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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 mRNA and protein are downregulated by EWS/FLI1 in the A673 Ewing sarcoma cell line.A) A673/TR/shEF cells were stimulated by different periods of time with doxycycline (DOX, 1 µg/ml) to induce the expression of an EWS/FLI1-specific shRNA. The levels of LOX, EWS/FLI1 and TBP (reference gene) mRNA were quantified by multiplex real time qRT-PCR. For each time point, LOX and EWS/FLI1 mRNA levels were normalized to that of TBP and referred to unstimulated cells. The figure shows the data (mean±SD) of one out of two independent experiments done in triplicate with equivalent results. B) LOX protein levels were also determined by western-blot in the A673/TR/shEF cells stimulated with doxycycline. The same blot was stripped and successively incubated with anti-FLI1 antibody to assess the expression of EWS/FLI1 and with anti-α-tubulin as a control for loading and transferring. LOX mRNA and protein levels were strongly downregulated by EWS/FLI1. C) LOX protein levels were determined by western-blot in 8 Ewing derived cell lines and in normal fibroblasts IMR-90, used here as a positive control of LOX expression. A673/TR/shEF cells stimulated with doxycycline for 48 hours were also included. The type of EWS/FLI1 (EF; EWS exon/FLI1 exon) and EWS/ERG fusion (EE; EWS exon/ERG exon) present in each Ewing cell line are indicated. LOX protein expression was nearly undetectable in Ewing derived cell lines. D) LOX mRNA levels were quantified by qRT-PCR in a set of Ewing's primary tumors (n = 21). Normal fibroblasts IMR-90 are used as a positive control. mRNA levels were normalized to that of TBP (mean±SD). LOX mRNA levels observed in Ewing tumors were very low compared to those observed in the control fibroblast cell line IMR-90.
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pone-0066281-g001: LOX mRNA and protein are downregulated by EWS/FLI1 in the A673 Ewing sarcoma cell line.A) A673/TR/shEF cells were stimulated by different periods of time with doxycycline (DOX, 1 µg/ml) to induce the expression of an EWS/FLI1-specific shRNA. The levels of LOX, EWS/FLI1 and TBP (reference gene) mRNA were quantified by multiplex real time qRT-PCR. For each time point, LOX and EWS/FLI1 mRNA levels were normalized to that of TBP and referred to unstimulated cells. The figure shows the data (mean±SD) of one out of two independent experiments done in triplicate with equivalent results. B) LOX protein levels were also determined by western-blot in the A673/TR/shEF cells stimulated with doxycycline. The same blot was stripped and successively incubated with anti-FLI1 antibody to assess the expression of EWS/FLI1 and with anti-α-tubulin as a control for loading and transferring. LOX mRNA and protein levels were strongly downregulated by EWS/FLI1. C) LOX protein levels were determined by western-blot in 8 Ewing derived cell lines and in normal fibroblasts IMR-90, used here as a positive control of LOX expression. A673/TR/shEF cells stimulated with doxycycline for 48 hours were also included. The type of EWS/FLI1 (EF; EWS exon/FLI1 exon) and EWS/ERG fusion (EE; EWS exon/ERG exon) present in each Ewing cell line are indicated. LOX protein expression was nearly undetectable in Ewing derived cell lines. D) LOX mRNA levels were quantified by qRT-PCR in a set of Ewing's primary tumors (n = 21). Normal fibroblasts IMR-90 are used as a positive control. mRNA levels were normalized to that of TBP (mean±SD). LOX mRNA levels observed in Ewing tumors were very low compared to those observed in the control fibroblast cell line IMR-90.

Mentions: To confirm microarray data, we used A673/TR/shEF Ewing cells, which in response to doxycycline express a specific shRNA directed against EWS/FLI1 mRNA, subsequently reducing the levels of EWS/FLI1 mRNA and protein [8], [9]. We isolated RNA from A673/TR/shEF cells incubated in absence or in presence of doxycycline and performed real time quantitative RT-PCR (qRT-PCR) analysis. Time-course experiments confirmed that LOX is a gene downregulated by EWS/FLI1, since LOX mRNA levels were significantly upregulated upon EWS/FLI1 knockdown in the A673 Ewing sarcoma cell line (Figure 1A). As shown in figure 1B, LOX protein was nearly undetectable in A673/TR/shEF cells in basal conditions. However, EWS/FLI1 knockdown produced a dramatic increase in LOX protein levels confirming that EWS/FLI1 downregulates LOX expression in these cells. To analyse if LOX expression downregulation is a common feature of Ewing cells, we analysed by western-blot the levels of LOX protein in a panel of Ewing derived cell lines (n = 8) harbouring different oncogenic fusion proteins. As shown in Figure 1C, LOX expression in these Ewing sarcoma cell lines was nearly undetectable, while high LOX expression was observed in IMR-90 normal fibroblasts, which were used here as a positive control of LOX expression. We then analysed the levels of LOX mRNA in Ewing primary tumors using qRT-PCR and compared them with the levels observed in the positive control IMR-90. Figure 1D shows that LOX mRNA levels are low in Ewing primary tumors compared to those observed in IMR90 cells. In addition, we searched public expression datasets in order to analyse the relative expression of LOX in Ewing sarcoma in relation to other tumor types. As shown in supplementary figure S1, LOX expression was low in Ewing sarcoma tumors, compared to other neoplasms.


