Regional methylation of the 5' end CpG island of BRCA1 is associated with reduced gene expression in human somatic cells.

In mammalians, demethylation of specific promoter regions often correlates with gene activation; inversely, dense methylation of CpG islands leads to gene silencing, probably mediated by methyl-CpG binding proteins. In cell lines and cancers, inhibition of tissue-specific genes and tumor suppressor genes expression seems to be related to such hypermethylation. The 5' end of the breast cancer predisposition gene BRCA1 is embedded in a large CpG island of approximately 2.7 kb in length. In human sporadic breast cancers, the down-regulation of BRCA1 does not seem to be related to BRCA1 gene alterations. Southern blot analysis and the bisulfite sequencing method indicate that the BRCA1 CpG island is regionally methylated in all human tissues analyzed and unmethylated in the gametes, suggesting a role for DNA methylation in the control of gene expression. We have therefore investigated the potential role of methyl-CpG binding proteins in the regulation of BRCA1 gene expression. In vitro, partial methylation of constructs containing this region strongly inhibits gene expression in the presence of MeCP2 protein. Moreover, in the five human cell lines analyzed, chemically induced hypomethylation is associated with BRCA1 gene activation. These data suggest that methyl-CpG binding proteins might be associated with the control of BRCA1 gene expression and that methyl-DNA binding proteins may participate in the regulation of gene expression in mammalian cells.     

Profile of methylation of certain tumor growth suppressing genes in non-small cell lung cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5' regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk-Rb-E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.     

Association of rs11801299 and rs1380576 polymorphisms at MDM4 with risk,clinicopathological features and prognosis in patients with retinoblastoma

Backgroundrs11801299 and rs1380576, two novel polymorphisms in MDM4 gene, have been investigated in several different cancer types. However, the role of these two polymorphisms in retinoblastoma (RB) remains unclear.MethodsA total of 126 patients with primary RB and 148 age-/gender-matched controls were included in this retrospective study. The frequency of rs11801299 and rs1380576 were determined between RB patients and controls. The association of these two polymorphisms with clinicopathological characteristics, prognosis were further evaluated.ResultsAA genotype at rs11801299 was significantly associated with an increased risk of developing RB (OR = 2.06, 95%CI 1.09–3.90). The possibility of developing RB was also significantly increased in individuals with A allele at rs11801299 (OR = 1.49, 95%CI 1.06–2.08). RB patients carrying AA genotype and A allele at rs11801299 were more likely to have tumor invasion and poor differentiation. As for rs1380576, a significantly lower risk of developing RB was observed in patients with G allele (CG + GG) compared with wild-type CC genotype (OR = 0.59, 95%CI 0.36–3.95). RB patients with GG genotype or G allele had a lower risk of developing highly aggressive cancer. Kaplan-Meier curves and log-rank results revealed that RB patients carrying AA genotype or A allele (AA + GA) at rs11801299 had significantly poorer prognosis. Multivariate COX analysis showed that the rs11801299 G allele was associated with decreased survival but was not an independent prognostic factor.Conclusionrs11801299 was significantly associated with RB risk, pathological differentiation, tumor aggressiveness and poor prognosis.     

RB loss contributes to aggressive tumor phenotypes in MYC-driven triple negative breast cancer

