Research Article: Methylomics analysis identifies a putative STAT3 target, SPG20, as a noninvasive epigenetic biomarker for early detection of gastric cancer

Date Published: June 13, 2019

Publisher: Public Library of Science

Author(s): Kuo-Liang Wei, Jian-Liang Chou, Yin-Chen Chen, Hongchuan Jin, Yu-Min Chuang, Cheng-Shyong Wu, Michael W. Y. Chan, Hiromu Suzuki.

http://doi.org/10.1371/journal.pone.0218338

Abstract

Gastric cancer is a leading cause of cancer worldwide. Our previous studies showed that aberrant activation of JAK/STAT3 signaling confer epigenetically silences STAT3 target genes in gastric cancer. To further investigate the clinical significance of this phenomenon, we performed Illumina 850K methylation microarray analysis in AGS gastric cancer cells, and cells depleted of STAT3. Integrative computational analysis identified SPG20 as a putative STAT3 epigenetic target, showing promoter hypomethylation in STAT3-depleted AGS cells. Bisulphite pyrosequencing and qRT-PCR confirmed that SPG20 is epigenetically silenced by promoter hypermethylation in a panel of gastric cancer cell lines including AGS cells, but not in immortalized gastric epithelial GES cells. Expression of SPG20 could be restored by the treatment with a DNMT inhibitor, further suggesting that SPG20 is epigenetically silenced by promoter methylation. Clinically, a progressive increase in SPG20 methylation was observed in tissues samples from gastritis (n = 34), to intestinal metaplasia (IM, n = 33), to gastric cancer (n = 53). Importantly, SPG20 methylation could be detected in cell-free DNA isolated from serum samples of gastritis, IM and gastric cancer patients, having a progressive similar to tissues. Taken together, SPG20, a potential STAT3 target, is frequently methylated in gastric cancer, representing a novel noninvasive biomarker for early detection of this deadly disease.

Partial Text

Gastric cancer is a leading cause of cancer deaths worldwide [1]. Despite advances in cancer therapy, gastric cancer patients still have a poor 5-year survival of less than 15% [2], likely due to a lack of biomarkers for early detection. Although endoscopic screening for early gastric cancer may improve patient’s survival, noninvasive, sensitive, and specific population screening assays are currently not available and are therefore urgently needed.

Our previous studies found that aberrant activation of JAK/STAT signaling could lead to epigenetic silencing of STAT3 targets in gastric cancer [12, 13]. We therefore hypothesized that binding of STAT3 to promoter-proximal CpG islands may affect their methylation status. In this regard, we performed Illumina 850K methylation microarray analysis in bisulphite-treated genomic DNA from AGS gastric cancer cells, and cells depleted of STAT3. Computational predictions were also performed to identify all STAT3-binding sites located in open chromatin regions (as demarcated by H3K4me1 and H3K27Ac) in close proximity to promoter CpG islands (Fig 1A). One probe (cg25179758, Fig 1B and 1C) within the promoter region of SPG20, showing differential hypomethylation in STAT3-depleted AGS cells, was identified. Bisulphite pyrosequencing further confirmed that knock-down of STAT3 decreased SPG20 methylation in AGS cells (Fig 1D).

DNA methylation, an “epigenetic” mode of transcriptional regulation, is altered in numerous pathologies. Consequently, due to its chemical uniqueness and stability (e.g., vs. RNA), disease-associated methylated DNA sequences represent promising tissue and liquid biomarkers. In the current study, by methylation microarray, we identified that a potential STAT3 target, SPG20, is differentially methylated in gastric cancer. Cell line studies further confirmed that SPG20 is epigenetically silenced, by DNA methylation, in gastric cancer cell lines. Clinical studies also demonstrated SPG20 hypermethylation in tissues and serum samples from IM and gastric cancer patients. Importantly, a progressive increase in SPG20 methylation, from gastritis to gastric cancer, was observed.

 

Source:

http://doi.org/10.1371/journal.pone.0218338

 

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