Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.

Disruption of circadian rhythms, daily oscillations in biological processes that are regulated by an endogenous clock, has been linked to tumorigenesis. Normal and malignant tissues often show asynchronies in cell proliferation and metabolic rhythms. Cancer chronotherapy takes biological time into account to improve the therapy. However, alterations of the circadian clock machinery genes have rarely been reported in human cancer. Herein, we show that the BMAL1 gene, a core component of the circadian clock, is transcriptionally silenced by promoter CpG island hypermethylation in hematologic malignancies, such as diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias. We also describe how BMAL1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas BMAL1 depletion by RNA interference in unmethylated cells enhances tumor growth. We also show that BMAL1 epigenetic inactivation impairs the characteristic circadian clock expression pattern of genes such as C-MYC, catalase, and p300 in association with a loss of BMAL1 occupancy in their respective promoters. Furthermore, the DNA hypermethylation–associated loss of BMAL1 also prevents the recruitment of its natural partner, the CLOCK protein, to their common targets, further enhancing the perturbed circadian rhythm of the malignant cells. These findings suggest that BMAL1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting the cellular circadian clock. [Cancer Res 2009;69(21):8447–54]

Aberrant activation and upregulation of the Wnt pathway is a key feature of many cancers. Wnt antagonists have recently attracted wide attention. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind to Wnt proteins directly and inhibit Wnt signaling pathway. It has been reported that WIF-1 expression is down regulated in several solid tumors and that WIF-1 is silenced by promoter hypermethylation in lung and colorectal cancer. By using RT–PCR, bisulfite sequence analysis, and methylation-specific PCR, we analysed expression and methylation of WIF-1 in cancer cell lines and freshly resected cancer tissues of the esophagus, stomach, colorectum, and pancreas. Downregulation of WIF-1 mRNA expression was observed in 61 (91.0%) of 67 cancer cell lines, 16 (80.0%) of 20 esophageal, 23 (74.2%) of 31 gastric, 41 (82.0%) of 50 colorectal, and six (75.0%) of eight pancreatic cancer tissues. Downregulation of WIF-1 expression was also observed at protein level. No significant association between WIF-1 downregulation and clinicopathological characteristics was found, suggesting that downregulation of WIF-1 expression is an early event in carcinogenesis of these cancers. Indeed, downregulation of WIF-1 expression was observed in 32 (72.7%) of 44 colorectal adenoma tissues and 18 (78.2%) of 23 early mucosal or submucosal colorectal carcinoma tissues. CpG island hypermethylation in the WIF-1 promoter region correlated with downregulation of WIF-1 expression in cancer cell lines and tissues. Treatment with demethylating agent, 5-aza-2′-deoxycytidine (5-aza-dC), restored WIF-1 expression in cancer cell lines. A combined treatment of 5-aza-dC and a histone deacetylase inhibitor, trichostatinA, restored WIF-1 expression synergistically, indicating the role of cytosine methylation and histone deacetylation in the silencing of the WIF-1 gene. Transfection of the WIF-1 gene construct into TE-1 esophageal cancer cell lines or SW48 colon cancer cell lines lacking WIF-1 expression resulted in a significant inhibition on colony formation, cell proliferation, anchorage-independent growth in soft agar. TOPflash assay showed WIF-1 inhibits Wnt canonical signaling in these cell lines. These results suggest tumor suppressive function of WIF-1, due to its ability to inhibit Wnt signaling. Our results suggest that WIF-1 silencing due to promoter hypermethylation is an important mechanism underlying aberrant activation of the Wnt signaling pathway in carcinogenesis of the digestive organs. Modulation of the Wnt pathway, through reversal of WIF-1 silencing by demethylating agents, is a potential target for treatment and/or prevention of gastrointestinal cancers.

The Kruppel-like factor (KLF) proteins are multitasked transcriptional regulators with an expanding tumor suppressor function. KLF2 is one of the prominent members of the family because of its diminished expression in malignancies and its growth-inhibitory, pro-apoptotic and anti-angiogenic roles. In this study, we show that epigenetic silencing of KLF2 occurs in cancer cells through direct transcriptional repression mediated by the Polycomb group protein Enhancer of Zeste Homolog 2 (EZH2). Binding of EZH2 to the 5′-end of KLF2 is also associated with a gain of trimethylated lysine 27 histone H3 and a depletion of phosphorylated serine 2 of RNA polymerase. Upon depletion of EZH2 by RNA interference, short hairpin RNA or use of the small molecule 3-Deazaneplanocin A, the expression of KLF2 was restored. The transfection of KLF2 in cells with EZH2-associated silencing showed a significant anti-tumoral effect, both in culture and in xenografted nude mice. In this last setting, KLF2 transfection was also associated with decreased dissemination and lower mortality rate. In EZH2-depleted cells, which characteristically have lower tumorigenicity, the induction of KLF2 depletion ‘rescued’ partially the oncogenic phenotype, suggesting that KLF2 repression has an important role in EZH2 oncogenesis. Most importantly, the translation of the described results to human primary samples demonstrated that patients with prostate or breast tumors with low levels of KLF2 and high expression of EZH2 had a shorter overall survival.