High-resolution oligonucleotide array-CGH pinpoints genes involved in cryptic losses in chronic lymphocytic leukemia

Although recurrent chromosomal alterations occur in chronic lymphocytic leukemia (CLL), relatively few affected tumor suppressors and oncogenes have been implicated. To improve genetic characterization of CLL, we performed high-resolution gene copy number analysis of 20 CLL patients using oligonucleotide array comparative genomic hybridization (aCGH). The most recurrent losses were observed in 13q and 11q with variable sizes. The 11q losses varied between 7.44 Mb and 41.72 Mb in size and targeted ATM among others. Lost regions in 13q were generally smaller, spanning from 0.79 Mb to 29.33 Mb. The minimal common region (158 kb) in 13q14.3, which was also homozygously deleted in some cases, harbored five genes: TRIM13, KCNRG, DLEU2, DLEU1, and FAM10A4. Additionally, two micro-RNA genes (MIRN15A and MIRN16-1) locate to the region. New cryptic losses were detected in 1q23.2-->q23.3, 3p21.31, 16pter-->p13.3, 17p13.3-->p13.2, 17q25.3-->qter, and 22q11.22. In conclusion, our oligonucleotide aCGH study revealed novel aberrations and provided detailed genomic profiles of the altered regions.     

Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

With the goal of studying epigenetic alterations in fibrolamellar hepatocellular carcinoma (FLC) and establish an associated DNA methylation signature, we analyzed LINE-1 methylation in a cohort of FLC and performed next-generation sequencing of DNA methylation in a training set of pure-FLCs and non-cirrhotic hepatocellular carcinomas (nc-HCC). DNA methylation was correlated with gene expression. Furthermore, we established and validated an epigenetic signature differentiating pure-FLC from other HCCs. LINE-1 methylation correlated with shorter recurrence-free survival and overall survival in resected pure-FLC patients. Unsupervised clustering using CG sites located in islands distinguished pure-FLC from nc-HCC. Major DNA methylation changes occurred outside promoters, mainly in gene bodies and intergenic regions located in the vicinity of liver developmental genes (i.e., SMARCA4 and RXRA). Partially methylated domains were more prone to DNA methylation changes. Furthermore, we identified several putative tumor suppressor genes (e.g., DLEU7) and oncogenes (e.g., DUSP4). While ∼70% of identified gene promoters gaining methylation were marked by bivalent histone marks (H3K4me3/H3K27me3) in embryonic stem cells, ∼70% of those losing methylation were marked by H3K4me3. Finally, we established a pure FLC DNA methylation signature and validated it in an independent dataset. Our analysis reveals a distinct epigenetic signature of pure FLC as compared to nc-HCC, with DNA methylation changes occurring in the vicinity of liver developmental genes. These data suggest new options for targeting FLC based on cancer epigenome aberrations.     

microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.     

Array-Based Approaches for the Identification of Epigenetic Silenced Tumor Suppressor Genes

Carcinogenesis involves the inactivation or inhibition of genes that function as tumor suppressors. Deletions, mutations, or epigenetic silencing of tumor suppressor genes can lead to altered growth, differentiation, and apoptosis. DNA methylation and histone modifications are important epigenetic mechanisms of gene regulation and play essential roles both independently and cooperatively in tumor initiation and progression. Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated discovery of novel tumor suppressor genes. One of the most useful of these approaches is an epigenetic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase (HDAC) inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Recently, using cancer cell lines treated with a DNA methylation inhibitor and/or a HDAC inhibitor in conjunction with cDNA microarray analysis, candidate tumor suppressor genes, which are subject to epigenetic silencing, have been identified in endometrial, colorectal, esophageal, and pancreatic cancers. An increasing number of studies have utilized epigenetic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

A novel deletion cluster at 13q14.2-q21.33 in an 80-year man with late onset leukemia: Clinical and molecular findings

Chromosomal deletions are among the most common genetic events observed in hematologic malignancies; loss of genetic material is regarded as a hallmark of putative tumor suppressor gene localization. We have identified an unusual cluster of deletions at 13q14.2-13q21.33 in an 80-year-old father of a monozygotic twin pair discordant for schizophrenia, who developed chronic leukemia (CLL) at age 69.

