Translationally controlled tumor protein (TCTP) is a multifunctional protein that plays an important role in cellular biological/physiological events such as immune responses, tumorigenicity, cell proliferation, gene regulation, stress responses, apoptosis, and cancer progression, including malignant transformation (Yoon et al., 2000; Bommer and Thiele, 2004; Jung et al., 2004; Liu et al., 2005; Chen et al., 2007; Telerman and Amson, 2009; Jung et al., 2011; Lucibello et al., 2011; Rho et al., 2011). TCTP has a variety of synonyms, including TPT-1, histamine-releasing factor, fortilin, P21, P23, and Q23 (Thomas et al., 1981; Yenofsky et al., 1982; Bohm et al., 1989; MacDonald et al., 1995; Li et al., 2001). It is a highly conserved cell survival factor in eukaryotic organisms and is widely expressed in various tissues and cell types (Nagano-Ito and Ichikawa, 2012; Acunzo et al., 2014).

In research on clinical samples, TCTP was shown to be associated with reduced cell survival in glioma patients and to induce glioma tumor cell growth by promoting the Wnt/β-catenin signaling cascade (Gu et al., 2014). TCTP was also found overexpressed in ovarian tumor tissues compared with normal tissues and to be significantly related to poor patient prognosis (Chen et al., 2015a). TCTP also interacts with many cellular proteins, including Na,K-ATPase (Jung et al., 2004), p53 (Rho et al., 2011), myeloid cell leukemia protein-1 (Mcl-1) (Li et al., 2001), tubulin (Tuynder et al., 2002), TSAP6 (Amzallag et al., 2004), translation elongation factors eEF1A and eEF1B-β (Langdon et al., 2004), Bcl-X_L (Yang et al., 2005), and actin (Tsarova et al., 2010). Overexpression of p53 induces apoptotic cell death in tumor cells. However, TCTP was observed to bind to p53 and prevent apoptosis by destabilizing the protein in a human A549 lung tumor cell line (Amson et al., 2011; Rho et al., 2011). In addition, p53 directly reduces the transcript level of TCTP. These findings strongly suggest that TCTP can promote transformation by suppressing p53 function (Rho et al., 2011; Nagano-Ito and Ichikawa, 2012).

On the other hand, TCTP has been reported to have an anti-apoptotic function, which may be associated with its interactions with the Mcl-1 protein and/or Bcl-X_L protein (Li et al., 2001; Liu et al., 2005). Previous studies reported that overexpression of TCTP can protect against stress-induced mammalian cell apoptosis. Conversely, inhibition of TCTP in malignant human tumor cells enhanced apoptotic cell death (Bommer and Thiele, 2004). Thus, TCTP may be involved in cell survival as a negative regulator of apoptotic cell death. Recent studies have added to these earlier findings by showing that levomepromazine and buclizine inhibit tumor cell growth by binding to TCTP and inducing cell differentiation (Seo and Efferth, 2016). TCTP is also a positive regulator of the epithelial to mesenchymal transition (EMT), and modulation of its expression has been shown to have potential in inhibiting the invasiveness and migration of tumor cells and the pathological processes associated with this, including metastasis (Bae et al., 2015).

Angiogenesis is the physiological and pathological process by which new blood vessels grow from pre-existing vessels. The walls of blood vessels are generally formed by vascular endothelial cells, which are associated with various diseases, including a range of tumors, cardiovascular disease, arthritis, diabetes, and Alzheimer’s disease (Folkman, 1995; Watanabe et al., 2004; Hicklin and Ellis, 2005; Poveshchenko and Konenkov, 2010; Ruf et al., 2010; Saharinen et al., 2011). Tumor metastasis is a major contributor to cancer-associated death, involving the spread of malignant cells from the original tumor to distant sites (Geiger and Peeper, 2009). This process is related to EMT, in that metastatic tumor cells activate the EMT program to achieve local invasion followed by dissemination into the circulation (Yang et al., 2004, 2006). Tumors typically stimulate blood vessel growth by several growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiopoietin (Ang1 and Ang2). For example, VEGF is a major contributor to the formation of blood vessel networks, which can in turn promote tumor growth (Yang et al., 2004; Yang et al., 2006; Geiger and Peeper, 2009). All members of the VEGF family induce cellular responses by binding to VEGF receptors on the cell surface. VEGF also plays a pivotal role in the progression of colon and ovarian tumors by controlling tumor growth via enhanced tumor angiogenesis (Hanahan and Folkman, 1996; Kumaran et al., 2009; Sarvaiya et al., 2013; Nagasaki et al., 2014; Riabov et al., 2014).

