CpG island methylator phenotypes in aging and cancer.

CpG islands are short stretches of CpG rich regions that are frequently associated with the promoter region of genes. Aberrant methylation of CpG islands is one mechanism of inactivating tumor suppressor genes (TSGs) in neoplasia, and there is growing evidence that altered cytosine methylation play important roles in cancer development. However, the differences in global CpG island methylation patterns between normal and cancer cells remain poorly understood. By examining a large number of loci in a series of cancers, global methylation profiles can be constructed. Such studies revealed that in colorectal cancer, there appears to be two types of methylation that are associated with cancer progression: type A (for age-related) methylation, and type C (for cancer-specific) methylation. Initially, type A methylation arises as a function of age in normal colorectal epithelial cells. By affecting genes that regulate the growth and/or differentiation of these cells, such methylation may result in a predisposition state that precedes tumor formation in the colon. Type C methylation, by contrast, was found exclusively in a subset of cancers, which display a CpG island methylator phenotype (CIMP). CIMP is a novel molecular instability pathway that appears to be responsible for most cases of aberrant TSG methylation in colorectal cancer, and which has important interactions with genetic pathways as well. In fact, CIMP+ tumors account for the majority of sporadic colorectal cancers with microsatellite instability, through methylation of the mismatch repair gene hMLH1. This model whereby age-related methylation increases cell-susceptibility to transformation and cancer-specific methylation results in neoplastic progression in a subset of cases may be applicable to many human neoplasms.     

DNA methylation biomarker candidates for early detection of colon cancer

Promoter CpG island hypermethylation of tumor suppressor genes is a common hallmark of all human cancers. Many researchers have been looking for potential epigenetic therapeutic targets in cancer using gene expression profiling with DNA microarray approaches. Our recent genome-wide platform of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that FBN2 and TCERG1L gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. In this study, promoter DNA hypermethylation of FBN2 and TCERG1L in CRC occurs as an early and cancer-specific event in colorectal cancer. Both genes showed high frequency of methylation in colon cancer cell lines (>80% for both of genes), adenomas (77% for FBN2, 90% for TCERG1L, n = 39), and carcinomas (86% for FBN2, 99% for TCERG1L, n = 124). Bisulfite sequencing confirmed cancer-specific methylation of FBN2 and TCERG1L of promoters in colon cancer cell line and cancers but not in normal colon. Methylation of FBN2 and TCERG1L is accompanied by downregulation in cell lines and in primary tumors as described in the Oncomine™ website. Together, our results suggest that gene silencing of FBN2 and TCERG1L is associated with promoter DNA hypermethylation in CRC tumors and may be excellent biomarkers for the early detection of CRC.     

Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype.

Aberrant methylation of 5' CpG islands is thought to play an important role in the inactivation of tumor suppressor genes in cancer. In colorectal cancer, a group of tumors is characterized by a hypermethylator phenotype termed CpG island methylator phenotype (CIMP), which includes methylation of such genes as p16 and hMLH1. To study whether CIMP is present in gastric cancer, the methylation status of five newly cloned CpG islands was examined in 56 gastric cancers using bisulfite-PCR. Simultaneous methylation of three loci or more was observed in 23 (41%) of 56 cancers, which suggests that these tumors have the hypermethylator phenotype CIMP. There was a significant concordance between CIMP and the methylation of known genes including p16, and hMLH1; methylation of p16 was detected in 16 (70%) of 23 CIMP+ tumors, 1 (8%) of 12 CIMP intermediate tumors, and 1 (5%) of 21 CIMP- tumors (P<0.0001). Methylation of the hMLH1 gene was detected in three of five tumors that showed microsatellite instability, and all three of the cases were CIMP+. The CIMP phenotype is an early event in gastric cancer, being present in the normal tissue adjacent to cancer in 5 of 56 cases. These results suggest that CIMP may be one of the major pathways that contribute to tumorigenesis in gastric cancers.     

18q loss of heterozygosity in microsatellite stable colorectal cancer is correlated with CpG island methylator phenotype-negative (CIMP-0) and inversely with CIMP-low and CIMP-high

Background:  

The CpG island methylator phenotype (CIMP) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, associated with microsatellite instability-high (MSI-high) and BRAF mutations. 18q loss of heterozygosity (LOH) commonly present in colorectal cancer with chromosomal instability (CIN) is associated with global hypomethylation in tumor cell. A recent study has shown an inverse correlation between CIN and CIMP (determined by MINTs, p16, p14 and MLH1 methylation) in colorectal cancer. However, no study has examined 18q LOH in relation to CIMP-high, CIMP-low (less extensive promoter methylation) and CIMP-0 (CIMP-negative), determined by quantitative DNA methylation analysis.     