MATERIALS AND METHODS:

The breakpoints for individual deletions in this cluster was identified by Affymetrix Human Array 6.0 screening.

RESULTS:

The deleted segments harbours a number of genes, most associated with cancer as well as a high concentration of LINEs, SINEs and related repeats. The derived chromosome represents an intra-chromosomal re-arrangement that quickly overtook blood progenitor cells probably before age 69 as a cause of CLL.

CONCLUSIONS:

The study highlights the role of ongoing de novo changes at susceptible sites, such as repeat rich regions, in the human genome. Also, it argues for the involvement of genes/deletions in the 13q(14.2-21.33) region in the development of CCL.     

microRNA-34b/c on chromosome 11q23 is aberrantly methylated in chronic lymphocytic leukemia

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence of 11q-deletion, indicating that methylation and del(11q) independently silence these miRs. Finally, 5-azacytidine and trichostatin A exposure synergistically increased the expression of miR-34b/c in CLL cells, and transfection of miR-34b or miR-34c into HG3 CLL cells significantly increased apoptosis. Altogether, our novel data suggest that miR-34b/c is a candidate tumor suppressor that is epigenetically silenced in CLL.     

Alterations in The Plasma Expression of mir-15b,mir-195 and the Tumor-Suppressor Gene DLEU7 in Patients with B-Cell Chronic Lymphocytic Leukemia

Background:Chronic lymphocytic leukemia (CLL) is one of the most prevalent forms of leukemia in adults. Inactivation of the DLEU7 gene is frequently observed in patients with CLL. Furthermore, microRNAs (miRNAs) have been observed to have a critical role in the pathogenesis of several cancers, including leukemia. Considering the tumor-suppressive role of DLEU7, as well as the tumor suppressor or oncogenic role of microRNAs (miRNAs), the aim of the present study was to evaluate the potential miRNAs targeting the DLEU7 gene in B-cells and explore expression changes these genes in the plasma of B-CLL patients. Methods:The miRNAs interacting with the DLEU7 gene were predicted and selected using bioinformatics tools. A total of 80 plasma samples were collected from 40 patients with B-cells and 40 healthy individuals, then subjected to RNA extraction and cDNA synthesis. The expression profiles of the predicted miRNAs and the DLEU7 gene in the plasma of B-CLL patients and healthy individuals were determined by RT-qPCR analysis. Results:The bioinformatics prediction indicated that miR-15b and miR-195 target the DLEU7 gene. The expression levels of miR-15b and miR-195 were significantly higher in the plasma of patients with B-CLL compared to the healthy individuals (91.6, p= 0.001) (169, p= 0.001). However, the expression level of the DLEU7 gene was found to be significantly lower in the patient group compared to healthy controls (0.304, p= 0.001).Conclusion:Both miR-15b and miR-195, have the potential to function as novel and non-invasive biomarkers in the diagnosis and prognosis of patients with B-CLL.Key Words: B-CLL, miRNA, Biomarker, DLEU7, RT-QPCR     

Aberrant methylation of tumor suppressor genes and allelic imbalance in cervical intraepithelial neoplasia

The methylation status of genes p16, MLH1, HIC1, MGMT, N33, and RB1 was determined in cervical smears of women without gynecological pathology, in biopsies from patients with high-grade cervical intraepithelial neoplasia (CIN3), and in samples of nondysplastic cervical tissue adjacent to CIn3. The level of methylation of these genes in normal smears was insignificant. In CIN3, methylation frequencies were as follows: 58% for p16, 51% for MLH1, 84% for HIC1, and 27% for N33. However, dysplastic tissues did not differ significantly from the control with respect to the methylation frequencies of the MGMT and RB1 genes (8 and 15%, respectively). The methylation frequency of the suppressor genes in nondysplastic, morphologically normal adjacent tissues was also high. We found that 21% of samples (9/42) had microsatellite instability at chromosomes 5q11.2–q14.3 (3/42) and 13q14–q14.3 (6/42). Loss of heterozygosity (LOH) at region 13q14 was detected in 7% of samples (3/42).     

