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TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival.

Chavali PL, Saini RK, Zhai Q, Vizlin-Hodzic D, Venkatabalasubramanian S, Hayashi A, Johansson E, Zeng ZJ, Mohlin S, Påhlman S, Hansford L, Kaplan DR, Funa K - Cell Death Dis (2014)

Bottom Line: We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation.In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues.Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

View Article: PubMed Central - PubMed

Affiliation: 1] Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, Gothenburg SE 40530, Sweden [2] Department of Oncology, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.

ABSTRACT
Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan-Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

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TLX promotes migration and invasion in IMR-32 cells. (a) Invasion and migration assays were performed as described in Materials and Methods using WT IMR-32, shRNA-control (ShCtrl) or Sh2 and Sh3 lines. Values depict the absorbance at 450 nm, representing the invasion/migration index values. (b) Graph depicting the increase of secreted MMP-2 levels in the conditioned media of WT, ShCtrl, Sh2 and Sh3 cells measured by ELISA. (c) Immunoblot analysis of MMP-2 and MMP-9 from conditioned media of control or shTLX cells. Remaining cells in the plate were lysed and used for GAPDH control. (d) Fold change in the MMP-2 and MMP-9 transcript, calculated by normalization against GAPDH in WT or TLX-silenced IMR-32 cells. (e) Migration assay in IMR-32 cells as described in (a), with the indicated transfections below
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fig5: TLX promotes migration and invasion in IMR-32 cells. (a) Invasion and migration assays were performed as described in Materials and Methods using WT IMR-32, shRNA-control (ShCtrl) or Sh2 and Sh3 lines. Values depict the absorbance at 450 nm, representing the invasion/migration index values. (b) Graph depicting the increase of secreted MMP-2 levels in the conditioned media of WT, ShCtrl, Sh2 and Sh3 cells measured by ELISA. (c) Immunoblot analysis of MMP-2 and MMP-9 from conditioned media of control or shTLX cells. Remaining cells in the plate were lysed and used for GAPDH control. (d) Fold change in the MMP-2 and MMP-9 transcript, calculated by normalization against GAPDH in WT or TLX-silenced IMR-32 cells. (e) Migration assay in IMR-32 cells as described in (a), with the indicated transfections below

Mentions: Staining of NB cell lines and NB-TIC xenograft tissues revealed the co- or juxtalocalization of TLX and MMP-2 and CD15, in particular at the edges of TLX-expressing tumor clusters, suggesting them to be migratory cells. As neural stem cells have a migratory capacity, we asked whether TLX could also promote NB cell migration and invasion. Using a colorimetry-based assay for quantifying migration and invasion separately, we observed that TLX-silenced IMR-32 cells had a two- to threefold reduced migration ability as compared with dispersed sphere-forming WT or control transfected IMR-32 cells (Figure 5a). Similar results were obtained in the invasion assays where the TLX-silenced cells showed a two- to threefold decrease as compared with WT or control cells. We then asked whether the secretion of MMPs known to be involved in migratory and invasive behavior of cancer cells is altered. Using ELISA, we observed a three- to fourfold reduction of secreted MMP-2 in the TLX-silenced cells (Figure 5b). These results were verified by blotting for MMP-2 and MMP-9 levels secreted in the conditioned media from shRNA-control or TLX-silenced cell lines (Figure 5c). This prompted us to investigate the possible role of TLX in gene regulation of MMP-2. To determine if TLX modulates the transcription of MMP-2, we performed RT-PCR analysis of the WT and TLX-silenced clones, and observed a 3.4-fold decrease in MMP-2 transcript levels (Figure 5d). We also observed a more moderate 1.8-fold decrease in MMP-9 mRNA expression. These results suggested the involvement of TLX in activating MMP-2 expression. To rule out a cell line-specific effect of TLX on MMP-2, we validated these results in SKN-BE2c cells. We performed rescue experiments with SKN-BE2c by simultaneous expression of siMMP-2 and TLX by western blot (Supplementary Figure 1).21 We observed a 1.8-fold increase in the pro-MMP-2 level upon TLX overexpression, and simultaneous expression of siMMP-2 and TLX rescued the decrease of MMP-2 level by the silencing effect. This is consistent with TLX being an activator of MMP-2 expression. To confirm the MMP-2-mediated promigratory role of TLX, we silenced MMP-2 with siRNA and after 24 h overexpressed TLX in IMR-32 cells. In the absence of MMP-2, TLX overexpression did not result in an significantly increased migratory activity seen with the control cells, indicating the dependence of TLX on MMP-2 for promoting the migration of NB cells (Figure 5e). In summary, TLX alters the migratory capacity of NB cells by inducing MMP-2 levels.


TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival.

