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TAZ promotes epithelial to mesenchymal transition via the upregulation of connective tissue growth factor expression in neuroblastoma cells.

Wang Q, Xu Z, An Q, Jiang D, Wang L, Liang B, Li Z - Mol Med Rep (2014)

Bottom Line: The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP).Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay.Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China.

ABSTRACT
Neuroblastoma (NB) is a neuroendocrine cancer that occurs most commonly in infants and young children. The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP). The effect of TAZ on the metastatic progression of neuroblastoma and the underlying mechanisms involved remain elusive. In the current study, it was determined by western blot analysis that the migratory and invasive properties of SK‑N‑BE(2) human neuroblastoma cells are associated with high expression levels of TAZ. Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay. In contrast, overexpression of TAZ in SK‑N‑SH human neuroblastoma cells was shown by Transwell migration and invasion assays, and western blot analysis, to result in epithelial‑mesenchymal transition (EMT) and increased invasiveness. Mechanistically, the overexpression of TAZ was demonstrated to upregulate the expression levels of connective tissue growth factor (CTGF), by western blot analysis and chromatin immunoprecipitation assay, while the knockdown of TAZ downregulated it. Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway. In conclusion, the present study is, to the best of our knowledge, the first to demonstrate that the overexpression of TAZ induces EMT, increasing the invasive abilities of neuroblastoma cells. This suggests that TAZ may serve as a potential target in the development of novel therapies for the treatment of neuroblastoma.

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Overexpression of TAZ upregulates connective tissue growth factor (CGTF) expression while knockdown of TAZ downregulates it. (A) Overexpression of TAZ increased the expression levels of CTGF in SK-N-SH cells compared with those in the controls. (B) Knockdown of TAZ repressed the expression of CTGF in SK-N-BE(2) cells compared with that in the control. (C) The indicated plasmids were coexpressed in 293 cells. Cells were harvested for measurement of luciferase activity 24 h after transfection. (D) A chromatin immunoprecipitation assay was performed with anti-human influenza hemagglutinin (HA) antibody using 293 cells expressing HA-TAZ. The presence of CTGF promoter was detected by polymerase chain reaction.
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f4-mmr-11-02-0982: Overexpression of TAZ upregulates connective tissue growth factor (CGTF) expression while knockdown of TAZ downregulates it. (A) Overexpression of TAZ increased the expression levels of CTGF in SK-N-SH cells compared with those in the controls. (B) Knockdown of TAZ repressed the expression of CTGF in SK-N-BE(2) cells compared with that in the control. (C) The indicated plasmids were coexpressed in 293 cells. Cells were harvested for measurement of luciferase activity 24 h after transfection. (D) A chromatin immunoprecipitation assay was performed with anti-human influenza hemagglutinin (HA) antibody using 293 cells expressing HA-TAZ. The presence of CTGF promoter was detected by polymerase chain reaction.

Mentions: To explore the mechanism by which TAZ induces EMT and increased invasiveness, the regulatory effect of TAZ on the expression of CTGF was investigated. As expected, ectopic TAZ expression clearly induced CTGF at the protein level (Fig. 4A). In contrast, TAZ suppression via RNAi reduced the CTGF expression level compared to that in the controls (Fig. 4B). Subsequently, it was assessed whether TAZ promotes the activity of the CTGF gene promoter. The CTGF luciferase reporter constructs (CTGF-Luc) were transiently transfected into HEK 293 cells with the TAZ expression plasmids. The luciferase reporter activity increased in a concentration-dependent manner in cells with ectopic TAZ expression compared with that in the mock control (Fig. 4C), suggesting that ectopic TAZ expression promotes the activity of CTGF. To determine if TAZ interacted with the endogenous CTGF promoter, ChIP assays were performed. CGTF promoter was detected in PCR-amplified DNA fragments immunoprecipitated with an anti-HA antibody but not in DNA fragments precipitated with an IgG control antibody, indicating that TAZ bound to the CTGF promoter (Fig. 4D).


