Aberrant methylation of promoter region CpG islands is associated with transcriptional inactivation of tumor-suppressor genes in neoplasia. To understand global patterns of CpG island methylation in colorectal cancer, we have used a recently developed technique called methylated CpG island amplification to examine 30 newly cloned differentially methylated DNA sequences. Of these 30 clones, 19 (63%) were progressively methylated in an age-dependent manner in normal colon, 7 (23%) were methylated in a cancer-specific manner, and 4 (13%) were methylated only in cell lines. Thus, a majority of CpG islands methylated in colon cancer are also methylated in a subset of normal colonic cells during the process of aging. In contrast, methylation of the cancer-specific clones was found exclusively in a subset of colorectal cancers, which appear to display a CpG island methylator phenotype (CIMP). CIMP؉ tumors also have a high incidence of p16 and THBS1 methylation, and they include the majority of sporadic colorectal cancers with microsatellite instability related to hMLH1 methylation. We thus define a pathway in colorectal cancer that appears to be responsible for the majority of sporadic tumors with mismatch repair deficiency.

Aberrant WNT pathway signaling is an early progression event in 90% of colorectal cancers 1 . It occurs through mutations mainly of APC and less often of CTNNB1 (encoding β-catenin) 1-3 or AXIN2 (encoding axin-2, also known as conductin) 4 . These mutations allow ligandindependent WNT signaling that culminates in abnormal accumulation of free β-catenin in the nucleus 1-3 . We previously identified frequent promoter hypermethylation and gene silencing of the genes encoding secreted frizzledrelated proteins (SFRPs) in colorectal cancer 5 . SFRPs possess a domain similar to one in the WNT-receptor frizzled proteins and can inhibit WNT receptor binding to downregulate pathway signaling during development 6-10 . Here we show that restoration of SFRP function in colorectal cancer cells attenuates WNT signaling even in the presence of downstream mutations. We also show that the epigenetic loss of SFRP function occurs early in colorectal cancer progression and may thus provide constitutive WNT signaling that is required to complement downstream mutations in the evolution of colorectal cancer.

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that, in cancer, expression of some miRNAs cells is silenced in association with CpG island hypermethylation. To identify epigenetically silenced miRNAs in colorectal cancer (CRC), we screened for miRNAs induced in CRC cells by 5-aza-2 ¶-deoxycytidine (DAC) treatment or DNA methyltransferase knockout. We found that miRNA-34b (miR-34b) and miR-34c, two components of the p53 network, are epigenetically silenced in CRC; that this down-regulation of miR-34b/c is associated with hypermethylation of the neighboring CpG island; and that DAC treatment rapidly restores miR-34b/c expression. Methylation of the miR34b/c CpG island was frequently observed in CRC cell lines (nine of nine, 100%) and in primary CRC tumors (101 of 111, 90%), but not in normal colonic mucosa. Transfection of precursor miR-34b or miR-34c into CRC cells induced dramatic changes in the gene expression profile, and there was significant overlap between the genes down-regulated by miR-34b/c and those down-regulated by DAC. We also found that the miR-34b/c CpG island is a bidirectional promoter which drives expression of both miR-34b/c and B-cell translocation gene 4 (BTG4); that methylation of the CpG island is also associated with transcriptional silencing of BTG4; and that ectopic expression of BTG4 suppresses colony formation by CRC cells. Our results suggest that miR-34b/c and BTG4 are novel tumor suppressors in CRC and that the miR-34b/c CpG island, which bidirectionally regulates miR34b/c and BTG4, is a frequent target of epigenetic silencing in

