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Lysyl oxidase is downregulated by the EWS/FLI1 oncoprotein and its propeptide domain displays tumor supressor activities in Ewing sarcoma cells.

Agra N, Cidre F, García-García L, de la Parra J, Alonso J - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Tumores Sólidos Infantiles, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.

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.

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LOX-PP interferes with ERK signalling.A673/TR/LOX-PP cells (clone 2) were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml, 48 hours) and the levels of activates P-AKT (Ser 473) and P-ERK (Tyr 204) determined by western-blot. The same blot was stripped and successively incubated with anti-AKT, anti-ERK and anti-α-tubulin as controls for loading and transferring.
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pone-0066281-g006: LOX-PP interferes with ERK signalling.A673/TR/LOX-PP cells (clone 2) were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml, 48 hours) and the levels of activates P-AKT (Ser 473) and P-ERK (Tyr 204) determined by western-blot. The same blot was stripped and successively incubated with anti-AKT, anti-ERK and anti-α-tubulin as controls for loading and transferring.

Mentions: Following, in an attempt to identify the pathways involved in the antiproliferative effect of LOX-PP we analysed by western-blot the status of the PI3K/AKT and ERK/MAPK pathways. As shown in Figure 6, induction of LOX-PP with doxycycline had no effect on the levels of activated Akt (P-Ser 473). By contrast, LOX-PP produced a marked inhibition of the levels of activated Erk (P-Tyr 204).


Lysyl oxidase is downregulated by the EWS/FLI1 oncoprotein and its propeptide domain displays tumor supressor activities in Ewing sarcoma cells.

Agra N, Cidre F, García-García L, de la Parra J, Alonso J - PLoS ONE (2013)

LOX-PP interferes with ERK signalling.A673/TR/LOX-PP cells (clone 2) were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml, 48 hours) and the levels of activates P-AKT (Ser 473) and P-ERK (Tyr 204) determined by western-blot. The same blot was stripped and successively incubated with anti-AKT, anti-ERK and anti-α-tubulin as controls for loading and transferring.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672102&req=5

pone-0066281-g006: LOX-PP interferes with ERK signalling.A673/TR/LOX-PP cells (clone 2) were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml, 48 hours) and the levels of activates P-AKT (Ser 473) and P-ERK (Tyr 204) determined by western-blot. The same blot was stripped and successively incubated with anti-AKT, anti-ERK and anti-α-tubulin as controls for loading and transferring.
Mentions: Following, in an attempt to identify the pathways involved in the antiproliferative effect of LOX-PP we analysed by western-blot the status of the PI3K/AKT and ERK/MAPK pathways. As shown in Figure 6, induction of LOX-PP with doxycycline had no effect on the levels of activated Akt (P-Ser 473). By contrast, LOX-PP produced a marked inhibition of the levels of activated Erk (P-Tyr 204).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Tumores Sólidos Infantiles, Área de Genética Humana, Instituto de Investigación de Enfermedades Raras, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.

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.

Show MeSH
Related in: MedlinePlus