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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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Induction of preLOX and LOX-PP inhibits cell proliferation in A673 Ewing sarcoma cells.A) A polyclonal population and a representative clone of A673/TR/empty (control cells), A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were maintained during 25 days in standard culture medium or in culture medium containing doxycycline (DOX, 1 µg/ml) to induce the expression of the corresponding proteins. Cumulative number of population doubling were determined by counting cells at different periods of time and plotted versus time. B) Representative clones of A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) for 5 days to induce the corresponding proteins and cell growth quantified by CellTiter-Fluor assay. The figure shows the mean±SD of one out of three independent experiments done in triplicate with similar results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005 versus unstimulated cells). C) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) and in the absence or in the presence of the irreversible antagonist of lysyl oxidase activity β-aminopropionitrile (β-APN, 500 µM). The figure shows the mean±SD of one out of two independent experiments done in triplicate with equivalent results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005, ns: not significant). D) A673 cells were incubated during 72 hours with conditioned media derived from A673/TR/LOX-PP cells cultured in absence of doxycycline (control) or conditioned media derived from the same cells cultured in presence of doxycycline to induce the expression of LOX-PP (LOX-PP rich media). The figure shows the mean±SD of two independent experiments done in triplicate. Data are shown as percentage versus cells incubated with control media, which were arbitrarily set to 100 (*P<0.05). LOX-PP rich media significantly reduced cell proliferation of A673 cells.
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pone-0066281-g004: Induction of preLOX and LOX-PP inhibits cell proliferation in A673 Ewing sarcoma cells.A) A polyclonal population and a representative clone of A673/TR/empty (control cells), A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were maintained during 25 days in standard culture medium or in culture medium containing doxycycline (DOX, 1 µg/ml) to induce the expression of the corresponding proteins. Cumulative number of population doubling were determined by counting cells at different periods of time and plotted versus time. B) Representative clones of A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) for 5 days to induce the corresponding proteins and cell growth quantified by CellTiter-Fluor assay. The figure shows the mean±SD of one out of three independent experiments done in triplicate with similar results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005 versus unstimulated cells). C) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) and in the absence or in the presence of the irreversible antagonist of lysyl oxidase activity β-aminopropionitrile (β-APN, 500 µM). The figure shows the mean±SD of one out of two independent experiments done in triplicate with equivalent results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005, ns: not significant). D) A673 cells were incubated during 72 hours with conditioned media derived from A673/TR/LOX-PP cells cultured in absence of doxycycline (control) or conditioned media derived from the same cells cultured in presence of doxycycline to induce the expression of LOX-PP (LOX-PP rich media). The figure shows the mean±SD of two independent experiments done in triplicate. Data are shown as percentage versus cells incubated with control media, which were arbitrarily set to 100 (*P<0.05). LOX-PP rich media significantly reduced cell proliferation of A673 cells.

Mentions: We next analyzed the effect of preLOX, LOX-PP and LOXenz on cell proliferation. As shown in Figure 4, induction of preLOX by doxycycline in A673/TR/preLOX cells resulted in a significant reduction (30%) in the number of population doubling accumulated during 25 days. This inhibitory effect on cell proliferation was clearly more pronounced (45%) when LOX-PP was induced in A673/TR/LOX-PP cells. Interestingly, the effect of LOXenz on cell proliferation was the opposite, with an increase in the number of population doubling upon LOXenz induction. No effect on cell proliferation was observed in A673 cells carrying the empty vector, both in absence and in presence of doxycycline. To confirm these effects on cell proliferation, we performed additional experiments using an available commercial kit designed to quantify viable cells (Cell-titer fluor cell viability assay). As shown in Figure 4B, induction of preLOX in two independent clones of A673/TR/preLOX cells produced a reduction (about 20%) in the number of viable cells. This inhibitory effect on cell number became again more pronounced (about 50%) when LOX-PP was induced in A673/TR/LOX-PP cells. By contrast, the induction of LOXenz resulted in an increase in the number of viable cells. Taken together, these results demonstrate that preLOX inhibits cell proliferation in Ewing cell lines and more interestingly, that this inhibitory effect on cell proliferation resides in the LOX propeptide. To additionally characterize these findings, we used the irreversible antagonist of lysyl oxidase activity β-amino-propionitrile (β-APN) [33]. As shown in Figure 4C, treatment with β-APN increased the inhibitory effect of preLOX on cell proliferation. Since preLOX expression results in the production of both the propeptide domain LOX-PP and the catalytic domain LOXenz, this result indicates that lysyl oxidase activity (which resides in LOXenz) is partially counteracting the cell proliferation inhibition mediated by LOX-PP. Furthermore, β-APN totally blocked the stimulatory effect of LOXenz on cell proliferation, demonstrating that this effect is entirely dependent on the lysyl oxidase catalytic activity. In concordance with this, β-APN had no effect on the cell proliferation inhibition observed upon LOX-PP induction. These results confirm that the inhibitory effect of LOX on cell proliferation resides in the LOX propeptide. Subsequently, we analyzed the effect of an exogenous source of LOX-PP on cell proliferation. For this purpose, we incubated A673 cells with LOX-PP rich media, obtained from conditioned media of A673/TR/LOX-PP cells stimulated with doxycycline during 72 hours. Control media, without LOX-PP, was obtained in parallel from the same cells incubated in absence of doxycycline for 72 hours. As shown in figure 4D, LOX-PP enriched media, reduced by 50% the cell number in A673 Ewing 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)

