TRAF4 Inhibits Bladder Cancer Progression by Promoting BMP/SMAD Signaling

Abstract

Patients with bladder cancer often have a poor prognosis due to the highly invasive and metastatic characteristics of bladder cancer cells. Epithelial-to-mesenchymal transition (EMT) has been causally linked to bladder cancer invasion. The E3 ubiquitin ligase, tumor necrosis factor receptor-associated factor 4 (TRAF4) has been implicated as a tumor promoter in a wide range of cancers. In contrast, here we show that low TRAF4 expression is associated with poor overall survival in patients with bladder cancer. We show that the TRAF4 gene is epigenetically silenced and that ERK mediates TRAF4 phosphorylation, resulting in lower TRAF4 protein levels in bladder cancer cells. In addition, we demonstrate that TRAF4 is inversely correlated with an EMT gene signature/protein marker expression. Functionally, by manipulating TRAF4 expression, we show that TRAF4 regulates EMT genes and epithelial and invasive properties in bladder cancer cells. Transcriptomic analysis of dysregulated TRAF4 expression in bladder cancer cell lines revealed that high TRAF4 expression enhances the bone morphogenetic protein (BMP)/SMAD and inhibits the NF-κB signaling pathway. Mechanistically, we show that TRAF4 targets the E3 ubiquitin ligase SMURF1, a negative regulator of BMP/SMAD signaling, for proteasomal degradation in bladder cancer cells. This was corroborated in patient samples where TRAF4 positively correlates with phospho-SMAD1/5, and negatively correlates with phospho-NFκb-p65. Lastly, we show that genetic and pharmacologic inhibition of SMURF1 inhibits the migration of aggressive mesenchymal bladder cancer cells.

Implications: Our findings identify E3 ubiquitin ligase TRAF4 as a potential therapeutic target or biomarker for bladder cancer progression.

Figure 1.

TRAF4 is downregulated in aggressive bladder tumors and mesenchymal bladder cancer cell lines. A, Kaplan–Meier plot showing the overall survival of patients with bladder cancer stratified by TRAF4 expression. Data were obtained and reproduced from TCGA (ref. ; obtained from Human Protein Atlas; ref. 25), and the median Fragments Per Kilobase of transcript per Million mapped reads (FKPM) value was taken as the TRAF4 expression cutoff, n = 203. B, Graph showing TRAF4 expression through scores obtained from IHC analysis of a tissue microarray; *, P ≤ 0.05 and **, P ≤ 0.01 calculated using one-way ANOVA; n.s. indicates a nonsignificant P value. C, Representative IHC images of TRAF4 expression (green) in the tissue microarray from stage 1–3 bladder tumors are shown. Scale bar, 400 μm. D, Violin plot showing the TRAF4 expression level (and distribution) in different subtypes of bladder cancer; Her2L: Her2-like (n = 253), Pap: papillary (n = 674), Lum: luminal (n = 107), Neu: neural (n = 448), SCC (n = 333) and Mes: mesenchymal (n = 308). The black bars in the middle of the distribution indicate the medians. The subtypes are arranged according to their EMT scores (26, 27). E, Plot showing the EMT scores in 59 bladder cancer cell lines; the light grey bars indicate cell lines with a negative EMT score, the dark grey bars indicate cell lines with a positive EMT score, and the red arrowheads indicate the cell lines that were used for further investigation (28). F, Regression plot of TRAF4 expression levels vs. EMT scores in 59 bladder cancer cell lines. G, Immunoblot analysis showing the expression of TRAF4 and other EMT marker proteins. GAPDH, loading control. Representative results of 2 independent biological replicates. H, Real-time PCR data showing TRAF4 mRNA expression in cell lines. The error bars indicate ± SD. Epithelial cell lines (dark grey bars) had significantly higher TRAF4 expression than mesenchymal cell lines (light grey bars). Performed in 3 technical repeats.

Figure 2.

TRAF4 is repressed in mesenchymal (bladder cancer) cell lines at the epigenetic and proteomic levels. A, Real-time PCR results showing changes in the TRAF4 mRNA level in mesenchymal cell lines after treatment with 5-AZA, performed in 3 technical repeats. B, Real-time PCR results showing changes in the CDH1 mRNA level in cell lines after treatment with 5-AZA; the error bars indicate ± SD, performed in 3 technical repeats. C, Immunoblot results showing the endogenous TRAF4 levels in the indicated cell lines after treatment with cycloheximide (CHX). GAPDH, loading control. The numbers indicate the relative quantitative TRAF4 levels with respect to the loading control GAPDH, n = 1 D, Schematic representation of TRAF4 showing the distinct domain structures and the candidate phosphorylated serine and threonine residues that were identified using mass spectrometric analysis. E, Immunoblot results from 293T cells transfected with expression constructs for either TRAF4 or the TRAF4 glutamic acid (E) mutant. GAPDH, loading control. The numbers indicate the relative quantitative TRAF4 levels with respect to the loading control GAPDH. F, Immunoblot results from 293T cells transfected with expression constructs for either TRAF4 or the TRAF4 alanine (A) mutant. GAPDH, loading control. The numbers indicate the relative quantitative TRAF4 levels with respect to the loading control GAPDH. G, Western blot analysis of ectopic Myc-TRAF4 WT and the S334E and S334A mutants in the UMUC3 cell line. GAPDH, loading control. H, Immunoprecipitation of Myc-TRAF4 with or without Flag-ERK1 overexpression, GAPDH, loading control. I, Western blot analysis of TRAF4 expression in T24 cells treated with CHX at the indicated times, in the presence of DMSO (control) or MEKi. GAPDH, loading control. The numbers indicate the relative quantitative TRAF4 levels for both DMSO and MEKi (PD0325901) treatment separately, with respect to the loading control GAPDH.

