AJTR Copyright © 2009-present, All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711
Am J Transl Res 2013;5(5):555-562

Original Article
Annexin A6 is down-regulated through promoter methylation in gastric
cancer

Xian Wang, Shengjie Zhang, Jianbin Zhang, Emily Lam, Xin Liu, Jie Sun, Lifeng Feng, Haiqi Lu, Jun Yu,
Hongchuan Jin

Department of Medical Oncology, Laboratory of Cancer Biology, Institute of Clinical Science, Sir Runrun Shaw
Hospital, Medical School of Zhejiang University, Hangzhou, China; Institute of Digestive Disease, The Chinese
University of Hong Kong, Hong Kong, China

Received July 1, 2013; Accepted August 2, 2013; Epub August 15, 2013; Published August 30, 2013

Abstract: Background: The aberrant activation of oncogenic signaling such as Ras/MAPK signaling is a frequent
event in human cancers. In addition to genetic changes, epigenetic silencing of inhibitors in Ras/MAPK signaling
contributes to the activation of Ras/MAPK signaling. Recently, ANXA6 has been shown to interact with Ras-GAP1
and inhibit Ras activation in human breast cancer. However, whether and how it is involved in human cancers
remain unknown. Methods: Real-time PCR was used to determine ANXA6 expression in gastric cancer cells and
primary gastric carcinomas. Next, we explored the methylation of ANXA6 promoter in cell lines and tumor tissues
with methylation-specific PCR and bisulfite genomic sequencing. We also investigated the function of ANXA6 in
gastric cancer cells with colony formation assay and western blotting analysis. Results: ANXA6 was
down-regulated in gastric cancer cells and primary gastric carcinomas. Ectopic ANXA6 expression inhibited the
growth of gastric cancer cells and the activity of Ras/MAPK signaling. Its expression was restored after
pharmaceutical demethylation. ANXA6 promoter was methylated in gastric cancer cell lines (6/6) and primary
gastric carcinoma tissues (29/156). Interestingly, the knockdown of oncoprotein Yin Yang 1 (YY1) also restored
ANXA6 expression and promoted the demethylation of ANXA6 promoter. However, ANXA6 methylation was not
associated with clinical parameters such as differentiation, and TNM staging. Neither Kaplan-Meier Curve nor
Cox regression analysis revealed a significant role of ANXA methylation to predict the survival of gastric cancer
patients. Conclusions: We firstly reported that ANXA6 is epigenetically silenced through promoter methylation in
human cancers and YY1 is important to initiate or maintain ANXA6 promoter methylation in gastric cancer cells.
ANXA6 functions as a tumor suppressor in gastric cancer cells through the inhibition of Ras/MAPK signaling.
ANXA6 methylation is not a prognostic factor for gastric cancer patients. (AJTR1307001).

Keywords: ANXA6, methylation, gastric cancer, Ras signaling

Address correspondence to: Dr. Hongchuan Jin, Department of Medical Oncology, Laboratory of Cancer Biology,
Institute of Clinical Science, Sir Runrun Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China.
Tel: +86 571 86006366; Fax: +86 571 86006145; E-mail: jinhc@zju.edu.cn