Against this background, the aim of this study is to clarify the role of TCTP in angiogenesis in ovarian tumor cells and to address its potential signaling targets, such as the VEGF receptor 2 (VEGFR-2)/Akt signaling pathway. However, the mechanisms by which TCTP contributes to ovarian angiogenesis and tumorigenesis are not fully understood. Given our observations suggesting a critical role for TCTP in tumor angiogenesis, we sought to determine the direct effects of the physiological functions of TCTP in ovarian tumorigenesis.

Cell culture and primary antibodies

A human ovarian tumor cell line (SKOV-3) and normal ovarian fibroblast cells (NOV-31) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). SKOV-3 cells were maintained in Dulbecco’s Modified Eagle’s Medium (Life Technologies, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS). Primary human umbilical vein endothelial cells (HUVECs) (Clonetics, Walkersville, MD, USA) were grown on 0.3% gelatin-coated dishes (Sigma, St. Louis, MO, USA) in EGM-2 BulletKit medium (Clonetics). All cells were incubated in a 37°C humidified atmosphere with 5% CO₂. The primary antibodies used in this study were anti-TCTP (Oncogene, San Diego, CA, USA), anti-VEGF, anti-HIF-1α, anti-VEGFR-1, anti-VEGFR-2, anti-phospho-VEGFR-2 (Tyr1175), anti-PI3K, anti-phospho-PI3K, anti-Akt, anti-phospho-Akt (Ser473 and Thr308), anti-PDK-1, anti-phospho-PDK-1 (Sre241), anti-mTOR, anti-phospho-mTOR (Ser2448), anti-TSC2, anti-phospho-TSC2 (Ser1462), anti-p70S6K, anti-phospho-p70S6K (Thr421) (all from Cell Signaling, Beverly, MA, USA), and β-actin (Sigma).

Endothelial cell migration and tube formation assay

Cell migration was measured using Transwell chambers (8 μm pore size; Corning Costar, Cambridge, MA, USA), in accordance with a previously reported protocol (Rho et al., 2023b; Zhou et al., 2023). In brief, the lower surface of the filter was coated with 10 μg gelatin. M199 containing 1% FBS with VEGF (10 ng/mL) was placed in the lower wells. A total of 1.5×10⁵ cells per well were grown in serum-free medium in the upper chamber. Cells were allowed to migrate in a 5% CO₂ incubator at 37°C for 24 h. After incubation, cells were fixed with 99% methanol and stained with hematoxylin and eosin (Sigma). Migrating cells were counted using an inverted microscope and photographed (Rho et al., 2023b; Zhou et al., 2023). For capillary-like tube formation analysis, growth factor-reduced Matrigel (200 μL 10 mg/mL) was added to a 24-well plate and then polymerized for 30 min at 37°C for 30 min. Untransfected (vector only/control), TCTP-, or short interfering (si)TCTP-transfected HUVECs (2×10⁵ cells) were seeded on the surface of the Matrigel. Seeded cells were then incubated with or without 10 ng/mL VEGF in M199 containing 1% FBS. Next, tubular structures were measured in accordance with a previously reported protocol (Rho et al., 2023b; Zhou et al., 2023).

[³H]thymidine incorporation assay

To assess endothelial cell proliferation, cells were seeded at a density of 2.5×10⁴ per well of gelatinized plates in standard medium on day 0. Next, [³H] thymidine incorporation analysis was performed as described previously (Rho et al., 2023b; Zhou et al., 2023).

