EWS/FLI1 target genes are downregulated by this oncogenic protein. The mechanism of this specific gene repression is only partially understood, and probably involves direct repression, upregulation of transcriptional repressors and epigenetic mechanisms. In addition, EWS/FLI1 has been also shown to regulate the expression of microRNAs that in turn are available to regulate the expression of other genes involved Ewing sarcoma tumorigenesis. Analysis of our gene expression profile dataset in the Ewing sarcoma cell line A673 upon EWS/FLI1 knockdown showed that one of the most strongly downregulated genes by EWS/FLI1 codes for the enzyme lysyl oxidase. LOX is the major member of a family of lysyl oxidases that share the enzyme catalytic domain. LOX is synthesized as a 50-KDa inactive pre-proenzyme which is secreted into the extracellular environment and then processed by proteolytic cleavage to a functional 32-KDa LOX enzyme and an 18-KDa propeptide. It is well characterized that the functional mature enzyme catalyses lysine-derived covalent MedChemExpress Halofuginone crosslinks required for normal structural integrity of the extracellular matrix and a huge amount of information on this function, both in physiological and pathological situations, has been accumulated during several decades. The role of the propeptide has been, however, much less studied, although recent reports suggest that LOX propeptide acts as a tumor suppressor in several contexts. Currently, no data are available about the possible contribution of LOX repression to the malignant phenotype of Ewing sarcoma. In this work, we show that EWS/FLI1 downregulates LOX expression and that, remarkably, LOX propeptide exhibits tumor suppressor activities in Ewing tumor cells. Ectopic expression of LOX propeptide in an Ewing sarcoma cell line reduced cell proliferation, cell migration, anchorage independent growth and tumor growth in vivo. These findings suggest that therapeutic strategies based in the administration of LOX propeptide or functional analogues could be useful for the treatment of this devastating paediatric cancer. Results During the last years we have used an inducible model of EWS/ FLI1 knockdown in combination 15864271 with whole gene expression analysis to identify and characterize EWS/FLI1 target genes relevant for Ewing sarcoma tumorigenesis. Review of these datasets indicated that one of the genes that showed a more intense and consistent downregulation by EWS/FLI1 was the enzyme lysyl oxidase. Consequently, we decided to study the regulation of LOX by EWS/FLI1 and the functional implications of this downregulation. To confirm microarray data, we used A673/TR/shEF Ewing cells, which in response to doxycycline express a specific shRNA directed against EWS/FLI1 mRNA, subsequently reducing the levels of EWS/FLI1 20383709 mRNA and protein. We isolated RNA from A673/TR/shEF cells incubated in absence or in presence of doxycycline and performed real time quantitative RT-PCR analysis. Time-course experiments confirmed that LOX is a gene downregulated by EWS/FLI1, since LOX mRNA levels LOX-PP Supresses Ewing Sarcoma Tumorigenesis As shown in figure 3D, LOX-PP was detected as a diffuse band of approximately 30 KDa in culture media of A673/TR/LOX-PP cells stimulated with doxycycline, suggesting that this protein was strongly glycosylated, as previously reported. In order to demonstrate that this diffuse band corresponds to secreted LOXPP highly glycosylated, we treated a fraction of the immunoprecipitated LOX-PP w
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