Prostaglandin E2 induces DNA hypermethylation in gastric cancer in vitro and in vivo

Abstract

Rationale: Prostaglandin E₂ (PGE₂) is a pro-inflammatory eicosanoid up-regulated in gastric cancer (GC). However, its impact on epigenetic dysfunction in the process of gastric carcinogenesis is unknown. In this study, we investigate the role of PGE₂ in DNA methylation in gastric epithelium in vitro, in mice, and humans. Methods: PGE₂-induced DNMT3B and DNA methylation was determined in gastric cell lines and COX-2 transgenic mice. Effect of COX-2 inhibition on DNA methylation was evaluated in a randomized controlled trial. Efficacy of combined COX-2/PGE₂ and DNMT inhibition on GC growth was examined in cell lines and mice models. Results: PCR array analysis of PGE₂-treated GC cells revealed the up-regulation of DNMT3B, a de novo DNA methyltransferase. In GC cells, PGE₂ induced DNMT3B expression and activity, leading to increased methylated cytosine (5mC) and promoter methylation of tumor suppressive genes (MGMT and CNR1). Consistently, Cox-2 (rate-limiting enzyme for PGE₂ biosynthesis) transgenic expression in mice significantly induced Dnmt3b expression, increased 5mC content, and promoted Mgmt promoter methylation. We retrospectively analyzed the 5mC content of 42 patients with intestinal metaplasia (a precancerous lesion of GC) treated with a COX-2 specific inhibitor Rofecoxib or placebo for 2 years, revealing that the COX-2 inhibitor significantly down-regulated 5mC levels (N=42, P=0.009). Collectively, these data indicate that PGE₂ is closely related to DNA hypermethylation in vitro and in vivo. Using genome-wide 450K methylation array, we identified chromosomal genes (POT1, ATM and HIST1H2AA) were preferentially methylated by PGE₂. Biofunctional work revealed that POT1 functions as a tumor suppressor. Finally, we demonstrated that combinatorial inhibition of COX-2 and DNMT using Celecoxib and Decitabine synergistically inhibited GC growth in vitro and in vivo. Conclusion: This study suggested that PGE₂ promotes DNA methylation in GC, and that co-targeting of PGE₂ and DNMT inhibits GC.

Keywords: COX-2 transgenic mice; DNA methylation; DNMT3B; Gastric cancer; Prostaglandin E2.

Figure 1

PGE₂ induced expression of DNA methyltransferase 3B (DNMT3B) in gastric cancer. (a) Epigenetic Chromatin Modification Enzymes PCR array analysis of PGE₂-treated KATOIII cells revealed alteration of various epigenetic regulators. (b) RT-PCR analysis of DNMT1 and DNMT3B mRNA expression after PGE₂ treatment in KATOIII cells. (c) Western blot showed that PGE₂ uniformly induced DNMT3B, but not DNMT1 or DNMT3A protein in GC cell lines. The relative expression was determined by densitometry analysis (Image Lab Software).

Figure 2

PGE₂ induced DNMT3B activity, 5mC levels and promoter DNA methylation. (a) PGE₂ increased nuclear DNMT3B activity in HGC27 and MKN1 cells, as determined by DNMT3B assay kit. (b) Knockdown of EP2 or EP4 in HGE27 cells abrogated the induction of DNMT3B expression by PGE₂. (c) PGE₂ promoted 5mC levels in HGC27 and MKN1 cells, as indicated by dot blot assay (d) PGE₂ induced promoter methylation of O⁶-Methylguanine-DNA Methyltransferase (MGMT) in gastric cancer cells, leading to down-regulation of MGMT mRNA expression. (e) PGE₂ induced promoter methylation of cannabinoid receptor type 1 (CNR1) in HGC27 and MKN1 cells as determined by BGS. CNR1 mRNA was also down-regulated by PGE₂ in HGC27 cells. MGMT and CNR1 promoter methylation were determined by Bisulfite sequencing.

Figure 3

PGE₂ induced DNMT3B expression and DNA methylation in vivo. (a) COX-2 transgenic mice were treated with water or MNU and gastric tissues were collected at 50 weeks. (b) Western blot showed that Dnmt3b, but not Dnmt3a, was to be overexpressed in mice with transgenic COX-2 overexpression. (c) Transgenic COX-2 mice revealed DNA hypermethylation, as evidenced by significantly increased 5mC levels (WT-vehicle, N=15; COX-2 TG-vehicle, N=11; WT-MNU, N=22; COX-2 TG-MNU, N=16). (d) Transgenic COX-2 mice showed increased Mgmt promoter as determined by BGS in both vehicle (WT, N=7; COX-2 TG, N=6) and MNU treatment groups (WT, N=13; COX-2 TG, N=11). (e) Correlation between levels of PTGS2 (COX-2), and DNMT1, DNMT3A and DNMT3B in human gastric cancer patients. PTGS2 mRNA is correlated with the expression levels of DNMT3A and DNMT3B (right), but not of DNMT1 (left).

Figure 4

COX-2 specific inhibitor slowed the elevation of 5mc levels in patients with intestinal metaplasia. (a) Representative IHC staining of 5mC in gastric tissues from intestinal metaplasia patients treated with placebo or rofecoxib for 2 years. (b) IHC scoring by a pathologist at baseline and year 3 revealed that placebo group had significantly increased 5mC score, but no statistically significant difference was shown in rofecoxib group. (c) Alteration in the 5mC levels between year 1 and 3 (Δ5mC) was significant decreased in rofecoxib group as compared to placebo.

Figure 5

Global profiling of PGE₂-induced DNA methylation. HGC27 and MKN1 cells treated with PGE₂ for 48 h and their global DNA methylation profiles were assessed by Illumina 450K methylation array. (a) PGE₂ treatment increased the overall methylation level (β-value) in all cell lines. (b) PGE₂ induced hypermethylated loci in GC cells is greater than that of hypomethylated loci. (c) Commonly hypermethylated and hypromethylated genes in HGC27 and MKN1 cells after PGE₂ treatment. (d) GO enrichment (WebGestalt) identified 3 biological process, chromosomal region, nuclear chromosome and spliceosomal complex, that were associated with hypermethylated loci. (e) POT1 promoter was methylated by PGE₂, leading to silencing of POT1 mRNA expression (f) POT1 was overexpressed in HGC27 and MKN1 cells. (g and h) POT1 overexpression inhibited GC cell proliferation, as indicated by MTT and colony formation assays. (i) POT1 overexpression induced apoptosis in MKN1 cells.

Figure 6

Simultaneous blockade of COX-2 (Celecoxib/Sulindac) and DNMT (Decitabine) synergistically inhibited gastric cancer growth. (a and b) Effect of Celecoxib/Sulindac, Decitabine or their combination on HGC27 and MKN1 cell proliferation, as assessed by cell growth curve assay. (c and d) Celecoxib/Sulindac, Decitabine or their combination on HGC27 and MKN1 cell apoptosis. In both assays, the drug combination more effectively inhibited cell growth as compared to single agents (p<0.0001).

Figure 7

(a) Celecoxib or (b) Sulindac plus Decitabine induced apoptosis in HGC27 and MKN1 cells more potently compared to either drug alone. (c) Combination of Celecoxib (10mg/kg) and Decitabine (5mg/kg) synergistically inhibited the growth HGC27 xenografts in nude mice (p<0.03).