Chavali PL, Saini RK, Zhai Q, Vizlin-Hodzic D, Venkatabalasubramanian S, Hayashi A, Johansson E, Zeng ZJ, Mohlin S, Påhlman S, Hansford L, Kaplan DR, Funa K - Cell Death Dis (2014)

TLX promotes migration and invasion in IMR-32 cells. (a) Invasion and migration assays were performed as described in Materials and Methods using WT IMR-32, shRNA-control (ShCtrl) or Sh2 and Sh3 lines. Values depict the absorbance at 450 nm, representing the invasion/migration index values. (b) Graph depicting the increase of secreted MMP-2 levels in the conditioned media of WT, ShCtrl, Sh2 and Sh3 cells measured by ELISA. (c) Immunoblot analysis of MMP-2 and MMP-9 from conditioned media of control or shTLX cells. Remaining cells in the plate were lysed and used for GAPDH control. (d) Fold change in the MMP-2 and MMP-9 transcript, calculated by normalization against GAPDH in WT or TLX-silenced IMR-32 cells. (e) Migration assay in IMR-32 cells as described in (a), with the indicated transfections below
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4237266&req=5

fig5: TLX promotes migration and invasion in IMR-32 cells. (a) Invasion and migration assays were performed as described in Materials and Methods using WT IMR-32, shRNA-control (ShCtrl) or Sh2 and Sh3 lines. Values depict the absorbance at 450 nm, representing the invasion/migration index values. (b) Graph depicting the increase of secreted MMP-2 levels in the conditioned media of WT, ShCtrl, Sh2 and Sh3 cells measured by ELISA. (c) Immunoblot analysis of MMP-2 and MMP-9 from conditioned media of control or shTLX cells. Remaining cells in the plate were lysed and used for GAPDH control. (d) Fold change in the MMP-2 and MMP-9 transcript, calculated by normalization against GAPDH in WT or TLX-silenced IMR-32 cells. (e) Migration assay in IMR-32 cells as described in (a), with the indicated transfections below
Mentions: Staining of NB cell lines and NB-TIC xenograft tissues revealed the co- or juxtalocalization of TLX and MMP-2 and CD15, in particular at the edges of TLX-expressing tumor clusters, suggesting them to be migratory cells. As neural stem cells have a migratory capacity, we asked whether TLX could also promote NB cell migration and invasion. Using a colorimetry-based assay for quantifying migration and invasion separately, we observed that TLX-silenced IMR-32 cells had a two- to threefold reduced migration ability as compared with dispersed sphere-forming WT or control transfected IMR-32 cells (Figure 5a). Similar results were obtained in the invasion assays where the TLX-silenced cells showed a two- to threefold decrease as compared with WT or control cells. We then asked whether the secretion of MMPs known to be involved in migratory and invasive behavior of cancer cells is altered. Using ELISA, we observed a three- to fourfold reduction of secreted MMP-2 in the TLX-silenced cells (Figure 5b). These results were verified by blotting for MMP-2 and MMP-9 levels secreted in the conditioned media from shRNA-control or TLX-silenced cell lines (Figure 5c). This prompted us to investigate the possible role of TLX in gene regulation of MMP-2. To determine if TLX modulates the transcription of MMP-2, we performed RT-PCR analysis of the WT and TLX-silenced clones, and observed a 3.4-fold decrease in MMP-2 transcript levels (Figure 5d). We also observed a more moderate 1.8-fold decrease in MMP-9 mRNA expression. These results suggested the involvement of TLX in activating MMP-2 expression. To rule out a cell line-specific effect of TLX on MMP-2, we validated these results in SKN-BE2c cells. We performed rescue experiments with SKN-BE2c by simultaneous expression of siMMP-2 and TLX by western blot (Supplementary Figure 1).21 We observed a 1.8-fold increase in the pro-MMP-2 level upon TLX overexpression, and simultaneous expression of siMMP-2 and TLX rescued the decrease of MMP-2 level by the silencing effect. This is consistent with TLX being an activator of MMP-2 expression. To confirm the MMP-2-mediated promigratory role of TLX, we silenced MMP-2 with siRNA and after 24 h overexpressed TLX in IMR-32 cells. In the absence of MMP-2, TLX overexpression did not result in an significantly increased migratory activity seen with the control cells, indicating the dependence of TLX on MMP-2 for promoting the migration of NB cells (Figure 5e). In summary, TLX alters the migratory capacity of NB cells by inducing MMP-2 levels.

Bottom Line: We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation.In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues.Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

View Article: PubMed Central - PubMed

Affiliation: 1] Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, Gothenburg SE 40530, Sweden [2] Department of Oncology, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.

ABSTRACT
Nuclear orphan receptor TLX (Drosophila tailless homolog) is essential for the maintenance of neural stem/progenitor cell self-renewal, but its role in neuroblastoma (NB) is not well understood. Here, we show that TLX is essential for the formation of tumor spheres in three different NB cell lines, when grown in neural stem cell media. We demonstrate that the knock down of TLX in IMR-32 cells diminishes its tumor sphere-forming capacity. In tumor spheres, TLX is coexpressed with the neural progenitor markers Nestin, CD133 and Oct-4. In addition, TLX is coexpressed with the migratory neural progenitor markers CD15 and matrix metalloproteinase-2 (MMP-2) in xenografts of primary NB cells from patients. Subsequently, we show the effect of TLX on the proliferative, invasive and migratory properties of IMR-32 cells. We attribute this to the recruitment of TLX to both MMP-2 and Oct-4 gene promoters, which resulted in the respective gene activation. In support of our findings, we found that TLX expression was high in NB patient tissues when compared with normal peripheral nervous system tissues. Further, the Kaplan-Meier estimator indicated a negative correlation between TLX expression and survival in 88 NB patients. Therefore, our results point at TLX being a crucial player in progression of NB, by promoting self-renewal of NB tumor-initiating cells and altering their migratory and invasive properties.

Show MeSH
Related in: MedlinePlus