TCTP promotes glioma cell proliferation in vitro and in vivo via enhanced β-catenin/TCF-4 transcription

Abstract

Background The translationally controlled tumor protein (TCTP) is a multifunctional protein that plays important roles in immune responses, cell proliferation, tumorigenicity and cell apoptosis. Here, we examined the clinical value of TCTP in glioma patient survival and investigated the functional roles and mechanism of TCTP in glioma development. Methods TCTP expression was determined through immunohistochemical staining, immunoblotting, and quantitative real-time PCR (qRT-PCR). TCTP or TCF-4 expression was silenced using short hairpin (sh) RNA. In vitro cell proliferation was detected using MTT, BrdU and colony formation assays, and in vivo tumor growth was performed using the xenograft model. TCTP/TCF-4/β-catenin association was detected using a co-immunoprecipitation (co-IP) assay. TCF-4 transcription activity was detected using a TOPflash/FOPflash report gene assay. Wnt/β-catenin-targeted gene expression was detected through Western blotting. Results TCTP protein levels were significantly elevated in high-grade gliomas compared with low-grade gliomas and normal brain tissues. Importantly, the expression of TCTP was significantly associated with poorer overall survival and disease-free survival, and TCTP also reduced the survival rate after treatment with radiotherapy and temozolomide (RT-TMZ) for glioma patients. The ectopic expression of TCTP enhanced glioma cell proliferation both in vitro and in vivo, whereas the knockdown of TCTP inhibited this effect. Similarly, the overexpression of TCTP increased β-catenin binding to TCF-4, TOPflash report gene transcription activity, and the expression of Wnt/β-catenin signaling target genes including c-Myc and cyclin D1; notably, the knockdown of TCTP reduced these effects. The knockdown of TCF-4 using shRNA rescued the enhanced cell proliferation induced by the overexpression of TCTP. Conclusion TCTP is associated with reduced survival of glioma patients and induces glioma tumor growth through enhanced Wnt/β-catenin signaling.

Keywords: TCF-4; TCTP; glioma; β-catenin.

Fig. 1.

Increased expression of TCTP in glioma. (A) The levels of TCTP in normal tissues compared with stage I-IV glioma tissues detected through immunostaining with the TCTP antibody. The normal brain tissues were obtained from automobile accident patients without glioma. Bar = 50 μm. (B) A positive percentage of TCTP expression in normal brain tissues and stage I-IV glioma. (C) The expression of TCTP was verified through qRT-PCR in normal tissues and glioma samples. Each box represents the 5th and 95th percentiles, the middle line represents the mean value, and the whiskers represent the standard deviation. (D) TCTP expression in human glioma cell lines detected through immunoblotting using an antibody to TCTP. Actin was used as a loading control. The results represent at least 3 separate experiments. (E) TCTP mRNA levels in normal brain tissues and glioma cell lines detected through qRT-PCR analysis. The results represent at least 3 separate experiments. Error bar: ± S.D. * P < .05, ** P < .01.

Fig. 2.

TCTP expression is correlated with low survival rate. (A and B) Kaplan–Meier analyses were performed according to TCTP protein expression scores in glioma participants. Scores of 0-2 and ≥3 were considered negative and positive staining, respectively. The overall survival of participants with positive TCTP expression was evidently shorter (A). The disease-free survival of participants with positive TCTP expression was evidently shorter (B). (C) and D) The 1-year survival rate after RT-TMZ treatment in glioma participants based on the immunoreactivity Scores in glioma participants. Scores of 0-2 and ≥3 were considered negative and positive staining, respectively. Survival rate of total TCTP-positive participants and TCTP-negative participants were compared in C. (D) To compare fairly, 46 TCTP-positive participants, including 18 WHO stage I glioma participants, 13 WHO stage II glioma participants, 9 WHO stage III glioma participants and 6 WHO stage IV glioma participants, as with the TCTP-negative participants, were selected randomly. Kaplan–Meier analyses were used here.

Fig. 3.

