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

Abstract

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.

RUNX3 is down‐regulated in glioma by Myc‐regulated miR‐4295

“…Prior reports have suggested that RUNX3 is frequently inactivated in human cancer cells and can be activated by hemizygous deletion of the RUNX3 gene, hypermethylation of the Runx3 promoter, or cytoplasmic sequestration of RUNX3 protein 32, 34. Furthermore, accumulating evidence demonstrated RUNX3 could inhibit the proliferation, tumourigenic and metastasis of glioma cells 21.…”

Runx3 at the interface of immunity, inflammation and cancer

“…As mentioned earlier, RUNX3 expression is regulated by two promoters. Interestingly, it was shown recently that the RUNX3 P1 promoter is heavily methylated in gastric epithelial cells from H. pylori uninfected humans, which do not express RUNX3, but is completely unmethylated in RUNX3-expressing immune cells [22]. It is thus possible that RUNX3 P1 heavy methylation is responsible for silencing its expression in normal gastric epithelium, providing an explanation for the lack of association between RUNX3 P2 hypermethylation in GC and its expression.…”

“…Similarly, proliferation historydependent DNA methylation events also underlie hematopoietic stem cell (HSC) aging and often occur at Polycomb PRC-2 complex-silenced target genes expressed in various other cell types, but not in HSC [90]. It is interesting to note that following H. pylori infection in mice, Runx3 P2 remains unmethylated in gastric tissue, even at the precancerous gastric intestinal metaplasia stage, but becomes mildly hypermethylated in GC tissue from gp130 F/F mice [22]. In addition, immortalization of mouse gastric epithelial cells from gp130 F/F mice by serial passage in culture is associated with increased Runx3 P2 methylation, and similarly the low methylation ratio of RUNX3 P2 in primary human GC samples (b 15%) is strongly increased to~90% in GC cell lines [22].…”

“…It is interesting to note that following H. pylori infection in mice, Runx3 P2 remains unmethylated in gastric tissue, even at the precancerous gastric intestinal metaplasia stage, but becomes mildly hypermethylated in GC tissue from gp130 F/F mice [22]. In addition, immortalization of mouse gastric epithelial cells from gp130 F/F mice by serial passage in culture is associated with increased Runx3 P2 methylation, and similarly the low methylation ratio of RUNX3 P2 in primary human GC samples (b 15%) is strongly increased to~90% in GC cell lines [22]. It can be concluded that RUNX3 P2 methylation in GC is (1) significantly lower than originally proposed; (2) does not correlate with RUNX3 expression level; and (3) it is not an early driving event in the development of gastric or other cancers.…”

“…Compelling evidence from several laboratories [6,12,[17][18][19][20][21][22][23][24][25][26][27][28][29][30] (Table 1) and the human proteome atlas portal (http: www.proteinatlas.org) demonstrating the lack of RUNX3 expression in normal GIT epithelium and other epithelial tissues has challenged the claim that RUNX3 is a TSG inactivated in GC or in other cancers. The large body of data disproving the claim that RUNX3 is a bona fide TSG and highlighting its functions in immunity and inflammation is the subject of this review.…”