Frequent epigenetic inactivation of KIBRA,an upstream member of the Salvador/Warts/Hippo (SWH) tumor suppressor network,is associated with specific genetic event in B-cell acute lymphocytic leukemia

The WW-domain containing protein KIBRA has recently been identified as a new member of the Salvador/Warts/Hippo (SWH) pathway in Drosophila and is shown to act as a tumor suppressor gene in Drosophila. This pathway is conserved in humans and members of the pathway have been shown to act as tumor suppressor genes in mammalian systems. We determined the methylation status of the 5′ CpG island associated with the KIBRA gene in human cancers. In a large panel of cancer cell lines representing common epithelial cancers KIBRA was unmethylated. But in pediatric acute lymphocytic leukemia (ALL) cell lines KIBRA showed frequent hypermethylation and silencing of gene expression, which could be reversed by treatment with 5-aza-2′-deoxycytidine. In ALL patient samples KIBRA was methylated in 70% B-ALL but was methylated in <20% T-ALL leukemia (p = 0.0019). In B-ALL KIBRA methylation was associated with ETV6/RUNX1 [t(12;21) (p13;q22)] chromosomal translocation (p = 0.0082) phenotype, suggesting that KIBRA may play an important role in t(12;21) leukemogenesis. In ALL paired samples at diagnosis and remission KIBRA methylation was seen in diagnostic but not in any of the remission samples accompanied by loss of KIBRA expression in disease state compared to patients in remission. Hence KIBRA methylation occurs frequently in B-cell acute lymphocytic leukemia but not in epithelial cancers and is linked to specific genetic event in B-ALL.Key words: KIBRA, methylation, ALL, SWH pathway, ETV6/RUNX1 translocation     

KIBRA gene methylation is associated with unfavorable biological prognostic parameters in chronic lymphocytic leukemia

Ras-association domain family (RASSF) members are a family of genes containing an RA domain in either the C-terminus (RASSF1-RASSF6) or in the N-terminus (RASSF7-RASSF10). Members of this gene family are core members of the Salvador/Warts/Hippo (SWH) tumor suppressor network and have been shown to be involved in human tumorigenesis. Among the RASSF genes, RASSF1A is one of the most frequently methylated genes in a wide range of epithelial cancers, and we previously demonstrated that RASSF6 and RASSF10 genes are frequently epigenetically inactivated in acute leukemias, particularly in those of the B cell type. We here determined the methylation profiles of all members of the RASSF gene family as well as two recently identified (KIBRA, CRB3) upstream members of the SWH pathway in the leukemic B cells obtained from a well-characterized cohort of 95 patients with chronic lymphocytic leukemia (CLL). Among the RASSF genes, RASSF10 (50%) was the most frequently methylated gene, followed by RASSF6 (16%). The remaining RASSF genes were either unmethylated or showed a frequency of methylation < 10%. The upstream SWH member KIBRA was also frequently methylated in CLL (35%) in contrast to CRB3. Interestingly, the analysis of clinical-pathological parameters showed that KIBRA methylation was associated with unfavorable biological prognostic parameters, including unmutated IGHV genes (p = 0.007) and high CD38 expression (p < 0.05).     

Frequent epigenetic inactivation of the SLIT2 gene in chronic and acute lymphocytic leukemia

Recently a mouse model of T/natural killer acute lymphoblastic leukemia was used to assess global promoter methylation across the mouse genome using the restriction landmark genomic scanning technique. One of the methylated mouse genes identified in this way was Slit2. There are three mammalian SLIT genes (SLIT1, SLIT2, SLIT3), that belong to a highly conserved family of axon guidance molecules. We have previously demonstrated that SLIT2 is frequently inactivated in lung, breast, colorectal and glioma tumors by hypermethylation of a CpG island in its promoter region, whilst inactivating somatic mutations are rare. Furthermore, we demonstrated that SLIT2 acts as a tumor suppressor gene in breast and colorectal cancer cells. In this report we determined the methylation status of the SLIT2 gene in leukemias (CLL and ALL). SLIT2 was methylated in all 10 leukemia cell lines analyzed (8 completely and 2 partially methylated). SLIT2 expression was restored after treating ALL lines with 5-aza-2dC. In primary ALL and CLL samples, SLIT2 was also frequently methylated, 58% (30/52) B-ALL; 83% (10/12) T-ALL and in 80% (24/30) CLL. Whilst DNA from peripheral blood and bone marrow from healthy control samples showed no SLIT2 methylation. Methylation results in leukemia cell lines and ALL and CLL primary samples were confirmed by direct sequencing of bisulfite modified DNA. Our results demonstrate that methylation of the SLIT2 5’ CpG island is conserved between mice and humans, and therefore is likely to be of functional importance.     

Epigenetic analysis of childhood acute lymphoblastic leukemia

We used a chromosome 3 wide NotI microarray for identification of epigenetically inactivated genes in childhood acute lymphoblastic leukemia (ALL). Three novel genes demonstrated frequent methylation in childhood ALL. PPP2R3A (protein phosphatase 2, regulatory subunit B'', alpha) was frequently methylated in T (69%) and B (82%)-ALL. Whilst FBLN2 (fibulin 2) and THRB (thyroid hormone receptor, beta) showed frequent methylation in B-ALL (58%; 56% respectively), but were less frequently methylated in T-ALL (17% for both genes). Recently it was demonstrated that BNC1 (Basonuclin 1) and MXS1 (msh homeobox 1) were frequently methylated across common epithelial cancers. In our series of childhood ALL BNC1 was frequently methylated in both T (77%) and B-ALL (79%), whilst MSX1 showed T-ALL (25%) specific methylation. The methylation of the above 5 genes was cancer specific and expression of the genes could be restored in methylated leukemia cell lines treated with 5-aza-2’-deoxycytidine. This is the first report demonstrating frequent epigenetic inactivation of PPP2R3A, FBLN2, THRB, BNC1 and MSX1 in leukemia. The identification of frequently methylated genes showing cancer specific methylation will be useful in developing early cancer detection screens and for targeted epigenetic therapies.     

