Limits...
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.

Show MeSH

Related in: MedlinePlus

Establishment of Ewing sarcoma cell lines expressing doxycycline-inducible preLOX, LOX-PP and LOXenz proteins.A) Schematic representation of preLOX, LOX-PP and LOXenz cDNAs cloned in the doxycycline-inducible lentiviral vector pLenti-TO-V5-DEST. SP: signal peptide. B) A673/TR Ewing cell line expressing high levels of the tetracycline repressor (TR) was infected with the doxycycline-inducible lentiviral vector encoding the different LOX cDNAs and stable clones were selected. The figure shows two independent clones for each construction stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) to induce the expression of the corresponding mRNAs. preLOX, LOX-PP and LOXenz mRNAs were detected by RT-PCR using LOX-specific (LOXF6 or LOXF9) and V5 tag-specific primers (V5R). EWS/FLI1 fusion mRNA remained unchanged upon LOX induction. TBP (TATA-binding protein) was used as an internal housekeeping control. C) Whole protein extracts were isolated from A673/TR/preLOX (clone 3), A673/TR/LOXenz (clone 1) and A673/TR/LOX-PP (clon 2) cells stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) and analysed by western-blot to detect the expression of preLOX, LOXenz and LOX-PP proteins, respectively, using an anti-V5 antibody. The same blot was stripped and incubated with anti-α-tubulin as a control for loading and transferring. preLOX and LOXenz, but not LOX-PP, were detected in whole cell extracts. D) Conditioned media derived from A673/TR/LOX-PP (clone 2) incubated with doxycycline (DOX, 1 µg/ml, 48 hours) was concentrated and immunoprecipitated with an anti-V5 antibody to confirm the secretion of LOX-PP in the culture media. An anti-α-tubulin or no antibody were used as negative controls. LOX-PP was detected in the culture media derived from A673/TR/LOX-PP stimulated with doxycycline indicating that LOX-PP was efficiently processed and secreted. E) Conditioned media derived from A673/TR/LOX-PP cells stimulated with doxycycline was immunoprecipitated and untreated or treated with the enzyme peptide-N-glycosidase (PNGase F). Glycosylated LOX-PP was observed as a diffused band of approximately 30 KDa in the untreated sample, that became a defined band of 18 KDa upon deglycosylation treatment. A673/TR/LOX-PP cells without doxycycline were used as a negative control of LOX-PP production.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672102&req=5

pone-0066281-g003: Establishment of Ewing sarcoma cell lines expressing doxycycline-inducible preLOX, LOX-PP and LOXenz proteins.A) Schematic representation of preLOX, LOX-PP and LOXenz cDNAs cloned in the doxycycline-inducible lentiviral vector pLenti-TO-V5-DEST. SP: signal peptide. B) A673/TR Ewing cell line expressing high levels of the tetracycline repressor (TR) was infected with the doxycycline-inducible lentiviral vector encoding the different LOX cDNAs and stable clones were selected. The figure shows two independent clones for each construction stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) to induce the expression of the corresponding mRNAs. preLOX, LOX-PP and LOXenz mRNAs were detected by RT-PCR using LOX-specific (LOXF6 or LOXF9) and V5 tag-specific primers (V5R). EWS/FLI1 fusion mRNA remained unchanged upon LOX induction. TBP (TATA-binding protein) was used as an internal housekeeping control. C) Whole protein extracts were isolated from A673/TR/preLOX (clone 3), A673/TR/LOXenz (clone 1) and A673/TR/LOX-PP (clon 2) cells stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) and analysed by western-blot to detect the expression of preLOX, LOXenz and LOX-PP proteins, respectively, using an anti-V5 antibody. The same blot was stripped and incubated with anti-α-tubulin as a control for loading and transferring. preLOX and LOXenz, but not LOX-PP, were detected in whole cell extracts. D) Conditioned media derived from A673/TR/LOX-PP (clone 2) incubated with doxycycline (DOX, 1 µg/ml, 48 hours) was concentrated and immunoprecipitated with an anti-V5 antibody to confirm the secretion of LOX-PP in the culture media. An anti-α-tubulin or no antibody were used as negative controls. LOX-PP was detected in the culture media derived from A673/TR/LOX-PP stimulated with doxycycline indicating that LOX-PP was efficiently processed and secreted. E) Conditioned media derived from A673/TR/LOX-PP cells stimulated with doxycycline was immunoprecipitated and untreated or treated with the enzyme peptide-N-glycosidase (PNGase F). Glycosylated LOX-PP was observed as a diffused band of approximately 30 KDa in the untreated sample, that became a defined band of 18 KDa upon deglycosylation treatment. A673/TR/LOX-PP cells without doxycycline were used as a negative control of LOX-PP production.

Mentions: As previously mentioned, LOX is synthesized as a 50-KDa inactive proenzyme (preLOX) which is secreted to the extracellular environment. There, it is processed by proteolytic cleavage to a functional 32-KDa LOX enzyme (LOXenz) and an 18-KDa propeptide (LOX-PP). We thus genetically modified the A673 Ewing sarcoma cell line to express LOX proenzyme (A673/TR/preLOX, aminoacides 1-415), its catalytic domain (A673/TR/LOXenz, aminoacides 166-415) and the LOX propeptide (A673/TR/LOX-PP, aminoacides 1-179) in an attempt to characterize the specific contribution of full-length LOX and of each one of the LOX-derived fragments to Ewing sarcoma tumorigenesis (Figure 3A). We have used a doxycycline-inducible system to express these proteins because, in our opinion, it has significant advantages to analyse genes that may be acting as tumor suppressors since constitutive expression of these genes is expected to produce deleterious effects on transformed 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)

