Abstract 3315: 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 (CSC) subset is well established in hepatocellular carcinoma (HCC). This subset can be characterized, among others, 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 study the 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. We validated the down-regulation of REX1 mRNA levels in sorted CD133+ HCC cell lines (n=4) and HCC clinical samples (n=46, p<0.05), as compared to CD133- counterparts and non-tumor liver samples. REX1 and CD133 expressions are also significantly negatively correlated in HCC cell lines (n=8, R2=0.65). To explore the hypothesis that promoter DNA hypermethylation modulates REX1 expression, we treated HCC cells with 5-aza. Only low-REX1-expressing cells re-expressed REX1. Results of pyrosequencing of a CG island of interest located in the REX1 promoter show that REX1 expression appears to be negatively correlated with the degree of DNA methylation in HCC cell lines (n=4); and that HCC samples (n=15) exhibit a predominantly hypermethylated state compared to non-tumor samples (n=16). To add onto the clinical relevance of REX1, HuMet450 BeadChip datasets of HCC sample cohorts were obtained from Gene Expression Omnibus and the Cancer Genome Atlas. In these cohorts, the REX1 promoter region is significantly hypermethylated in HCC samples and this methylation is correlated to poor prognosis as well as increased expression of stemness markers. In addition to DNA methylation, we also discovered other regulatory mechanisms for REX1 expression involving histone methylation and acetylation. Functional studies with stable REX1 over-expressing and knock-down HCC cells suggest REX1 expression to negatively correlate with the cells' abilities to migrate, invade, and form foci in vitro; and initiate tumor formation, metastasize and self-renew in vivo. In summary, REX1 is a tumor-suppressor gene that is frequently down-regulated in human HCCs. The multiplicity of REX1 deactivating mechanisms together facilitates REX1 down-regulation to mediate various cancer and stem-like properties in the disease. 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 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3315.