DAB2 suppresses gastric cancer migration by regulating the Wnt/β-catenin and Hippo-YAP signaling pathways

Abstract

Background: Disabled-2 (DAB2), a potential tumor suppressor, plays an in important role in cancer development and cellular differentiation. Its lower expression levels have founded in many cancers. In addition, DAB2 is involved in multiple signaling pathways, including TGF-β and Wnt signal pathways. Gastric cancer (GC) is a common gastrointestinal malignant tumor. Nonetheless, the role of DAB2 in GC remains unclear.

Methods: Thirty-seven clinical specimens of GC tissues and adjacent non-tumor tissues were examined by immunohistochemistry. Proteins were extracted from two of them to perform Western blot analysis. Then, CMV-MCS-3FLAG-SV40-DAB2 and si-DAB2 were transfected into MGC and SGC cell line, respectively. The migration of GC cells was evaluated by transwell migration assay. And, the expression of migration related proteins was detected by Western blot and immunofluorescence (IF).

Results: Eighty-six percent (32/37) of patients DAB2 staining was reduced in GC tissues compared to adjacent normal tissues. Further studies showed that in six human GC cell lines, the level of DAB2 expression was lower than normal gastric epithelial cells, and that DAB2 was closely related to cell migration in vitro. In DAB2 silenced cells, the Wnt/β-catenin signaling was increased and the Hippo-YAP pathway was affected. In addition, lower DAB2 level led to nuclear translocation of β-catenin and Yap.

Conclusions: The lower expression of DAB2 regulates cell migration in GC via interfering with the Wnt and Hippo signaling pathway. Our findings suggested that DAB2 played an important role in the migration of GC.

Keywords: Gastric cancer (GC); Hippo-YAP; Wnt/β-catenin; disabled-2 (DAB2); migration.

Figure 1

DAB2 expression level was detected in GC and paired adjacent non-tumor tissues, in normal gastric epithelial cells and six GC cell lines by immunohistochemistry and Western blot. (A) Representative immunohistochemical staining of DAB2 was shown in an adjacent normal tissue (left) and GC tissue (right) (×100). (B,C) In two pairs of GC patients and adjacent non-tumor tissues, the expression of DAB2 was diminished in GC tissues as detected by Western blot. (D,E) DAB2 protein expression was lower in all six GC cell lines than in GES-1. GC, gastric cancer; DAB2, disabled-2.

Figure 2

The expression of DAB2 was detected in SGC cells transfected by si-DAB2 and in MGC cells transfected by CMV-MCS-3FLAG-SV40-DAB2. (A,B) DAB2 protein expression was obviously decreased in si-DAB2 cells compared with that of the control. (C,D) CMV-MCS-3FLAG-SV40-DAB2 could be up-regulated the expression of DAB2 in MGC cells (**, P<0.01). DAB2, disabled-2.

Figure 3

Aberrant DAB2 expression influences cell migration. (A,B) In DAB2-silenced SGC cells, the migratory ability of cell was enhanced as compared with the control SGC cells (HE, ×200). (C,D) DAB2 overexpression reduced the migratory ability of MGC cells as compared with the control MGC cells (HE, ×200). (E-H) Western blot demonstrated MMP-9, as well as MMP-2 and E-cadherin expression levels in si-DAB2 cells and in DAB2-overexpressed DAB2 cells, respectively (*, P<0.05; **, P<0.01). DAB2, disabled-2.

Figure 4

DAB2 expression affected the Wnt/β-catenin signaling pathway. (A,B) Western blot analyzed Wnt canonical gene targets in DAB2-knockout cells and showed that CyclinD1, β-catenin and p-GSK3β were significantly increased in DAB2-silencing cells. (C,D) Western blot analyzed Wnt canonical gene targets in DAB2-overexpressed cells (*, P<0.05). (E,F) IF assay using anti-β-catenin (red) antibodies was performed in DAB2-knockout cells and DAB2-overexpressed cells. Hoechst was used to visualized the cell nucleus (blue) (×600). DAB2, disabled-2; IF, immunofluorescence.

Figure 5

DAB2 regulated the Hippo-YAP pathway. (A,B) The expression level of Yap, p-Yap, LATS1 and p-Lats were examined by Western blot in DAB2-knockout cells and DAB2-overexpressed cells. (C) The ratios of p-Yap/Yap and p-LATS1/LATS1 were analyzed in t DAB2-knockout SGC cells and DAB2-overexpressed MGC cells, respectively. (D,E,F,G) IF assay using anti-p-YAP (red) and anti-YAP (red) antibodies was performed in DAB2-knockout cells and DAB2-overexpressed cells, respectively. Hoechst was used to visualized the cell nucleus (blue) (×600).