Yeast two-hybrid analysis

For bait plasmid construction using human TCTP, cDNA encoding full-length human TCTP was subcloned into the EcoRI and XhoI restriction enzyme sites of the pGilda/LexA yeast shuttle vector. The bait pGilda/LexA-TCTP plasmid was transformed into the yeast strain EGY48 using a modified version of the lithium acetate method (Rho et al., 1996, 2023a). cDNAs encoding full-length human VEGFR-1 and VEGFR-2 were introduced into the multi-cloning sites of the pJG4-5 shuttle vector, which includes B42 fusion proteins (Clontech, Palo Alto, CA, USA). The VEGFR-1 or VEGFR-2 cDNA encoding pJG4-5 fusion proteins was transformed into yeast competent cells that had already been transformed with pGilda/LexA-TCTP, and the successful transformants were selected based on their tryptophan prototrophy (plasmid marker) on a synthetic medium (Ura, His, Trp) containing 2% (w/v) glucose. The binding activity of the interaction was calculated as described previously (Rho et al., 2023a).

Coimmunoprecipitation (Co-IP) and immunoblotting

Co-IP assays were performed as described previously (Kim et al., 2015). In brief, cells were trypsinized and then centrifuged. Cell pellets were rinsed in cold PBS and resuspended in lysis buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail containing 1 μg/mL leupeptin, 1 μg/mL pepstatin, 2 μg/mL aprotinin, and 200 μg/mL phenylmethylsulfonyl fluoride). The cell lysates were incubated with anti-Flag antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then precipitated with protein A–agarose (Amersham, Little Chalfont, UK). Approximately 20-30 μg precipitated proteins were separated by 10-12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon P membranes (Millipore Corporation, Billerica, MA, USA). After blocking, the membranes were incubated with the indicated specific primary antibodies, including anti-TCTP and anti-VEGFR-2. The membranes were washed three times in TBST washing buffer for 5 min and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The blots were visualized using an ECL detection system (Amersham).

Gelatin zymography analysis

Gelatin zymography assays were employed to determine matrix metalloproteinase (MMP)-2 and MMP-9 activities. Cells (1×10⁶ per well) were grown in six-well plates for 24 h, and their MMP-2 and MMP-9 activities were measured by gelatin zymography. In brief, equal volumes of protein were subjected to 10% SDS-PAGE with polymerization using 0.1% gelatin as a substrate (Invitrogen, Carlsbad, CA, USA). After electrophoresis, the gels were renatured by washing in 2.5% Triton X-100 solution (including 50 mM Tris-HCl [pH 7.4], 5 mM CaCl₂, and 1 μM ZnCl₂ in distilled water) twice for 30 min at room temperature to remove all of the SDS solution. Next, the gels were incubated in visualizing solution (50 mM Tris-HCl, 5 mM CaCl₂, 1 μM ZnCl₂, and 0.02% Brij-35 in distilled water) for 20 h. The gels were stained with Coomassie brilliant blue stain R-250 and destained using 20% methanol and 7% acetic acid in distilled water until clear bands developed. The bands representing MMP activity were quantified using densitometry.

Statistical analysis

All data are expressed as means ± standard deviation (SD) and were evaluated by Student’s t-test and analysis of variance according to the number of groups compared. Significant differences (at p<0.05) are depicted with asterisks in each figure. The analyses were performed using SPSS 20 software (Statistical Product and Service Solutions, Chicago, IL, USA).

TCTP promotes VEGF-induced endothelial cell migration and tubular structure formation and proliferation in vitro

VEGF is a major pro-angiogenic factor that controls multiple key steps of angiogenesis and induces a signaling cascade in endothelial cells. Endothelial cell activity plays an important role in regulating various vascular-related physiological functions and diseases, including tumor growth and maintenance. To explore the possibility that TCTP regulates the effects of VEGF on cell migration in HUVECs, angiogenesis was examined according to cell proliferation, migration, and capillary-like tubular structure formation in the endothelial cells. The regulatory effects of TCTP on VEGF-induced endothelial cell migration were estimated using the Transwell migration assay system. As presented in Fig. 1A, VEGF notably enhanced the migration of the untransfected cells and control (empty-insert plasmid)-transfected cells compared with uninduced cells. Results from the cell migration assay showed that HUVEC movement was markedly activated in the presence of TCTP compared with that in the control groups, while TCTP knockdown (using siTCTP) disrupted it. We performed further transient transfections of siTCTP at various concentrations. As indicated in Fig. 1B, knockdown of TCTP gradually suppressed VEGF-induced cell migration in a dose-dependent manner. Subsequently, we examined the pro-angiogenic effects of TCTP on the formation of VEGF-induced capillary-like tubular structures on Matrigel using an in vitro HUVEC angiogenesis model system. As presented in Fig. 1C, untreated or control cells incubated with VEGF formed capillary-like structures on Matrigel. The overexpression of TCTP significantly promoted VEGF-induced tubular structure formation. Meanwhile, the regulatory effect of TCTP on VEGF-induced capillary-like tubular structures was conspicuously inhibited by siTCTP transient transfection. Finally, the effects of TCTP on VEGF-induced proliferation of endothelial cells were estimated using a [³H]thymidine incorporation assay system. VEGF increased DNA synthesis in the untransfected cells and the control (empty-insert plasmid)-transfected cells compared with the unstimulated endothelial cells (Plate et al., 1992). siTCTP dramatically inhibited VEGF-induced DNA synthesis in endothelial cells (Fig. 1D). Therefore, TCTP overexpression activates a key event in VEGF-induced angiogenesis in vitro, including endothelial cell migration and proliferation. Collectively, our results indicate that TCTP promotes angiogenesis by inducing cell migration and tube formation, as well as the proliferation of HUVECs.

