Yunsheng Yang

Epigenetic silencing of DACH1 induces loss of transforming growth factor‐β1 antiproliferative response in human hepatocellular carcinoma

Human dachshund homolog 1 (DACH1) is a major component of the Retinal Determination Gene Network (RDGN) and functions as a tumor suppressor. However, the regulation of DACH1 expression and its function in hepatocellular carcinoma (HCC) remain unclear. In this study, epigenetic changes of DACH1 were analyzed in HCC cell lines and primary cancers. We found that promoter region hypermethylation was correlated with loss or reduction of DACH1 expression, and restoration of DACH1 expression was induced by 5‐aza‐2′‐deoxycytidine (5‐AZA) in HCC cell lines. Promoter region methylation was found in 42% of primary HCC. Reduced expression of DACH1 was associated with poor differentiation of HCC nodules and higher serum aspartate aminotransferase/alanine aminotransferase ratio. DACH1 suppressed cellular growth by reactivating transforming growth factor beta (TGF‐β) signaling. Ectopic expression of DACH1 enhanced chemosensitivity to 5‐fluorouracil (5‐FU) by inducing p21 expression in HCC cells. Conclusion: DACH1 is frequently methylated in HCC and DACH1 expression is regulated by promoter hypermethylation. Down‐regulation of DACH1 is a novel mechanism for gaining resistance to the antiproliferative signaling of TGF‐β1 and 5‐FU resistance. (Hepatology 2013; 58:2012–2022)

Epigenetic regulation of DACT2, a key component of the Wnt signalling pathway in human lung cancer

Dapper, Dishevelled‐associated antagonist of β‐catenin (DACT), is involved in Xenopus embryonic development. Human DACT2 is localized on chromosome 6q27, a region of frequent loss of heterozygosity (LOH) in human cancers. However, the function and regulation of DACT2 in human lung cancer remain unclear. DNA sequencing, methylation‐specific PCR (MSP), semi‐quantitative RT‐PCR, western blotting, and xenograft models were employed in this study. Eight lung cancer cell lines, 106 cases of primary lung cancer, four specimens of normal lung from patients without cancer, and 99 blood samples from healthy individuals were examined. We found that while there was no SNP related to lung cancer, the DACT2 promoter region is frequently methylated in human lung cancer. DACT2 is silenced by promoter region hypermethylation and re‐expressed by 5‐aza‐2′‐deoxyazacytidine treatment of lung cancer cell lines. Methylation of DACT2 was associated with poor differentiation of lung cancer. Loss of DACT2 expression was associated with promoter region hypermethylation in primary lung cancer, and was associated with increased β‐catenin expression. Restoration of DACT2 expression suppressed tumour proliferation both in vitro and in vivo. DACT2 expression was down‐regulated by siRNA knockdown in H727 cells. DACT2 inhibited T‐cell factor/lymphoid enhancer factor (TCF/LEF) and its downstream genes. In conclusion, DACT2 methylation is a potential lung cancer detection marker. DACT2 is regulated by promoter region hypermethylation. DACT2 inhibits lung cancer proliferation by suppressing the Wnt signalling pathway in lung cancer. Copyright

Epigenetic regulation of the Wnt signaling inhibitor DACT2 in human hepatocellular carcinoma

DACT2 (Dapper, Dishevelled-associated antagonist of β-catenin homolog 2) is a member of the DACT family involved in the regulation of embryonic development. Human DACT2 is localized on 6q27, a region of frequent loss of heterozygosity in human cancers. However, the regulation of DACT2 expression and function in hepatocellular carcinoma (HCC) remains unclear. In this study, genetic and epigenetic changes of DACT2 were analyzed in HCC cell lines and primary cancer. We found no single-nucleotide polymorphism (SNP) associated with HCC. Promoter region methylation was correlated with loss or reduction of DACT2 expression, and restoration of DACT2 expression was induced by 5-aza-2’-deoxycytidine (5-AZA) in HCC cell lines. Promoter region methylation was found in 54.84% of primary HCC. Reduction of DACT2 expression was associated with promoter hypermethylation, and expression of DACT2 was inversely related to β-catenin expression in primary HCC. DACT2 suppressed cell proliferation, induced G2-M arrest in cell lines and inhibited tumor growth in xenograft nude mice. The transcriptional activity of TCF-4 and the expression of Wnt signaling downstream genes were suppressed by DACT2 re-expression and reactivated by depletion of DACT2. In conclusion, DACT2 is frequently methylated in HCC and its expression is regulated by promoter hypermethylation. DACT2 suppresses HCC by inhibiting Wnt signaling in human HCC.

AKT signaling pathway activated by HIN-1 methylation in non-small cell lung cancer

The purpose of this study is to determine the epigenetic changes and function of High in Normal-1 (HIN-1) in non-small cell lung cancer (NSCLC). HIN-1 expression was examined by semiquantitative RT-PCR before and after 5-aza-2′-deoxycytidine (5-aza) treatment in NSCLC cell lines. Promoter methylation status of HIN-1 was tested by methylation-specific PCR (MSP). Effect of forced expression of HIN-1 on different key molecules of AKT signaling pathway was tested by Western Blot analysis in H157 and H23 cell lines. Promoter methylations are inversely correlated with expression of HIN-1 in eight (H23, H157, 95D, H1299, H358, H1752, H460, A549) of ten NSCLC cell lines and re-expression was observed by 5-aza treatment. We then tested promoter methylation of HIN-1 in primary NSCLC tissues. Methylation was detected in 73 out of 152 (48%) NSCLC cases. Forced expression of HIN-1in NSCLC cell lines inhibited colony formation and induce apoptosis. Furthermore, overexpression of HIN-1 reduces the expression of phosphorated-AKT (p-AKT), c-myc, Bcl-2 and cyclinD1 while Bax was increased. Our data suggest that HIN-1 is a potential tumor suppressor gene in NSCLC, silenced by promoter hypermethylation and negatively regulate AKT signaling pathway.

