Inhibition of TPL2 by interferon-α suppresses bladder cancer through activation of PDE4D

Abstract

Background: Drugs that inhibit the MEK/ERK pathway have therapeutic benefit in bladder cancer treatment but responses vary with patients, for reasons that are still not very clear. Interferon-α (IFN-α) is also used as a therapeutic agent for bladder cancer treatment but the response rate is low. It was found that IFN-α could enhance the cytotoxic effect of MEK inhibition. However, the potential mechanisms of that are still unclear. Understanding of the cross-talk between the IFN-α and MEK/ERK pathway will help enhance the efficacy of IFN-α or MEK inhibitors on bladder cancer.

Methods: Immunoprecipitation and pull-down assay were used to reveal the formation of signaling complex. The protein expressions were detected by western blot and immunohistochemistry. The cAMP level, Phosphodiesterase 4D (PDE4D) activity and Prostaglandin E₂ (PGE₂) concentration in cells, serum and tissues were detected by enzyme-linked immunosorbent assay. The role of PDE4D in bladder tumorigenesis in vivo was examined by the xenograft model. Tissue microarray chips were used to investigate the prognostic roles of PDE4D and tumor progression locus 2 (TPL2) in bladder cancer patients.

Results: IFN-α down-regulated the cyclooxygenase-2 (COX-2) expression in bladder cancer cells through the inhibition of TPL2/NF-κB pathway; IFN-α also inhibited COX-2 expression by suppressing cAMP signaling through TPL2-ERK mediated PDE4D activity. Reduction of the intracellular cAMP level by PDE4D potentiated the antitumor effect of IFN-α against bladder cancer in vitro and in vivo. Further analysis of clinical samples indicated that low PDE4D expression and high level of TPL2 phosphorylation were correlated to the development and poor prognosis in bladder cancer patients.

Conclusions: Our data reveal that IFN-α can exert its antitumor effect through a non-canonical JAK-STAT pathway in the bladder cancer cells with low activity of IFN pathway, and the TPL2 inhibition is another function of IFN-α in the context of bladder cancer therapy. The antitumor effects of IFN-α and MEK inhibition also depend on the PDE4D-mediated cAMP level in bladder cancer cells. Suppression of the TPL2 phosphorylation and intracellular cAMP level may be possible therapeutic strategies for enhancing the effectiveness of IFN-α and MEK inhibitors in bladder cancer treatment.

Keywords: COX-2; Interferon; PDE4D; TPL2; cAMP.

Fig. 1

Suppression of COX-2 expression by IFN-α via inhibition of the TPL2 and cAMP/CREB. (a) T24 cells were treated with IFN-α (1 × 10⁴ U/mL) for specific time points; or treated using various concentrations of IFN-α for 24 h. The cell lysates were immunoblotted with COX-2 antibody. β-Tubulin staining is shown as a loading control. (b) T24 cells were treated with IFN-α (1 × 10⁴ U/mL) for specific time points. The TPL2, p-TPL2, IKKα/β, p- IKKα/β, IκBα and p-IκBα were analyzed by performing western blotting. (c) T24 cells were treated with IFN-α (1 × 10⁴ U/ml), TPL2i (2 μM), and PD98059 (40 μM) for 12 h. The COX-2, TPL2, p-TPL2, ERK, p- ERK, IKKα/β, p-IKKα/β, IκBα and p-IκBα were analyzed by performing western blotting. β-Tubulin staining is shown as a loading control. (d) The intracellular cAMP level was detected after T24 cells were treated with IFN-α (1 × 10⁴ U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. (e) T24 cells were treated with IFN-α (1 × 10⁴ U/mL), TPL2i (2 μM), PD98059 (40 μM), or forskolin (50 μM) for 24 h. The expression levels of COX-2, CREB, and p-CREB were analyzed by western blotting. The β-tubulin was used as the loading control. (f) T24 cells were treated with IFN-α (1 × 10⁴ U/mL), PD98059 (40 μM), and EGF (25 ng/mL) for 12 h. The COX-2 expression was analyzed by performing western blotting. (g) Cell viability was detected after T24 cells were treated using forskolin (50 μM), TPL2i (2 μM), and PD98059 (40 μM) for 72 h. Data represent the results of three independent experiments. Error bars indicate mean ± SD. *, P < 0.05; **, P < 0.01; #, P < 0.05 (t-test)

Fig. 2

Regulation of PDE4D activity at IFNAR2 by TPL2. (a) T24 cells were treated with IFN-α (1 × 10⁴ U/mL) for specific time points. The levels of phosphorylated and total TPL2 bound to IFNAR2 or IFNAR1 were detected by performing western blotting after co-immunoprecipitation using IFNAR2 or IFNAR1 antibodies. (b) T24 cells were treated with IFN-α (1 × 10⁴ U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. The levels of phosphorylated and total TPL2 bound to IFNAR2 or IFNAR1 were detected by performing western blotting after co-immunoprecipitation using IFNAR2 or IFNAR1 antibodies. (c) T24 cells were treated with IFN-α (1 × 10⁴ U/mL) for specific time points. The levels of RACK1 and PDE4D that interacted with IFNAR2 or IFNAR1 were detected after co-immunoprecipitation using IFNAR2 or IFNAR1 antibodies. (d) T24 cells were treated with IFN-α (1 × 10⁴ U/mL) for specific time points. The intracellular cAMP level, activity of total PDE4D, and activity of PDE4D that interacted with IFNAR2 were detected after co-immunoprecipitation using IFNAR2 or PDE4D antibodies. (e) T24 cells were treated with IFN-α (1 × 10⁴ U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. The activity of total PDE4D was detected after immunoprecipitation using PDE4D antibody. (f) T24 cells were treated with IFN-α (1 × 10⁴ U/mL), TPL2i (2 μM), and PD98059 (40 μM) for 4 h. The PDE4D that interacted with IFNAR2 and their activity were detected after co-immunoprecipitation using IFNAR2 antibody. Data represent the results of five independent experiments. Error bars indicate mean ± SD. *, P < 0.05; **, P < 0.01 (t-test)

