Abstract
Zinc-finger protein 471 (ZNF471) was preferentially methylated in gastric cancer using promoter methylation array. The role of ZNF471 in human cancer is unclear. Here we elucidated the functional significance, molecular mechanisms and clinical impact of ZNF471 in gastric cancer. ZNF471 mRNA was silenced in 15 out of 16 gastric cancer cell lines due to promoter hypermethylation. Significantly higher ZNF471 promoter methylation was also observed in primary gastric cancers compared to their adjacent normal tissues (P < 0.001). ZNF471 promoter CpG-site hypermethylation correlated with poor survival of gastric cancer patients (n = 120, P = 0.001). Ectopic expression of ZNF471 in gastric cancer cell lines (AGS, BGC823, and MKN74) significantly suppressed cell proliferation, migration, and invasion, while it induced apoptosis in vitro and inhibited xenograft tumorigenesis in nude mice. Transcription factor AP-2 Alpha (TFAP2A) and plastin3 (PLS3) were two crucial downstream targets of ZNF471 demonstrated by bioinformatics modeling and ChIP-PCR assays. ZNF471 directly bound to the promoter of TFAP2A and PLS3 and transcriptionally inhibited their expression. TFAP2A and PLS3 showed oncogenic functions in gastric cancer cell lines. Moreover, ZNF471 recruited KAP1 to the promoter of the target genes, thereby inducing H3K9me3 enrichment for transcriptional repression and inhibition of oncogenic TFAP2A and PLS3. In conclusion, ZNF471 acts as a tumor suppressor in gastric cancer by transcriptionally inhibiting downstream targets TFAP2A and PLS3. KAP1 is a co-repressor of ZNF471 at the promoter of the target genes. The promoter CpG-site methylation is an independent prognostic factor for overall survival of gastric cancer patients.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Gastric cancer remains the fifth most common malignancy all over the world, after cancers of lung, breast, colorectum, and prostate [1]
Bisulfite genomic sequencing (BGS) results indicate that all the gastric cancer cell lines but MKN1 demonstrated hypermethylation at CpG sites in Zinc-finger protein 471 (ZNF471) promoter region, whereas very low methylation was found in GES1 and normal gastric samples (Fig. 1b, c), consistent with ZNF471 expression in cell lines and gastric tissues
A negative association was displayed between ZNF471 promoter methylation and the matched mRNA expression (n = 56, r = −0.5945, P < 0.0001) (Fig. 1g), which was further validated in TCGA data (Pearson’s r = −0.64, P = 8.6e−40) (Figure S1B)
Summary
Zinc-finger proteins (ZFPs), which compose the largest family of transcription factors, tend to be silenced by promoter hypermethylation in gastric cancer [5]. In combination of our in-house gastric cancer DNA methylation (450k) data (gastric cancer cell lines AGS, MGC803, MKN45, normal gastric tissue, and normal gastric epithelial GES1; data unpublished) and TCGA gastric cancer DNA methylation (450k) data, we found that ZNF471 is one the most significantly hypermethylated zinc-finger protein genes, with low mRNA expression in gastric cancer as lines. E The methylation status of ZNF471 promoter in paired patient samples (n = 15). F The mRNA expression of ZNF471 in paired patient samples (n = 56). H Kaplan–Meier curves of patients with gastric cancer, stratified by CpG-site 3 in ZNF471 promoter methylation status.
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