Induction of preLOX and LOX-PP inhibits cell proliferation in A673 Ewing sarcoma cells.A) A polyclonal population and a representative clone of A673/TR/empty (control cells), A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were maintained during 25 days in standard culture medium or in culture medium containing doxycycline (DOX, 1 µg/ml) to induce the expression of the corresponding proteins. Cumulative number of population doubling were determined by counting cells at different periods of time and plotted versus time. B) Representative clones of A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) for 5 days to induce the corresponding proteins and cell growth quantified by CellTiter-Fluor assay. The figure shows the mean±SD of one out of three independent experiments done in triplicate with similar results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005 versus unstimulated cells). C) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) and in the absence or in the presence of the irreversible antagonist of lysyl oxidase activity β-aminopropionitrile (β-APN, 500 µM). The figure shows the mean±SD of one out of two independent experiments done in triplicate with equivalent results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005, ns: not significant). D) A673 cells were incubated during 72 hours with conditioned media derived from A673/TR/LOX-PP cells cultured in absence of doxycycline (control) or conditioned media derived from the same cells cultured in presence of doxycycline to induce the expression of LOX-PP (LOX-PP rich media). The figure shows the mean±SD of two independent experiments done in triplicate. Data are shown as percentage versus cells incubated with control media, which were arbitrarily set to 100 (*P<0.05). LOX-PP rich media significantly reduced cell proliferation of A673 cells.
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Related In: Results  -  Collection

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

pone-0066281-g004: Induction of preLOX and LOX-PP inhibits cell proliferation in A673 Ewing sarcoma cells.A) A polyclonal population and a representative clone of A673/TR/empty (control cells), A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were maintained during 25 days in standard culture medium or in culture medium containing doxycycline (DOX, 1 µg/ml) to induce the expression of the corresponding proteins. Cumulative number of population doubling were determined by counting cells at different periods of time and plotted versus time. B) Representative clones of A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) for 5 days to induce the corresponding proteins and cell growth quantified by CellTiter-Fluor assay. The figure shows the mean±SD of one out of three independent experiments done in triplicate with similar results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005 versus unstimulated cells). C) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were incubated in the absence or in the presence of doxycycline (DOX, 1 µg/ml) and in the absence or in the presence of the irreversible antagonist of lysyl oxidase activity β-aminopropionitrile (β-APN, 500 µM). The figure shows the mean±SD of one out of two independent experiments done in triplicate with equivalent results. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, **P<0.005, ns: not significant). D) A673 cells were incubated during 72 hours with conditioned media derived from A673/TR/LOX-PP cells cultured in absence of doxycycline (control) or conditioned media derived from the same cells cultured in presence of doxycycline to induce the expression of LOX-PP (LOX-PP rich media). The figure shows the mean±SD of two independent experiments done in triplicate. Data are shown as percentage versus cells incubated with control media, which were arbitrarily set to 100 (*P<0.05). LOX-PP rich media significantly reduced cell proliferation of A673 cells.
Mentions: We next analyzed the effect of preLOX, LOX-PP and LOXenz on cell proliferation. As shown in Figure 4, induction of preLOX by doxycycline in A673/TR/preLOX cells resulted in a significant reduction (30%) in the number of population doubling accumulated during 25 days. This inhibitory effect on cell proliferation was clearly more pronounced (45%) when LOX-PP was induced in A673/TR/LOX-PP cells. Interestingly, the effect of LOXenz on cell proliferation was the opposite, with an increase in the number of population doubling upon LOXenz induction. No effect on cell proliferation was observed in A673 cells carrying the empty vector, both in absence and in presence of doxycycline. To confirm these effects on cell proliferation, we performed additional experiments using an available commercial kit designed to quantify viable cells (Cell-titer fluor cell viability assay). As shown in Figure 4B, induction of preLOX in two independent clones of A673/TR/preLOX cells produced a reduction (about 20%) in the number of viable cells. This inhibitory effect on cell number became again more pronounced (about 50%) when LOX-PP was induced in A673/TR/LOX-PP cells. By contrast, the induction of LOXenz resulted in an increase in the number of viable cells. Taken together, these results demonstrate that preLOX inhibits cell proliferation in Ewing cell lines and more interestingly, that this inhibitory effect on cell proliferation resides in the LOX propeptide. To additionally characterize these findings, we used the irreversible antagonist of lysyl oxidase activity β-amino-propionitrile (β-APN) [33]. As shown in Figure 4C, treatment with β-APN increased the inhibitory effect of preLOX on cell proliferation. Since preLOX expression results in the production of both the propeptide domain LOX-PP and the catalytic domain LOXenz, this result indicates that lysyl oxidase activity (which resides in LOXenz) is partially counteracting the cell proliferation inhibition mediated by LOX-PP. Furthermore, β-APN totally blocked the stimulatory effect of LOXenz on cell proliferation, demonstrating that this effect is entirely dependent on the lysyl oxidase catalytic activity. In concordance with this, β-APN had no effect on the cell proliferation inhibition observed upon LOX-PP induction. These results confirm that the inhibitory effect of LOX on cell proliferation resides in the LOX propeptide. Subsequently, we analyzed the effect of an exogenous source of LOX-PP on cell proliferation. For this purpose, we incubated A673 cells with LOX-PP rich media, obtained from conditioned media of A673/TR/LOX-PP cells stimulated with doxycycline during 72 hours. Control media, without LOX-PP, was obtained in parallel from the same cells incubated in absence of doxycycline for 72 hours. As shown in figure 4D, LOX-PP enriched media, reduced by 50% the cell number in A673 Ewing 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