DACH1Was Regulated By Promoter Region Hypermethylation And Was Involved InTGF-pand Wnt Signaling Pathway In Colorectal Cancer

Background Colorectal cancer (CRC) is the second leading cause of cancer-relateddeaths in most Western countries. The incidence of CRC is increasing very fast with thechanging of diet and life style in China. Epigenetic changes were regarded as animportant factor of human colorectal cancer. Both Wnt and TGF-β signaling pathwayswere regarded to play important roles in the development of colorectal cancer. Recentstudies have found that the TGF-β and Wnt signaling pathways are crosstalking duringbreast cancer carcinogenesis and other situations. Reduced expression of DACH1wasreported in various cancers including endometrial cancer, breast and prostate cancer.DACH1was found can inhibit TGF-β signaling pathway in breast and ovarian cancer.The epigenetic regulation and the function of DACH1in colorectal cancer remainunclear.Objective The methylation status of DACH1was analyzed in colorectal cancer celllines and tissue samples to evaluate it as CRC detection marker. The epigeneticregulation of DACH1expression and the effect of DACH1on colorectal carcinogenesiswere evaluated to understand CRC development. The role of DACH1in TGF-β&Wntsignaling pathways was analyzed to find new approaches for prvention and treatment ofCRC.Methods Semi-quantitative RT-PCR was used to examine DACH1expression incolorectal cancer cell lines before and after5-aza-deoxycytidine treatment. Methylationspecific PCR (MSP) was employed to detect DACH1methylation status in5colorectalcancer cell lines,8cases of normal mucosa,15cases of colon polyps and100cases ofprimary colorectal cancer. The association of DACH1methylation and clinical factors of CRC patients was analyzed by SPSS13.0software, p<0.05was regarded assignificant difference. Expression of DACH1, CylinD1and β-catenin were detected byimmunohistochemistry (IHC) in30cases of available matched colorectal cancer andadjacent tissue samples. Dual-Luciferase reporter assay was applied to evaluate theeffect of DACH1on TGF-β&WNT signaling pathway. The effect of DACH1onTGF-β&WNT downstream targets(c-myc, CyclinD1, etc) were analyzed by westernblot. Colony formation, cell proliferation assay, flow cytometry analysis and mousexenograft model were employed to analyze the function of DACH1in colorectalccarcinogenesis.Results Complete promoter region methylation and loss of DACH1expression werefound in1of5colorectal cancer cell lines (HCT116). Re-expression of DACH1wasinduced by5-aza-2’-deoxycytidine treatment in HCT116, but no expression changeswas found in the other4unmethylated CRC cell lines before and after5-aza-2’-deoxycytidine treatment. DACH1methylation was found in48%(48/100) ofcolorectal cancer,6.67%of colonic polyps (1/15), and no methylation was detected innormal colorectal mucosa. Methylation of DACH1was associated with late tumor stage,poor differentiation and lymph node metastasis significantly in CRC (p<0.05).Dual-luciferase assay showed that DACH1can inhibit the activity of both the TGF-βand Wnt signaling pathways. Re-expression of DACH1in HCT116cell line inhibitedcolony formation and cell proliferation, and induced cell cycle arrest in G2/M, but noeffect was found on apoptosis. Re-expression of DACH1inhibited the expression ofCyclinD1, c-myc and cdc2in HCT116cell line. Inhibition of tumorigenecity was foundin xenograft mice by re-expression of DACH1in HCT116cells.Conclusion DACH1was silenced by promoter region hypermethylation in colorectalcancer. DACH1may serve as a tumor suppressor in colorectal cancer via inhibitingTGF-β and Wnt signaling pathways. Methylation of DACH1may serve as a potentialCRC detection marker.