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 mRNA and protein are downregulated by EWS/FLI1 in the A673 Ewing sarcoma cell line.A) A673/TR/shEF cells were stimulated by different periods of time with doxycycline (DOX, 1 µg/ml) to induce the expression of an EWS/FLI1-specific shRNA. The levels of LOX, EWS/FLI1 and TBP (reference gene) mRNA were quantified by multiplex real time qRT-PCR. For each time point, LOX and EWS/FLI1 mRNA levels were normalized to that of TBP and referred to unstimulated cells. The figure shows the data (mean±SD) of one out of two independent experiments done in triplicate with equivalent results. B) LOX protein levels were also determined by western-blot in the A673/TR/shEF cells stimulated with doxycycline. The same blot was stripped and successively incubated with anti-FLI1 antibody to assess the expression of EWS/FLI1 and with anti-α-tubulin as a control for loading and transferring. LOX mRNA and protein levels were strongly downregulated by EWS/FLI1. C) LOX protein levels were determined by western-blot in 8 Ewing derived cell lines and in normal fibroblasts IMR-90, used here as a positive control of LOX expression. A673/TR/shEF cells stimulated with doxycycline for 48 hours were also included. The type of EWS/FLI1 (EF; EWS exon/FLI1 exon) and EWS/ERG fusion (EE; EWS exon/ERG exon) present in each Ewing cell line are indicated. LOX protein expression was nearly undetectable in Ewing derived cell lines. D) LOX mRNA levels were quantified by qRT-PCR in a set of Ewing's primary tumors (n = 21). Normal fibroblasts IMR-90 are used as a positive control. mRNA levels were normalized to that of TBP (mean±SD). LOX mRNA levels observed in Ewing tumors were very low compared to those observed in the control fibroblast cell line IMR-90.
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Related In: Results  -  Collection

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

pone-0066281-g001: LOX mRNA and protein are downregulated by EWS/FLI1 in the A673 Ewing sarcoma cell line.A) A673/TR/shEF cells were stimulated by different periods of time with doxycycline (DOX, 1 µg/ml) to induce the expression of an EWS/FLI1-specific shRNA. The levels of LOX, EWS/FLI1 and TBP (reference gene) mRNA were quantified by multiplex real time qRT-PCR. For each time point, LOX and EWS/FLI1 mRNA levels were normalized to that of TBP and referred to unstimulated cells. The figure shows the data (mean±SD) of one out of two independent experiments done in triplicate with equivalent results. B) LOX protein levels were also determined by western-blot in the A673/TR/shEF cells stimulated with doxycycline. The same blot was stripped and successively incubated with anti-FLI1 antibody to assess the expression of EWS/FLI1 and with anti-α-tubulin as a control for loading and transferring. LOX mRNA and protein levels were strongly downregulated by EWS/FLI1. C) LOX protein levels were determined by western-blot in 8 Ewing derived cell lines and in normal fibroblasts IMR-90, used here as a positive control of LOX expression. A673/TR/shEF cells stimulated with doxycycline for 48 hours were also included. The type of EWS/FLI1 (EF; EWS exon/FLI1 exon) and EWS/ERG fusion (EE; EWS exon/ERG exon) present in each Ewing cell line are indicated. LOX protein expression was nearly undetectable in Ewing derived cell lines. D) LOX mRNA levels were quantified by qRT-PCR in a set of Ewing's primary tumors (n = 21). Normal fibroblasts IMR-90 are used as a positive control. mRNA levels were normalized to that of TBP (mean±SD). LOX mRNA levels observed in Ewing tumors were very low compared to those observed in the control fibroblast cell line IMR-90.
Mentions: To confirm microarray data, we used A673/TR/shEF Ewing cells, which in response to doxycycline express a specific shRNA directed against EWS/FLI1 mRNA, subsequently reducing the levels of EWS/FLI1 mRNA and protein [8], [9]. We isolated RNA from A673/TR/shEF cells incubated in absence or in presence of doxycycline and performed real time quantitative RT-PCR (qRT-PCR) analysis. Time-course experiments confirmed that LOX is a gene downregulated by EWS/FLI1, since LOX mRNA levels were significantly upregulated upon EWS/FLI1 knockdown in the A673 Ewing sarcoma cell line (Figure 1A). As shown in figure 1B, LOX protein was nearly undetectable in A673/TR/shEF cells in basal conditions. However, EWS/FLI1 knockdown produced a dramatic increase in LOX protein levels confirming that EWS/FLI1 downregulates LOX expression in these cells. To analyse if LOX expression downregulation is a common feature of Ewing cells, we analysed by western-blot the levels of LOX protein in a panel of Ewing derived cell lines (n = 8) harbouring different oncogenic fusion proteins. As shown in Figure 1C, LOX expression in these Ewing sarcoma cell lines was nearly undetectable, while high LOX expression was observed in IMR-90 normal fibroblasts, which were used here as a positive control of LOX expression. We then analysed the levels of LOX mRNA in Ewing primary tumors using qRT-PCR and compared them with the levels observed in the positive control IMR-90. Figure 1D shows that LOX mRNA levels are low in Ewing primary tumors compared to those observed in IMR90 cells. In addition, we searched public expression datasets in order to analyse the relative expression of LOX in Ewing sarcoma in relation to other tumor types. As shown in supplementary figure S1, LOX expression was low in Ewing sarcoma tumors, compared to other neoplasms.

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