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Translationally controlled tumor protein induces epithelial to mesenchymal transition and promotes cell migration, invasion and metastasis.

Bae SY, Kim HJ, Lee KJ, Lee K - Sci Rep (2015)

Bottom Line: We found that overexpression of TCTP in a porcine renal proximal tubule cell line, LLC-PK1, induced EMT-like phenotypes with the expected morphological changes and appearance of EMT related markers.TCTP overexpression also enhanced cell migration via activation of mTORC2/Akt/GSK3β/β-catenin, and invasiveness by activating MMP-9.Moreover, TCTP depletion in melanoma cells significantly reduced pulmonary metastasis by inhibiting the development of mesenchymal-like phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Translationally controlled tumor protein (TCTP), is a highly conserved protein involved in fundamental processes, such as cell proliferation and growth, tumorigenesis, apoptosis, pluripotency, and cell cycle regulation. TCTP also inhibits Na,K-ATPase whose subunits have been suggested as a marker of epithelial-to-mesenchymal transition (EMT), a crucial step during tumor invasiveness, metastasis and fibrosis. We hypothesized that, TCTP might also serve as an EMT inducer. This study attempts to verify this hypothesis. We found that overexpression of TCTP in a porcine renal proximal tubule cell line, LLC-PK1, induced EMT-like phenotypes with the expected morphological changes and appearance of EMT related markers. Conversely, depletion of TCTP reversed the induction of these EMT phenotypes. TCTP overexpression also enhanced cell migration via activation of mTORC2/Akt/GSK3β/β-catenin, and invasiveness by activating MMP-9. Moreover, TCTP depletion in melanoma cells significantly reduced pulmonary metastasis by inhibiting the development of mesenchymal-like phenotypes. Overall, these findings support our hypothesis that TCTP is a positive regulator of EMT and suggest that modulation of TCTP expression is a potential approach to inhibit the invasiveness and migration of cancer cells and the attendant pathologic processes including metastasis.

No MeSH data available.


Related in: MedlinePlus

Ectopic overexpression of TCTP promotes EMT and enhances cell migration.(a) Phase-contrast microscopic images of LLC-PK1 cells infected with control Ad-GFP virus (Cont) or Ad-TCTP-GFP virus (TCTP). Images were taken with ×100 magnification. scale bar 200μm (b) Expression levels of epithelial marker as well as mesenchymal markers were examined by immunoblotting of control and TCTP overexpressed cells. (c) Expression levels of transcription repressors of E-cadherin were examined by immunoblotting. β-actin was used as a loading control. (d, e) Fluorescence microscopic staining of E-cadherin (d) and F-actin (e) were performed with control and TCTP overexpressed cell (red). Nuclear DNA was stained with DAPI (blue). (f) Wound healing assays were performed with control and TCTP overexpressed cells for 48 hours. Representative images were taken at 0 h and 48 h after wounding, as indicated. Images were taken with ×100 magnification using. Quantification was carried out by measuring the distance migrated compared with the controls. Values are means ± S. E. M. of three experiments. *P < 0.05, **P < 0.01 (g) Transwell migration assays were performed with LLC-PK1 cells infected with adeno-GFP virus or adeno-TCTP-GFP virus over 18 h. Experiments were done in triplicate and representative images were taken with ×100 magnification using. Quantification was carried by counting the number of migratory cells that had infiltrated the filter. Values are means ± S. E. M. **P < 0.01.
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f1: Ectopic overexpression of TCTP promotes EMT and enhances cell migration.(a) Phase-contrast microscopic images of LLC-PK1 cells infected with control Ad-GFP virus (Cont) or Ad-TCTP-GFP virus (TCTP). Images were taken with ×100 magnification. scale bar 200μm (b) Expression levels of epithelial marker as well as mesenchymal markers were examined by immunoblotting of control and TCTP overexpressed cells. (c) Expression levels of transcription repressors of E-cadherin were examined by immunoblotting. β-actin was used as a loading control. (d, e) Fluorescence microscopic staining of E-cadherin (d) and F-actin (e) were performed with control and TCTP overexpressed cell (red). Nuclear DNA was stained with DAPI (blue). (f) Wound healing assays were performed with control and TCTP overexpressed cells for 48 hours. Representative images were taken at 0 h and 48 h after wounding, as indicated. Images were taken with ×100 magnification using. Quantification was carried out by measuring the distance migrated compared with the controls. Values are means ± S. E. M. of three experiments. *P < 0.05, **P < 0.01 (g) Transwell migration assays were performed with LLC-PK1 cells infected with adeno-GFP virus or adeno-TCTP-GFP virus over 18 h. Experiments were done in triplicate and representative images were taken with ×100 magnification using. Quantification was carried by counting the number of migratory cells that had infiltrated the filter. Values are means ± S. E. M. **P < 0.01.

