Quantitative assessment of <i>RUNX3</i> methylation in neoplastic and non‐neoplastic gastric epithelia using a DNA microarray

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

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“…In agreement, recent work found that P2 methylation levels of <10% can be reported as ‘hypermethylated’ by MSP. 45 …”

Section: Discussionmentioning

confidence: 99%

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer

Kurklu

Ong

et al. 2014

Oncogene

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“…showed that RUNX3 expression was decreased in 60% of primary human gastric tumors; with nearly 90% of late stage tumors that represent highly metastatic tumors showing reduced RUNX3 levels. An analysis of clinical tissue samples from peritoneal metastases arising from gastric cancers showed that RUNX3 expression decreased significantly in the metastatic tissue, compared to normal gastric mucosa or primary main tumors 19, 20. Therefore, RUNX3 deficiency is closely associated with gastric cancer metastasis.…”

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confidence: 99%

Runx3 suppresses gastric cancer metastasis through inactivation of MMP9 by upregulation of TIMP‐1

Chen

Wei

Guo

et al. 2011

Intl Journal of Cancer

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Recent studies have suggested that loss of RUNX3 expression is involved with gastric tumor metastasis. However, the precise mechanism of RUNX3-mediated suppression of tumor metastasis remains elusive. We aimed to clarify the effect of RUNX3 on tumor metastasis in gastric cancer cell lines and tumors. Immunohistochemistry revealed that RUNX3 was significantly decreased in metastatic gastric cancer. Gelatin zymography and Western blot showed that instead of regulating matrix metalloproteinase 9 (MMP9) expression, RUNX3 expression inhibited MMP9 enzyme activity, and this was consistent with the upregulation of tissue inhibitor of metalloproteinases 1 (TIMP1) by RUNX3. TIMP1 siRNA treatment impaired RUNX3-mediated suppression of gastric cancer cell invasion. Reporter assays demonstrated regulation of TIMP-1 by RUNX3. Two RUNX3 binding sites were identified in the TIMP-1 promoter and direct interaction of RUNX3 with the TIMP-1 promoter was confirmed in vitro and in vivo. These findings provide evidence for RUNX3-mediated suppression of gastric cancer invasion and metastasis and define a novel molecular mechanism that for the metastasis-inhibiting activity of RUNX3. These data may be applied in the development of RUNX3 for gastric cancer metastasis diagnostics and therapeutics.Gastic cancer is second only to lung cancer as a cause of cancer-related deaths worldwide, and poor prognosis and high mortality continue to be problems with this disease. 1-3 Approximately 20% of patients survive to 5 years, 4 and metastasis is a major cause of mortality in gastric cancer patients. Clearly, improvements in treatment depend on understanding the molecular mechanisms of gastric cancer metastasis. In general, cancer metastasis consists of a series of sequential, interrelated events involving growth factors, cell-adhesion molecules, matrix-degradation enzymes and motility factors. These molecules induce not only cell growth but also the extracellular matrix (ECM) degradation and angiogenesis that are required for tumor invasion and proliferation. 5 Previous research on the molecular pathology of gastric cancer metastasis identified a variety of key oncogenes and tumor suppressor genes in this process, such as c-erbB2, c-met, p27 and pRb. [6][7][8][9][10][11] Recently, deficiency in the Runt-related gene RUNX, which is a candidate tumor suppressor, was found to be causally related with gastric cancer. 12 The RUNX family members, RUNX1, RUNX2 and RUNX3, encode DNA-binding a subunits that bind a common b subunit, CBFb, to generate heterodimeric transcription regulators. 13 All three RUNX family members play important roles in normal developmental processes and carcinogenesis. 14-18 Recent studies showed that RUNX3 expression levels are downregulated in gastric cancer, either from hemizygous deletion or from promoter methylation of the RUNX3 gene. Furthermore, a decrease in RUNX3 protein expression is significantly associated with decreased survival of gastric cancer patients. Li et al. 12 showed that RUNX3 expression was decreased in ...

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“…The successful identification of differentially methylated regions (DMRs) associated with aging in studies of candidate loci (12)(13)(14) has stimulated interest in mapping all such sites * To whom correspondence should be addressed at: Center for Biomarker Research and Personalized Medicine, McGuire Hall, Medical College of Virginia campus, Virginia Commonwealth University, 1112 East Clay Street, Richmond, VA 23298, USA. Tel: +1 8048283428; Fax: +1 8046283991; Email: jlmcclay@vcu.edu genome-wide.…”

Section: Introductionmentioning

confidence: 99%

A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects

McClay¹,

Åberg²,

Clark³

et al. 2013

Human Molecular Genetics

150

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The central importance of epigenetics to the aging process is increasingly being recognized. Here we perform a methylome-wide association study (MWAS) of aging in whole blood DNA from 718 individuals, aged 25 -92 years (mean 5 55). We sequenced the methyl-CpG-enriched genomic DNA fraction, averaging 67.3 million reads per subject, to obtain methylation measurements for the ∼27 million autosomal CpGs in the human genome. Following extensive quality control, we adaptively combined methylation measures for neighboring, highly-correlated CpGs into 4 344 016 CpG blocks with which we performed association testing. Eleven age-associated differentially methylated regions (DMRs) passed Bonferroni correction (P-value < 1.15 3 10 28 ). Top findings replicated in an independent sample set of 558 subjects using pyrosequencing of bisulfite-converted DNA (min P-value < 10 230 ). To examine biological themes, we selected 70 DMRs with false discovery rate of <0.1. Of these, 42 showed hypomethylation and 28 showed hypermethylation with age. Hypermethylated DMRs were more likely to overlap with CpG islands and shores. Hypomethylated DMRs were more likely to be in regions associated with polycomb/regulatory proteins (e.g. EZH2) or histone modifications H3K27ac, H3K4m1, H3K4m2, H3K4m3 and H3K9ac. Among genes implicated by the top DMRs were protocadherins, homeobox genes, MAPKs and ryanodine receptors. Several of our DMRs are at genes with potential relevance for age-related disease. This study successfully demonstrates the application of next-generation sequencing to MWAS, by interrogating a large proportion of the methylome and returning potentially novel age DMRs, in addition to replicating several loci implicated in previous studies using microarrays.

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Quantitative assessment of RUNX3 methylation in neoplastic and non‐neoplastic gastric epithelia using a DNA microarray

Cited by 24 publications

References 13 publications

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer

Runx3 suppresses gastric cancer metastasis through inactivation of MMP9 by upregulation of TIMP‐1

A methylome-wide study of aging using massively parallel sequencing of the methyl-CpG-enriched genomic fraction from blood in over 700 subjects

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