Overexpression of Indoleamine 2,3-Dioxygenase 1 Promotes Epithelial-Mesenchymal Transition by Activation of the IL-6/STAT3/PD-L1 Pathway in Bladder Cancer

Abstract

Indoleamine 2,3-dioxygenase 1 (IDO1) is a key enzyme in tryptophan metabolism and plays an important role in tumor cell immunosuppression and angiogenesis. The molecular mechanisms of IDO1 and epithelial-mesenchymal transition (EMT) have not been elucidated or studied in bladder cancer. Therefore, the aims of this study were to detect IDO1 expression in bladder cancer tissues and then to investigate the role of IDO1 in bladder cancer cell EMT and malignant phenotypes as well as the underlying molecular mechanisms. By immunohistochemistry, Western blot, and quantitative reverse transcription-polymerase chain reaction experiments, IDO1 was found to be overexpressed in bladder cancer tissues and cell lines compared to the noncancerous ones. In addition, knockdown of IDO1 expression was shown to inhibit bladder cancer cell growth, migration, invasion, and EMT. Furthermore, we demonstrated that IDO1 may promote EMT by activation of the interleukin 6/signal transducer and activator of transcription 3/programmed cell death ligand 1 signaling pathway. Collectively, these data suggest that IDO1 may play an important role in bladder cancer and may be a novel therapeutic target for patients with bladder cancer.

Figure 1

IDO1 is overexpressed human bladder cancer tissues and cell lines. (A) IHC staining of IDO1 in bladder cancer tissues (T) and adjacent normal tissues (ANT). The representative images are shown. Scale bar = 200 μm for 10× and 100 μm for 20×. (B) Kaplan-Meier survival analysis of patients with gastric cancer according to the IDO1 expression status. Kaplan-Meier analysis of cumulative survival in the low–IDO1 expression group (25 patients) and high–IDO1 expression group (43 patients). The P value was based on the log-rank test. Patients with a high IDO1 expression had a worse survival (P < .05). (C) and mRNA (D) levels of IDO1 in bladder cancer tissues (tumor) and adjacent normal tissues detected by Western blot and qPCR, respectively.

Figure 2

IDO1 is overexpressed human bladder cancer cell lines. Relative protein (A) and mRNA (B) levels of IDO in bladder cancer cell lines (T24, UMUC3, and 5637) and the immortalized human normal bladder epithelial cell line SV-HUC-1 detected by Western blot and qRT-PCR, respectively. (C) mRNA and (D) protein levels of IDO1 in transfected T24 and UMUC3 cells were analyzed using RT-qPCR and Western blot analysis, respectively. GAPDH and β-actin served as the internal controls, respectively. *P < .05.

Figure 3

Silencing of IDO1 inhibits cell growth of T24 and UMUC3 cells. (A, B) Cell proliferation was detected at 24, 48, 72, and 96 hours after IDO1 knockdown as determined by CCK8 assay. (C, D) The left panel is a representative image of clone formation of cells after transfection. The right panel shows the number of colonies of cloned cells (Student’s t test, *P < .05).

Figure 4

Effects of IDO1 knockdown on the inhibition of bladder cancer cell migration, invasion, and EMT. Migration abilities of T24 and UMUC3 cells after IDO1 knockdown detected by cell scratch assays (A) and Transwell tumor cell migration assays (B), respectively. (C) Invasion abilities of T24 and UMUC3 cells after IDO1 knockdown detected by Transwell tumor cell invasion assays. (D) Expression levels of EMT-associated markers in T24 and UMUC3 cells after IDO1 knockdown were evaluated by Western blot analysis.

Figure 5

IDO1 affects EMT through IL6/STAT3/PD-L1 in T24 and UMUC3 cells. (A) Expression of IL6 in T24 and UMUC3 cells after IDO1 knockdown detected by ELISA assays. (B) STAT3, P-STAT3, and PD-L1 were evaluated by Western blot analysis in T24 and UMUC3 cells after IDO1 knockdown. (C and D) T24 and UMUC3 cells after IDO1 knockdown were pretreated with 100 ng/ml of IL-6 for 48 hours. The protein levels of STAT3, P-STAT3, PD-L1, and EMT-associated markers were determined by Western blot analysis.

Figure 6

AG490 blocks the IL6/STAT3/PD-L1 signaling pathway and is more pronounced after knockdown of IDO1. (A) T24 and UMUC3 cells after IDO1 knockdown were treated with STAT3 specific inhibitor AG490. The protein levels of STAT3, P-STAT3, and PD-L1 were measured by Western blot analysis. (B) EMT-associated markers were measured after treatment with AG490 by Western blot analysis.

Figure 7

The proposed model is a graphical representation of the mechanism of interaction of IDO1 and IL6/STAT3/PD-L1 in EMT regulation in bladder tumor cells.