NR4A3 regulates anoikis resistance and metastasis of bladder cancer through EWSR1

Abstract

Bladder cancer (BLCA) is a common urinary malignancy with high metastatic potential. However, the mechanisms underlying its progression remain unclear. This study aimed to investigate the role and regulatory mechanisms of NR4A3, a nuclear receptor involved in apoptosis and tumor suppression, in BLCA progression, particularly its impact on anoikis resistance and metastasis. NR4A3 expression levels were analyzed using the GEPIA database. Functional studies were conducted by overexpressing NR4A3 in adherent and suspension-cultured BLCA cells. Apoptosis, invasion, migration, and ER stress marker (Bip and CHOP) expression were evaluated. Subcutaneous and lung metastasis models in BALB/c nude mice were used for in vivo validation. GEPIA analysis showed that NR4A3 is significantly downregulated in BLCA. NR4A3 overexpression increased apoptosis, reduced invasion and migration, and upregulated Bip and CHOP expression. In vivo, NR4A3 overexpression significantly reduced lung metastasis in BALB/c nude mice (n = 8 per group, p < .001). Mechanistically, NR4A3 promoted ER stress by regulating the EWSR1/Ezrin pathway, thereby suppressing anoikis resistance. NR4A3 functions as a tumor suppressor in BLCA by enhancing endoplasmic reticulum stress and inhibiting anoikis resistance through the EWSR1/Ezrin pathway. It may serve as a promising therapeutic target for metastatic BLCA.

Keywords: Bladder cancer; EWSR1; NR4A3; anoikis; endoplasmic reticulum stress.

Figure 1.

NR4A3 level in BLCA and its relationship with prognosis. (a). NR4A3 was low-expressed in BLCA. (b). Expression levels of NR4A3 in different TNM stages of BLCA. The relationship between NR4A3 level and the overall survival (c) and disease-free survival (d) of BLCA patients. (e). Representative immunohistochemical staining images of NR4A3 in BLCA tumor tissues and adjacent tissues. (f). Statistical graph of the expression level of NR4A3 in tumor and normal tissues. (g). The expression of NR4A3 in four BLCA cell lines and human ureteral epithelial immortalized cell line was evaluated by Western blot. *p < 0.05, **p < 0.01, **p < 0.001.

Figure 2.

NR4A3 inhibits the anoikis resistance and metastasis ability of BLCA cells. (a). The infection efficiency of ad-NR4A3 and ad-NC in 5637 and T24 cells was verified by detecting GFP fluorescence and RT-qPCR assay. Scale bar = 100 μm. The infected 5637 and T24 cells were treated with or without poly-HEMA, the apoptosis rate and the expression of Cleaved-caspase3 and Bcl-2 were determined by flow cytometry (b) and Western blot (c), respectively. (d). The migration and invasion ability of the infected cells were detected by transwell assay. Scale bar = 50 μm. **p < 0.01.

Figure 3.

NR4A3 inhibits anoikis resistance by promoting ER stress. (a). The mRNA and protein expressions of Bip and CHOP were evaluated through RT-qPCR and Western blot. The infected 5637 and T24 cells were treated with or without 4-PBA, and the expression of Cleaved-caspase3 and Bcl-2 was determined by Western blot (b), the apoptosis rate was detected by flow cytometry (c), and the migration and invasion were detected by transwell assay (d). Scale bar = 50 μm. *p < 0.05, **p < 0.01.

Figure 4.

Overexpression of NR4A3 inhibits the growth and metastasis of BLCA in vivo. (a). Images of subcutaneous xenograft tumor model nude mice injected with ad-NC and ad-NR4A3 and the isolated tumor. Arrows indicate the location of the tumors. (b). Tumor weight and volume of the nude mice in ad-NC and ad-NR4A3 group. (c). in vivo imaging of lung metastases in two groups of nude mice. (d). Number of pulmonary tumor in nude mice. (e). HE staining of lung tissue slices of mice in two groups. Scale bar = 200 μm. (f). The statistics of survival of two groups of mice. TUNEL staining (g, scale bar = 20 μm) and immunohistochemical staining (h, scale bar = 50 μm) of the tumors. *p < .05, **p < .01.

Figure 5.

NR4A3 regulates the EWSR1/Ezrin pathway in BLCA. (a). The expression level of EWSR1 in 404 BLCA tumor tissues and 28 adjacent normal tissues from TCGA database was analyzed. The correlation between EWSR1 and NR4A3 expression levels in BLCA clinical samples was analyzed. (b). The protein level of EWSR1 in 5637 and T24 cells that infected with ad-NR4A3/ad-NC was determined by Western blot. The mRNA and protein levels of EWSR1, Ezrin in 5637 and T24 cells that overexpressing NR4A3 or knocking down NR4A3, and Ezrin in 5637 and T24 cells that overexpressing EWSR1, were detected by RT-qPCR (c) and Western blot (d), respectively. **p < 0.01.

Figure 6.

The expression of EWSR1 (a), Ezrin (b) in 5637 and T24 cells that overexpressing NR4A3 or knocking down NR4A3, and Ezrin in 5637 and T24 cells that overexpressing EWSR1 (c), were visualized by immunofluorescent staining. Scale bar = 20 μm. **p < 0.01, ***p < 0.001.

Figure 7.

NR4A3 affects the ER stress by regulating EWSR1/Ezrin pathway. 5637 and T24 cells were divided into four groups: a: ad-NC + ad-NC + si-NC, b: ad-NR4A3 + ad-NC + si-NC, c: ad-NR4A3 + ad-EWSR1 + si-NC, and d: ad-NR4A3 + ad-EWSR1 + si-Ezrin group. The relative mRNA expression and protein expression levels of Bip and CHOP in 5637 and T24 cells in these four groups were assessed by RT-qPCR (a) and Western blot (b). *p < 0.05, **p < 0.01.

Figure 8.

NR4A3 affects the ER stress by regulating EWSR1/Ezrin pathway and thus interferes with anoikis resistance and metastasis. 5637 and T24 cells were divided into four groups: a: ad-NC + ad-NC + si-NC, b: ad-NR4A3 + ad-NC + si-NC, c: ad-NR4A3 + ad-EWSR1 + si-NC, and d: ad-NR4A3 + ad-EWSR1 + si-Ezrin group. (a). Cell apoptosis of cells treated with or without poly-HEMA was detected. (b). The migration and invasion of cells in these four groups were detected by transwell assay. Scale bar = 50 μm. *p < 0.05, **p < 0.01, **p < 0.001.

Figure 9.

Schematic illustration of the proposed mechanism by which NR4A3 contributes to disease pathogenesis via the EWSR1/Ezrin signaling pathway.