Triple negative breast cancer (TNBC) is characterized by multiple genetic events occurring in concert to drive pathogenic features of the disease. Here we interrogated the coordinate impact of p53, RB, and MYC in a genetic model of TNBC, in parallel with the analysis of clinical specimens. Primary mouse mammary epithelial cells (mMEC) with defined genetic features were used to delineate the combined action of RB and/or p53 in the genesis of TNBC. In this context, the deletion of either RB or p53 alone and in combination increased the proliferation of mMEC; however, the cells did not have the capacity to invade in matrigel. Gene expression profiling revealed that loss of each tumor suppressor has effects related to proliferation, but RB loss in particular leads to alterations in gene expression associated with the epithelial-to-mesenchymal transition. The overexpression of MYC in combination with p53 loss or combined RB/p53 loss drove rapid cell growth. While the effects of MYC overexpression had a dominant impact on gene expression, loss of RB further enhanced the deregulation of a gene expression signature associated with invasion. Specific RB loss lead to enhanced invasion in boyden chambers assays and gave rise to tumors with minimal epithelial characteristics relative to RB-proficient models. Therapeutic screening revealed that RB-deficient cells were particularly resistant to agents targeting PI3K and MEK pathway. Consistent with the aggressive behavior of the preclinical models of MYC overexpression and RB loss, human TNBC tumors that express high levels of MYC and are devoid of RB have a particularly poor outcome. Together these results underscore the potency of tumor suppressor pathways in specifying the biology of breast cancer. Further, they demonstrate that MYC overexpression in concert with RB can promote a particularly aggressive form of TNBC.     

Common breast cancer-predisposition alleles are associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers

Germline mutations in BRCA1 and BRCA2 confer high risks of breast cancer. However, evidence suggests that these risks are modified by other genetic or environmental factors that cluster in families. A recent genome-wide association study has shown that common alleles at single nucleotide polymorphisms (SNPs) in FGFR2 (rs2981582), TNRC9 (rs3803662), and MAP3K1 (rs889312) are associated with increased breast cancer risks in the general population. To investigate whether these loci are also associated with breast cancer risk in BRCA1 and BRCA2 mutation carriers, we genotyped these SNPs in a sample of 10,358 mutation carriers from 23 studies. The minor alleles of SNP rs2981582 and rs889312 were each associated with increased breast cancer risk in BRCA2 mutation carriers (per-allele hazard ratio [HR] = 1.32, 95% CI: 1.20-1.45, p(trend) = 1.7 x 10(-8) and HR = 1.12, 95% CI: 1.02-1.24, p(trend) = 0.02) but not in BRCA1 carriers. rs3803662 was associated with increased breast cancer risk in both BRCA1 and BRCA2 mutation carriers (per-allele HR = 1.13, 95% CI: 1.06-1.20, p(trend) = 5 x 10(-5) in BRCA1 and BRCA2 combined). These loci appear to interact multiplicatively on breast cancer risk in BRCA2 mutation carriers. The differences in the effects of the FGFR2 and MAP3K1 SNPs between BRCA1 and BRCA2 carriers point to differences in the biology of BRCA1 and BRCA2 breast cancer tumors and confirm the distinct nature of breast cancer in BRCA1 mutation carriers.     

DCC (deleted in colorectal carcinoma) gene variants confer increased susceptibility to gallbladder cancer (Ref. No.: Gene-D-12-01446)

Background and aim

GBC is a lethal and multifaceted disease. Deleted in colorectal carcinoma (DCC) is a well known tumor suppressor gene. Recently a small genome-wide association study (GWAS) identified DCC to be significantly associated with gallbladder cancer (GBC) susceptibility in a Japanese population sample. However, the study sample size was small and lacked independent replication. Therefore, the present study was carried out to replicate the association of two GWAS identified DCC SNPs (A>G_rs4078288, C>T_rs7504990) and two other SNPs (C>G_rs2229080 and A>G_rs714) previously associated with various cancers.

Methodology

The study was accomplished in 406 GBC cases and 260 healthy control samples from North India. Genotyping was carried out by PCR-RFLP and Taqman genotyping assays. Statistical analysis was performed by using SPSS ver16 and functional prediction of these variants was carried out using Bioinformatics tools (FAST-SNP, F-SNP).

Result

We did not observe association with GWAS-identified SNPs of DCC but other SNPs showed significant association. In addition, a DCC haplotype G_rs2229080-A_rs4078288-C_rs7504990-A_rs714 conferred high risk of GBC in India. The haplotype associated risk was independent of gallstone, sex or tobacco usages which are well-known modifiers of GBC risk. Further analysis suggested DCC A>G_rs714 as a major risk conferring SNP in the Indian population.