Figure 1. Translationally controlled tumor protein (TCTP) promotes vascular endothelial growth factor (VEGF)-induced endothelial cell migration, tubular structure formation, and cell proliferation in vitro. (A) The pro-angiogenic effect of human TCTP on human umbilical vein endothelial cell (HUVEC) migration. Cells transfected with TCTP were evaluated using Matrigel-coated Transwells as a migration assay system, followed by activation for 48 h in the absence or presence of VEGF. The number of cells that migrated was calculated under a light microscope. Each bar represents the mean ± standard deviation (SD) of three independent experiments that yielded similar results. *p<0.05. (B) After treatment with VEGF, HUVECs were transfected with short interfering (si)TCTP constructs at various concentrations. All transfected cells were fixed and then stained with hematoxylin and eosin. The number of migrated cells was measured under a light microscope and is presented as the mean ± SD. *p<0.05 versus control group. Three independent experiments were performed in triplicate. (C) Effects of human TCTP and siTCTP on the capillary-like tubular structure formation of HUVECs. Each transfectant was grown on growth factor-reduced Matrigel and then treated with/without VEGF. Quantification of the newly formed tubule networks was determined based on photographs taken from an inverted microscope. Data are presented as means ± SD from three independent experiments. Significant differences (p<0.05) are depicted with asterisks in each graph. (D) The regulatory effect of TCTP on endothelial cell proliferation. Cells were grown for 3 days with/without VEGF. The counts per min values of [³H]thymidine were determined using a liquid scintillation counter. The data are presented as means ± SD of three independent experiments. *p<0.05 compared with the control group.

TCTP can promote VEGF-induced VEGFR-2 phosphorylation via interaction with VEGFR-2, but not with VEGFR-1