Mentions: Several studies showed that TCTP levels increase in colon cancer14, prostate cancer15 and hepatocellular carcinoma (HCC)16. In addition, a strong correlation between the expression levels of TCTP and degree of metastasis was observed in ovarian cancer17, colon cancer cell2, and human glioma18. It has been well established that TCTP acts as an anti-apoptotic protein and contributes to malignancy19. Although TCTP is clearly associated with cancer progression and metastasis, the exact role of TCTP on cancer metastasis is unclear. We tested our hypothesis that TCTP increases metastasis by inducing EMT, employing LLC-PK1- renal proximal tubular epithelial cells transiently altered by adenoviral vector to overexpress TCTP. Phase contrast microscopic studies indicated that the TCTP-overexpressing cells lost cell-cell contacts and acquired dispersed appearance, which are hallmarks of cellular/morphologic changes during EMT (Figure 1a)20. Immunoblotting studies demonstrated alterations in the epithelial and mesenchymal markers in these cells. We also observed reduction in the epithelial marker; E-cadherin, and increases in the mesenchymal markers, fibronectin, vimentin, α-smooth muscle actin (α-SMA) and N-cadherin, hallmarks of EMT induced by ectopic expression of TCTP (Figure 1b). Because of the demonstrated role of transcriptional repressors in the loss of E-cadherin21, we also examined the expression levels of E-cadherin transcription repressors such as ZEB1, slug and twist, by immunoblotting, and found that these repressors were elevated by TCTP overexpression (Figure 1c). Furthermore, we also confirmed that TCTP induces the expression of mesenchymal markers in cancer cell lines, A549 and HeLa cells (Figure S1). Next, we immunostained E-cadherin and mapped the changes in the localization of E-cadherin caused by TCTP overexpression. E-cadherin was found localized in areas of cell-cell contact in the control cells, in contrast TCTP overexpressing cells which showed reduced membrane localization of E-cadherin (Figure 1d).


Translationally controlled tumor protein induces epithelial to mesenchymal transition and promotes cell migration, invasion and metastasis.

Bae SY, Kim HJ, Lee KJ, Lee K - Sci Rep (2015)

Ectopic overexpression of TCTP promotes EMT and enhances cell migration.(a) Phase-contrast microscopic images of LLC-PK1 cells infected with control Ad-GFP virus (Cont) or Ad-TCTP-GFP virus (TCTP). Images were taken with ×100 magnification. scale bar 200μm (b) Expression levels of epithelial marker as well as mesenchymal markers were examined by immunoblotting of control and TCTP overexpressed cells. (c) Expression levels of transcription repressors of E-cadherin were examined by immunoblotting. β-actin was used as a loading control. (d, e) Fluorescence microscopic staining of E-cadherin (d) and F-actin (e) were performed with control and TCTP overexpressed cell (red). Nuclear DNA was stained with DAPI (blue). (f) Wound healing assays were performed with control and TCTP overexpressed cells for 48 hours. Representative images were taken at 0 h and 48 h after wounding, as indicated. Images were taken with ×100 magnification using. Quantification was carried out by measuring the distance migrated compared with the controls. Values are means ± S. E. M. of three experiments. *P < 0.05, **P < 0.01 (g) Transwell migration assays were performed with LLC-PK1 cells infected with adeno-GFP virus or adeno-TCTP-GFP virus over 18 h. Experiments were done in triplicate and representative images were taken with ×100 magnification using. Quantification was carried by counting the number of migratory cells that had infiltrated the filter. Values are means ± S. E. M. **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4306963&req=5

