Epigenetic silencing of DACH1 induces the invasion and metastasis of gastric cancer by activating TGF-β signalling

Abstract

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non-tumour tissues were examined. Methylation-specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re-expression was induced by 5-Aza-2'-deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non-tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial-mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)-β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1-expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF-β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.

Keywords: DACH1; DNA methylation; chemosensitive marker; docetaxel; gastric cancer; invasion; metastasis.

Fig. 1

Down-regulation of DACH1 expression by DNA methylation in gastric cancer cell lines. (A) Expression of DACH1 was analysed by semi-quantitative RT-PCR in gastric cancer cell lines (BGC823, AGS, NCI-N87, MKN45, NUGC3, MGC803 and SGC7901), the immortalized human gastric mucosal cell line GES-1 and a case of normal control gastric mucosa (NC). GAPDH: Internal control of cDNA quality. (B) MSP results of DACH1 in gastric cancer cell lines, GES-1 and normal control gastric mucosa (NC). IVD: in vitro methylated DNA serves as methylation positive control, NL: normal blood lymphocyte DNA, serves as unmethylation control. U: unmethylated alleles; M: methylated alleles. (C) Sodium bisulfite sequencing results of DACH1 promoter region (−426 bp to −140 bp). BGC823 and NCI-N87: gastric cancer cell lines; Normal: normal gastric mucosa. Double-headed arrow: MSP product site, spanned 130 bp. Filled circles represent methylated CpG sites, and open circles denote unmethylated CpG sites. (D) Expression level of DACH1 detected by semi-quantitative RT-PCR; (−): absence of 5-Aza, (+): presence of 5-Aza.

Fig. 2

DACH1 expression and methylation status in primary gastric cancer. (A) Representative MSP results of DACH1 methylation status in gastric cancer tissues (GC), normal gastric mucosa (NG) and adjacent non-cancerous tissues (ANT). (B) Representative images of DACH1 protein expression in gastric cancer tissues and their adjacent non-tumour tissues determined by immunohistochemistry (IHC). (up, ×200; below, ×400). (C) DACH1 expression levels are shown as box plots, the horizontal lines represent the median score; the bottom and top of the boxes representing the 25th and 75th percentiles respectively; and the vertical bars representing the range of expression level (**P < 0.01). (D) The correlation of DNA methylation and loss/reduced DACH1 expression in 32 available matched primary GCs (**P < 0.01).

Fig. 3

Effect of DACH1 on TGF-β signalling. (A) Luciferase activity was detected in BGC823 and AGS cells transfected with empty vector, DACH1 (200 ng/well), DS (40 ng/well) or ΔDS (160 ng/well), with 10 ng/ml TGF-β1 addition in each group (*P < 0.05, **P < 0.01). (B) Expression of TGF-β signalling downstream genes in DACH1 -unexpressed and -re-expressed BGC823 and AGS cells detected by Western blot; (−): absence of TGF-β1, (+): presence of TGF-β1 (T-: total; p-: phosphorylated). (C) The level of T-SMAD2 and p-SMAD2 detected by Western blot before and after knocking down DACH1 in GES-1 cells.

Fig. 4

The effect of DACH1 on gastric cancer EMT and metastasis. (A) Trans-well assay results: the number of migrated cells' in empty vector group, empty vector with TGF-β1 addition group and re-expression of DACH1 plus addition of TGF-β1 group (*P < 0.05, **P < 0.01). (B) Invasion assay results: the number of invasive cells' in empty vector group, empty vector with TGF-β1 addition group and re-expression of DACH1 plus addition of TGF-β1 group (*P < 0.05, **P < 0.01). (C) The expression of E-cadherin and vimentin detected by Western blot in DACH1 -unexpressed and -re-expressed BGC823 and AGS cells. (D) The left panel shows the expression of MMP-2 and MMP-9 tested by Western blot in DACH1 -unexpressed and -re-expressed BGC823/AGS cells; the right panel shows the expression of MMP-2 and MMP-9 in GES-1 cells before and after DACH1 knocking down.

Fig. 5

Effect of DACH1 on tumour growth and cell cycle in gastric cancer cells. (A) Colony formation results of DACH1 -unexpressed and -re-expressed gastric cancer cells. The left panel: the representative colony formation results of DACH1 -unexpressed and -re-expressed BGC823 and AGS cells. Right panel: colony numbers (*P < 0.05, **P < 0.01). (B) Growth curves: cell viability was tested by CCK-8 kit in BGC823 and AGS cells (*P < 0.05, **P < 0.01). (C) Representative cell cycle and flow cytometry data (*P < 0.05, **P < 0.01). (D) The expression of cyclinB1 and cdc2 detected by Western blot in DACH1 -unexpressed and -re-expressed BGC823 and AGS cells.

Fig. 6

The chemosensitivity of DACH1 -unexpressed and -re-expressed BGC823 and AGS cells to docetaxel. (A) The responsive curves of DACH1 -unexpressed and -re-expressed BGC823 cells in different concentration of docetaxel (0, 2.5, 10, 20, 40, 160 μg/ml). (B) The dose–response curves of DACH1 -unexpressed and -re-expressed AGS cells in different concentrations of docetaxel (0, 0.02, 0.2, 2, 10, 100 μg/ml). The viability of cells was measured by CCK-8 after docetaxel treatment for 48 hrs.

Fig. 7

The effect of DACH1 on gastric cancer cell xenograft. (A) Representative picture of xenograft tumour of DACH1 -unexpressed (up) and -re-expressed BGC823 cells (low). (B) Subcutaneous tumour growth curve in xenograft mice with or without DACH1 re-expression (**P < 0.01). (C) Histogram represents average weight of xenograft in DACH1 -unexpressed and -re-expressed groups (**P < 0.01). (D) Immunohistochemical (IHC) staining in xenograft (×400).