Abstract 4352: Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis

Abstract

Abstract The existence of a cancer stem cell (CSCs) subset is well established in hepatocellular carcinoma (HCC). This subset can be characterized by its CD133 expression. Since the discovery of this marker, studies have revealed deregulated pathways and epigenetic alterations associated with this subset, but few have focused on changes in DNA methylation. This study aims to fill this research gap by discovering aberrant DNA methylation patterns governing CD133+ CSC-driven HCC. Results from Infinium HumanMethylation450 BeadChip (HuMet450 BeadChip) have led to the discovery of differential methylation patterns between CD133+ liver CSCs and matched CD133- differentiated samples. ZFP42/REX1 is one of the top-ranking differentially methylated candidates. Probes corresponding to its promoter are extensively hypermethylated in the CD133+ samples. To explore the hypothesis that promoter DNA hypermethylation modulates REX1 expression, REX1 mRNA levels were first validated to be down regulated in sorted CD133+ HCC cell lines (n=2) and HCC samples (n=46, p<0.05) by RT-qPCR. Upon 5-AZA treatment, only low-REX1-expressing cells re-expressed REX1. By in silico prediction, a CpG island of interest located in the REX1 promoter region was selected for further investigation. Results of pyrosequencing of this CpG island show that REX1 expression appears to be negatively correlated with the degree of DNA methylation in HCC cell lines (n=3); and that HCC samples (n=13) exhibit a predominantly hypermethylated state compared to the matched non-tumor samples. To add onto the clinical relevance of REX1, two HuMet450 BeadChip datasets of HCC sample cohorts (GSE54503 and GSE60753) were obtained from Gene Expression Omnibus (GEO). In these cohorts, the CpG probes corresponding to the REX1 promoter region are significantly hypermethylated (p<0.01) in HCC samples compared to the adjacent non-tumor tissues. Lentiviral-based approaches were adopted to establish stable REX1 overexpressing and knockdown cells in HCC cell lines. REX1 expression appears to be negatively correlated with the cells’ abilities to migrate, invade, and form foci in vitro; and tumorigenicity in vivo. ChIP-seq has been adopted to study the DNA-binding property of REX1 in HCC cells. These findings suggest that REX1 may play a part in mediating various cancer properties in HCC. Citation Format: Steve Tin-Chi Luk, Man Tong, Kai Yu Ng, Kevin Yuk-Lap Yip, Xin Yuan Guan, Stephanie Ma. Identification of ZFP42/REX1 as a regulator of cancer stemness in CD133+ liver cancer stem cells by genome-wide DNA methylation analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4352. doi:10.1158/1538-7445.AM2017-4352