Colon cancer has been viewed as the result of progressive accumulation of genetic and epigenetic abnormalities. However, this view does not fully reflect the molecular heterogeneity of the disease. We have analyzed both genetic (mutations of BRAF, KRAS, and p53 and microsatellite instability) and epigenetic alterations (DNA methylation of 27 CpG island promoter regions) in 97 primary colorectal cancer patients. Two clustering analyses on the basis of either epigenetic profiling or a combination of genetic and epigenetic profiling were performed to identify subclasses with distinct molecular signatures. Unsupervised hierarchical clustering of the DNA methylation data identified three distinct groups of colon cancers named CpG island methylator phenotype (CIMP) 1, CIMP2, and CIMP negative. Genetically, these three groups correspond to very distinct profiles. CIMP1 are characterized by MSI (80%) and BRAF mutations (53%) and rare KRAS and p53 mutations (16% and 11%, respectively). CIMP2 is associated with 92% KRAS mutations and rare MSI, BRAF, or p53 mutations (0, 4, and 31% respectively). CIMP-negative cases have a high rate of p53 mutations (71%) and lower rates of MSI (12%) or mutations of BRAF (2%) or KRAS (33%). Clustering based on both genetic and epigenetic parameters also identifies three distinct (and homogeneous) groups that largely overlap with the previous classification. The three groups are independent of age, gender, or stage, but CIMP1 and 2 are more common in proximal tumors. Together, our integrated genetic and epigenetic analysis reveals that colon cancers correspond to three molecularly distinct subclasses of disease.classification ͉ DNA methylation ͉ genetic alterations C olorectal cancer (CRC) is the second and fourth most common cancer in men and women, respectively (1). Approximately 70% of colorectal cancers are sporadic, with no inherited predisposition. A stepwise progression model involving two distinct genetic pathways has been proposed to explain the etiology of colon cancer from benign neoplasm to adenocarcinoma (2). One class of genetic alterations involves mutations of oncogenes and tumor-suppressor genes that directly control cell birth and death, such as APC, KRAS, and p53. Another involves mutations of DNA mismatch repair genes.In addition to these genetic alterations, cancer initiation and promotion can occur by epigenetic mechanisms (3). CpG methylation is the best characterized epigenetic change in the mammalian genome. Whereas CpG dinucleotides are underrepresented in the mammalian genome, approximately half of all human genes contain a CpG-rich region called a ''CpG island'' in the 5Ј area, often encompassing the promoter and transcription start site of the associated gene (4, 5). Gene silencing by hypermethylation of CpG islands (including tumor-suppressor genes) is a common event in tumors. Further, hypermethylation of specific genes such as ER␣, MYOD1, and N33 occurs in the normal colon tissue of aging individuals (6, 7), and hypermethylation of the secreted frizzled-relat...

Fusobacterium species are part of the gut microbiome in humans. Recent studies have identified over-representation of Fusobacterium in colorectal cancer (CRC) tissues but it is not yet clear whether this is pathogenic or simply an epiphenomenon. In this study, we evaluated the relationship between Fusobacterium status and molecular features in CRCs through quantitative real-time PCR in 149 CRC tissues, 89 adjacent normal appearing mucosae and 72 colonic mucosae from cancer-free individuals. Results were correlated with CpG island methylator phenotype (CIMP) status, microsatellite instability (MSI) and mutations in BRAF, KRAS, TP53, CHD7 and CHD8. Whole exome capture sequencing data were also available in 11 cases. Fusobacterium was detectable in 111/149 (74%) CRC tissues and heavily enriched in 9% (14/149) of the cases. As expected, Fusobacterium was also detected in normal appearing mucosae from both cancer and cancer-free individuals but the amount of bacteria was much lower compared to CRC tissues (a mean of 250-fold lower for Pan-fusobacterium). We found the Fusobacterium-high CRC group (FB-high) to be associated with CIMP positivity (p=0.001), TP53 wild type (p=0.015), hMLH1 methylation positivity (p=0.0028), MSI (p=0.018) and CHD7/8 mutation positivity (p=0.002). Among the 11 cases where whole exome sequencing data was available, two that were FB-high cases also had the highest number of somatic mutations (a mean of 736 per case in FB-high vs. 225 per case in all others). Taken together, our findings show that Fusobacterium enrichment is associated with specific molecular subsets of CRCs, offering support for a pathogenic role in CRC for this gut microbiome component