Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-β Signaling

et al. (2014) Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-b Signaling. PLoS ONE 9(4): e95509. doi:10.1371/journal.pone.0095509 Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-b Signaling Liang Wu 0 1 James G. Herman 0 1 Malcolm V. Brock 0 1 Kongming Wu 0 1 Gaoping Mao 0 1 Wenji Yan 0 1 Yan Nie 0 1 Hao Liang 0 1 Qimin Zhan 0 1 Wen Li 0 1 Mingzhou Guo 0 1 Dajun Deng, Peking University Cancer Hospital and Institute, China 0 Competing Interests: JGH is a consultant to MDxHealth, however, this does not alter the authors' adherence to PLOS ONE policies on sharing data and materials 1 1 Department of Gastroenterology & Hepatology, Chinese PLA General Hospital , Beijing , China , 2 Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University , Baltimore , Maryland, United States of America, 3 Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology , Wuhan , China , 4 Department of Gastroenterology, General Air Force Hospital , Beijing , China , 5 Department of Gastroenterology & Hepatology, The Affiliated Hainan Hospital of the Chinese PLA General Hospital , Hai Tang wan, Sanya , China , 6 State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College , Beijing , China Human Dachshund homologue 1 (DACH1) is a major component of the Retinal Determination Gene Network. Loss of DACH1 expression was found in breast, prostate, lung, endometrial, colorectal and hepatocellular carcinoma. To explore the expression, regulation and function of DACH1 in human esophageal cancer, 11 esophageal cancer cell lines, 10 cases of normal esophageal mucosa, 51 cases of different grades of dysplasia and 104 cases of primary esophageal squamous cancer were employed. Methylation specific PCR, immunohistochemistry, western blot, flow cytometry, small interfering RNAs, colony formation techniques and xenograft mice model were used. We found that DACH1 expression was regulated by promoter region hypermethylation in esophageal cancer cell lines. 18.8% (6 of 32) of grade 1, 42.1% (8 of 19) of grade 2 and grade 3 dysplasia (ED2,3), and 61.5% (64 of 104) of esophageal cancer were methylated, but no methylation was found in 10 cases of normal esophageal mucosa. The methylation was increased in progression tendency during esophageal carcinogenesis (P,0.01). DACH1 methylation was associated with poor differentiation (P,0.05) and late tumor stage (P, 0.05). Restoration of DACH1 expression inhibited cell growth and activated TGF-b signaling in KYSE150 and KYSE510 cells. DACH1 suppressed human esophageal cancer cell tumor growth in xenograft mice. In conclusion, DACH1 is frequently methylated in human esophageal cancer and methylation of DACH1 is involved in the early stage of esophageal carcinogenesis. DACH1 expression is regulated by promoter region hypermethylation. DACH1 suppresses esophageal cancer growth by activating TGF-b signaling. - Esophageal cancer is the fifth most malignant disease and has been ranked as the fourth leading cause of cancer related deaths in China. [1] Esophageal squamous cell carcinoma (ESCC) is the predominant histological type of esophageal cancer, and accounts for approximately 90% of esophageal cancer cases in the northern and central China. [2] Despite the development of multimodal therapies, the 5 year overall survival remains below 20%. [3] The mechanisms of esophageal carcinogenesis remain unclear. Multiple genetic and epigenetic alterations were regarded as important factors for developing esophageal cancer [46]. Dachshund homolog 1 (DACH1), a major component of the Retinal Determination Gene Network, is widely expressed in epithelial cells. Reduction of DACH1 expression was associated with poor prognosis in breast, prostate, lung, endometrial, colorectal and hepatic cancer. [713] The expression of DACH1 was regulated by promoter region hypermethylation in endometrial, colorectal and hepatocellular cancer. [1113] DACH1 suppressed human hepatocellular carcinoma by activating TGFb signaling. [13] While the epigenetic changes and the function of DACH1 in human ESCC remain unclear. In this study, we mainly analyzed the epigenetic changes and the mechanism of DACH1 on esophageal carcinogenesis. Materials and Methods Ethics Statement The study protocols were approved by the Ethics Committee of the Chinese PLA General Hospital (Permit Number: 20090701015), and written informed consent was obtained from the participants. All procedures of animal research were approved by the Animal Ethics Committee of the Chinese PLA General Hospital (Permit Number: 2013-X8-40) and all efforts were made Figure 1. Representative results of DACH1 expression and methylation in esophageal cancer cells. (A) DACH1 expression level detected by RT-PCR in esophageal cancer cell lines. (B) Methylation status in promoter region; IVD: in vitro methylated DNA, used as methylation (...truncated)