Role of miR-15/16 in CLL

B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia. The most common chromosomal abnormalities detectable by cytogenetics include deletion at 13q (55%), 11q (18%), trisomy 12 (12–16%) and 17p (8%). In 2002, we discovered that a microRNA cluster miR-15a/miR-16-1 (miR-15/16) is the target of 13q deletions in CLL. MicroRNAs encoded by the miR-15/16 locus (miR-15 and miR-16) function as tumor suppressors. Expression of these miRNAs downregulated in CLL, melanoma, colorectal cancer, bladder cancer and other solid tumors. miR-15/16 cluster targets multiple oncogenes, including BCL2, Cyclin D1, MCL1 and others. The most important target of miR-15/16 in CLL is arguably BCL2, as BCL2 is overexpressed in almost all CLLs. In this review, we discuss the discovery, functions, clinical relevance and treatment opportunities related to miR-15/16.     

Physical Interaction between MYCN Oncogene and Polycomb Repressive Complex 2 (PRC2) in Neuroblastoma: FUNCTIONAL AND THERAPEUTIC IMPLICATIONS*

CLU (clusterin) is a tumor suppressor gene that we have previously shown to be negatively modulated by the MYCN proto-oncogene, but the mechanism of repression was unclear. Here, we show that MYCN inhibits the expression of CLU by direct interaction with the non-canonical E box sequence CACGCG in the 5′-flanking region. Binding of MYCN to the CLU gene induces bivalent epigenetic marks and recruitment of repressive proteins such as histone deacetylases and Polycomb members. MYCN physically binds in vitro and in vivo to EZH2, a component of the Polycomb repressive complex 2, required to repress CLU. Notably, EZH2 interacts with the Myc box domain 3, a segment of MYC known to be essential for its transforming effects. The expression of CLU can be restored in MYCN-amplified cells by epigenetic drugs with therapeutic results. Importantly, the anticancer effects of the drugs are ablated if CLU expression is blunted by RNA interference. Our study implies that MYC tumorigenesis can be effectively antagonized by epigenetic drugs that interfere with the recruitment of chromatin modifiers at repressive E boxes of tumor suppressor genes such as CLU.     

Differential epigenetic profiles induced by sodium selenite in breast cancer cells

ObjectivesSelenium (Se) was a potential anticancer micronutrient with proposed epigenetic effect. However, the Se-induced epigenome in breast cancer cells was yet to be studied.MethodsThe profiles of DNA methylation, microRNA (miRNA), long non-coding RNA (lncRNA), and message RNA (mRNA) in breast cancer cells treated with sodium selenite were examined by microarrays. We verified the epigenetic modifications by integrating their predicted target genes and differentially expressed mRNAs. The epigenetically regulated genes were further validated in a breast cancer cohort by associating with tumor progression. We conducted a series of bioinformatics analyses to assess the biological function of these validated genes and identified the critical genes.ResultsThe Se-induced epigenome regulated the expression of 959 genes, and 349 of them were further validated in the breast cancer cohort. Biological function analyses suggested that these validated genes were enriched in several cancer-related pathways, such as PI3K/Akt and metabolic pathways. Based on the degrees of expression change, hazard ratio difference, and connectivity, NEDD4L and FMO5 were identified as the critical genes.ConclusionsThese results confirmed the epigenetic effects of sodium selenite and revealed the epigenetic profiles in breast cancer cells, which would help understand the mechanisms of Se against breast cancer.