Effect of HER2 on bladder cancer by regulating PI3K-Akt signaling pathway via YWHAE and its mechanism
10.3760/cma.j.cn121382-20250623-00047
- VernacularTitle:HER2通过YWHAE调控PI3K-Akt信号通路对膀胱癌的影响及其作用机制
- Author:
Long YANG
1
;
Jing CUI
;
Fan WANG
;
Dan LI
;
Qi ZHANG
;
Yanlei LI
Author Information
1. 天津医科大学总医院泌尿外科，天津 300052
- Keywords:
Bladder cancer;
Human epidermal growth factor receptor 2;
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon peptide;
Phospho
- From:
International Journal of Biomedical Engineering
2025;48(4):365-376
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of human epidermal growth factor receptor 2 (HER2) on bladder cancer by regulating phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway via tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon peptide (YWHAE) and to examine its mechanism.Methods:The gene expression profiling interactive analysis (GEPIA) database was used to analyze HER2 expression in 408 bladder cancer tissues and 19 adjacent normal tissues. HER2 expression was then compared between 215 tumor protein 53 ( TP53) mutant and 193 TP53 non-mutant bladder cancer tissues. Tissue samples were obtained from patients who underwent surgical resection for bladder cancer in Tianjin Medical University General Hospital between June 2010 and March 2015. Immunohistochemistry and Western blotting were performed to validate HER2 and p53 protein expression, as well as analyze their correlation. Bladder cancer T24 cells were transfected with short hairpin RNA targeting HER2 (shHER2) control (shCon) or shHER2, designated as shCon and shHER2 groups. Bladder cancer UMUC3 cells were transfected with overexpression control (oeCon), HER2 overexpression (oeHER2), oeYWHAE, or short hairpin RNA targeting murine double minute 2 (MDM2) (shMDM2), and were designated as the oeCon, oeHER2, oeYWHAE and shMDM2 groups, respectively. UMUC3 cells were then treated with either 0.1% dimethyl sulfoxide or 100 mmol/L dihydrotestosterone and designated as the solvent control and dihydrotestosterone groups, respectively. Additionally, oeCon and oeYWHAE UMUC3 cells were treated with the PI3K inhibitor LY294002 (25 μmol/L), designated as the LY294002 and LY294002+oeYWHAE groups. On this basis, shHER2 was transfected into the oeCon and oeYWHAE groups, which were then designated as the shHER2-2 and shHER2-2+oeYWHAE groups. The relative expression levels of HER2, YWHAE mRNA, and HER2, p53, YWHAE, MDM2, phosphorylated Akt (p-Akt), and Akt proteins were determined using quantitative reverse transcription PCR and Western blotting. Cell Counting Kit-8, Transwell, and wound-healing assays were performed to evaluate the impact of HER2 on the proliferation, invasion, and migration of bladder cancer cells. Mass spectrometry and co-immunoprecipitation assays were performed to confirm the interaction between YWHAE and HER2, and immunofluorescence was used to detect p53 expression. BALB/c nude mice were subcutaneously injected with 5×10 6 UMUC3 cells in the scapular region. According to the random number table method, they were divided into negative the control group and the transfection group, with 3 mice in each group, and transfected with oeCon and oeHER2, respectively. Tumor volume and weight were measured and calculated, and HER2 and p53 protein expression in bladder cancer tissues was validated by immunohistochemistry and Western blotting. Independent sample t test or Mann-Whitney U test was used to compare the two groups. One-way analysis of variance or Kruskal-Wallis test was used for comparison of multiple groups. Results:GEPIA database analysis demonstrated significantly higher levels of HER2 expression in bladder cancer tissues and in TP53 mutant bladder cancers compared with adjacent normal tissues (both P<0.01). HER2 expression was inversely correlated with p53 expression ( r=?0.6). Immunohistochemistry and Western blotting confirmed that p53 expression level in the bladder cancer tissues (5.32±0.11) was higher than that in the adjacent normal tissues (2.00±0.01), while HER2 expression level in the bladder cancer tissues (1.13±0.02) was lower than that in the adjacent normal tissues (6.20±0.06) (both P<0.01). HER2 mRNA and protein expression, absorbance