TCTP promotes glioma cell proliferation in vitro and in vivo. (A) U-251 and H-4 cells transfected with plain vector (V) or plasmid encoding TCTP (TCTP). The cell lysates were immunoblotted with the TCTP antibody, and actin was used as a loading control. (B) In vitro growth of U-251/V (V) and U-251/TCTP (TCTP) cells measured using MTT assays. (C) In vitro growth of H-4/V (V) and H-4/TCTP (TCTP) cells measured using MTT assays. (D) In vitro growth of U-251/V (V) and U-251/TCTP (TCTP) cells measured using BrdU assays. (E) In vitro growth of H-4/V (V) and H-4/TCTP (TCTP) cells measured using BrdU assays. (F) The colony formation assay was performed using U-251/V (V) and U-251/TCTP (TCTP) cells treated with or without irradiation. (G) The colony formation assay was performed using H-4/V (V) and H-4/TCTP (TCTP) cells treated with or without irradiation. (H) Average tumor volume (left) and tumor TCTP/Ki67 staining (5 weeks, right) in athymic nude mice subcutaneously inoculated with U-251/V or U-251/TCTP cells. (I) Average tumor volume (left) and tumor TCTP/Ki67 staining (5 weeks, right) in athymic nude mice subcutaneously inoculated with H-4/V (V) or H-4/TCTP (TCTP) cells. For H and I, n = 10 mice/group. Error bar: ± S.D. * P < .05.

Fig. 4.

TCTP knockdown inhibits glioma cell proliferation in vitro and in vivo. (A) U-138 and HS-683 cells transfected with control shRNA (Ctrl shRNA) or TCTP shRNA1. The TCTP levels were detected through immunostaining with the TCTP antibody, and actin was used as a loading control. (B) In vitro growth of U-138/Ctrl shRNA and U-138/TCTP shRNA1 cells measured using MTT assays. (C) In vitro growth of HS-683/Ctrl shRNA and HS-683/TCTP shRNA1 cells measured using MTT assays. (D) In vitro growth of U-138/Ctrl shRNA and U-138/TCTP shRNA1 cells measured using BrdU assays. (E) In vitro growth of HS-683/Ctrl shRNA and HS-683/TCTP shRNA1 cells measured using BrdU assays. (F) The colony formation assay was performed using U-138/Ctrl shRNA and U-138/TCTP shRNA1 cells treated with or without irradiation. (G) The colony formation assay was performed using HS-683/Ctrl shRNA and HS-683/TCTP shRNA1 cells treated with or without irradiation. (H) Average tumor volume (left) and tumor TCTP/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with U-138/Ctrl shRNA or U-138/TCTP shRNA1 cells. (I) Average tumor volume (left) and tumor TCTP/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with HS-683/Ctrl shRNA or HS-683/TCTP shRNA1 cells. (J) U-138 and HS-683 cells transfected with control shRNA (Ctrl shRNA) or TCTP shRNA2. TCTP levels were detected through immunostaining with the TCTP antibody, and actin was used as a loading control. (K) In vitro growth of U-138/Ctrl shRNA and U-138/TCTP shRNA2 cells measured using MTT assays. (L) In vitro growth of HS-683/Ctrl shRNA and HS-683/TCTP shRNA2 cells measured using MTT assays. (M) In vitro growth of U-138/Ctrl shRNA and U-138/TCTP shRNA2 cells measured using BrdU assays. (N) In vitro growth of HS-683/Ctrl shRNA and HS-683/TCTP shRNA2 cells measured using BrdU assays. (O) The colony formation assay was performed using U-138/Ctrl shRNA and U-138/TCTP shRNA2 cells treated with or without irradiation. (P) The colony formation assay was performed using HS-683/Ctrl shRNA and HS-683/TCTP shRNA2 cells treated with or without irradiation. (Q) Average tumor volume (left) and tumor TCTP/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with U-138/Ctrl shRNA or U-138/TCTP shRNA2 cells. (R) Average tumor volume (left) and tumor TCTP/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with HS-683/Ctrl shRNA or HS-683/TCTP shRNA2 cells. For (A-G and K-P); the results represent at least 3 separate experiments. For H, I, Q, and R, n = 10 mice/group. Error bar: ± S.D. *P < .05.

Fig. 5.

