Aberrant CpG methylation is a universal epigenetic trait of cancer cell genomes. However, human cancer samples or cell lines preclude the investigation of epigenetic changes occurring early during tumour development. Here, we have used MeDIP-seq to analyse the DNA methylome of APCMin adenoma as a model for intestinal cancer initiation, and we present a list of more than 13,000 recurring differentially methylated regions (DMRs) characterizing intestinal adenoma of the mouse. We show that Polycomb Repressive Complex (PRC) targets are strongly enriched among hypermethylated DMRs, and several PRC2 components and DNA methyltransferases were up-regulated in adenoma. We further demonstrate by bisulfite pyrosequencing of purified cell populations that the DMR signature arises de novo in adenoma cells rather than by expansion of a pre-existing pattern in intestinal stem cells or undifferentiated crypt cells. We found that epigenetic silencing of tumour suppressors, which occurs frequently in colon cancer, was rare in adenoma. Quite strikingly, we identified a core set of DMRs, which is conserved between mouse adenoma and human colon cancer, thus possibly revealing a global panel of epigenetically modified genes for intestinal tumours. Our data allow a distinction between early conserved epigenetic alterations occurring in intestinal adenoma and late stochastic events promoting colon cancer progression, and may facilitate the selection of more specific clinical epigenetic biomarkers.

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The short isoform of single-minded 2 (SIM2s), a basic helix-loop-helix/PAS (bHLH/PAS) transcription factor, is upregulated in pancreatic and prostate tumours; however, a mechanistic role for SIM2s in these cancers is unknown. Microarray studies in prostate DU145 cells identified the pro-cell death gene, BNIP3 (Bcl-2/adenovirus E1B 19 kDa interacting protein 3), as a novel putative target of SIM2s repression. Further validation showed BNIP3 repression in several prostate and pancreatic carcinoma-derived cell lines with ectopic expression of human SIM2s. BNIP3 levels are enhanced in prostate carcinoma cells upon short interfering (si)RNA-mediated knockdown of endogenous SIM2s. Chromatin immunoprecipitation and promoter studies show that SIM2s represses BNIP3 through its activities at the proximal promoter hypoxia response element (HRE), the site through which the bHLH/PAS family member, hypoxia-inducible factor 1a (HIF1a), induces BNIP3. SIM2s attenuates BNIP3 hypoxic induction via the HRE, and increased hypoxic induction of BNIP3 occurs with siRNA knockdown of endogenous SIM2s in prostate PC3AR þ cells. BNIP3 is implicated in hypoxia-induced cell death processes. Prolonged treatment of PC3AR þ cells with hypoxia mimetics, DP and DMOG, confers hypoxia-induced autophagy, measured by enhanced LC3-II levels and SQSTM1/p62 turnover. We show that PC3AR þ cells expressing ectopic SIM2s have enhanced survival in these conditions. Induction of LC3-II and turnover of SQSTM1/p62 are attenuated in PC3AR þ / SIM2s DMOG and hypoxia-treated cells, suggesting that SIM2s may attenuate autophagic cell death processes, perhaps through BNIP3 repression. These data show, for the first time, SIM2s cross-talk on an endogenous HRE. SIM2s' functional interference with HIF1a activities on BNIP3 may indicate a novel role for SIM2s in promoting tumourigenesis.

Cellular hierarchies and signals that govern stemness and differentiation of intestinal adenoma cells are not well defined. In this study, we used organotypic culture to investigate the impact of b-catenin and BMP signals in cells that form intestinal adenoma in the mouse. We found that activation of b-catenin signaling by loss of APC or transgenic induction of oncogenic mutant b-catenin (Ctnnb1 mut ) initiates the conversion of untransformed intestinal cells to tumor cells. These tumor cells display cancer stem cell (CSC) traits such as increased expression of the CSC markers Cd133 and Cd44, a high capacity for self-renewal and unlimited proliferative potential. Subsequent inactivation of transgenic Ctnnb1 mut results in the reversion of tumor cells to normal intestinal stem cells, which immediately reinstall the cellular hierarchy of the normal intestinal epithelium. Our data demonstrate that oncogenic activation of b-catenin signaling initiates the early steps of intestinal cellular transformation in the absence of irreversible genetic or epigenetic changes. Interestingly, we found that tumor cells in culture and in adenoma produce BMP4, which counteracts CSC-like traits by initiating irreversible cellular differentiation and loss of self-renewal capacity. We conclude that the opposition of stemness-maintaining oncogenic b-catenin signals and autocrine differentiating BMP signals within the adenoma cell provides a rationale for the formation of cellular hierarchies in intestinal adenoma and may serve to limit adenoma growth.In the intestine, recurring oncogenic mutations play key roles in tumor initiation and progression. 1 Such mutations often target components of conserved signaling pathways that underlie the maintenance of a cellular hierarchy in the intestinal epithelium. For instance, activity of the Wnt/b-catenin signaling pathway is in the normal intestine restricted to the stem cell compartment and provides cues for stem cell maintenance and cellular proliferation. 2 In intestinal adenoma, which is a benign precursor of carcinoma, b-catenin signaling is generally activated by oncogenic mutations in APC, which controls b-catenin stability, or in CTNNB1 (coding for b-catenin) itself.3,4 Mutations in Apc or Ctnnb1 also induce formation of intestinal adenoma in mice, such as in Apc Min mice. 5-8Active BMP signaling, on the other hand, antagonizes Wnt/b-catenin and is normally restricted to the differentiated cell compartment of the intestine, and excluded from the stem cell niche. [9][10][11] Inactivating mutations in components of the BMP signaling pathway are often found in sporadic human carcinoma and in Juvenile Polyposis. Studies in mice show that loss of BMP signals can initiate intestinal tumorigenesis and contribute to tumor progression (for review, Ref. 9).In contrast to cellular hierarchies of the normal intestine, the perturbed hierarchies formed by mutated intestinal tumor cells have not been well defined. Morphological and gene expression heterogeneities, such as unequal distribution of cell ...