Epigenetic Silencing Of DACH1 Induces The Invasion And Metastasis Of Gastric Cancer By Activating TGF-β Signalling

Background Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide and a leading cause of cancer related death. An estimated 951,600 new stomach cancer cases and 723,100 deaths occurred in 2012. Most of GC patients die of recurrence and metastasis, with low 5-year survival rate. TGF-β signaling functions in several biological processes, including cell proliferation, differentiation, migration and apoptosis and plays a critical but paradoxical role in the predisposition and progression of cancer. During the later stages of carcinogenesis, TGF-β signaling promotes the invasion and metastasis of cancer through its induction of epithelial-mesenchymal transition (EMT). Reduced expression of DACH1 was found in breast, prostate, endometrial cancer, hepatic and colorectal cancer. The expression of DACH1 was regulated by promoter region methylation in hepatic and colorectal cancer. In addition, the inhibiting effect of DACH1 on TGF-β signaling was found in human cancers such as colorectal and breast cancer.Objective To examine whether epigenetic changes in DACH1 occurred in GCs and explore the role of DACH1 in tumour growth, invasion, metastasis and chemosensitivity in human GC.Methods MSP (Methylation specific PCR) was used to detect methylation status of DACH1 in GC cell lines, normal gastric mucosa,98 cases of primary GC and 50 cases of adjacent non-tumour tissues. RT-PCR was applied to detect expression of DACH1 in gastric cancer cells with or without 5-aza (5-aza-2’-deoxycytidine) treatment. SPSS 15.0 software was employed to analyze the correlation of DACH1 methylation and clinical factors of gastric cancer patients. IHC (immunohistochemistry) was applied to detect the DACH1 expression in 32 cases of gastric cancer and adjacent normal tissues. Dual-Luciferase reporter assay was used to detect the activity of TGF-β signaling. Western blot was employed to detect the expression of TGF-β downstream targets (Smad2/3, p-Smad2/3, E-cadherin, vimentin, MMP2 and MMP9, etc) with or without DACH1 re-expression. Cell proliferation assay, flow cytometry analysis, colony formation and xenograft mice model were applied to analyze the effect of DACH1 on stomach tumor growth.Results Loss of DACH1 expression correlates with DNA methylation in gastric cancer cell lines, and restoration of DACH1 expression was induced by 5-aza. The DACH1 is methylated in 63.3%(62/98) of primary GC and 38%(19/50) of adjacent non-tumour tissues. In contrast, all 8 cases of normal gastric mucosa were unmethylated. Methylation of DACH1 in primary GC 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 inhibited EMT (epithelial-mesenchymal transition) and metastasis by inhibiting 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.Conclusion 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-b signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells’ resistance to docetaxel.