VEGFR-2 is an essential signal transduction factor involved in pathological/physiological angiogenesis. To address the possible mechanism involved in the promotion of angiogenesis by TCTP, we employed a yeast two-hybrid protein interaction screening assay in vivo and a Co-IP analysis system in vitro. We first examined the regulatory effect of TCTP on VEGF-stimulated VEGFR-2 protein expression in HUVECs. The overexpression of siTCTP markedly suppressed the phosphorylation of VEGFR-2, while TCTP reversed this effect (Fig. 2A). Because VEGF rapidly activates angiogenesis in tumor metastasis as well as tumorigenesis, we evaluated the effect of TCTP on VEGF protein expression in SKOV-3 tumor cells. Knockdown of TCTP markedly inhibited VEGF expression, while TCTP reversed this effect (Fig. 2B). We also demonstrated the effect of TCTP on the protein expression of HIF-1α, a transcription factor and important regulator of VEGF expression. As indicated in Fig. 2B, siTCTP notably decreased the expression of HIF-1α. The inhibitory effect of siTCTP was recovered completely by TCTP transient transfection. These observations suggest that VEGF-induced protein expression is completely suppressed by siTCTP. Direct interactions among many proteins are crucial for the majority of cellular biological/physiological functions. For instance, signaling from the exterior to the interior of a cell is controlled via protein–protein interactions. Thus, signal transduction plays a major role in cellular processes, as well as in aggressive solid tumors and many disease types. Therefore, protein–protein interactions are important for the majority of processes in the microenvironment of living cells (Kim et al., 2016). Next, we examined the cellular interaction between VEGFR-2 and TCTP proteins in vivo and in vitro. As shown in Fig. 2C, the binding activity between TCTP and VEGFR-2 was high (91.08 ± 0.77 unit), which was not the case with the empty-insert plasmid (vector only) (2.51 ± 0.73 unit) or VEGFR-1 (2.43 ± 0.69 unit). Therefore, VEGFR-1 was used as a negative control in subsequent experiments. To confirm this direct interaction between TCTP and VEGFR-2 identified in the yeast two-hybrid assay system, we used Co-IP analysis. Bait constructs of TCTP (pcDNA3.1/TCTP) and VEGFR-2 (pcDNA3.1/Flag-VEGFR-2) or pcDNA3.1/Flag-VEGFR-2 and expression plasmid only (pcDNA3.1) were co-transfected into HEK293T cells. Simultaneously, immunoprecipitation was performed using an anti-Flag primary antibody with whole lysates from both transfected cell types. After immunoprecipitation, the precipitated proteins were immunoblotted using either anti-TCTP or anti-VEGFR-2 primary antibodies. As presented in Fig. 2D, pcDNA3.1-VEGFR-2 coimmunoprecipitated with pcDNA3.1/Flag-TCTP (lane 2 in the upper right panel), but not with pcDNA3.1 (plasmid only) or VEGFR-1 (lane 1 in the upper left panel). We next examined the interaction between endogenous TCTP and VEGFR-2. The tumor stimulator TCTP was bound strongly to VEGFR-2 (right panel), but not to VEGFR-1 (left panel) (Fig. 2E). These results clearly suggest a specific protein–protein interaction between endogenous VEGFR-2 and TCTP in the cells.

Figure 2. Translationally controlled tumor protein (TCTP) directly regulates vascular endothelial growth factor receptor 2 (VEGFR-2) phosphorylation and binds to VEGFR-2. (A) SKOV-3 ovarian cancer cells were treated with VEGF and then transfected with control (empty-insert plasmid), TCTP, or TCTP plus short interfering (si)TCTP. Knockdown of TCTP effectively inhibited the phosphorylation (Tyr1175) of VEGFR-2 induced by VEGF. Phosphorylation of VEGFR-2 was visualized using a specific primary antibody. Levels of unphosphorylated VEGFR-2 and β-actin were measured to confirm equal sample loading. (B) SKOV-3 cells were incubated with 10 ng/ml VEGF and then transfected with control (empty-insert plasmid), TCTP, or TCTP plus siTCTP. HIF-1α and VEGF expression levels were determined using the indicated primary antibodies and an immunoblotting assay system. Three independent experiments were performed in triplicate. (C) Positive interactions were observed by monitoring cell growth on a medium lacking leucine for the formation of blue colonies on X-gal plates containing 2% galactose. β-galactosidase activity (unit), estimated by adding o-nitrophenyl β-_D-galactopyranoside, is shown below the corresponding lanes. (D) Coimmunoprecipitation (Co-IP) of TCTP with VEGFR-1 or VEGFR-2. Immunoprecipitation (IP) was performed using anti-FLAG antibodies in lysates from transfected HEK293T cells, followed by immunoblotting with anti-TCTP, anti-VEGFR-1, and anti-VEGFR-2 antibodies. (E) Endogenous proteins in whole lysates from HEK293T cells were developed to Co-IP with an antibody as indicated followed by Western blotting (WB) with an anti-TCTP or anti-VEGFR-2 antibodies. A rabbit IgG and VEGFR-1 were employed as IP negative controls. The input (non-IP) WB data indicated the integrity of the lysates used for IP. All experiments were performed at least three times with similar results.