at 450 nm wavelength ( A450) values, and cell invasion number and cell migration distance in the shHER2 group were all lower than those in the shCon group [0.25±0.01 vs 1.00±0.05, 1.00± 0.01 vs 3.26±0.09, 1.36±0.04 vs 1.65±0.06, (107.00±5.51) vs (202.70±11.61) cells, and (298.70±6.94) vs (454.30±7.84) μm] ( P<0.05, 0.01). HER2 mRNA and protein expression, absorbance ( A450) values, and cell invasion number and cell migration distance in the oeHER2 group were all higher than those in the oeCon group [0.78±0.02 vs 0.46±0.01, 2.05±0.02 vs 1.00±0.00, 1.23±0.06 vs 0.78±0.03, (136.30±5.24) vs (59.00±5.51) cells, and (153.70±7.27) vs (66.33±33.84) μm] ( P<0.05, 0.01). HER2 protein expression level in the dihydrotestosterone group was higher than that in the solvent control (1.83±0.19 vs 1.00±0.00), while p53 protein expression level in the dihydrotestosterone group was lower than that in the solvent control group (1.10±0.10 vs 1.53±0.15) (both P<0.01). The differentially expressed protein between the dihydrotestosterone group and solvent control group was YWHAE. The expression levels of YWHAE mRNA and protein in the dihydrotestosterone group (1.10±0.12 and 3.05±0.03) were higher than those in the solvent control group (0.30±0.12 and 1.00±0.00) (both P<0.01). YWHAE protein expression level in the oeHER2 group was higher than that in the oeCon group (1.37±0.08 vs 1.00±0.00) ( P<0.01) and YWHAE expression level in the bladder cancer tissues was higher than that in the adjacent normal tissues ( P<0.01). YWHAE expression positively correlated with HER2 expression ( r=0.4). Co-immunoprecipitation confirmed direct binding between HER2 and YWHAE. Overexpression of YWHAE significantly reduced p53 expression. The relative expression level of MDM2 protein in the oeYWHAE group (2.73±0.09) was lower than that in the oeCon group (3.43±0.12) ( P<0.01). The relative expression level of MDM2 protein in the shMDM2 group (1.00±0.00) was lower than that in the oeYWHAE group, and the relative expression level of p53 protein (2.00±0.00) was higher than that in the oeYWHAE group (1.07±0.07) (both P<0.01). The relative expression levels of YWHAE and p-Akt protein in the oeYWHAE group (1.23±0.09, 3.00±0.06) were higher than those in the oeCon group (1.00±0.00, 1.13±0.03) ( P<0.05, 0.01). The relative expression level of p-Akt protein in LY294002 group (2.20±0.06) was lower than that in the oeCon group (3.30±0.10), and the relative expression level of p53 protein (2.10±0.06) was higher than that in the oeCon group (1.00±0.00) (both P<0.01). The relative expression level of p-Akt protein in LY294002+oeYWHAE group (2.00±0.06) was lower than that in the oeYWHAE group (3.53±0.14), and the relative expression level of p53 protein (2.10±0.06) was higher than that in the oeYWHAE group (1.00±0.06) (both P<0.01). The relative expression levels levels of YWHAE, p-Akt and MDM2 protein in the shHER2-2 group (1.60±0.15, 1.70±0.06, 0.80±0.06) were lower than those in the oeCon group (2.30±0.06, 2.30±0.06, 1.13±0.09), and the relative expression level of p53 protein (1.83±0.12) was higher than that in the oeCon group (1.00±0.00) ( P<0.05, 0.01). The relative expression level of YWHAE protein in the shHER2-2+oeYWHAE group (2.00±0.06) was lower than that in the oeCon group ( P<0.01), and the relative expression levels of MDM2 and p53 protein (2.63±0.15, 1.13±0.03) were higher than those in the oeCon group ( P<0.05, 0.01). The tumor volume, tumor weight, and relative expression levels of HER2, YWHAE, p-Akt, and MDM2 proteins on day 28 in the transfection group [(5 133.0±185.6) mm 3, (0.65±0.12) g, 2.23±0.02, 4.00±0.12, 3.33±0.06 and 2.24±0.02] were higher than those in the negative control group [(2 633.0±88.2) mm 3, (0.33±0.07) g, 0.98±0.02, 1.27±0.03, 1.29±0.02 and 1.46±0.06] (all P<0.01). The relative expression level of p53 protein (1.21±0.04) was lower than that in the negative control group (3.29±0.04) ( P<0.01). Conclusions:HER2 may promote the malignant progression of bladder cancer by regulating the PI3K-Akt pathway via YWHAE, thereby facilitating MDM2 nuclear translocation and p53 degradation. This ultimately enhances the proliferative, migratory, and invasive capacities of bladder cancer cells.
