Aberrant methylation and silencing of ARHI,an imprinted tumor suppressor gene in which the function is lost in breast cancers

ARHI is a maternally imprinted tumor suppressor gene that maps to a site on chromosome 1p31 where loss of heterozygosity has been observed in 40% of human breast and ovarian cancers. ARHI is expressed in normal ovarian and breast epithelial cells, but ARHI expression is lost in a majority of ovarian and breast cancers. Expression of ARHI from the paternal allele can be down-regulated by multiple mechanisms in addition to loss of heterozygosity. This article explores the role of DNA methylation in silencing ARHI expression. There are three CpG islands in the ARHI gene. CpG islands I and II are located in the promoter region, whereas CpG island III is located in the coding region. Consistent with imprinting, we have found that all three CpG islands were partially methylated in normal human breast epithelial cells. Additional confirmation of imprinting has been obtained by studying DNA methylation and ARHI expression in murine A9 cells that carry either the maternal or the paternal copy of human chromosome 1. All three CpG islands were methylated, and ARHI was not expressed in A9 cells that contained the maternal allele. Conversely, CpG islands were not methylated and ARHI was expressed in A9 cells that contained the paternal allele of human chromosome 1. Aberrant methylation was found in several breast cancer cell lines that exhibited decreased ARHI expression. Hypermethylation was detected in 67% (6 of 9) of breast cancer cell lines at CpG island I, 33% (3 of 9) at CpG island II, and 56% (5 of 9) at CpG island III. Hypomethylation was observed in 44% (4 of 9) of breast cancer cell lines at CpG island II. When methylation of CpG islands was studied in 20 surgical specimens, hypermethylation was not observed in CpG island I, but 3 of 20 cases exhibited hypermethylation in CpG island II (15%), and 4 of 20 cases had hypermethylation in CpG island III (20%). Treatment with 5-aza-2'-deoxycytidine, a methyltransferase inhibitor, could reverse aberrant hypermethylation of CpG island I, II and III and partially restore ARHI expression in some, but not all of the cell lines. Treatment with 5-aza-2'-deoxycytidine partially reactivated ARHI expression in cell lines with hypermethylation of CpG islands I and II but not in cell lines with partial methylation or hypomethylation of these CpG islands. To test the impact of CpG island methylation on ARHI promoter activity more directly, constructs were prepared with the ARHI promoter linked to a luciferase reporter and transfected into SKBr3 and human embryo kidney 293 cells. Methylation of the entire construct destroyed promoter activity. Selective methylation of CpG island II alone or in combination with CpG island I also abolished ARHI promoter activity. Methylation of CpG I alone partially inhibited promoter activity of ARHI. Thus, hypermethylation of CpG island II in the promoter region of ARHI is associated with the complete loss of ARHI expression in breast cancer cells. Other epigenetic modifications such as hypermethylation in CpG island III may also contribute to the loss of ARHI expression.     

Germ-line variants in methyl-group metabolism genes and susceptibility to DNA methylation in normal tissues and human primary tumors

Aberrant DNA methylation is recognized as being a common feature of human neoplasia.CpG island hypermethylation and global genomic hypomethylation occur simultaneously in the cancer cell. However, very little is known about the interindividual inherited susceptibility to these epigenetic processes. To address this matter, we have genotyped in 233 cancer patients (with colorectal, breast, or lung tumors), four germ-line variants in three key genes involved in the metabolism of the methyl group, methylene-tetrahydrofolate reductase, methionine synthase, and cystathionine beta-synthase, and analyzed their association with DNA methylation parameters. The epigenetic features analyzed were the 5-methylcytosine content in the genome of the tumors and their normal counterparts, and the presence of CpG island hypermethylation of tumor suppressor genes (p16(INK4a), p14(ARF), hMLH1, MGMT, APC, LKB1, DAPK, GSTP1, BRCA1, RAR beta 2, CDH1, and RASSF1). Two positive associations were found. First, carriers of genotypes containing the methylene-tetrahydrofolate reductase 677T allele show constitutive low levels of 5-methylcytosine in their genomes (P = 0.002), and tumors in these patients do not achieve severe degrees of global hypomethylation (P = 0.047). Second, tumors occurring in homozygous carriers of the methionine synthase 2756G allele show a lower number of hypermethylated CpG islands of tumor suppressor genes (P = 0.029). The existence of these associations may provide another example of the interplay between genetic and epigenetic factors in the cancer cell.     