Characteristic methylation profile in CpG island methylator phenotype‐negative distal colorectal cancers

Aberrant DNA methylation is involved in colon carcinogenesis. Although the CpG island methylator phenotype (CIMP) is defined as a subset of colorectal cancers (CRCs) with remarkably high levels of DNA methylation, it is not known whether epigenetic processes are also involved in CIMP‐negative tumors. We analyzed the DNA methylation profiles of 94 CRCs and their corresponding normal‐appearing colonic mucosa with 11 different markers, including the five classical CIMP markers. The CIMP markers were frequently methylated in proximal CRCs (p < 0.01); however, RASSF1A methylation levels were significantly higher in distal CRCs, the majority of which are CIMP‐negative (p < 0.05). Similarly, methylation levels of RASSF1A and SFRP1 in the normal‐appearing mucosae of distal CRC cases were significantly higher than those in the proximal CRC cases (p < 0.05). They were also positively correlated with age (RASSF1A, p < 0.01; SFRP1, p < 0.01). Microarray‐based genome‐wide DNA methylation analysis of 18 CRCs revealed that 168 genes and 720 genes were preferentially methylated in CIMP‐negative distal CRCs and CIMP‐positive CRCs, respectively. Interestingly, more than half of the hypermethylated genes in CIMP‐negative distal CRCs were also methylated in the normal‐appearing mucosae, indicating that hypermethylation in CIMP‐negative distal CRCs is more closely associated with age‐related methylation. By contrast, more than 60% of the hypermethylated genes in CIMP‐positive proximal CRCs were cancer specific (p < 0.01). These data altogether suggest that CpG island promoters appear to be methylated in different ways depending on location, a finding which may imply the presence of different mechanisms for the acquisition of epigenetic changes during colon tumorigenesis.     

Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature review

A cyclin‐dependent kinase inhibitor CDKN2A (p16/Ink4a) is a tumor suppressor and upregulated in cellular senescence. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colon cancer. However, prognostic significance of CDKN2A methylation or loss of CDKN2A (p16) expression independent of CIMP status remains uncertain. Using a database of 902 colorectal cancers in 2 independent cohort studies (the Nurses' Health Study and the Health Professionals Follow‐up Study), we quantified CDKN2A promoter methylation and detected hypermethylation in 269 tumors (30%). By immunohistochemistry, we detected loss of CDKN2A (p16) expression in 25% (200/804) of tumors. We analyzed for LINE‐1 hypomethylation and hypermethylation at 7 CIMP‐specific CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of TP53 (p53), CTNNB1 (β‐catenin), CDKN1A (p21), CDKN1B (p27), CCND1 (cyclin D1), FASN (fatty acid synthase) and PTGS2 (cyclooxygenase‐2). CDKN2A promoter methylation and loss of CDKN2A (p16) were associated with shorter overall survival in univariate Cox regression analysis [hazard ratio (HR): 1.36, 95% CI: 1.10–1.66, p = 0.0036 for CDKN2A methylation; HR: 1.30, 95% CI: 1.03–1.63, p = 0.026 for CDKN2A (p16) loss] but not in multivariate analysis that adjusted for clinical and tumor variables, including CIMP, MSI and LINE‐1 methylation. Neither CDKN2A promoter methylation nor loss of CDKN2A (p16) was associated with colorectal cancer‐specific mortality in uni‐ or multivariate analysis. Despite its well‐established role in carcinogenesis, CDKN2A (p16) promoter methylation or loss of expression in colorectal cancer is not independently associated with patient prognosis.     

Examination of a CpG island methylator phenotype and implications of methylation profiles in solid tumors

The CpG island methylator phenotype (CIMP), thoroughly described in colorectal cancer and to a lesser extent in other solid tumors, is important in understanding epigenetics in carcinogenesis and may be clinically useful for classification of neoplastic disease. Therefore, we investigated whether this putative phenotype exists in exposure-related solid tumors, where somatic gene alterations and enhanced clonal growth are selected for by carcinogens, and examined the ability of methylation profiles to classify malignant disease. We studied promoter hypermethylation of 16 tumor suppressor genes and 3 MINT loci (acknowledged classifiers of CIMP) in 344 bladder cancers, 346 head and neck squamous cell carcinomas (HNSCC), 146 non-small-cell lung cancer (NSCLC), and 71 malignant pleural mesotheliomas (MPM). We employed rigorous statistical methods to examine the distribution of promoter methylation and the usefulness of these profiles for disease classification. In bladder cancer, HNSCC, and NSCLC, there was a significant correlation (P < 0.0001) between methylation of the three MINT loci and methylation index, although the distribution of methylated loci varied significantly across these disease. Although there was a significant (P < 0.001) association between gene methylation profile and disease, rates of misclassification of each disease by their methylation profile ranged from 28% to 32%, depending on the classification scheme used. These data suggest that a form of CIMP exists in these solid tumors, although its etiology remains elusive. Whereas the gene profiles of hypermethylation among examined loci could not unequivocally distinguish disease type, the existence of CIMP and the relative preponderance of hypermethylation in these cancers suggest that methylation analysis may be clinically useful as a targeted screening tool.     

