Abstract 2169: FHL1 on chromosome X is a single-hit gastrointestinal tumor-suppressor gene and contributes to the formation of an epigenetic field defect
Authors: Asada Kiyoshi , Ando Takayuki , Ushijima Toshikazu 
Published: August 2012

Abstract
Tumor-suppressor genes on chromosome X can be inactivated by a single hit, any of point mutations, chromosomal loss, and aberrant DNA methylation. Among these mechanisms, aberrant DNA methylation can be present not only in tumor tissue but also in normal-appearing tissues. Levels of aberrant DNA methylation in non-cancerous tissues are known to correlate with cancer risk for gastric cancers and other cancers (epigenetic field defect). However, only a limited number of genes that functionally contribute to the field defect have been identified. To identify a tumor-suppressor gene on chromosome X that contributes to the formation of an epigenetic field defect, we here searched for genes on chromosome X whose expression was up-regulated by treatment of AGS gastric cancer cell line with a DNA demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC). Among the 495 genes up-regulated at 4-fold or more by treatment with 5-aza-dC, 69 genes were located on chromosome X. Among the 69 genes, 11 genes had low expression (signal intensity &lt; 200) in non-treated AGS cells and high expression (signal intensity &gt; 500) in a pool of gastric mucosae of three healthy volunteers. Among the 11 genes, FHL1 was frequently methylation-silenced in gastric and colon cancer cell lines, and in primary gastric (11/58) and colon (5/50) cancers. In HCT116 cells, knock-down of FHL1 accelerated cell growth (shRNA-1, 243 % of control cells at 120 hours, P &lt; 0.001, and shRNA-2, 191%, P &lt; 0.001) and sizes of xenografts in nude mice (shRNA-1, 2.7-fold larger tumors than that of control cells, P &lt; 0.001). Also in HSC39 cells, knock-down of FHL1 significantly increased cell growth. Expression of exogenous FHL1 in a non-expressing AGS cell line significantly reduced its growth (72.2% of control cells, P &lt; 0.05). A somatic mutation (G642T; Lys214Asn) was identified in one of 144 colon cancer specimens, and the mutant FHL1 was shown to lack a growth-suppressive effect. Among the healthy volunteers, FHL1 methylation was elevated only in H. pylori-positive individuals (0/16 vs. 10/16, P &lt; 0.001). Since potent methylation induction by H. pylori can mask a difference in H. pylori-positive individuals, FHL1 methylation levels were compared between healthy volunteers and gastric cancer patients among the H. pylori-negative individuals, and FHL1 methylation levels were shown to be elevated only in gastric cancer patients (0/16 vs. 5/26, P = 0.06). These data showed that FHL1 is a methylation-silenced tumor-suppressor gene on chromosome X in gastrointestinal cancers, and that its silencing contributes to the formation of an epigenetic field for cancerization.
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 2169. doi:1538-7445.AM2012-2169