Methylation of ZNF331 is an independent prognostic marker of colorectal cancer and promotes colorectal cancer growth

BackgroundZNF331 was reported to be a transcriptional repressor. Methylation of the promoter region of ZNF331 has been found frequently in human esophageal and gastric cancers. The function and methylation status of ZNF331 remain to be elucidated in human colorectal cancer (CRC).MethodsSix colorectal cancer cell lines, 146 cases of primary colorectal cancer samples, and 10 cases of noncancerous colorectal mucosa were analyzed in this study using the following techniques: methylation specific PCR (MSP), qRT-PCR, siRNA, flow cytometry, xenograft mice, MTT, colony formation, and transfection assays.ResultsLoss of ZNF331 expression was found in DLD1 and SW48 cells, reduced expression was found in SW480, SW620, and HCT116 cells, and high level expression was detected in DKO cells. Complete methylation of the ZNF331 in the promoter region was found in DLD1 and SW48 cells, partial methylation was found in SW480, SW620, and HCT116 cells, and unmethylation was detected in DKO cells. Loss of/reduced expression of ZNF331 is correlated with promoter region methylation. Restoration of ZNF331 expression was induced by 5-aza-2′-deoxycytidine (DAC) in DLD1 and SW48 cells. These results suggest that ZNF331 expression is regulated by promoter region methylation in CRC cells. ZNF331 was methylated in 67.1% (98/146) of human primary colorectal cancer samples. Methylation of ZNF331 was significantly associated with tumor size, overall survival (OS), and disease-free survival (DFS) (p < 0.01, p < 0.01, p < 0.05). Methylation of ZNF331 was an independent poor prognostic marker for 5-year OS and 5-year DFS (both p < 0.05). ZNF331 suppressed cell proliferation and colony formation in CRC cells and suppressed human CRC cell xenograft growth in mice.ConclusionsZNF331 is frequently methylated in human colorectal cancer, and the expression of ZNF331 is regulated by promoter region methylation. Methylation of ZNF331 is a poor prognostic marker of CRC.

Abstract 2974: Epigenetic silencing DACH1 activated both TGF-β and Wnt signaling in colorectal cancer.

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 W 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

Abstract 3993: MiRNA-141 and methylation regulation of WNT signal pathway by suppressing SOX17 i n esophageal cancer

Purpose: WNT/β-catenin gene SOX17 was reported frequently methylated in colonic and hepatic cancer. To explore epigenetic changes of SOX17 as esophageal cancer marker and therapeutic target, epigenetic regulation and tumor suppressor function of SOX17 were analyzed in study. Experimental Design: Methylation specific PCR (MSP) was employed to examine SOX17 methylation in 8 esophageal cancer cell lines and 9 normal mucosa, 60 dysplasia and 169 cancer tissue. Semi-quantitative RT-PCR method was used to examine SOX17 expression before and after 5-aza-2′-deoxycytidine treatment of esophageal cancer cells. Expression of SOX17 and β-catenin were detected by immunohistochemistry (IHC) in esophageal cancer and adjacent tissue. Luciferase reporter assay, colony formation, transfection and western blotting were applied to analyze SOX17 function in WNT signal pathway. Prediction software and miRNAs related detection methods were preformed to evaluate miRNA regulation of SOX17. Results: SOX17 was silenced by promoter region methylation and reactivated by 5-aza-2′-deoxycytidine treatment in esophageal cancer cell lines. Loss of SOX17 expression and accumulation of β-catenin in nuclear and cytoplasm were associated with promoter region hypermethylation in esophageal cancer(test, P=0.0359, P=0.0105). Restoration of SOX17 expression inhibited β-catenin/TCF-dependent transcription and colony formation. The presence of SOX17 methylation correlated significantly with alcohol history in esophageal cancer(test, P=0.0057). MiR-141 was also found to down-regulate SOX17 expression and activated the WNT signal pathway. Conclusions: SOX17 is frequently methylated in esophageal cancer, and SOX17 expression is regulated both by miR-141 and promoter region methylation. Loss of SOX17 removes the normal inhibition of WNT signaling and promotes esophageal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3993. doi:1538-7445.AM2012-3993

Abstract LB-90: Sox17 antagonizes Wnt/ß-catenin signaling pathway in HCC

Background: SRY-box containing gene 17 (Sox17) is a candidate tumor suppressor gene, which is indispensable for the embryonic development by antagonizing the canonical Wnt/s-catenin signaling pathway. We investigated the function and epigenetic regulation of Sox17 in human hepatocellular carcinoma (HCC). Methods: Methylation and expression of Sox17 were analyzed in 62 HCC tissues and HCC cell lines by MSP and RT-PCR. Inhibitory effects of Sox17 in Wnt/s-catenin pathway was detected by IHC and luciferase reporter assay. Function as tumor suppressor gene was tested via colony formation assay. Results: Methylation of Sox17 promoter region occurs in 82.26% of HCC tissues and is associated with nucleus location of -catenin. Restoration of Sox17 in HepG2 cells inhibits colony formation and s-catenin/TCF-dependent transcription with the presence of HMG box. Conclusions: Sox17 negatively regulates canonical Wnt/s-catenin signaling pathway, and inhibits human HCC cells growth through epigenetic mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-90.