In summary, we show that colon cancer cDMRs are generally involved in the common solid tumors of adulthood, lung, breast, thyroid, and colon cancer, and the most common solid tumor of childhood, Wilms tumor, with tight clustering of methylation levels in normal tissues, and marked stochastic variation in cancers. Efforts to exploit DNA methylation for cancer screening focus on identifying narrowly defined cancer-specific profiles17
. Our data suggests future efforts might instead be directed at defining the cancer epigenome as the departure from a narrowly defined normal profile.
Surprisingly, two-thirds of all methylation changes in colon cancer involve hypomethylation of large blocks, with consistent locations across samples, comprising more than half of the genome. The functional relevance is supported by the fact that genes in colon blocks not in fibroblast blocks tend to be silenced in colon and not in fibroblasts and vice-versa.
The most variably expressed genes in cancer are enriched in the blocks, and involve genes associated with tumor heterogeneity and progression, including three matrix metalloproteinase genes, MMP3
, and MMP1018
, and a fourth, SIM2
, which acts through metalloproteinases to promote tumor invasion19
. Another, STC1
, helps mediate the Warburg effect of reprogramming tumor metabolism20
encodes a secreted glycoprotein associated with inflammatory responses and poor prognosis in multiple tumor types including colon21
genes are targets of Wnt-1 thought to contribute to tissue invasion in breast and colon cancer22
. Our gene ontology enrichment analysis15
of genes associated with hypervariable expression in blocks (FDR<0.05)showed enrichment for categories including extracellular matrix remodeling genes (Supplementary Table 13
). One cautionary note raised by these findings is that treatment of cancer patients with nonspecific DNA methylation inhibitors could have unintended consequences in the activation of tumor-promoting genes in hypomethylated blocks. It is also important to note that while previous studies23,24
have shown large-region hypermethylation or no regional methylation change, this study is based on whole-genome bisulfite sequencing. Nevertheless, future studies are needed to show whether block hypomethylation is a feature of cancer epigenomes in general.
Small DMRs, while representing a relatively small fraction of the genome (0.3%), are numerous (10,125), and frequently involve loss of boundaries of DNA methylation at the edge of CpG islands, shifting of DNA methylation boundaries, or the creation of novel hypomethylated regions in CG-dense regions that are not canonical islands. These data underscore the importance of hypomethylated CpG island shores in cancer since shores associated with hypomethylation and gene overexpression in cancer are enriched for cell cycle related genes, suggesting a role in the unregulated growth that characterizes cancer.
We propose a model relating tissue-specific DMRs to the sites of methylation hypervariability in cancer. Normal pluripotency might require stochastic gene expression at some loci, allowing for differentiation along alternative pathways in response to external stimuli or even intrinsically. The epigenome could collaborate to create a permissive state by changing its physical configuration to relax the stringency of epigenetic marks, since variance increases away from the extremes, and a similar process may occur in cancer. One way is by altering LOCKs/LADs/blocks, which could involve a change in the chromatin packing density or proximity to the nuclear lamina. Similarly, subtle shifts in DNA methylation boundaries near CpG islands may drive normal chromatin organization and tissue-specific gene expression. Given the importance of boundary regions for both small DMRs and large blocks identified in this study, it will be important to focus future epigenetic investigations on the boundaries of blocks and CpG islands (shores), and on genetic or epigenetic changes in genes encoding factors that interact with them.
The increased methylation and expression variability in each cancer type is consistent with the potential selective value of increased epigenetic plasticity in a varying environment first suggested for evolution but applicable to the strong but variable selective forces under which a cancer grows, such as varying oxygen tension or metastasis to a distant site25
. Thus, increased epigenetic heterogeneity in cancer at cDMRs (which we show are also tDMRs) could underlie the ability of cancer cells to adapt rapidly to changing environments, such as increased oxygen with neovascularization, then decreased oxygen with necrosis; or metastasis to a new intercellular milieu.