f1: Ectopic overexpression of TCTP promotes EMT and enhances cell migration.(a) Phase-contrast microscopic images of LLC-PK1 cells infected with control Ad-GFP virus (Cont) or Ad-TCTP-GFP virus (TCTP). Images were taken with ×100 magnification. scale bar 200μm (b) Expression levels of epithelial marker as well as mesenchymal markers were examined by immunoblotting of control and TCTP overexpressed cells. (c) Expression levels of transcription repressors of E-cadherin were examined by immunoblotting. β-actin was used as a loading control. (d, e) Fluorescence microscopic staining of E-cadherin (d) and F-actin (e) were performed with control and TCTP overexpressed cell (red). Nuclear DNA was stained with DAPI (blue). (f) Wound healing assays were performed with control and TCTP overexpressed cells for 48 hours. Representative images were taken at 0 h and 48 h after wounding, as indicated. Images were taken with ×100 magnification using. Quantification was carried out by measuring the distance migrated compared with the controls. Values are means ± S. E. M. of three experiments. *P < 0.05, **P < 0.01 (g) Transwell migration assays were performed with LLC-PK1 cells infected with adeno-GFP virus or adeno-TCTP-GFP virus over 18 h. Experiments were done in triplicate and representative images were taken with ×100 magnification using. Quantification was carried by counting the number of migratory cells that had infiltrated the filter. Values are means ± S. E. M. **P < 0.01.
Mentions: Several studies showed that TCTP levels increase in colon cancer14, prostate cancer15 and hepatocellular carcinoma (HCC)16. In addition, a strong correlation between the expression levels of TCTP and degree of metastasis was observed in ovarian cancer17, colon cancer cell2, and human glioma18. It has been well established that TCTP acts as an anti-apoptotic protein and contributes to malignancy19. Although TCTP is clearly associated with cancer progression and metastasis, the exact role of TCTP on cancer metastasis is unclear. We tested our hypothesis that TCTP increases metastasis by inducing EMT, employing LLC-PK1- renal proximal tubular epithelial cells transiently altered by adenoviral vector to overexpress TCTP. Phase contrast microscopic studies indicated that the TCTP-overexpressing cells lost cell-cell contacts and acquired dispersed appearance, which are hallmarks of cellular/morphologic changes during EMT (Figure 1a)20. Immunoblotting studies demonstrated alterations in the epithelial and mesenchymal markers in these cells. We also observed reduction in the epithelial marker; E-cadherin, and increases in the mesenchymal markers, fibronectin, vimentin, α-smooth muscle actin (α-SMA) and N-cadherin, hallmarks of EMT induced by ectopic expression of TCTP (Figure 1b). Because of the demonstrated role of transcriptional repressors in the loss of E-cadherin21, we also examined the expression levels of E-cadherin transcription repressors such as ZEB1, slug and twist, by immunoblotting, and found that these repressors were elevated by TCTP overexpression (Figure 1c). Furthermore, we also confirmed that TCTP induces the expression of mesenchymal markers in cancer cell lines, A549 and HeLa cells (Figure S1). Next, we immunostained E-cadherin and mapped the changes in the localization of E-cadherin caused by TCTP overexpression. E-cadherin was found localized in areas of cell-cell contact in the control cells, in contrast TCTP overexpressing cells which showed reduced membrane localization of E-cadherin (Figure 1d).

Bottom Line: We found that overexpression of TCTP in a porcine renal proximal tubule cell line, LLC-PK1, induced EMT-like phenotypes with the expected morphological changes and appearance of EMT related markers.TCTP overexpression also enhanced cell migration via activation of mTORC2/Akt/GSK3β/β-catenin, and invasiveness by activating MMP-9.Moreover, TCTP depletion in melanoma cells significantly reduced pulmonary metastasis by inhibiting the development of mesenchymal-like phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea.

ABSTRACT
Translationally controlled tumor protein (TCTP), is a highly conserved protein involved in fundamental processes, such as cell proliferation and growth, tumorigenesis, apoptosis, pluripotency, and cell cycle regulation. TCTP also inhibits Na,K-ATPase whose subunits have been suggested as a marker of epithelial-to-mesenchymal transition (EMT), a crucial step during tumor invasiveness, metastasis and fibrosis. We hypothesized that, TCTP might also serve as an EMT inducer. This study attempts to verify this hypothesis. We found that overexpression of TCTP in a porcine renal proximal tubule cell line, LLC-PK1, induced EMT-like phenotypes with the expected morphological changes and appearance of EMT related markers. Conversely, depletion of TCTP reversed the induction of these EMT phenotypes. TCTP overexpression also enhanced cell migration via activation of mTORC2/Akt/GSK3β/β-catenin, and invasiveness by activating MMP-9. Moreover, TCTP depletion in melanoma cells significantly reduced pulmonary metastasis by inhibiting the development of mesenchymal-like phenotypes. Overall, these findings support our hypothesis that TCTP is a positive regulator of EMT and suggest that modulation of TCTP expression is a potential approach to inhibit the invasiveness and migration of cancer cells and the attendant pathologic processes including metastasis.

No MeSH data available.


Related in: MedlinePlus