Predicting clinical outcome of 5-fluorouracil-based chemotherapy for colon cancer patients: is the CpG island methylator phenotype the 5-fluorouracilresponsive subgroup?

The CpG island methylator phenotype (CIMP+) of colorectal cancer (CRC) occurs predominantly in the proximal colon and is characterized by frequent hypermethylation of gene promoter regions. In this review, we present evidence suggesting CIMP+ represents the subgroup of colon cancers that are responsive to 5-fluorouracil (5-FU)-based treatments. CIMP+ has been associated with survival benefit from 5-FU in a clinical study of CRC, with additional evidence coming from studies on gastric cancer and tumor cell lines. Elevated concentrations of 5-10-methylene tetrahydrofolate (CH₂FH₄) occur in CIMP+ tumors and are probably due to low expression levels for γ-glutamyl hydrolase (GGH). Clinical and in vitro work has previously shown that high CH₂FH₄ and low GGH expression levels correlate with good response to 5-FU. Methylation-induced silencing of dihydropyrimidine dehydrogenase, the rate-limiting enzyme in 5-FU degradation, may also provide a link between CIMP+ and good response to 5-FU. The CIMP+-related phenotype referred to as microsatellite instability (MSI+) has been widely investigated as a predictive marker of response to 5-FU, with contradictory results. The interpretation of these studies is likely to be confounded by the fact that some MSI+ tumors occur in the background of CIMP+, but a significant proportion of others do not. Further studies on tumors from randomized clinical trials are required to confirm the value of CIMP+ and associated molecular features for the prediction of clinical outcome to 5-FU-based chemotherapy.     

CpG island methylator phenotype associates with low-degree chromosomal abnormalities in colorectal cancer

PURPOSE: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. EXPERIMENTAL DESIGN: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. RESULTS: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). CONCLUSION: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.     

CpG island hypermethylation and tumor suppressor genes: a booming present,a brighter future

We have come a long way since the first reports of the existence of aberrant DNA methylation in human cancer. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as an important mechanism for gene inactivation. CpG island hypermethylation has been described in almost every tumor type. Many cellular pathways are inactivated by this type of epigenetic lesion: DNA repair (hMLH1, MGMT), cell cycle (p16(INK4a), p15(INK4b), p14(ARF)), apoptosis (DAPK), cell adherence (CDH1, CDH13), detoxification (GSTP1), etc em leader However, we still know little of the mechanisms of aberrant methylation and why certain genes are selected over others. Hypermethylation is not an isolated layer of epigenetic control, but is linked to the other pieces of the puzzle such as methyl-binding proteins, DNA methyltransferases and histone deacetylase, but our understanding of the degree of specificity of these epigenetic layers in the silencing of specific tumor suppressor genes remains incomplete. The explosion of user-friendly technologies has given rise to a rapidly increasing list of hypermethylated genes. Careful functional and genetic studies are necessary to determine which hypermethylation events are truly relevant for human tumorigenesis. The development of CpG island hypermethylation profiles for every form of human tumors has yielded valuable pilot clinical data in monitoring and treating cancer patients based in our knowledge of DNA methylation. Basic and translational will both be needed in the near future to fully understand the mechanisms, roles and uses of CpG island hypermethylation in human cancer. The expectations are high.     

Identification of GABRA1 and LAMA2 as new DNA methylation markers in colorectal cancer

Aberrant methylation of CpG islands in the promoter region of genes is a common epigenetic phenomenon found in early cancers. Therefore conducting genome-scale methylation studies will enhance our understanding of the epigenetic etiology behind carcinogenesis by providing reliable biomarkers for early detection of cancer. To discover novel hypermethylated genes in colorectal cancer by genome-wide search, we first defined a subset of genes epigenetically reactivated in colon cancer cells after treatment with a demethylating agent. Next, we identified another subset of genes with relatively down-regulated expression patterns in colorectal primary tumors when compared with normal appearing-adjacent regions. Among 29?genes obtained by cross-comparison of the two gene-sets, we subsequently selected, through stepwise subtraction processes, two novel genes, GABRA1 and LAMA2, as methylation targets in colorectal cancer. For clinical validation pyrosequencing was used to assess methylation in 134 matched tissue samples from CRC patients. Aberrant methylation at target CpG sites in GABRA1 and LAMA2 was observed with high frequency in tumor tissues (92.5% and 80.6%, respectively), while less frequently in matched tumor-adjacent normal tissues (33.6% for GABRA1 and 13.4% for LAMA2). Methylation levels in primary tumors were not significantly correlated with clinico-pathological features including age, sex, survival and TNM stage. Additionally, we found that ectopic overexpression of GABRA1 in colon cancer cell lines resulted in strong inhibition of cell growth. These results suggest that two novel hypermethylated genes in colorectal cancer, GABRA1 and LAMA2, may have roles in colorectal tumorigenesis and could be potential biomarkers for the screening and the detection of colorectal cancer in clinical practice.     

