Abstract 2974: Epigenetic silencing DACH1 activated both TGF-β and Wnt signaling in colorectal cancer.
Authors: Guo Mingzhou , Yan Wenji , Yang Yunsheng 
Published: August 2014

Background Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western countries. The incidence of CRC is increasing very fast with the changing of diet and life style in China. Both Wnt and TGF-β signaling pathways were regarded to play important roles in the development of colorectal cancer. Recent studies have found that the TGF-β and Wnt signaling pathways are crosstalking during breast cancer carcinogenesis and other situations. DACH1 was found inhibiting TGF-β signaling in breast and ovarian cancer. The epigenetic regulation and the function of DACH1 in colorectal cancer remain unclear.

Objective To evaluate the possibility of DACH1 methylation serving as CRC detection marker and the regulation of DACH1 expression in colorectal cancer, the role of DACH1 in TGF-β &Wnt signaling pathways was also analyzed in CRC in this study.

Methods Semi-quantitative RT-PCR was used to detect DACH1 expression. Methylation specific PCR (MSP) was employed to detect DACH1 methylation in 5 cell lines, 8 cases of normal mucosa, 15 cases of colon polyps and 100 cases of primary colorectal cancer. Expression of DACH1, CylinD1 and β-catenin were detected by immunohistochemistry (IHC) in 30 cases of available matched colorectal cancer and adjacent tissue samples. Dual-Luciferase reporter assay was applied to evaluate the effect of DACH1 on TGF-β &WNT signaling pathway. The effect of DACH1 on TGF-β &WNT downstream targets(c-myc, CyclinD1, etc) were analyzed by western blot. Colony formation, cell proliferation assay, flow cytometry and mouse xenograft model were employed to analyze the function of DACH1.

Results Complete promoter region methylation and loss of DACH1 expression were found in 1 of 5 colorectal cancer cell lines (HCT116). Re-expression of DACH1 was induced by 5-aza-2′-deoxycytidine treatment in HCT116, but no expression changes was found in 4 unmethylated cell lines before and after 5-AZ treatment. DACH1 methylation was found in 48% (48/100) of colorectal cancer, 6.67% of colonic polyps (1/15), and no methylation was detected in normal colorectal mucosa. Methylation of DACH1 was associated with late tumor stage, poor differentiation and lymph node metastasis significantly (p&lt;0.05). Dual-luciferase assay showed that DACH1 can inhibit the activity of both the TGF-β and Wnt signaling pathways. Re-expression of DACH1 inhibited colony formation and cell proliferation, and induced cell cycle arrest in G2/M, but no effect was found on apoptosis in HCT116 cells. Re-expression of DACH1 inhibited the expression of CyclinD1, c-myc and cdc2. Inhibition of tumorigenecity was found in DACH1 expressed HCT116 cell xenograft.

Conclusion DACH1 was silenced by promoter region hypermethylation in colorectal cancer, methylation of DACH1 may serve as a potential CRC detection marker. DACH1 may serve as a tumor suppressor by inhibiting both TGF-β and Wnt signaling in colorectal cancer.

Citation Format: Mingzhou Guo, Wenji Yan, Yunsheng Yang. Epigenetic silencing DACH1 activated both TGF-β and Wnt signaling in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2974. doi:10.1158/1538-7445.AM2013-2974