FBXW2 inhibits the progression of gastric cancer via promoting β-catenin ubiquitylation

Abstract

Background: F-box and WD-repeat-containing protein 2 (FBXW2), an E3 ubiquitin ligase, may play a crucial role in tumorigenesis. However, its function in gastric cancer remains unknown. Methods: The expression levels of FBXW2 and β-catenin in gastric cancer samples were analyzed using RT-PCR and immunohistochemistry, with Pearson correlation analysis to assess their relationship. AGS and HGC-27 gastric cancer cells were transfected with sh-FBXW2, and their viability was evaluated using the CCK8 assay, while invasion ability was assessed via the transwell assay. Western blotting was performed to measure the expression levels of FBXW2, β-catenin, GSK3β, and Axin2 in AGS cells. Additionally, a ubiquitination assay was conducted to examine the effect of sh-FBXW2 on β-catenin ubiquitination. Immunoprecipitation was used to determine the potential interaction between FBXW2 and β-catenin. Results: FBXW2 expression was downregulated, whereas β-catenin expression was upregulated in gastric cancer tissues compared to adjacent normal tissues, showing a significant negative correlation (r = -0.52, P < 0.001). Knockdown of FBXW2 (sh-FBXW2) promoted gastric cancer cell viability and invasion while increasing β-catenin expression and reducing GSK3β and Axin2 levels. Furthermore, FBXW2 was found to bind β-catenin and facilitate its ubiquitination, leading to enhanced nuclear translocation of β-catenin. Conclusions: FBXW2 suppresses gastric cancer progression by promoting β-catenin ubiquitination, highlighting its potential as a therapeutic target.

Keywords: FBXW2; GSK3β; gastric cancer; ubiquitylation; β-catenin.

Figure 1

FBXW2 was down-regulation in gastric cancer. (A and B) FBXW2 expression in tumor and adjacent normal tissues of gastric cancer (GC) patients was assayed with RT-qPCR (n = 74). violin plot. *** P < 0.001 from t-test. (C) Receiver operating characteristic (ROC) analysis of FBXW2 expression in gastric cancer. (D) Representative Western blots of FBXW2 between adjacent normal and tumor tissues of gastric cancer patients. (E) Overall survival during 5 years in gastric cancer patients with the low or high expressions of FBXW2 mRNA levels according to the median of mRNA expressions normalized to adjacent tissues.

Figure 2

The expressions correlation between FBXW2 and β-catenin in gastric cancer. (A and B) The relative staining intensities of FBXW2 and β-catenin in adjacent normal and tumor tissues of gastric cancer patients. n = 74 for each group. Violin plot. *** P < 0.001 from paired t-test. (C) Pearson analysis of the relative staining intensities of β-catenin and FBXW2 in tumor tissues of gastric cancer patients. n = 74 for each group.

Figure 3

Inhibition of FBXW2 promoted gastric cancer viability and invasion. Sh-FBXW2 was transfected into AGS cells (A) or HGC-27 cells (B). Cell viability was measured by CCK8 after 24, 48, 72, and 96 hours after sh-FBXW2 transfection. ***P<0.001 compared to control. Brown-Forsythe ANOVA analysis was followed by Dunn's multiple comparisons test. (C) The colony formation assay of sh-FBXW2 transfected AGS cells was conducted 10 days after transfection and the number of colonies were calculated. (D) The invasion characteristics of sh-FBXW2 transfected AGS cells was observed by the transwell assay and invaded cells number was counted. Data were shown with mean ± SD. **P < 0.01, ***P < 0.001 compared to control. Mann Whitney test.

Figure 4

Inhibition of FBXW2 activated Wnt/β-catenin signaling in AGS cells. After sh-FBXW2 transfection for 72 hours, Western blotting was utilized to measure FBXW2, GSK3β, β-catenin, and Axin2 expression in AGS cells (A). The relative expression was normalized to control (B-E). Data were shown with mean ± SD. **P < 0.01, ***P < 0.001 compared to control. Mann Whitney test.

Figure 5

FBXW2 can bind with β-catenin to accelerate the ubiquitination of β-catenin. (A) Indicated plasmids transfected AGS cells were lysed and pull-down with nickel-nitrilotriacetic acid (Ni-NTA)-beads. Ubiquitination level of β-catenin was detected by Western blotting. (B) Binding of FBXW2 with β-catenin was determined by Western blotting.

Figure 6

FBXW2 inhibited β-catenin nuclear translocation. (A) AGS cells were transfected with sh-FBXW2 for 72 hours. The expressions of β-catenin in cytoplasm and nucleus (A) were detected with Western blotting. β-actin and laminB1 were utilized as loading controls and β-catenin expression was normalized to control (B and C). Data were shown with mean ± SD. **p < 0.01, ***p < 0.001 compared to control. Mann Whitney test.