Frequent inactivation of SPARC by promoter hypermethylation in colon cancers

Epigenetic modification of gene expression plays an important role in the development of human cancers. The inactivation of SPARC through CpG island methylation was studied in colon cancers using oligonucleotide microarray analysis and methylation specific PCR (MSP). Gene expression of 7 colon cancer cell lines was evaluated before and after treatment with the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) by oligonucleotide microarray analysis. Expression of SPARC was further examined in colon cancer cell lines and primary colorectal cancers, and the methylation status of the SPARC promoter was determined by MSP. SPARC expression was undetectable in 5 of 7 (71%) colorectal cancer cell lines. Induction of SPARC was demonstrated after treatment with the demethylating agent 5Aza-dC in 5 of the 7 cell lines. We examined the methylation status of the CpG island of SPARC in 7 colon cancer cell lines and in 20 test set of colon cancer tissues. MSP demonstrated hypermethylation of the CpG island of SPARC in 6 of 7 cell lines and in all 20 primary colon cancers, when compared with only 3 of 20 normal colon mucosa. Immunohistochemical analysis showed that SPARC expression was downregulated or absent in 17 of 20 colon cancers. A survival analysis of 292 validation set of colorectal carcinoma patients revealed a poorer prognosis for patients lacking SPARC expression than for patients with normal SPARC expression (56.79% vs. 75.83% 5-year survival rate, p = 0.0014). The results indicate that epigenetic gene silencing of SPARC is frequent in colon cancers, and that inactivation of SPARC is related to rapid progression of colon cancers.     

MicroRNA‐31 expression in relation to BRAF mutation,CpG island methylation and colorectal continuum in serrated lesions

The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP‐high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA‐31 (miR‐31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP‐high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR‐31 and epigenetic features in serrated pathway, and hypothesized that miR‐31 supports the “colorectal continuum” concept. We evaluated miR‐31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non‐serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR‐31 expression and CIMP‐high status in serrated lesions with BRAF mutation (p = 0.0001). In contrast, miR‐31 was slightly but insignificantly associated with CIMP status in the cases with wild‐type BRAF. miR‐31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high‐grade dysplasia. The frequency of miR‐31, BRAF mutation CIMP‐high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR‐31 expression was associated with CIMP‐high status in serrated lesions with BRAF mutation. Our data also suggested that miR‐31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum.     

DNA methylation of multiple genes in gastric carcinoma: association with histological type and CpG island methylator phenotype

Hypermethylation of CpG islands is associated with silencing of various tumor suppressor genes. Recent studies on colorectal and gastric cancer have identified a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. For determination of association between DNA methylation pattern or histological type and CIMP status in gastric carcinoma, CpG islands in the promoters of hMLH1 and CDH1 genes, CpG islands overlapping exon 1 of MGMT and p16^INK4a genes, and a non-CpG island in exon 1 of the RAR-β gene were studied. The presence of the CIMP was determined by monitoring five methylated in tumor (MINT) loci in 103 gastric carcinomas. Among the 103 gastric carcinomas, DNA hypermethylation was detected in the following frequencies: 14 (14%) for hMLH1 , 26 (25%) for MGMT , 26 (25%) for p16^INK4a , 54 (52%) for CDH1 , and 53 (52%) for RAR-β. Forty-two (41%) of 103 gastric carcinomas were positive for the CIMP. CIMP and hypermethylation of p16^INK4a gene were found more frequently in intestinal and diffuse-adherent types than in diffuse-scattered type ( P =0.013 and 0.017, respectively). In contrast, hypermethylation of the CDH1 and RAR-β genes was more common in the diffuse-scattered type than in the other types ( P =0.008 and 0.007, respectively). In intestinal- and diffuse-adherent-type gastric carcinomas, we found significant associations between the presence of the CIMP and hypermethylation of several genes: hMLH1 ( P =0.006), p16^INK4a ( P =0.018), CDH1 ( P =0.024), and RAR-β ( P =0.044). Our overall results suggest that in some intestinal- and diffuse-adherent-type gastric carcinomas, DNA hypermethylation affects non-specific gene promoters concordantly, at least in part, whereas in diffuse-scattered-type gastric carcinoma, DNA hyper-methylation affects specific genes such as CDH1 and RAR-β.