Knockdown of Gab1 Inhibits Cellular Proliferation, Migration, and Invasion in Human Oral Squamous Carcinoma Cells

Abstract

Grb2-associated binder 1 (Gab1) is often aberrant in cancerous cells and tissues, whose alteration is responsible for aggressive phenotypes. In this study, we examined the Gab1 expression in human oral squamous cell carcinoma (OSCC) tissues and investigated the cellular and molecular effect of Gab1 on migration, invasion, and cell growth of the OSCC cell lines SCC15 and SCC25. We found that Gab1 was overexpressed in OSCC tissues and cells, which is related to the protein levels of various molecules associated with cellular proliferation, migration, and invasion. Functional assays identified that Gab1 overexpression promoted cell proliferation and invasion of OSCC cells and inhibited cell apoptosis in the SCC15 and SCC25 cell lines. On the other hand, Gab1 silencing affected the proliferation and invasion of OSCC cells and induced cell apoptosis. Western blot assay identified that Gab1 overexpression suppressed the expression of Cdc20 homolog 1 (Cdh1) and then promoted cell invasion in OSCC cells. Furthermore, Gab1-mediated Cdh1 downregulation was significantly reversed when the cells were subjected to an inhibitor of p-Akt. In conclusion, these results suggested that Gab1 induced malignant progression of OSCC cells probably via activation of the Akt/Cdh1 signaling pathway. Thus, Gab1 may be a potential therapeutic target in the treatment of OSCC patients.

Figure 1

The expression of growth factor receptor bound protein 2 (Grb2)-associated binder 1 (Gab1) in oral squamous cell carcinoma (OSCC) tissues. (a) Western blot analysis was used to assess the protein levels of Gab1 in 30 pairs of OSCC tissues and adjacent normal oral epithelial tissues. (b) The expression of Gab1 in metastasis and nonmetastasis OSCC tissues was assessed. Relative Gab1 expression was normalized to β-actin. Results represent mean ± SEM. *p < 0.001 compared to normal tissues or nonmetastasis. Statistical significance was assessed using an unpaired two-tailed Student’s t-test.

Figure 2

The expression of Gab1 in OSCC cells and the expression of malignant progression-related protein markers. (a) Determination of endogenous expression of Gab1 mRNA by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR) in OSCC cells lines SCC15, SCC25, HN4, and HN6, and the human nontumor cell line NHOK (normal human oral keratinocytes). (b) Western blot analysis was used to assess the protein levels of Gab1 in OSCC cells lines SCC15, SCC25, HN4, and HN6, and the human nontumor cell line NHOK. (c) Western blot analysis was used to assess the protein levels of Ki-67, B cell lymphoma 2 (Bcl-2), cyclin D1, Bcl-2-associated X protein (Bax), tissue inhibitor of matrix metalloproteinases 2 (TIMP2), and Cdc20 homolog 1 (Cdh1) in SiHa, SCC25, and the human nontumor line NHOK. *p < 0.001 compared to NHOK cells. Statistical significance was assessed using one-way ANOVA followed by the Dunnett’s multiple comparisons test for between-group analysis.

Figure 3

Gab1 expression promoted the proliferation of SCC15 and SCC25 cells. (a) Total cell number assays were performed using cell counting kit-8. Cells were seeded in full serum, and the total cell number was counted every 12 h. (b) Flow cytometry assays showed a statistically significant increase in apoptosis when Gab1 was knocked down in SCC15 and SCC25 cells. (c) Western blot analysis was used to assess the protein levels of Bcl-2 and Bax in SCC15 and SCC25 cells transfected with small interfering RNA for Gab1 (si-Gab1) or si-control. Results represent mean ± SEM. *p < 0.001. Statistical significance was assessed using (p < 0.05 was considered as significant compared to si-control, using an unpaired two-tailed Student’s t-test) SPSS 17.0.

Figure 4

Gab1 regulates cell migration and invasion of OSCC cells. Transwell migration (a) and invasion (b) assays were carried out in SCC15 and SCC25 cells with depleted expression of Gab1 (si-Gab1). Each bar represents the mean ± SEM of three independent experiments. *p < 0.001, compared with si-control, using an unpaired two-tailed Student’s t-test.

Figure 5

Gab1 induces protein kinase B (Akt)/cell division cycle 20 (Cdc20) homolog 1 (Cdh1) pathways in SCC15 cells. (a) Western blot analysis was used to assess the levels of Gab1, Cdh1, phosphorylated (p)-Akt, and total (t)-Akt in OSCC SCC15 cells with depleted expression of Gab1. (b) Western blot analysis was used to assess the levels of Gab1, Cdh1, p-Akt, and Akt in OSCC SCC15 cells in the presence of Akt activity inhibitor LY294002. (c) Western blot analysis was used to assess the levels of Gab1, Cdh1, p-Akt, and Akt in OSCC SCC15 cells with depleted expression of Cdh1.

Figure 6

Gab1/Akt/Cdh1 regulates cell migration and invasion of OSCC SCC15 cells. (a–c) Transwell migration and invasion assays were carried out in SCC15 and SCC25 cells with different treatments as mentioned in Figure 4. Each bar represents the mean ± SEM of three independent experiments. *p < 0.001, compared with si-control or dimethyl sulfoxide (DMSO), using an unpaired two-tailed Student’s t-test.