the metastasis-associated MKL/SRF target gene MYL9 in serum-deprived conditions. Exposure of ES cells to a small molecule antagonist of Rho-MKL signaling inhibited ES cell motility.

Conclusions: Serum deprivation causes reversible transformation of ES cells to a more migratory phenotype. This is associated with nuclear translocation of MKL and induction of MYL9, a gene that encodes a regulatory light chain required for myosin stability. We propose a model of ES metastasis in which changes to the microenvironment that result in growth-factor deprivation (e.g. increased tumor size or cytotoxic therapy) induce expression of Rho-MKL transcriptional targets that contribute to cell migration and invasion. The Rho-MKL axis may thus be a novel therapeutic target for prevention of ES metastasis.

Citation Format: Merlin Airik, Melanie Krook, Christopher Scannell, Andrew Haak, Richard Neubig, Elizabeth Lawlor. Serum deprivation induces Ewing Sarcoma cells to adopt a more invasive phenotype and to upregulate RHO-MKL transcriptional activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2013-5030