Genetic and epigenetic analysis of CHEK2 in sporadic breast, colon, and ovarian cancers.

PURPOSE: Germ-line variants in CHEK2 have been associated with increased breast, thyroid, prostate, kidney, and colorectal cancer risk; however, the prevalence of somatic inactivation of CHEK2 in common cancer types is less clear. The aim of this study was to determine if somatic mutation and/or epigenetic modification play a role in development of sporadic breast, colon, or ovarian cancers. EXPERIMENTAL DESIGN: We undertook combined genetic and epigenetic analysis of CHEK2 in sporadic primary breast, ovarian, and colon tumors [all exhibiting chromosome 22q loss of heterozygosity (LOH)] and cancer cell lines. Expression of Chk2 was assessed by immunohistochemistry in 119 ovarian tumors. RESULTS: Two novel germ-line variants were identified; however, none of the primary tumors harbored somatic mutations. Two CpG clusters previously implicated in CHEK2 silencing were investigated for evidence of hypermethylation. No methylation was detected at the distal CpG island. The proximal CpG cluster was methylated in all tumor and normal DNA, suggesting that this might not represent a true CpG island and is not relevant in the control of CHEK2 expression. Twenty-three percent of ovarian tumors were negative for Chk2 protein by immunohistochemistry, but there was no significant correlation between LOH across the CHEK2 locus and intensity of Chk2 staining (P = 0.12). CONCLUSIONS: LOH across the CHEK2 locus is common in sporadic breast, ovarian, and colorectal cancers, but point mutation or epigenetic inactivation of the retained allele is uncommon. Loss of Chk2 protein in ovarian cancer was not associated with allelic status, suggesting that inactivation does not occur as a consequence of haploinsufficiency.     

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.     

Germ-line variants in methyl-group metabolism genes and susceptibility to DNA methylation in human breast cancer

Aberrant CpG island hypermethylation in gene promoter regions may be an important epigenetic event in human neoplasias, including breast cancer. Dietary and genetic factors that alter DNA methylation levels in normal and tumour tissues could therefore influence both the susceptibility to this disease and tumour phenotype, respectively. In the present study of 227 breast cancers, we investigated whether common polymorphisms in 6 key genes involved in methyl group metabolism (thymidylate synthase, methylene tetrahydrofolate reductase, cystathione beta-synthase, DNA methyltransferase 3B, methylene tetrahydrofolate dehydrogenase, and methionine synthase) were associated with major pathological features of this disease or the frequency of CpG island hypermethylation. No associations were observed between any of the polymorphisms and patient age, tumour size, histological grade or patient outcome. However, tumours from patients who were homozygous for the methionine synthase A2756G polymorphism showed strikingly lower estrogen and progesterone hormone receptor concentrations compared to wild-type homozygotes. Moreover, patients who were homozygous for the methylene tetrahydrofolate dehydrogenase G1958A polymorphism showed a significantly higher frequency of tumour CpG island hypermethylation compared to wild-type homozygotes. Our results show that polymorphisms in two genes involved in methyl group metabolism are associated with hormone receptor content and DNA methylation frequency in breast cancer, however these observations are unlikely to be linked.     

Differential methylation hybridization array of endometrial cancers reveals two novel cancer-specific methylation markers.