Conclusion

This study re-affirms the role of plausible tumor suppressor DCC variants, in gallbladder carcinogenesis and the risk haplotype may be explored as a useful marker for GBC susceptibility.     

Monozygotic twins discordant for constitutive BRCA1 promoter methylation, childhood cancer and secondary cancer

We describe monozygotic twins discordant for childhood leukemia and secondary thyroid carcinoma. We used bisulfite pyrosequencing to compare the constitutive promoter methylation of BRCA1 and several other tumor suppressor genes in primary fibroblasts. The affected twin displayed an increased BRCA1 methylation (12%), compared with her sister (3%). Subsequent bisulfite plasmid sequencing demonstrated that 13% (6 of 47) BRCA1 alleles were fully methylated in the affected twin, whereas her sister displayed only single CpG errors without functional implications. This between-twin methylation difference was also found in irradiated fibroblasts and untreated saliva cells. The BRCA1 epimutation may have originated by an early somatic event in the affected twin: approximately 25% of her body cells derived from different embryonic cell lineages carry one epigenetically inactivated BRCA1 allele. This epimutation was associated with reduced basal protein levels and a higher induction of BRCA1 after DNA damage. In addition, we performed a genome-wide microarray analysis of both sisters and found several copy number variations, i.e., heterozygous deletion and reduced expression of the RSPO3 gene in the affected twin. This monozygotic twin pair represents an impressive example of epigenetic somatic mosaicism, suggesting a role for constitutive epimutations, maybe along with de novo genetic alterations in recurrent tumor development.     

Functional interactions between retinoblastoma and c-MYC in a mouse model of hepatocellular carcinoma

Inactivation of the RB tumor suppressor and activation of the MYC family of oncogenes are frequent events in a large spectrum of human cancers. Loss of RB function and MYC activation are thought to control both overlapping and distinct cellular processes during cell cycle progression. However, how these two major cancer genes functionally interact during tumorigenesis is still unclear. Here, we sought to test whether loss of RB function would affect cancer development in a mouse model of c-MYC-induced hepatocellular carcinoma (HCC), a deadly cancer type in which RB is frequently inactivated and c-MYC often activated. We found that RB inactivation has minimal effects on the cell cycle, cell death, and differentiation features of liver tumors driven by increased levels of c-MYC. However, combined loss of RB and activation of c-MYC led to an increase in polyploidy in mature hepatocytes before the development of tumors. There was a trend for decreased survival in double mutant animals compared to mice developing c-MYC-induced tumors. Thus, loss of RB function does not provide a proliferative advantage to c-MYC-expressing HCC cells but the RB and c-MYC pathways may cooperate to control the polyploidy of mature hepatocytes.     

DNA Methylation Differences Associated with Tumor Tissues Identified by Genome Scanning Analysis

Most investigations on the role of DNA methylation in cancer have focused on epigenetic changes associated with known tumor suppressor genes. This may have led to an underestimation of the number of CpG islands altered by DNA methylation, since it is possible that a subset of unknown genes relevant to cancer development may preferentially be affected by epigenetic rather than genetic means and would not be identified as familial deletions, mutations, or loss of heterozygosity. We used a recently developed screening procedure (methylation-sensitive arbitrarily primed-polymerase chain reaction to scan genomic DNA for CpG islands methylated in white blood cells (WBCs) and in tumor tissues. DNA methylation pattern analysis showed little interindividual differences in the WBCs and normal epithelium (adjacent to colon, bladder, and prostate cancer cells), but with some tissue-specific differences. Cancer cells showed marked methylation changes that varied considerably between different tumors, suggesting variable penetrance of the methylation phenotype in patients. Direct sequencing of 8 of 45 bands altered in these cancers showed that several of them were CpG islands, and 2 of these sequences were identified in GenBank. Surprisingly, three of the bands studied corresponded to transcribed regions of genes. Thus, hypermethylation of CpG islands in cancer cells is not confined to the promoters of growth regulatory genes but is also found in actively transcribed regions.     

