Given the increasing evidence for a functional contribution of stromal myofibroblasts in the progression of human carcinomas, it is important to understand the genomic changes that accompany the conversion of normal myofibroblasts into cancer-associated myofibroblasts. Myofibroblasts are prominent elements of the tumor stroma in most types of human carcinomas, along with tumor-associated blood vessels and inflammatory cells. Their exact origin is an area of active research and candidate precursor cells include fibroblasts, smooth muscle cells and bone marrow derived stem cells (4
). In the intestine, for example, myofibroblasts share several properties with the smooth muscle cells of the muscularis mucosae and histologically appear to arise from that layer (30
), and in the invasive gastric carcinomas (intestinal type) that we have examined the histology also suggests that at least some of these cells have proliferated from this layer. In this study we have defined myofibroblasts as spindle-shaped cells in the mucosa and muscularis mucosae that express αSMA and are not part of blood vessel walls. Smooth muscle cells of the muscularis propria are also SMA-positive, but here we have specifically dealt with areas of the gastric cancers that had not invaded this deeper layer. Intraepithelial myofibroblasts are rare but detectable in normal non-inflamed mucosa (16
) and we have used both types of ASMA-positive cells (muscularis mucosae and intraepithelial) for comparing to cancer-associated myofibroblasts. It has been shown that their number (both the cellular thickness of the muscularis mucosae and the intraepithelial component) increases in pre-neoplastic tissue and in advanced cancer and that these cells come to constitute a significant portion of the tumor volume (31
). Relevant to the controls in our study, myofibroblasts were originally described in non malignant wound healing and as such are also seen in gastric ulcers.
In a research area that is still evolving, recent studies have suggested that cancer-associated stromal cells undergo specific genetic and epigenetic changes. Our data shown here, from multiple modalities including MSNP for genomic methylation profiling, bisulfite sequencing, an in vitro radiolabeled cytosine incorporation assay, and anti-5-methyl-C IHC on primary gastric cancers and dysplasias, together indicate that a major phenotypic change in cancer-associated myofibroblasts is a global reduction in DNA methylation. This phenomenon parallels the overall loss of DNA methylation that has been well documented in the malignant epithelial component of multiple types of human carcinomas (32
), including gastric carcinomas (data shown here, and prior studies, for example (36
)). However, the cause of genomic demethylation in cancer cells remains unresolved, and likewise additional research will be needed to understand the mechanisms underlying this phenomenon in stromal myofibroblasts. Our data suggest that reduced expression of DNMTs probably does not explain this loss of methylation. Alternatively it has been suggested that a deficiency in tissue or circulating methyl donors may be contribute to demethylation in cancer.
A number of hypotheses have also been put forward concerning the downstream consequences of loss of CpG methylation in neoplastic epithelial cells, including effects on gene expression and genome stability. Our MSNP data for the 2 cases of short term primary cultures of myofibroblasts isolated from within gastric cancers to cultures of myofibroblasts isolated from histologically uninvolved gastric mucosa distant from the cancers showed no evidence for genomic instability in terms of altered chromosomal or sub-chromosomal copy number, and no evidence for loss of heterozygosity (LOH). It remains to be determined whether the cause and the consequence of global hypomethylation are distinct between the epithelial or neoplastic cell population and the cancer associated stroma cells.
Emerging evidence suggests that expression of cytokines, proteolytic enzymes and their endogenous inhibitors (MMPs and TIMPs), and growth factors, are essential for the cross talk between cancer-associated myofibroblasts and malignant epithelial cells, including in gastric cancers (16
). Several of these genes are up-regulated in cancer-associated myofibroblasts during the phenotypic change that accompanies a transformation from normal resting fibroblasts. Based on experiments in tissue culture with demethylating drugs, some of these genes have been suggested to be potentially up-regulated via hypomethylation (41
). In general support of this observation is the finding that different patterns of gene expression are observed between cultured cancer-associated and non-cancer-associated myofibroblasts (30
). In considering the functional correlates of global loss of methylation in these stromal cells, it is interesting that a recent study in a different organ system has found that decitabine treatment (inhibition of DNA methyltransferases) inhibits myofibroblast transdifferentiation from hepatic stellate cells (42
). One of the most intriguing aspects of global hypomethylation in gastric cancer-associated myofibroblasts, shown by our analysis in the transgenic mouse model, is its occurrence early in cancer progression – at the dysplastic pre-invasive stage. Thus, scoring this phenomenon by IHC may prove useful in diagnosis of early gastric lesions.
Mechanistically, loss of DNA methylation may have several causes. For example, it could be a consequence of abnormal cellular proliferation, a local deficiency of methyl donors in dysplastic gastric mucosa, or both. Early studies have suggested that certain premalignant conditions may lead to decreased tissue folate content and thus a local deficiency in methyl donors (43
). This same question has persisted for many years in the context of the parallel loss of methylation seen in malignant epithelial cells, and the answer may have ramifications for chemopreventive and therapeutic strategies that deliberately seek to increase or decrease cellular DNA methylation. Supplementation with methyl donors will need to be done taking into account the balance between reversing hypomethylation and potentially increasing tumor suppressor gene hypermethylation, so it will be important to define the time course of hypo- and hypermethylation in cancer initiation and progression, which may allow a more targeted approach to chemoprevention. If, as suggested previously (44
), genomic hypomethylation is an early event that precedes at least some instances of gene specific hypermethylation, the optimal timing of methyl supplementation (i.e. folate, choline, betaine, selenomethionine and S-adenosylmethionine) may be different from the optimal timing for administering methyltransferase inhibitors (i.e. EGCG, decitabine). This will be a challenging problem, as pathological gain of DNA methylation can occur very early in the tumorigenic pathway, sometimes as early as fetal organogenesis (35
). More optimistically, our findings of reduced genomic methylation in cancer-associated stromal cells raise the possibility that demethylating drugs like decitabine might be able to exert anti-cancer activity in solid tumors not only via effects on the neoplastic cells, but also possibly by provoking a hypomethylation crisis in the supporting stromal myofibroblasts.