Fig. 3

Induction of PDE4D by roflumilast potentiates anti-proliferation effect of IFN-α in vitro. (a) T24 cells were treated using the specific concentrations of roflumilast for 24 h. (b) T24 cells were treated with roflumilast (1 μM) for specific time points. (c) T24 cells were treated with roflumilast (1 μM) for specific time points. Intracellular cAMP levels and activity of immunoprecipitated PDE4D were detected. (d) T24 cells were treated with IFN-α (1 × 10⁴ U/mL) and roflumilast (1 μM) either individually or in combination for 24 h. The level of PDE4D that interacted with IFNAR2 or IFNAR1 was detected by performing western blotting after co-immunoprecipitation using IFNAR2 or IFNAR1 antibodies. The expression levels of total PDE4D and β-tubulin in the cell lysates were used as loading control. (e) T24 cells were treated with IFN-α (1 × 10⁴ U/mL) and roflumilast (1 μM) either individually or in combination for 24 h. The intracellular cAMP level and activity of total PDE4D were detected after immunoprecipitation using PDE4D antibody. (f, g) The cell viability (f) and PGE₂ production (g) were detected after T24 cells were treated with IFN-α (1 × 10⁴ U/mL) and roflumilast (1 μM) either individually or in combination for 72 h. Data represent the results of three independent experiments. Error bars indicate mean ± SD. *, P < 0.05; **, P < 0.01; #, P < 0.05 (t-test)

Fig. 4

Roflumilast potentiated the anti-tumor effect of IFN-α in vivo. 5637 cells (5 × 10⁶ cells/mouse) were subcutaneously injected into BALB/c nude mice. When the tumor size was ~ 100 mm³, mice were treated with phosphate buffered saline (control), roflumilast (5 mg/kg/day, oral administration), and IFN-α (1 × 10⁴ U/mouse/2 days, intraperitoneal injection) either individually or in combination for 24 days before sacrifice. The tumor volumes were measured every 4 days. (a) The tumor growth curves of all the treatment groups. Each data point indicates the mean of tumor volume (n = 6 per group). (b) Image of the tumors in all the treatment groups. (c) The tumor weights in all the treatment groups (n = 6 per group). (d, e) The lysis of tumor tissues in all treatment groups were used to detected the cAMP levels (d) and expressions of PDE4D (e). (f, g) IHC and difference analyses of PDE4D (f) and pTPL2 (g) expressions (Histochemistry-Score) among the T24 tumor tissues of the indicated groups. Error bars indicate mean ± SD (n = 7). *, P < 0.05; **, P < 0.01; #, P < 0.05 (t-test and Mann-Whitney test)

Fig. 5

Correlations of PDE4D expression and TPL2 phosphorylation with human MIBC development. (a) H&E and IHC staining of PDE4D in the representative bladder tumor tissues and the adjacent normal bladder tissues. (Scale bar: 200 μm). (b) Statistical data of PDE4D staining in the bladder tumor tissues and adjacent normal bladder tissues. (c) Because all specimens had the same positive-staining scores of PDE4D, we used the staining intensity score to replace the staining index. All the specimens were segregated into two groups based on their staining index (lower expression < staining index 2; higher expression ≥ staining index 2) and compared to observe the variations. (d) Kaplan-Meier survival curves based on the PDE4D expression levels to demonstrate the prognostic importance of PDE4D. (e) H&E and IHC staining of p-TPL2 in the representative bladder tumor tissues and adjacent normal bladder tissues. (Scale bar: 200 μm). (f) Statistical data of p-TPL2 staining in the bladder tumor tissues and adjacent normal bladder tissues. (g) All specimens were segregated into two groups based on their staining index (high: ≥ 4 and low: < 4) and compared to observe the variations. (h) Kaplan-Meier survival curves based on the p-TPL2 levels to demonstrate the prognostic importance of pTPL2. (i, j) The data derived from TCGA database were analyzed and PDE4D mRNA levels were significantly down-regulated in the bladder tumor compared to the bladder normal tissue (i) and correlated with the poor prognosis (j). Error bars indicate mean ± SEM. Statistical significances of differences between experimental groups were evaluated using the Wilcoxon signed rank test (c and g), unpaired Wilcoxon test (i), and log-rank test (d, h, and j). P < 0.01 was considered as statistically significant value

Fig. 6

Synergistic antitumor effect of IFN-α and roflumilast on MIBC. (a) In MIBC, TPL2 is phosphorylated and activates IKK complexes. Therefore, NF-κB is activated and results in COX-2 overexpression that promotes the MIBC development. Moreover, TPL2 induces COX-2 expression by the enhancement of cAMP/CREB signaling through ERK-mediated inhibition of PDE4D activity. (b) IFN-α-induced TPL2 inhibition leads to down-regulation of COX-2 expression and exerts the anti-tumor effect in MIBC treatment. PDE4D induction by roflumilast synergizes with IFN-α activity to inhibit COX-2 expression through the reduction of cAMP level and potentiates the anti-tumor effect of IFN-α on MIBC