TCTP regulates ovarian tumor metastasis through modulation of MMP expression and cell cycle-related proteins

To explore the potential mechanism by which TCTP regulates ovarian tumor metastasis, we examined the expression of well-known molecules associated with tumor metastasis and angiogenesis. Specifically, we focused on MMPs, which play important roles in tissue remodeling, tumor metastasis, angiogenesis, hemostasis, and wound healing, including tumor growth and spread. MMPs are frequently overexpressed in most human tumor types (Kessenbrock et al., 2010). We first examined their expression levels in response to TCTP and siTCTP in SKOV-3 ovarian cancer cells. Our data showed that TCTP knockdown (using siTCTP) significantly inhibited such expression, while TCTP overexpression dramatically increased the levels of MMP-2 and MMP-9 mRNA expression compared with levels in the respective controls (Fig. 3A).

Figure 3. Effect of the expression of translationally controlled tumor protein (TCTP) on matrix metalloproteinase (MMP)-2, MMP-9, and cell cycle-related genes. (A) mRNA expression was measured after transfection with various concentrations of TCTP (upper panel). MMP-2 and -9 activities were estimated using reverse transcription polymerase chain reaction (lower panel). The results shown are representative of at least three independent experiments. (B) SKOV-3 cells were transfected with TCTP or short interfering TCTP and compared with untransfected cells (the control). After 48 h, the cells were harvested and treated with lysis buffer. Whole cell lysates were immunoblotted to determine the expression of cell cycle regulatory genes using the indicated specific antibodies. All experiments were performed at least three times with similar results.

Control of the eukaryotic cell cycle is essential for cell survival and maintenance, including cell division and DNA replication. This event is controlled by the regulatory molecules cyclins and cyclin-dependent kinases (CDKs) (Nigg, 1995). Thus, we evaluated how TCTP expression affects the expression of these cell cycle-related proteins using immunoblot analysis. As shown in Fig. 3B, TCTP overexpression significantly promoted the expression of cyclin D1 and CDK4 proteins, whereas the expression of p21 and p27 proteins, well-known CDK inhibitors, was suppressed. To confirm these results, SKOV-3 cells were transfected with TCTP-specific siRNA (siTCTP). Cyclin D1 and CDK4 expression was downregulated, while that of p21 and p27 was upregulated, by TCTP knockdown (Fig. 3B). Collectively, these results indicate that TCTP promotes tumor metastasis by stimulating cell proliferation and MMPs in ovarian tumorigenesis.

Knockdown of TCTP causes a decrease in PI3K/Akt/mTOR phosphorylation

PI3K/Akt phosphorylation is an essential step in the regulation of cellular physiological processes involved in tumor angiogenesis and growth. Akt, a key downstream molecule of PI3K, stimulates mTOR through a number of cellular functions, including phosphorylation and inactivation of apoptosis-related proteins (Downward, 1995; Khwaja, 1999; Guertin and Sabatini, 2005). Therefore, to explore the detailed regulatory mechanism underlying its effects, we examined the involvement of Akt, mTOR, and TSC-2, which are up- and downstream regulators of Akt. Cell lysates from VEGF-expressing cells (control) and transfected cells were subjected to immunoblotting analysis. As shown in Fig. 4A, VEGF-induced PI3K and Akt phosphorylation was activated by TCTP. In contrast, the inhibitory effect on VEGF-induced phosphorylation was completely abolished by transient transfection of siTCTP. These results indicated that siTCTP specifically inhibits VEGF-induced PI3K/Akt phosphorylation in ovarian carcinoma cells. We then assessed the effects of phosphorylation on up- and downstream signaling components of the PI3K/Akt cascade that control tumor endothelial cell suppression of angiogenesis. An example of these components is p70 ribosomal protein S6 kinase (p70S6K), a major regulator of protein synthesis that plays a pivotal role in cell growth, survival, and differentiation. As shown in Fig. 4B, TCTP enhanced VEGF-induced phosphorylation of PI3K/Akt signaling pathway molecules, including PDK-1, mTOR, TSC-2, and p70S6K. On the other hand, siTCTP overexpression dramatically inhibited the phosphorylation levels of these proteins. Collectively, these results indicate that siTCTP promotes apoptotic cell death, as well as suppresses tumor angiogenesis, through simultaneous inactivation of VEGF-induced phosphorylation of essential components of the VEGFR-2/Akt/mTOR signaling cascade.