TAZ promotes epithelial to mesenchymal transition via the upregulation of connective tissue growth factor expression in neuroblastoma cells.

Wang Q, Xu Z, An Q, Jiang D, Wang L, Liang B, Li Z - Mol Med Rep (2014)

Overexpression of TAZ upregulates connective tissue growth factor (CGTF) expression while knockdown of TAZ downregulates it. (A) Overexpression of TAZ increased the expression levels of CTGF in SK-N-SH cells compared with those in the controls. (B) Knockdown of TAZ repressed the expression of CTGF in SK-N-BE(2) cells compared with that in the control. (C) The indicated plasmids were coexpressed in 293 cells. Cells were harvested for measurement of luciferase activity 24 h after transfection. (D) A chromatin immunoprecipitation assay was performed with anti-human influenza hemagglutinin (HA) antibody using 293 cells expressing HA-TAZ. The presence of CTGF promoter was detected by polymerase chain reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262480&req=5

f4-mmr-11-02-0982: Overexpression of TAZ upregulates connective tissue growth factor (CGTF) expression while knockdown of TAZ downregulates it. (A) Overexpression of TAZ increased the expression levels of CTGF in SK-N-SH cells compared with those in the controls. (B) Knockdown of TAZ repressed the expression of CTGF in SK-N-BE(2) cells compared with that in the control. (C) The indicated plasmids were coexpressed in 293 cells. Cells were harvested for measurement of luciferase activity 24 h after transfection. (D) A chromatin immunoprecipitation assay was performed with anti-human influenza hemagglutinin (HA) antibody using 293 cells expressing HA-TAZ. The presence of CTGF promoter was detected by polymerase chain reaction.
Mentions: To explore the mechanism by which TAZ induces EMT and increased invasiveness, the regulatory effect of TAZ on the expression of CTGF was investigated. As expected, ectopic TAZ expression clearly induced CTGF at the protein level (Fig. 4A). In contrast, TAZ suppression via RNAi reduced the CTGF expression level compared to that in the controls (Fig. 4B). Subsequently, it was assessed whether TAZ promotes the activity of the CTGF gene promoter. The CTGF luciferase reporter constructs (CTGF-Luc) were transiently transfected into HEK 293 cells with the TAZ expression plasmids. The luciferase reporter activity increased in a concentration-dependent manner in cells with ectopic TAZ expression compared with that in the mock control (Fig. 4C), suggesting that ectopic TAZ expression promotes the activity of CTGF. To determine if TAZ interacted with the endogenous CTGF promoter, ChIP assays were performed. CGTF promoter was detected in PCR-amplified DNA fragments immunoprecipitated with an anti-HA antibody but not in DNA fragments precipitated with an IgG control antibody, indicating that TAZ bound to the CTGF promoter (Fig. 4D).

Bottom Line: The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP).Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay.Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang 150001, P.R. China.

ABSTRACT
Neuroblastoma (NB) is a neuroendocrine cancer that occurs most commonly in infants and young children. The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP). The effect of TAZ on the metastatic progression of neuroblastoma and the underlying mechanisms involved remain elusive. In the current study, it was determined by western blot analysis that the migratory and invasive properties of SK‑N‑BE(2) human neuroblastoma cells are associated with high expression levels of TAZ. Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay. In contrast, overexpression of TAZ in SK‑N‑SH human neuroblastoma cells was shown by Transwell migration and invasion assays, and western blot analysis, to result in epithelial‑mesenchymal transition (EMT) and increased invasiveness. Mechanistically, the overexpression of TAZ was demonstrated to upregulate the expression levels of connective tissue growth factor (CTGF), by western blot analysis and chromatin immunoprecipitation assay, while the knockdown of TAZ downregulated it. Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway. In conclusion, the present study is, to the best of our knowledge, the first to demonstrate that the overexpression of TAZ induces EMT, increasing the invasive abilities of neuroblastoma cells. This suggests that TAZ may serve as a potential target in the development of novel therapies for the treatment of neuroblastoma.

Show MeSH
Related in: MedlinePlus