Role of REX1 down-regulation in promoting cancer and stemness properties in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC), a deadly disease with poor prognosis, is often complicated by challenges including late diagnosis, frequent tumor relapse, and the chemoresistant nature of the tumor. Since HCC is especially prevalent in our locality, the importance and urgency for the identification of more effective treatment regimens and diagnostic markers cannot be undermined. The existence of cancer stem cells (CSCs) is confirmed in many cancers including HCC. These cells are important players in the hepatocarcinogenesis process and have been implicated in tumor relapse and drug resistance. They can be characterized, among others, by their surface CD133 expression. Although many studies have revealed deregulated pathways and epigenetic alterations in these 〖CD133〗^+ liver CSCs, there has not been any prior study that focused on global DNA methylation changes in this subset. Results from HuMet450 BeadChip have led to the discovery of differential methylation patterns between 〖CD133〗^+ liver CSCs and CD133- non-CSCs. By analysing commonly differentially methylated CpG islands, REX1 was one of the top-ranking candidates. REX1 downregulation was present in 〖CD133〗^+ HCC cell lines and clinical specimens, and said downregulation was negatively correlated to CD133 expression in HCC cell lines. HuMet450 BeadChip datasets of HCC cohorts extracted from Gene Expression Omnibus revealed that REX1 promoter was significantly hypermethylated in HCC specimens when comparing to non-tumor tissue. Modulation of REX1 expression by promoter DNA hypermethylation was explored by 5-AZA treatment and pyrosequencing of a CpG island of interest in the REX1 promoter. Results demonstrated a negative correlation between REX1 expression and its DNA methylation status in HCC cell lines; and that 〖CD133〗^+ HCC cells and HCC tissue exhibited a predominantly hypermethylated state. In addition, modulation of REX1 expression by histone modifications including changes in H3Ac, H4Ac, and H3K27me3 marks was also discovered. Functional studies using stable REX1 overexpressing and knockdown HCC cells suggested 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 light of this, REX1 overexpressing HCC cells were analyzed by RNA-Seq. DAVID and IPA identified potential deregulated pathways. REX1 was found to bind directly to the MKK6 promoter, leading to its downregulation and subsequent inactivation of its downstream target p38 MAPK and NRF2-mediated oxidative response, and an elevation of intracellular reactive oxygen species. Suppression of p38 MAPK by specific inhibitor SB202190 or suppression of NRF2 by specific inhibitor ML385 could revert the enhancement of migratory and foci-forming abilities observed in REX1-silenced HCC cells. In summary, frequent downregulation of REX1 in human HCCs by multiple deactivating mechanisms mediates various cancer and stem-like properties in the disease via an activated MKK6/p38 MAPK/NRF2/ROS axis.