TCTP binds to TCF-4 and enhances β-catenin/TCF-4 transcription activity. (A) Identification of TCF-4 as a binding partner to TCTP. Full-length pAS2-1-TCTP was used as the bait for screening the Human Fetal Brain cDNA Library (BD Clontech). The cDNA inserts were sequenced and identified as N-terminal fragments of TCF-4 encoding amino acid residues 25-288. The direct interaction was confirmed through the cotransformation of GAL4AD-TCF-4 and pAS2-1-TCTP constructs into yeast strain Y190 cultured in the SD/-Leu-Trp-His triple-deficient medium for positive β-galactosidase activity. (B) TCTP binding to TCF-4 in U-138 cells. TCTP was immunoprecipitated from the cell lysates followed by immunoblotting with antibodies to TCF-4 and TCTP. Anti-rIgG was used as the immunoprecipitation negative control antibody. Sample loading controls are shown (5% input). (C) TCF-4 bound more β-catenin and TCTP when TCTP was overexpressed in U-251 cells, as determined using the co-immunoprecipitation (co-IP) assay. (D) TCF-4 bound more β-catenin and TCTP when TCTP was overexpressed in normal rat astrocyte DI TNC1 cells, as determined using the co-IP assay. (E) TCF-4 bound less β-catenin and TCTP when TCTP was knocked down using shRNA1 in U-138 cells, as determined using the co-IP assay. (F) TCTP increased TCF-4 targeted promoter activities (TOPflash) in U-251 cells. FOPflash was used as a negative report gene control. (G) TCTP increased TCF-4 targeted promoter activities (TOPflash) in normal rat astrocyte DI TNC1 cells. FOPflash was used as a negative report gene control. (H) Knockdown of TCTP decreased TCF-4-targeted promoter activities (TOPflash). FOPflash was used as a negative control. (I) The protein levels of the Wnt/β-catenin target genes c-Myc and cyclin D1 were upregulated after the overexpression of TCTP in U-251 cells and normal rat astrocyte DI TNC1 cells, and downregulated after the knockdown of TCTP using shRNA1 in U-138 cells. Error bar: ± S.D. **, P < .01. The results represent at least 3 separate experiments.

Fig. 6.

Knockdown of TCF-4 inhibits TCTP-induced glioma cell proliferation in vitro and in vivo. (A) TCF-4 was knocked down in U-251/TCTP and H-4/TCTP cells. U-251/TCTP cells and H-4/TCTP cells were transfected with control shRNA (Ctrl shRNA) or TCF-4 shRNA1. TCTP levels were detected through immunostaining with the TCTP antibody, and actin was used as a loading control. (B) In vitro growth of U-251/TCTP cells treated with nonsense control shRNA (Ctrl shRNA), or TCF-4 shRNA1 was measured using MTT assays. (C) In vitro growth of H-4/TCTP cells treated with nonsense control shRNA (Ctrl shRNA), or TCF-4 shRNA1 was measured using MTT assays. (D) In vitro growth of U-251/V cells treated with nonsense control shRNA (Ctrl shRNA) or TCF-4 shRNA1 was measured using MTT assays. (E) In vitro growth of H-4/V cells treated with nonsense control shRNA (Ctrl shRNA), or TCF-4 shRNA1 was measured using MTT assays. (F) Average tumor volume (left) and tumor TCF-4/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with U-251/TCTP treated with nonsense control shRNA (Ctrl shRNA) or TCF-4 shRNA1. (G) Average tumor volume (left) and tumor TCF-4/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with H-4/TCTP treated with nonsense control shRNA (Ctrl shRNA) or TCF-4 shRNA1. (H) Another TCF-4 shRNA called TCF-4 shRNA2 was used to knock down TCF-4. U-251/TCTP, and H-4/TCTP cells were transfected with control shRNA (Ctrl shRNA) or TCF-4 shRNA2. TCTP levels were detected through immunostaining with TCTP antibody, and actin was used as a loading control. (I) In vitro growth of U-251/TCTP cells treated with nonsense control shRNA (Ctrl shRNA), or TCF-4 shRNA2 was measured using MTT assays. (J) In vitro growth of H-4/TCTP cells treated with nonsense control shRNA (Ctrl shRNA), or TCF-4 shRNA2 was measured using MTT assays. (K) In vitro growth of U-251/V cells treated with nonsense control shRNA (Ctrl shRNA) or TCF-4 shRNA2 was measured using MTT assays. (L) In vitro growth of H-4/V cells treated with nonsense control shRNA (Ctrl shRNA), or TCF-4 shRNA2 was measured using MTT assays. (M) Average tumor volume (left) and tumor TCF-4/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with U-251/TCTP treated with nonsense control shRNA (Ctrl shRNA) or TCF-4 shRNA2. (N) Average tumor volume (left) and tumor TCF-4/Ki67 staining (5 weeks after implantation, right) in athymic nude mice subcutaneously inoculated with H-4/TCTP treated with nonsense control shRNA (Ctrl shRNA) or TCF-4 shRNA2. For A–E and H­L), the results represent at least 3 separate experiments. For F, G, M, and N, n = 10 mice/group. Error bar: ±S.D.