Demethylation of the RB1 promoter concomitant with reactivation of TET2 and TET3 impairs gastric carcinogenesis in K19-Wnt1/C2mE transgenic mice

Abstract

Aberrant methylation of promoter CpG islands (CGIs) can inactivate the expression of many tumor suppressor genes and play an important role in the carcinogenesis of gastric cancer. The tumor suppressor gene RB1, which encodes a cell cycle regulator, is hypermethylated and downregulated in multiple kinds of cancer. Activation of RB1 expression through DNA demethylation is a potential strategy for the treatment of gastric cancer. Herein, we found that the methylation status of the RB1 promoter was negatively related to the development of gastric tumors, while its expression was positively correlated with TET2 and TET3 expression. Further reactivation of RB1 expression by curcumin could inhibit gastric cell viability and carcinogenesis both in vitro and in vivo. Molecular docking and other studies confirmed that curcumin could bind to and upregulate the expression of TET2 and TET3 with hydrogen bonds and arene-H bonds, suggesting that demethylation of RB1 was attributed to reactivation of the demethylation enzymes TET2 and TET3 after curcumin treatment. Thus, our findings reveal a promising therapeutic strategy for gastric cancer prevention and treatment through RB1 demethylation and reactivation.

Introduction

Gastric cancer is the third leading cause of cancer-related death and the sixth most common malignancy in the world [1]. Gastric cancer is a consequence of multiple genetic and epigenetic alterations and the aberrant activation of oncogenes or inactivation of tumor suppressor genes [2]. Currently, the prevention and treatment of cancer is predominantly focused on targeting oncogenes, while less effort has been focused on activating tumor suppressor genes.

Increasing evidence indicates that the hypermethylation of CpG islands within tumor suppressor genes is an important mechanism for inactivating gene expression and has been recognized as one of the hallmarks of cancer [3]. The RB1 gene encodes a 110-kDa nuclear phosphoprotein that functions as a cell cycle regulator [4]. RB1 is downregulated in multiple kinds of cancer [5], and the methylation of RB1 is considered a prognostic indicator of bladder cancer progression [6]. DNA hypermethylation is induced not only by the aberrant activation DNA methyltransferases (DNMTs) but also by the silencing of ten-eleven translocation (TET) family members. DNMTs, including DNMT1, DNMT3a and DNMT3b, which are responsible for establishing and maintaining DNA methylation patterns [7], were expressed at significantly higher levels in gastric cancer samples than in paired control samples [8]. The TET family members, including TET1, TET2 and TET3, remove methyl groups from methylated DNA [9]. Considering that DNA methylation is relatively reversible and dynamic, the pharmacological activation of RB1 by demethylation of its promoter offers an opportunity to enhance its tumor suppressor function.

Although the DNA methylation inhibitors 5-azacytidine/VIDAZA (AZA) and 5-aza-2′-deoxycytidine/DACOGEN (DAC) have been approved by the FDA for the treatment of MDS [10], off-target toxicities limit their application and development for other tumors. Dietary components have emerged as promising sources with the ability to reverse the DNA methylation and regulate the gene expression implicated in tumorigenesis. Curcumin (C21H20O6, MW: 368.4), which is a key polyphenolic component in turmeric roots, exhibits anticancer effects on a wide variety of cancer cells but not on normal cells [11]. In clinical trials, curcumin has been used either alone or in combination with other agents, and 44 clinical trials and five animal studies have indicated its therapeutic benefits [12]. Curcumin acts as a potent DNA hypomethylating agent that downregulates DNMTs in different cancer models [13]. However, whether TETs are also influenced by curcumin has not been studied in vitro or in vivo thus far.

Chemically, curcumin is diferuloyl methane, and a methylene bridge links two ferulic acid residues in the structure. Curcumin can exist in many different conformations, which allows curcumin to bind directly to several target molecules to modulate their biological activities. Over the years, some computational methods, such as molecular docking, have been developed to predict the putative binding targets and affinities between ligands and receptors [14]. Although multiple kinases have been indicated as targets to which curcumin binds with high affinity, whether curcumin binds to the epigenetic DNMT and TET enzymes has not yet been determined.

In this study, we explored the methylation levels of RB1 in gastric tumors and confirmed that a high methylation level was associated with a risk of lymph node metastasis. Moreover, we explored the ability of the demethylation reagent curcumin to prevent gastric tumorigenesis and development both in gastric cancer cell lines in vitro and in transgenic gastric tumor-carrying K19-Wnt1/C2mE mice in vivo. In addition, whether the epigenetic enzymes were molecular targets of curcumin was predicted chemically. This research will provide an experimental basis for the development of curcumin as a new therapeutic strategy.