Establishment of Ewing sarcoma cell lines expressing doxycycline-inducible preLOX, LOX-PP and LOXenz proteins.A) Schematic representation of preLOX, LOX-PP and LOXenz cDNAs cloned in the doxycycline-inducible lentiviral vector pLenti-TO-V5-DEST. SP: signal peptide. B) A673/TR Ewing cell line expressing high levels of the tetracycline repressor (TR) was infected with the doxycycline-inducible lentiviral vector encoding the different LOX cDNAs and stable clones were selected. The figure shows two independent clones for each construction stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) to induce the expression of the corresponding mRNAs. preLOX, LOX-PP and LOXenz mRNAs were detected by RT-PCR using LOX-specific (LOXF6 or LOXF9) and V5 tag-specific primers (V5R). EWS/FLI1 fusion mRNA remained unchanged upon LOX induction. TBP (TATA-binding protein) was used as an internal housekeeping control. C) Whole protein extracts were isolated from A673/TR/preLOX (clone 3), A673/TR/LOXenz (clone 1) and A673/TR/LOX-PP (clon 2) cells stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) and analysed by western-blot to detect the expression of preLOX, LOXenz and LOX-PP proteins, respectively, using an anti-V5 antibody. The same blot was stripped and incubated with anti-α-tubulin as a control for loading and transferring. preLOX and LOXenz, but not LOX-PP, were detected in whole cell extracts. D) Conditioned media derived from A673/TR/LOX-PP (clone 2) incubated with doxycycline (DOX, 1 µg/ml, 48 hours) was concentrated and immunoprecipitated with an anti-V5 antibody to confirm the secretion of LOX-PP in the culture media. An anti-α-tubulin or no antibody were used as negative controls. LOX-PP was detected in the culture media derived from A673/TR/LOX-PP stimulated with doxycycline indicating that LOX-PP was efficiently processed and secreted. E) Conditioned media derived from A673/TR/LOX-PP cells stimulated with doxycycline was immunoprecipitated and untreated or treated with the enzyme peptide-N-glycosidase (PNGase F). Glycosylated LOX-PP was observed as a diffused band of approximately 30 KDa in the untreated sample, that became a defined band of 18 KDa upon deglycosylation treatment. A673/TR/LOX-PP cells without doxycycline were used as a negative control of LOX-PP production.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0066281-g003: Establishment of Ewing sarcoma cell lines expressing doxycycline-inducible preLOX, LOX-PP and LOXenz proteins.A) Schematic representation of preLOX, LOX-PP and LOXenz cDNAs cloned in the doxycycline-inducible lentiviral vector pLenti-TO-V5-DEST. SP: signal peptide. B) A673/TR Ewing cell line expressing high levels of the tetracycline repressor (TR) was infected with the doxycycline-inducible lentiviral vector encoding the different LOX cDNAs and stable clones were selected. The figure shows two independent clones for each construction stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) to induce the expression of the corresponding mRNAs. preLOX, LOX-PP and LOXenz mRNAs were detected by RT-PCR using LOX-specific (LOXF6 or LOXF9) and V5 tag-specific primers (V5R). EWS/FLI1 fusion mRNA remained unchanged upon LOX induction. TBP (TATA-binding protein) was used as an internal housekeeping control. C) Whole protein extracts were isolated from A673/TR/preLOX (clone 3), A673/TR/LOXenz (clone 1) and A673/TR/LOX-PP (clon 2) cells stimulated with doxycycline (DOX, 1 µg/ml, 48 hours) and analysed by western-blot to detect the expression of preLOX, LOXenz and LOX-PP proteins, respectively, using an anti-V5 antibody. The same blot was stripped and incubated with anti-α-tubulin as a control for loading and transferring. preLOX and LOXenz, but not LOX-PP, were detected in whole cell extracts. D) Conditioned media derived from A673/TR/LOX-PP (clone 2) incubated with doxycycline (DOX, 1 µg/ml, 48 hours) was concentrated and immunoprecipitated with an anti-V5 antibody to confirm the secretion of LOX-PP in the culture media. An anti-α-tubulin or no antibody were used as negative controls. LOX-PP was detected in the culture media derived from A673/TR/LOX-PP stimulated with doxycycline indicating that LOX-PP was efficiently processed and secreted. E) Conditioned media derived from A673/TR/LOX-PP cells stimulated with doxycycline was immunoprecipitated and untreated or treated with the enzyme peptide-N-glycosidase (PNGase F). Glycosylated LOX-PP was observed as a diffused band of approximately 30 KDa in the untreated sample, that became a defined band of 18 KDa upon deglycosylation treatment. A673/TR/LOX-PP cells without doxycycline were used as a negative control of LOX-PP production.
Mentions: As previously mentioned, LOX is synthesized as a 50-KDa inactive proenzyme (preLOX) which is secreted to the extracellular environment. There, it is processed by proteolytic cleavage to a functional 32-KDa LOX enzyme (LOXenz) and an 18-KDa propeptide (LOX-PP). We thus genetically modified the A673 Ewing sarcoma cell line to express LOX proenzyme (A673/TR/preLOX, aminoacides 1-415), its catalytic domain (A673/TR/LOXenz, aminoacides 166-415) and the LOX propeptide (A673/TR/LOX-PP, aminoacides 1-179) in an attempt to characterize the specific contribution of full-length LOX and of each one of the LOX-derived fragments to Ewing sarcoma tumorigenesis (Figure 3A). We have used a doxycycline-inducible system to express these proteins because, in our opinion, it has significant advantages to analyse genes that may be acting as tumor suppressors since constitutive expression of these genes is expected to produce deleterious effects on transformed 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