Figure 4. Short interfering translationally controlled tumor protein (siTCTP) reduced the phosphorylation of essential players in vascular endothelial growth factor receptor 2 (VEGFR-2)/Akt-mediated signaling pathways. (A) siTCTP transfection was performed to inhibit phospho-PI3K and phospho-Akt in SKOV-3 ovarian cancer cells. Cells were incubated with 10 ng/ml VEGF and transfected with the control (empty-insert plasmid only), TCTP, or TCTP plus siTCTP. Phosphorylation of PI3K and Akt (Ser473 and Thr308) was then detected by immunoblotting. Unphosphorylated PI3K and Akt were used as loading controls. Three independent experiments were carried out in triplicate. (B) The phosphorylation effects of siTCTP on PDK-1 (Ser241), mTOR (Ser2448), TSC-2 (Ser1462), and p70S6K (Thr421) signaling regulator components. After transfection with TCTP or siTCTP, equal amounts of total proteins (20-25 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting with the indicated specific primary antibodies (p-PDK-1, p-mTOR, p-TSC-2, and p-p70S6K). Immunoblotting of unphosphorylated PDK-1, mTOR, TSC-2, and p70S6K was used to confirm equal sample loading. All experiments were carried out at least three times with similar results.

High TCTP expression is associated with poor prognosis in ovarian cancer patients, and its expression is increased in advanced ovarian cancer

As a first step to investigate the impact of TCTP (TPT1) expression on ovarian cancer patients, we used ovarian cancer (OV) dataset from Clinical Proteome Tumor Analysis Consortium (CPTAC) (Li et al., 2023). The analysis of expression in ovarian tumor versus normal tissues indicated that TPT1 expression levels were significantly higher in patients with primary OV than in normal tissues (consisting of 100 tumors and 20 normal samples) as depicted in Fig. 5A. Furthermore, we assessed the changes of TPT1 in the different stages classified by AJCC using TCGA dataset. The mRNA expression of TPT1 significantly increased from stage I to stage IV (Fig. 5B). The overall survival (OS) analysis showed that the OS hazard ratio is 1.6 (n=424, p=0.00043) (Fig. 5C, left) and that of disease free survival is 1.4 (n=424, p=0.0052) (Fig. 5C, right). These results suggested that TPT1 might be the prognostic marker for ovarian cancer.

Figure 5. Prognostic Value of TCTP (TPT1) in Ovarian Cancer Patients. (A) Protein expression levels of TPT1 in ovarian cancer samples (n=100) compared to normal tissue samples (n=20) from the CPTAC dataset. (B) Expression levels of TPT1 in ovarian cancer (OV) categorized by individual cancer stages. (C) Kaplan-Meier plot (KM plot) analysis of TPT1 expression in ovarian cancer patients, illustrating Overall Survival (OS) (left) and Disease-Free Survival (DFS) (right).

In females, ovarian cancer is the leading cause of death from a gynecologic tumor and is generally initiated by malignant transformation of epithelial cells. The progression of an ovarian tumor involves a cascade of various physiological events, including tumor cell EMT, angiogenesis, and metastasis. During tumor angiogenesis, high expression of pro-angiogenic factors in tumor cells overrides the influence of anti-angiogenic components (Al-Alem and Curry, 2015; Chen et al., 2016). Recent studies have reported that TCTP is a positive regulator of EMT. Ectopic expression of TCTP promoted cell motility and invasion via EMT in epithelial LLC-PK1 cells (Seo and Efferth, 2016). Jin et al. (2015) confirmed that RNA interference-mediated knockdown of TCTP remarkably suppressed proliferation and invasion and induced apoptotic cell death of glioma cells. These results indicate that TCTP may be important in glioma development and metastasis.

In ovarian tumors and HUVECs, the underlying physiological mechanisms associated with TCTP in promoting tumor angiogenesis are not fully understood. Here, using a tumor in vitro model system to shed light on this issue, we identified a new critical molecular mechanism of TCTP as a novel pro-angiogenic factor targeting the VEGFR-2/Akt/mTOR signaling pathways. Knockdown of TCTP markedly inhibited VEGF-induced cell migration of HUVECs and completely inhibited VEGF-stimulated capillary-like tubular structures of endothelial cells (Fig. 1). Collectively, these results strongly indicate that TCTP specifically controls VEGF-induced angiogenic process in vitro. In addition, the expression of cyclin D1 and CDK4 was down-regulated, while that of p21 and p27 was up-regulated, by TCTP knockdown (Fig. 3B). Collectively, our results indicate that TCTP promotes tumor metastasis by stimulating endothelial cell proliferation in ovarian tumorigenesis.

