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

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.

Figure 1. 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.

Figure 2. Knockdown of TCTP reverses EMT phenotypes and suppresses cell migration.

(a) Phase-contrast microscopic images of LLC-PK1 cells transfected with control shRNA (shCont) or TCTP shRNA (shTCTP). Images were taken with ×100 magnification. scale bar 200 μm (b) EMT phenotypic markers including E-cadherin and mesenchymal markers were examined by immunoblotting in both LLC-PK1 shCont and shTCTP cells. β-actin was used as a loading control. (c) Expression levels of transcription repressors of E-cadherin in shCont and shTCTP cells were examined by immunoblotting. β-actin was used as a loading control. (d) Transwell migration assays were performed with shCont and shTCTP cells. 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 < 0.001.

Figure 3. TCTP enhances cell invasion through MMP-9 activation.

(a) Transwell invasion assay was performed with LLC-PK1 cells infected with adeno-GFP virus or adeno-TCTP-GFP virus over 24 hr. 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. (b, c) The expression levels of MMP-2 and MMP-9 in cell lysates were examined by immunoblotting. (d) Secreted MMP-9 was detected in conditioned media of control and TCTP overexpressed cells by immunoblotting. (e) Conditioned media were collected at indicated time course from adenoviral infection. Gelatinolytic activity of pro and active MMP-9 were visible as a clear area on the gel.

Figure 4. TCTP promotes cell migration through mTORC2/Akt/GSK3β/β-catenin pathway.

(a) Phosphorylation states of Akt (Ser⁴⁷³) and GSK3β (Ser⁹) in control and TCTP overexpressed cells were examined by immunoblotting. (b) The nuclear translocation of β-catenin was evaluated by immunoblotting after subcellular fractionation. The purity of each fraction was monitored by the respective markers, GAPDH for the cytosolic fraction, and lamin A/C for the nuclear fraction. (c) Fluorescent microscopic visualization of β-catenin (green) in control and TCTP overexpressed cells. Nuclear DNA was stained with DAPI (blue). (d) Both control and TCTP overexpressed LLC-PK1 cells treated with 10 μM of LY294002, 100 nM of rapamycin, and 5 μM of PP242 for 18 h were subjected to transwell migration assay. 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, ***P<0.001.

Figure 5. Silencing TCTP promotes MET process and suppresses pulmonary metastasis of mouse melanoma cell line.

(a) Phase-contrast microscopic images of B16F10 cells infected with lentiviral control shRNA (shCont) or shRNA vector targeting TCTP (shTCTP #1 and #2). Images were taken with ×100 magnification. scale bar 200 μm (b) Expression levels of mesenchymal markers were determined by immunoblotting. GAPDH was used as a loading control. (c) The expression levels of MMP-2 and MMP-9 in cell lysates were examined by immunoblotting. (d, e) Fluorescence microscopic staining of N-cadherin (d) and F-actin (e) were performed with B16F10-shCont, shTCTP #1 and #2 (red). Nuclear DNA was stained with DAPI (blue). (f) The nuclear translocation of β-catenin was evaluated by immunoblotting after subcellular fractionation. GAPDH was used for the cytosolic fraction, and lamin A/C was used for the nuclear fraction. (g, h) B16F10-shCont, B16F10-shTCTP #1 and #2 cells were subjected to the transwell migration (g) and transwell invasion (h) assay. 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<0.05, **P<0.01, ***P<0.001. (i) Effect of TCTP depletion on the pulmonary metastasis was determined. Both B16F10-shCont and shTCTP #1 cells were injected intravenously into the tail vein of male BALB C/nude mice with 1 × 10⁶ cells (n = 6/group). After 11days, the lungs were extirpated and areas of nodules were calculated. Three representatives per each set are shown. Normal lung indicates control injected with PBS.