Evolutionary dynamics of BRCA1 alterations in breast tumorigenesis

Cancer results from the accumulation of alterations in oncogenes and tumor suppressor genes. Tumor suppressors are classically defined as genes which contribute to tumorigenesis if their function is lost. Genetic or epigenetic alterations inactivating such genes may arise during somatic cell divisions or alternatively may be inherited from a parent. One notable exception to this rule is the BRCA1 tumor suppressor that predisposes to hereditary breast cancer when lost. Genetic alterations of this gene are hardly ever observed in sporadic breast cancer, while individuals harboring a germline mutation readily accumulate a second alteration inactivating the remaining allele—a finding which represents a conundrum in cancer genetics. In this paper, we present a novel mathematical framework of sporadic and hereditary breast tumorigenesis. We study the dynamics of genetic alterations driving breast tumorigenesis and explore those scenarios which can explain the absence of somatic BRCA1 alterations while replicating all other disease statistics. Our results support the existence of a heterozygous phenotype of BRCA1 and suggest that the loss of one BRCA1 allele may suppress the fitness advantage caused by the inactivation of other tumor suppressor genes. This paper contributes to the mathematical investigation of breast tumorigenesis.     

MCPH1 protein expression and polymorphisms are associated with risk of breast cancer

Microcephalin 1 (MCPH1) has a crucial role in the DNA damage response by promoting the expression of checkpoint kinase 1 (CHK1) and breast cancer susceptibility gene 1 (BRCA1). MCPH1 containing BRCT domain has been suggested as a tumor suppressor in breast and ovarian cancers. We analyzed the effect of both protein expression and MCPH1 polymorphisms in breast cancer patients. Low nuclear expression of microcephalin was present in 52.4% of breast cancers and was associated with allele T in rs2912010 (p = 0.046). However, cytoplasmic microcephalin expression increased with increasing grade (p = 0.010). An association between low nucleus microcephalin expression and allele T was identified in rs2912010 (p = 0.046). After data analysis, allele distribution of the MCPH1 polymorphisms was not different between breast cancer patients and healthy controls. But the polymorphism was associated with negative status for ER (rs2912010/C2302T; p = 0.032, rs1057090/C2358T; p = 0.027, rs2912016/C2494A; p = 0.024), and allele T in both rs2912010 and rs1057090 was associated with increasing tumor grade (rs2912010; p = 0.040, rs1057090; p = 0.043) in breast cancer. We are first to report that association of MCPH1 protein expression and its polymorphisms in breast cancer. The MCPH1 polymorphisms and protein expression were associated with tumorigenesis in breast cancer and may be a useful biomarker for identification of the aggressive types of breast cancer.     

Senescence and epigenetic dysregulation in cancer

Mammalian cells have a finite proliferative lifespan, at the end of which they are unable to enter S phase in response to mitogenic stimuli. They undergo morphological changes and synthesize an altered repertoire of cell type-specific proteins. This non-proliferative state is termed replicative senescence and is regarded as a major tumor suppressor mechanism. The ability to overcome senescence and obtain a limitless replicative potential is called immortalization, and considered to be one of the prerequisites of cancer formation. While senescence mainly represents a genetically governed process, epigenetic changes in cancer have received increasing attention as an alternative mechanism for mediating gene expression changes in transformed cells. DNA methylation of promoter-containing CpG islands has emerged as an epigenetic mechanism of silencing tumor suppressor genes. New insights are being gained into the mechanisms causing aberrant methylation in cancer and evidence suggests that aging is accompanied by accumulation of cells with aberrant CpG island methylation. Aberrant methylation may contribute to many of the physiological and pathological changes associated with aging including tumor development. Finally, we describe how genes involved in promoting longevity might inhibit pathways promoting tumorigenesis.     

