Abstract
Ewing sarcoma is the second most common bone malignancy in children and young adults. It is driven by oncogenic fusion proteins (i.e. EWS/FLI1) acting as aberrant transcription factors that upregulate and downregulate target genes, leading to cellular transformation. Thus, identificating these target genes and understanding their contribution to Ewing sarcoma tumorigenesis are key for the development of new therapeutic strategies. In this study we show that lysyl oxidase (LOX), an enzyme involved in maintaining structural integrity of the extracellular matrix, is downregulated by the EWS/FLI1 oncoprotein and in consequence it is not expressed in Ewing sarcoma cells and primary tumors. Using a doxycycline inducible system to restore LOX expression in an Ewing sarcoma derived cell line, we showed that LOX displays tumor suppressor activities. Interestingly, we showed that the tumor suppressor activity resides in the propeptide domain of LOX (LOX-PP), an N-terminal domain produced by proteolytic cleavage during the physiological processing of LOX. Expression of LOX-PP reduced cell proliferation, cell migration, anchorage-independent growth in soft agar and formation of tumors in immunodeficient mice. By contrast, the C-terminal domain of LOX, which contains the enzymatic activity, had the opposite effects, corroborating that the tumor suppressor activity of LOX is mediated exclusively by its propeptide domain. Finally, we showed that LOX-PP inhibits ERK/MAPK signalling pathway, and that many pathways involved in cell cycle progression were significantly deregulated by LOX-PP, providing a mechanistic explanation to the cell proliferation inhibition observed upon LOX-PP expression. In summary, our observations indicate that deregulation of the LOX gene participates in Ewing sarcoma development and identify LOX-PP as a new therapeutic target for one of the most aggressive paediatric malignancies. These findings suggest that therapeutic strategies based on the administration of LOX propeptide or functional analogues could be useful for the treatment of this devastating paediatric cancer.
Highlights
Ewing sarcoma is an aggressive neoplasm that mainly affects child and young adults in the first and second decade of life
Time-course experiments confirmed that lysyl oxidase (LOX) is a gene downregulated by EWS/FLI1, since LOX mRNA levels were significantly upregulated upon EWS/FLI1 knockdown in the A673 Ewing sarcoma cell line (Figure 1A)
To analyse if LOX expression downregulation is a common feature of Ewing cells, we analysed by western-blot the levels of LOX protein in a panel of Ewing derived cell lines (n = 8) harbouring different oncogenic fusion proteins
Summary
Ewing sarcoma is an aggressive neoplasm that mainly affects child and young adults in the first and second decade of life. It mainly occurs in bones a small percentage of these tumors arise in soft tissues. The most common of these translocations, observed in approximately 85% of the cases, is t(11;22) that fuse the EWS gene to the FLI1 transcription factor resulting in the EWS/FLI1 fusion protein. Important efforts have been made to identify gene targets of the EWS/FLI1 oncoprotein in Ewing sarcoma cells (reviewed in [2,3,4,5,6]). Many of these target genes have been shown to regulate cell proliferation, invasiveness, metastasis or responsiveness to oxidative stress in Ewing sarcoma cells (reviews above and [7])
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