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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 induction inhibits growth in soft agar, cell migration and tumor growth in vivo.A) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were grown in soft agar in the absence or in the presence of doxycycline (DOX, 1 µg/ml)) for 25 days. Culture dishes were then photographed and colony number was calculated. The figure shows the mean±SEM of three independent experiments done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). B) Two 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) during 48 hours to induce the expression of the corresponding proteins. Afterwards, cells were placed in the upper compartment of a transwell and allowed to migrate through the membrane in response to serum. Migrating cells were quantified by crystal violet staining. The figure shows the mean±SEM of one experiment done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). C) Nude mice were injected with A673/TR/empty clone 1 (n = 7) or A673/TR/LOX-PP clone 2 (n = 8) cells and split in two groups, one of which was given doxycycline (DOX, 1 mg/ml) in the drinking water to induce the expression of the corresponding protein. The figure shows the evolution of tumor volume (mean±SEM of 3–4 animals per group) versus time.
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pone-0066281-g005: LOX-PP induction inhibits growth in soft agar, cell migration and tumor growth in vivo.A) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were grown in soft agar in the absence or in the presence of doxycycline (DOX, 1 µg/ml)) for 25 days. Culture dishes were then photographed and colony number was calculated. The figure shows the mean±SEM of three independent experiments done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). B) Two 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) during 48 hours to induce the expression of the corresponding proteins. Afterwards, cells were placed in the upper compartment of a transwell and allowed to migrate through the membrane in response to serum. Migrating cells were quantified by crystal violet staining. The figure shows the mean±SEM of one experiment done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). C) Nude mice were injected with A673/TR/empty clone 1 (n = 7) or A673/TR/LOX-PP clone 2 (n = 8) cells and split in two groups, one of which was given doxycycline (DOX, 1 mg/ml) in the drinking water to induce the expression of the corresponding protein. The figure shows the evolution of tumor volume (mean±SEM of 3–4 animals per group) versus time.

Mentions: Following, we analysed the effect of preLOX, LOX-PP and LOXenz on cell migration and the ability to grow in an anchorage-independent manner. In Figure 5A are shown the effects of preLOX, LOX-PP and LOXenz on cell migration through porous membranes in response to serum. Induction of preLOX with doxycycline in the A673/TR/preLOX cells did not produce a significant difference in the number of cells migrating through the membrane. However, the induction of LOX-PP in A673/TR/LOX-PP cells decreased cell migration through the porous membranes by 35%, indicating that LOX-PP is also active in inhibiting cell migration. In this case, LOXenz also showed the opposite effect when compared to LOX-PP. Thus, LOXenz increased the migration of A673 cells by 25%. Afterwards, we analysed the effect of preLOX, LOX-PP and LOXenz on the ability of A673 cells to grow in soft agar. As shown in Figure 5B, preLOX and LOX-PP induction decreased the number of colonies grown in soft agar by approximately 25% and 30%, respectively. By contrast, LOXenz increased the number of colonies by about 50%.


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 induction inhibits growth in soft agar, cell migration and tumor growth in vivo.A) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were grown in soft agar in the absence or in the presence of doxycycline (DOX, 1 µg/ml)) for 25 days. Culture dishes were then photographed and colony number was calculated. The figure shows the mean±SEM of three independent experiments done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). B) Two 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) during 48 hours to induce the expression of the corresponding proteins. Afterwards, cells were placed in the upper compartment of a transwell and allowed to migrate through the membrane in response to serum. Migrating cells were quantified by crystal violet staining. The figure shows the mean±SEM of one experiment done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). C) Nude mice were injected with A673/TR/empty clone 1 (n = 7) or A673/TR/LOX-PP clone 2 (n = 8) cells and split in two groups, one of which was given doxycycline (DOX, 1 mg/ml) in the drinking water to induce the expression of the corresponding protein. The figure shows the evolution of tumor volume (mean±SEM of 3–4 animals per group) versus time.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672102&req=5

pone-0066281-g005: LOX-PP induction inhibits growth in soft agar, cell migration and tumor growth in vivo.A) A673/TR/empty, A673/TR/preLOX, A673/TR/LOX-PP and A673/TR/LOXenz cells were grown in soft agar in the absence or in the presence of doxycycline (DOX, 1 µg/ml)) for 25 days. Culture dishes were then photographed and colony number was calculated. The figure shows the mean±SEM of three independent experiments done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). B) Two 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) during 48 hours to induce the expression of the corresponding proteins. Afterwards, cells were placed in the upper compartment of a transwell and allowed to migrate through the membrane in response to serum. Migrating cells were quantified by crystal violet staining. The figure shows the mean±SEM of one experiment done in triplicate. Data are shown as percentage versus unstimulated cells, which were arbitrarily set to 100 (*P<0.05, versus unstimulated cells). C) Nude mice were injected with A673/TR/empty clone 1 (n = 7) or A673/TR/LOX-PP clone 2 (n = 8) cells and split in two groups, one of which was given doxycycline (DOX, 1 mg/ml) in the drinking water to induce the expression of the corresponding protein. The figure shows the evolution of tumor volume (mean±SEM of 3–4 animals per group) versus time.
Mentions: Following, we analysed the effect of preLOX, LOX-PP and LOXenz on cell migration and the ability to grow in an anchorage-independent manner. In Figure 5A are shown the effects of preLOX, LOX-PP and LOXenz on cell migration through porous membranes in response to serum. Induction of preLOX with doxycycline in the A673/TR/preLOX cells did not produce a significant difference in the number of cells migrating through the membrane. However, the induction of LOX-PP in A673/TR/LOX-PP cells decreased cell migration through the porous membranes by 35%, indicating that LOX-PP is also active in inhibiting cell migration. In this case, LOXenz also showed the opposite effect when compared to LOX-PP. Thus, LOXenz increased the migration of A673 cells by 25%. Afterwards, we analysed the effect of preLOX, LOX-PP and LOXenz on the ability of A673 cells to grow in soft agar. As shown in Figure 5B, preLOX and LOX-PP induction decreased the number of colonies grown in soft agar by approximately 25% and 30%, respectively. By contrast, LOXenz increased the number of colonies by about 50%.

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