Detection method for methylation density on microarray using methyl-CpG-binding domain protein

A method for determining methylation density of target CpG islands has been established. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite-converted sample DNAs. The PCR products on the microarray were treated by SssI methyltransferase and labeled with TAMRA fluorescence. A recombinant, antibody-like methyl-CpG-binding protein labeled with Cy5 fluorescence was used to identify symmetrical methyl-CpG dinucleotide of the PCR products on the microarray. By use of a standard curve with control mixtures, the ratio of two fluorescence signals can be converted into percentage values to assess methylation density of targeted fragments. We obtained the methylation density of six CpG islands on the two tumor suppressor genes of CDK2A and CDK2B from seven cancer cell line samples and two normal blood samples. The validity of this method was tested by bisulfite sequencing. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples.     

cis-Expression QTL analysis of established colorectal cancer risk variants in colon tumors and adjacent normal tissue

Genome-wide association studies (GWAS) have identified 19 risk variants associated with colorectal cancer. As most of these risk variants reside outside the coding regions of genes, we conducted cis-expression quantitative trait loci (cis-eQTL) analyses to investigate possible regulatory functions on the expression of neighboring genes. Forty microsatellite stable and CpG island methylator phenotype-negative colorectal tumors and paired adjacent normal colon tissues were used for genome-wide SNP and gene expression profiling. We found that three risk variants (rs10795668, rs4444235 and rs9929218, using near perfect proxies rs706771, rs11623717 and rs2059252, respectively) were significantly associated (FDR q-value ≤0.05) with expression levels of nearby genes (<2 Mb up- or down-stream). We observed an association between the low colorectal cancer risk allele (A) for rs10795668 at 10p14 and increased expression of ATP5C1 (q = 0.024) and between the colorectal cancer high risk allele (C) for rs4444235 at 14q22.2 and increased expression of DLGAP5 (q = 0.041), both in tumor samples. The colorectal cancer low risk allele (A) for rs9929218 at 16q22.1 was associated with a significant decrease in expression of both NOL3 (q = 0.017) and DDX28 (q = 0.046) in the adjacent normal colon tissue samples. Of the four genes, DLGAP5 and NOL3 have been previously reported to play a role in colon carcinogenesis and ATP5C1 and DDX28 are mitochondrial proteins involved in cellular metabolism and division, respectively. The combination of GWAS findings, prior functional studies, and the cis-eQTL analyses described here suggest putative functional activities for three of the colorectal cancer GWAS identified risk loci as regulating the expression of neighboring genes.     

Methylation Profile of Several Tumor Suppressor Genes in Non-Small-Cell Lung Cancer

Multiplex methylation-sensitive PCR was employed in studying the methylation of CpG islands in the RB1, p16/CDKN2A, p15/CDKN2B, p14/ARF, CDH1, HIC1, and N33 5 regions in non-small cell lung cancer (51 tumors). Methylation was observed for the two suppressor genes involved in controlling the cell cycle through the Cdk–Rb–E2F signaling pathway, RB1 (10/51, 19%) and p16 (20/51, 39%). The highest methylation frequencies were established for CDH1 (72%) and HIC1 (82%). The CpG islands of p14 and p15 proved to be nonmethylated. At least one gene was methylated in 90% (46/51) tumors and no gene, in 10% (5/51) tumors. In addition, the genes were tested for methylation in peripheral blood lymphocytes of healthy subjects. Methylation frequency significantly differed between tumors and normal cells in the case of RB1, p16, CDH1, HIC1, and N33. Gene methylation frequency was tested for association with histological type of the tumor and stage of tumor progression. Methylation index of a panel of tumor suppressor genes was established for groups of tumors varying in clinical and morphological parameters.