Figure 3.

Knockdown of TRAF4 in epithelial (bladder cancer) cell lines leads to loss of epithelial integrity and changes in EMT marker expression. A, Real-time PCR results from RT4 cells showing the mRNA expression levels of the indicated genes; the error bars indicate ± SD, performed in 3 technical repeats. B, Immunoblot results showing changes in EMT marker protein expression in RT4 cells upon TRAF4 knockdown. GAPDH, loading control. Representative result of 2 biological replicates. C, RT4 cell colonies visualized by brightfield imaging (top) or after staining with CellMask Orange Plasma membrane stain (bottom); scale bar: 25 μm. Representative of n = 5. D, Images showing RT4 spheroids formed from control (empty pLKO vector) and TRAF4 knockdown (sh5) cells; scale bar: 200 μm. The graph shows circularities calculated from five independent spheroids of different sizes (n = 5). The error bars indicate ± SD; **, P ≤ 0.01 calculated using two-tailed Student t test. E, Real-time PCR results from HT1376 cells showing the mRNA expression levels of the indicated genes; the error bars indicate ± SD performed in 3 technical repeats. F, Immunoblot results showing EMT marker protein expression levels in HT1376 cells with or without TRAF4 knockdown. GAPDH, loading control. Representative results of 2 biological repeats. G, Representative images of Transwell assays performed on HT1376 cells are shown. Cells were stained with crystal violet; scale bar: 200 μm. H, Quantification of the number of migrated cells in four random fields; the error bars indicate ± SD; ***, P ≤ 0.001 calculated using two-tailed Student t test, 4 technical repeats.

Figure 4.

Ectopic expression of TRAF4 in mesenchymal cells inhibits their migration and invasion. A, Immunoblot showing T24 cells stably expressing either control vector (Myc-tag), TRAF4 or the catalytically inactive TRAF4 mutant (C/A: cysteine substituted with alanine at residue C18). GAPDH, loading control. B, Representative images of Transwell assays performed on T24 cells stably expressing TRAF4 or the catalytically inactive TRAF4 mutant (C/A) are shown. Cells were stained with crystal violet; scale bar: 100 μm. C, Quantification of the number of migrated cells in four random fields. The error bars indicate ± SD; **, P ≤ 0.01 and ****, P ≤ 0.0001 calculated using one-way ANOVA, 4 technical repeats. D, Graph showing the relative wound widths as determined with an IncuCyte system. Representative results from ten independent experiments are shown; the error bars indicate ± SEM; ***, P ≤ 0.001 calculated using two-tailed Student t test (n = 10). E, Representative images related to the graph shown in D; the brown area represents the cell coverage, and the gray area indicates the initial wound produced and the remaining wound after 12 hours. F, MTS cell viability/proliferation assay performed with either control T24 cells or T24 cells stably expressing TRAF4. The absorbance was measured at the indicated time points; the error bars indicate ± SD from three sample replicates; ***, P ≤ 0.001 calculated using two-tailed Student t test (n = 3). G, Immunoblot results for MBT-2 cells stably expressing either control vector (empty vector with a Myc-tag) or Myc-TRAF4. GAPDH, loading control. H, Representative images of Transwell assays performed on control and TRAF4-overexpressing MBT-2 cells stained with crystal violet; scale bar: 200 μm. I, Quantification of the number of migrated cells in four random fields. The error bars indicate ± SD; ***, P ≤ 0.001 calculated using two-tailed Student t test, 4 technical repeats.

Figure 5.