PURPOSE: To identify novel endometrial cancer-specific methylation markers and to determine their association with clinicopathologic variables and survival outcomes. EXPERIMENTAL DESIGN: Differential methylation hybridization analysis (DMH) was done for 20 endometrioid endometrial cancers using normal endometrial DNA as a reference control. Combined bisulfite restriction analysis (COBRA) was used to verify methylation of sequences identified by DMH. Bisulfite sequencing was undertaken to further define CpG island methylation and to confirm the reliability of the COBRA. The methylation status of newly identified markers and the MLH1 promoter was evaluated by COBRA in a large series of endometrioid (n=361) and non-endometrioid uterine cancers (n=23). RESULTS: DMH and COBRA identified two CpG islands methylated in tumors but not in normal DNAs: SESN3 (PY2B4) and TITF1 (SC77F6/154). Bisulfite sequencing showed dense methylation of the CpG islands and confirmed the COBRA assays. SESN3 and TITF1 were methylated in 20% and 70% of endometrioid tumors, respectively. MLH1 methylation was seen in 28% of the tumors. TITF1 and SESN3 methylation was highly associated with MLH1 methylation (P<0.0001). SESN3 and TITF1 were methylated in endometrioid and non-endometrioid tumors, whereas MLH1 methylation was restricted to endometrioid tumors. Methylation at these markers was not associated with survival outcomes. CONCLUSIONS: The 5' CpG islands for SESN3 and TITF1 are novel cancer-specific methylation markers. Methylation at these loci is strongly associated with aberrant MLH1 methylation in endometrial cancers. SESN3, TITF1 and MLH1 methylation did not predict overall survival or disease-free survival in this large cohort of patients with endometrioid endometrial cancer.     

BRCA1 Promoter Hypermethylation Signature for Early Detection of Breast Cancer in the Vietnamese Population

Breast cancer, a leading cause of death among women in most countries worldwide, is rapidly increasing inincidence in Vietnam. One of biomarkers is the disruption of the genetic material including epigenetic changeslike DNA methylation. With the aim of finding hypermethylation at CpG islands of promoter of BRCA1 gene,belonged to the tumor suppressor gene family, as the biomarker for breast cancer in Vietnamese population,sensitive methyl specific PCR (MSP) was carried out on 115 samples including 95 breast cancer specimens and20 normal breast tissues with other diseases which were obtained from Ho Chi Minh City Medical Hospital,Vietnam. The result indicated that the frequency of BRCA1 hypermethylation reached 82.1% in the cases(p<0.001). In addition, the DNA hypermethylation of this candidate gene increased the possibility to be breastcancer with high incidence via calculated odd ratios (p<0.05). In conclusion, hypermethylation of this candidategene could be used as the promising biomarker application with Vietnamese breast cancer patients.     

Mismatch repair deficiency and CpG island hypermethylation in sporadic colon adenocarcinomas.

Many studies have documented CpG island hypermethylation in human colon adenocarcinomas. Several of these reports have additionally found such CpG island hypermethylation to be more extensive in tumors with a mismatch-repair deficiency, as revealed by microsatellite instability (MSI+). Because the source of samples used in these prior studies may not have been representative of the general population, we have reinvestigated this issue using samples from a population-based study. A total of 15 MSI+ tumors were identified, and they were compared with 47 MSI- tumors that were similar in distribution by age, sex, and race. Microdissected tumor and normal adjacent mucosal DNA samples from each patient were subjected to a quantitative DNA methylation analysis at 13 separate CpG dinucleotides located in five CpG islands in four different genes [APC, ESR1 (ER), CDKN2A (p16; promoter and exon 2), and MLH1]. Four of five CpG islands showed a statistically significantly increased level of methylation in tumor tissue compared with adjacent normal mucosa. In contrast to previous studies, we did not find any statistically significant correlations between MSI status and methylation levels of any of the CpG islands other than MLH1. Furthermore, we observed a positive correlation between MLH1 methylation and CDKN2A methylation (P = 0.03), whereas no association was noted between MSI positivity and CDKN2A methylation (P = 0.95). The latter results suggest a possible defect in the protection against CpG island hypermethylation shared between CDKN2A and MLH1 and do not support the notion of a functional association between CDKN2A methylation and the phenotype of mismatch repair deficiency.