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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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Effect of the histone deacetylases inhibitor SAHA and the demethylating agent 5-aza-2′-deoxycytidine on LOX mRNA levels.A) A673 cells were incubated in absence (C) or presence of SAHA 1 µM for 24 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by multiplex real time qRT-PCR. LOX mRNA levels were normalized to that of TBP and referred to unstimulated control (C) cells, which were arbitrary set to 1. The figure shows the data (mean±SD) of three independent experiments done in triplicate. The figure shows the mean±SD of one out of two independent experiments done in duplicate (*P<0.01 vs control). B) A673 cells were incubated in the absence (C) or presence of 5-aza-2′-deoxycytidine 1 µM for 24, 48, 72 or 96 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by qRT-PCR and analysed as above. The figure shows the mean±SD of two independent experiments done in duplicate (*P<0.01 vs control). Both SAHA and 5-aza-2′-deoxycytidine produce a significant increase in the levels of LOX mRNA in A673 cells.
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pone-0066281-g002: Effect of the histone deacetylases inhibitor SAHA and the demethylating agent 5-aza-2′-deoxycytidine on LOX mRNA levels.A) A673 cells were incubated in absence (C) or presence of SAHA 1 µM for 24 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by multiplex real time qRT-PCR. LOX mRNA levels were normalized to that of TBP and referred to unstimulated control (C) cells, which were arbitrary set to 1. The figure shows the data (mean±SD) of three independent experiments done in triplicate. The figure shows the mean±SD of one out of two independent experiments done in duplicate (*P<0.01 vs control). B) A673 cells were incubated in the absence (C) or presence of 5-aza-2′-deoxycytidine 1 µM for 24, 48, 72 or 96 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by qRT-PCR and analysed as above. The figure shows the mean±SD of two independent experiments done in duplicate (*P<0.01 vs control). Both SAHA and 5-aza-2′-deoxycytidine produce a significant increase in the levels of LOX mRNA in A673 cells.

Mentions: Next, we analysed if epigenetic mechanisms, known to repress gene expression, could be involved in the negative regulation of LOX expression observed in Ewing sarcoma cells. We first studied the effect of Vorinostat (SAHA), a class I/II histone deacetylase inhibitor approved for clinical use in cancer patients ([37]). A673 cells were incubated in presence or absence of SAHA for 24 hours and LOX mRNA levels were quantified by qRT-PCR. As shown in Figure 2A, incubation of A673 Ewing cells with SAHA produced a 5-fold increase in LOX mRNA levels. Following, we analysed the effect of 5-aza-cytidine (5-aza), a potent inhibitor of DNA methyltransferase 1 (DNMT1) that induces demethylation and reactivation of silenced genes [38] on LOX expression. As shown in figure 2B, incubation of A673 Ewing cells with 5-aza during 72 hours produced a 10-fold increase in LOX mRNA levels. These results suggest that both histone acetylation status and DNA methylation could be involved in the negative regulation of LOX expression in A673 cells.


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)

Effect of the histone deacetylases inhibitor SAHA and the demethylating agent 5-aza-2′-deoxycytidine on LOX mRNA levels.A) A673 cells were incubated in absence (C) or presence of SAHA 1 µM for 24 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by multiplex real time qRT-PCR. LOX mRNA levels were normalized to that of TBP and referred to unstimulated control (C) cells, which were arbitrary set to 1. The figure shows the data (mean±SD) of three independent experiments done in triplicate. The figure shows the mean±SD of one out of two independent experiments done in duplicate (*P<0.01 vs control). B) A673 cells were incubated in the absence (C) or presence of 5-aza-2′-deoxycytidine 1 µM for 24, 48, 72 or 96 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by qRT-PCR and analysed as above. The figure shows the mean±SD of two independent experiments done in duplicate (*P<0.01 vs control). Both SAHA and 5-aza-2′-deoxycytidine produce a significant increase in the levels of LOX mRNA in A673 cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672102&req=5

pone-0066281-g002: Effect of the histone deacetylases inhibitor SAHA and the demethylating agent 5-aza-2′-deoxycytidine on LOX mRNA levels.A) A673 cells were incubated in absence (C) or presence of SAHA 1 µM for 24 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by multiplex real time qRT-PCR. LOX mRNA levels were normalized to that of TBP and referred to unstimulated control (C) cells, which were arbitrary set to 1. The figure shows the data (mean±SD) of three independent experiments done in triplicate. The figure shows the mean±SD of one out of two independent experiments done in duplicate (*P<0.01 vs control). B) A673 cells were incubated in the absence (C) or presence of 5-aza-2′-deoxycytidine 1 µM for 24, 48, 72 or 96 hours and the levels of LOX and TBP (reference gene) mRNAs quantified by qRT-PCR and analysed as above. The figure shows the mean±SD of two independent experiments done in duplicate (*P<0.01 vs control). Both SAHA and 5-aza-2′-deoxycytidine produce a significant increase in the levels of LOX mRNA in A673 cells.
Mentions: Next, we analysed if epigenetic mechanisms, known to repress gene expression, could be involved in the negative regulation of LOX expression observed in Ewing sarcoma cells. We first studied the effect of Vorinostat (SAHA), a class I/II histone deacetylase inhibitor approved for clinical use in cancer patients ([37]). A673 cells were incubated in presence or absence of SAHA for 24 hours and LOX mRNA levels were quantified by qRT-PCR. As shown in Figure 2A, incubation of A673 Ewing cells with SAHA produced a 5-fold increase in LOX mRNA levels. Following, we analysed the effect of 5-aza-cytidine (5-aza), a potent inhibitor of DNA methyltransferase 1 (DNMT1) that induces demethylation and reactivation of silenced genes [38] on LOX expression. As shown in figure 2B, incubation of A673 Ewing cells with 5-aza during 72 hours produced a 10-fold increase in LOX mRNA levels. These results suggest that both histone acetylation status and DNA methylation could be involved in the negative regulation of LOX expression in A673 cells.

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