TRAF4 targets SMURF1 for ubiquitination and degradation. A, Immunoprecipitation of SMURF1 followed by Western blot analysis of TRAF4 in HT1376 cells. B, Immunoblot results in control (empty pLKO vector) and TRAF4 knockdown (sh4 and sh5) RT4 cells probed with the indicated antibodies. GAPDH, loading control. The numbers indicate the relative quantitative SMURF1 levels with respect to the loading control GAPDH. C, Immunoblot results in control (empty pLKO vector) and TRAF4 knockdown (sh4) HT1376 cells probed with the indicated antibodies. The numbers indicate the relative quantitative SMURF1 levels with respect to the loading control GAPDH. D, Real-time PCR results showing SMURF1 mRNA expression levels in RT4 and HT1376 (control and TRAF4 knockdown) cells; the error bars indicate ± SD, performed in 3 technical repeats. E, A ubiquitination assay was performed with anti-Flag antibodies in 293T cells overexpressing the indicated plasmids. Cells were treated with MG132 (2 μmol/L) overnight prior to lysis. Representative results from three independent experiments are shown. F, Immunoblot results in 293T cells transfected with the indicated plasmids. GAPDH, loading control. G, Representative images of Transwell assays performed on control and Smurf1 knockdown MBT-2 cells stained with crystal violet; scale bar: 200 μm. H, Quantification of the number of migrated cells in four random fields. The error bars indicate ± SD; ***, P ≤ 0.001 and ****, P ≤ 0.0001 calculated using one-way ANOVA, 4 technical repeats. I, Graph showing relative wound widths as determined with an IncuCyte system. Images were acquired every hour after wounding. T24 cells were treated with either DMSO or the SMURF1 inhibitor A01 (10 μmol/L); the error bars indicate ± SEM; ***, P ≤ 0.001 calculated using two-tailed Student t test. Representative results from ten independent experiments are shown (n = 10). J, Representative images related to the graph in I. The brown area represents the cell coverage, and the gray area indicates the wound initially produced and remaining after 12 hours. K, An MTS assay was performed on T24 cells treated with either control (DMSO) or the SMURF1i A01 at 5 μmol/L. The absorbance was measured at the indicated time points; the error bars indicate ± SD from three sample replicates; *, P ≤ 0.05 calculated using two-tailed Student t test (n = 3).

Figure 6.

Dysregulated expression of TRAF4 in bladder cancer cell lines affects BMP/SMAD- and NF-κb-responsive genes. A, Graph showing differences in enrichment scores when TRAF4 was overexpressed in T24 cells. Gene signatures of eleven major cancer-associated signaling pathways were considered for analysis. B, Venn diagram showing the numbers of genes that were upregulated in HT1376 cells transfected with two independent shRNAs targeting TRAF4 (pink) and downregulated in T24 cells with stable overexpression of TRAF4 (green) compared with the corresponding control cells. The 252 genes in the middle are the reciprocally affected common genes. The results were obtained from four independent replicates for each sample (n = 4). C, Venn diagram showing the numbers of genes that were downregulated in HT1376 cells transfected with two independent shRNAs targeting TRAF4 (green) and upregulated in T24 cells with stable overexpression of TRAF4 (pink) compared with the corresponding control cells. The 96 genes in the middle represent the reciprocally affected common genes. The results were obtained from four independent replicates for each sample (n = 4). D, Heatmap showing the common dysregulated genes in the BMP,NF-κB and EMT gene signatures in both cell lines. E, Real-time PCR results showing the mRNA expression levels of the indicated genes in control (empty vector with a Myc tag) vs. TRAF4-overexpressing T24 cells upon stimulation with BMP6 (50 ng/mL) for 1 hour. The error bars indicate ± SD, performed in 3 technical repeats. F, Real-time PCR results showing the mRNA expression levels of ID1, ID2, and ID3 in the indicated cell lines, performed in 3 technical repeats. G, A luciferase reporter assay was conducted in 293T cells transfected with the BRE-luciferase reporter, SV40 Renilla and either empty vector control or TRAF4. Transfected cells were stimulated overnight with BMP6 (50 ng/mL) and/or TNFα (10 ng/mL) where indicated. The error bars indicate ± SD; **, P ≤ 0.01 and ****, P ≤ 0.0001 calculated using two-way ANOVA; n.s. indicates a nonsignificant P value. Representative results from three independent experiments are shown (n = 3). H, Real-time PCR results showing the mRNA expression levels of the indicated genes in control (empty vector with a Myc tag) vs. TRAF4-overexpressing T24 cells upon stimulation with BMP6 (50 ng/mL) and/or TNFα (10 ng/mL) as indicated for 1 hour. The error bars indicate ± SD, performed in 3 technical repeats.

Figure 7.

TRAF4 expression correlates positively with pSMAD1/5/8 levels and negatively with the p-p65 level in bladder tumors. A, Regression analysis showing the correlations between the TRAF4 expression level (score) and phospho (p)SMAD1/5/8 scores in patients with bladder cancer. Pearson χ² test was used to determine the correlations between the TRAF4 and pSMAD1/5/8 scores. B, Regression analysis showing the correlations between the TRAF4 expression level (score) and phospho (p)-p65 score in bladder cancer patients. Pearson χ² test was used to determine the correlations between the TRAF4 and p-p65 scores. C, Representative images of continuous sections of tissue microarray samples probed with the indicated antibodies using fluorescent IHC. The magnified insets for pSMAD1/5/8 show nuclear staining. Scale bar: 400 μm. D, Schematic representation of TRAF4 signaling dynamics in epithelial-like and mesenchymal-like bladder cancer cells. Ub denotes ubiquitin and P stands for phosphorylation of serine 334.