The adenosine A2b receptor promotes tumor progression of bladder urothelial carcinoma by enhancing MAPK signaling pathway

Yihong Zhou, Xi Chu, Fei Deng, Liang Tong, Guoxiong Tong, Ye Yi, Jianye Liu, Jin Tang, Yuxin Tang, Yang Xia, Yingbo Dai, Yihong Zhou, Xi Chu, Fei Deng, Liang Tong, Guoxiong Tong, Ye Yi, Jianye Liu, Jin Tang, Yuxin Tang, Yang Xia, Yingbo Dai

Abstract

The adenosine A2b receptor (A2bR) was considered to play an oncogenic role in many human malignancies. However, the expression and molecular function of A2bR in bladder urothelial carcinoma (BUC) have not been well elucidated. Herein, we found that the expression of A2bR was higher than other adenosine receptors in BUC tissues and cells, and it was upregulated in BUC tissues compared with matched normal bladder tissues. Furthermore, high expression of A2bR was associated with poor prognosis of patients with BUC. In addition, suppression of A2bR inhibited the proliferation, migration and invasion of BUC cells and arrested the cell cycle at the G1 phase. Finally, we demonstrated that downregulation of A2bR inhibited the proliferation, migration and invasion of BUC in part via the MAPK signaling pathway, increasing the levels of P21 but decreasing the levels of cyclin B1, D, E1, MMP-2 and MMP-9. Overexpression of MMP-2 could rescue BUC cells migration and invasion. Thus, the present study indicates that A2bR may play a potential oncogenic role in BUC progression and act as a potential biomarker to identify BUC patients with poor clinical outcomes.

Keywords: MAPK signaling; adenosine A2b receptor; bladder urothelial carcinoma; prognosis.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that they have no conflicts of interest.

Figures

Figure 1. A2bR is the main subtype…
Figure 1. A2bR is the main subtype of ARs and upregulated in BUC
A. mRNA expression of four subtypes of ARs in BUC tissues (T) and adjacent normal tissues (N). B. mRNA expression of four subtypes of ARs in three BUC cell lines. C. Protein expression of A2bR in 12 pairs of fresh BUC tissues and their matched normal urothelial bladder epithelial tissues. D. Protein expression of A2bR in three BUC cell lines and normal human urinary tract epithelial cell line. E., F. and G. qRT-PCR and western blot analyses of knockdown efficiency with shRNA transfection in EJ and T24 cells. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01.
Figure 2. High expression of A2bR in…
Figure 2. High expression of A2bR in BUC is associated with poorer prognosis
A. Representative images of A2bR expression in normal urothelial bladder epithelial tissue and BUC specimens examined by IHC (200×). The normal urothelial bladder epithelial tissue was stained negatively (a), and the BUC tissues showed low (b) and high (c and d) expression of A2bR. B. Kaplan-Meier analysis of survival in patients between low expression of A2bR group (n = 104) and high expression of A2bR group (n = 56).
Figure 3. Downregulation of A2bR inhibits cell…
Figure 3. Downregulation of A2bR inhibits cell growth of BUC cells in vitro, leads to a G0/G1 phase cell cycle arrest and reduces the level of cyclin family and P21
A. and B. Suppression of A2bR inhibits cell growth as detected by MTT (A) and colony formation assays (B). C. and D. Flow cytometry analysis shows that knockdown of A2bR expression increases the percentage of cells in G0/G1 phase and decreases the percentage of cells in G2/M phase. E. Downregulation of A2bR upregulates P21 but downregulates cyclin B1, D and E1. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01, *P < 0.05.
Figure 4. Downregulation of A2bR inhibits cell…
Figure 4. Downregulation of A2bR inhibits cell migration and invasion of BUC cells in vitro and reduces the level of MMP-2 and MMP-9
A. Wound healing assay shows EJ and T24 cells with A2bR knockdown migrate into the scratching area more slowly than control cells. B. and C. Suppression of A2bR reduces EJ and T24 cells migratory (B) and invasive (C) ability in a transwell assay without/with Matrigel. (D) Suppression of A2bR downregulates MMP-2 and MMP-9. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01.
Figure 5. Overexpression of MMP-2 rescues cell…
Figure 5. Overexpression of MMP-2 rescues cell migration and invasion of BUC cells in vitro
A. After A2bR knockdown cells are stable constructed, MMP-2 is overexpressed in EJ and T24 cells detected by western blot analyses. B. Wound healing assay shows EJ and T24 cells with MMP-2 overexpression migrate into the scratching area faster than control cells. C. MMP-2 overexpression reduces EJ and T24 cells migratory and invasive ability in a transwell assay without/with Matrigel. The graph shows the mean ± SD; ***P < 0.001, **P < 0.01.
Figure 6. Downregulation of A2bR inhibits tumor…
Figure 6. Downregulation of A2bR inhibits tumor growth of T24 cells in vivo, and regulates expression of MAPK pathway proteins in EJ and T24 cells
A. and B. Tumors were removed at the 35th day after nude mice received subcutaneous injection (n = 5 for each group). C. Tumor growth curve. D. HE staining (200×) and IHC (200×) for A2bR, Ki-67, MMP-2 and MMP-9 in BUC xenografts. E. Suppression of A2bR decreases the phosphorylation levels of P38, JNK and ERK. The graph shows the mean ± SD; ***P < 0.001.
Figure 7. An effect and mechanism model…
Figure 7. An effect and mechanism model that A2bR knockdown inhibits tumor growth and invasion through MAPK signaling pathway in BUC
Suppression of A2bR expression reduces the phosphorylation levels of P38, JNK and ERK. Moreover, it upregulates P21 but downregulates cyclin B1, D, E1, MMP-2 and MMP-9, which leads to inhibition of BUC cells proliferation, migration and invasion.

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