VEGF activates various steps in tumor angiogenesis, such as endothelial cell proliferation, invasion, and tubular structure formation, and is induced in many solid tumor types (Leung et al., 1989; Elson et al., 2000). The VEGF family consists of five members, VEGF-A–D and placental growth factor. VEGF-A is the main factor participating in tumor angiogenesis, and a high VEGF-A level is associated with tumor progression. VEGF-A exhibits angiogenic properties via the binding and activation of receptor tyrosine kinases (RTKs), including VEGFR-2. To date, three VEGF RTKs have been identified: VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Flt-4) (Hoeben et al., 2004; Olsson et al., 2006). VEGFR-2 is the primary modulator of the VEGF-induced signaling cascade, being the most important receptor in VEGF-induced angiogenesis (Takahashi et al., 2001; Kearney et al., 2002; Ferrara and Kerbel, 2005).

Phosphorylation of VEGFR-2 (Tyr1175) is important for both endothelial cell proliferation and migration. Phospho-Tyr1175 induces Src kinase, leading to the phosphorylation of FAK and cell migration. In addition, VEGFR-2 phosphorylation triggers downstream signaling pathways in endothelial cells, such as focal adhesion kinase, Src, phosphoinositide 3-kinase, protein kinase B, and extracellular signal-regulated kinases. VEGF and its associated receptors appear to be the main pro-angiogenic regulators of tumor neovascularization, including vascular development (Zhang et al., 2010; Simons, 2012; Chen et al., 2015b). Additionally, tumor metastasis is facilitated by remodeling of the extracellular matrix by a family of zinc-dependent proteolytic enzymes known as MMPs. MMPs play an important role in multiple biological/physiological processes requiring tissue remodeling, such as angiogenesis, wound healing, embryogenesis, and ovulation (Vihinen and Kahari, 2002; Stamenkovic, 2003; Page-McCaw et al., 2007; Schropfer et al., 2010). In ovarian tumors, MMP-2 and MMP-9 play pivotal roles in metastasis and invasion (Lamar et al., 2008; Hadler-Olsen et al., 2013). As indicated in Fig. 2, our study showed that TCTP overexpression significantly increased the expression levels of VEGF and HIF-1α, which are well known angiogenic inducers, during ovarian tumor progression through binding to VEGFR-2. In addition, the expression of MMP-2 and MMP-9 mRNA was increased in TCTP-overexpressing cells (Fig. 3A). The PI3K/Akt signaling pathway is frequently promoted in various human cancer types, and it plays a key role in cellular physiological conditions such as cell migration, tumor cell proliferation, metastasis, and resistance to apoptotic induction in tumorigenesis. As a result, the inhibition and targeting of PI3K or Akt phosphorylation are critical aspects of cancer therapy (West et al., 2002; Fresno Vara et al., 2004; Hennessy et al., 2005). In addition, knockdown of TCTP markedly inhibited VEGF-induced phosphorylation of PI3K/Akt signaling cascade components, including PDK-1, mTOR, TSC-2, and p70S6K (Fig. 4).

Therefore, siTCTP might target the VEGFR-2/Akt/mTOR signaling pathway in ovarian tumorigenesis, thereby suppressing tumor angiogenesis and metastasis (Fig. 6). In conclusion, our findings revealed a critical role of TCTP in ovarian tumor angiogenesis. TCTP enhanced ovarian tumor metastasis and tumor angiogenesis. Importantly, siTCTP inhibition of angiogenesis may result from the direct binding of TCTP to VEGFR-2 and downregulation of VEGFR2-mediated Akt and mTOR phosphorylation. Taken together, our findings should support the future development of TCTP-based therapies targeting the ovarian tumor signaling cascade.

Figure 6. Proposed scheme about mechanism of TCTP-Induced angiogenesis via VEGFR2 signaling in ovarian tumors.

This work was supported by a grant from the National Cancer Center, Korea (NCC-2210450-2 and 2310590-1) and the Basic Science Research Program through the NRF (NRF-2020R1A2C3004973, NRF-2018R1A5A2023127).

The authors declare no conflict of interest.