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.     

RASSF2 methylation is a strong prognostic marker in younger age patients with Ewing sarcoma

Ras-association domain family of genes consist of 10 members (RASSF1-RASSF10), all containing a Ras-association (RA) domain in either the C- or the N-terminus. Several members of this gene family are frequently methylated in common sporadic cancers; however, the role of the RASSF gene family in rare types of cancers, such as bone cancer, has remained largely uninvestigated. In this report, we investigated the methylation status of RASSF1A and RASSF2 in Ewing sarcoma (ES). Quantitative real-time methylation analysis (MethyLight) demonstrated that both genes were frequently methylated in Ewing sarcoma tumors (52.5% and 42.5%, respectively) as well as in ES cell lines and gene expression was upregulated in methylated cell lines after treatment with 5-aza-2′-deoxcytidine. Overexpression of either RASSF1A or RASSF2 reduced colony formation ability of ES cells. RASSF2 methylation correlated with poor overall survival (p = 0.028) and this association was more pronounced in patients under the age of 18 y (p = 0.002). These results suggest that both RASSF1A and RASSF2 are novel epigenetically inactivated tumor suppressor genes in Ewing sarcoma and RASSF2 methylation may have prognostic implications for ES patients.     

Assessing combined methylation–sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.     

Epigenetic inactivation of the hsa-miR-203 in haematological malignancies

miR-203 is a tumour suppressor microRNA (miRNA). We studied the methylation of hsa-miR-203 in 150 samples including acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL) and non-Hodgkin's lymphoma (NHL) by methylation-specific PCR, and miRNA expression by stem-loop RT-qPCR. hsa-miR-203 promoter was unmethylated in normal controls but homozygously methylated in two AML and four lymphoma cell lines, in which 5-Aza-2'-deoxycytidine treatment led to promoter demethylation and miR-203 re-expression. Restoration of miR-203 expression in lymphoma cells inhibited cellular proliferation and increased cell death, suggesting an inherent tumour suppressor activity. In primary samples, hsa-miR-203 methylation was absent in CML but detected in 5.0% ALL, 10.0% AML, 42.0% CLL and 38.8% of NHL (including six [60.0%] natural killer-cell, nine [40.9%] B-cell and four [23.5%] T cell NHL). Moreover, hsa-miR-203 methylation was associated with hypermethylation of hsa-miR-34a, -124a and -196b in NHL but not CLL. In CLL, hsa-miR-203 methylation was associated with a higher presenting Hb level (P = 0.033). The projected 10 year overall survival of the CLL patients was 58.2%, which was impacted by Rai stage and high-risk karyotypes but not hsa-miR-203 methylation. hsa-miR-203 was more frequently methylated in lymphoid than myeloid malignancies (P = 0.002). In conclusion, miR-203, a tumour suppressor gene, was hypermethylated in a tumour-specific manner with gene silencing. hsa-miR-203 was more frequently hypermethylated in lymphoid than myeloid malignancies. In NHL, hsa-miR-203 methylation was associated with concomitant methylation of other tumour suppressor miRNAs. The frequent hsa-miR-203 methylation in lymphoid malignancies suggested a pathogenetic role of hsa-miR-203 methylation.     

The effect of decitabine on the expression and methylation of the PPP1CA,BTG2, and PTEN in association with changes in miR-125b,miR-17, and miR-181b in NALM6 cell line

Precursor B-cell acute lymphoblastic leukemia (B-ALL) is the most prevalent pediatric cancer. DNA methylation and changes in the microRNAs (miRNAs) expression are known to be important causes of B-ALL. Decitabine as a DNA methyltransferase inhibitor agent is able to induce hypomethylation in several tumor suppressor genes. Much evidence has proven BTG2, PPP1CA, and PTEN act as tumor suppressor genes in many malignancies. In this case control study, the messenger RNA (mRNA) expression of PPP1CA, BTG2, and PTEN genes using quantitative real-time polymerase chain reaction (rRT-PCR) in Nalm6 cell line and five patients suffer from ALL with mean age 5.6 years were determined in compare with seven normal healthy donors age and sex matched. qRT-PCR analysis revealed that the expression levels of PPP1CA, BTG2, and PTEN genes were significantly decreased in Nalm6 ([FC] = 0.46, [FC] = 0.046, [FC] = 0.54) and according to the Methylation-specific PCR (MSP) analysis, these genes were hypermethylated in Nalm6. In next step, the effects of decitabine treatment on the methylation and expression of these genes in association with changes in miR-125b, miR-17, and miR-181b expression levels were evaluated in optimal concentration 2.5 µM of decitabine. Our data showed that decitabine is able to restore the expression levels of aforementioned genes and downregulate expression levels of oncomiRs; including miR-125b, miR-17, and miR-181b in Nalm6 cell line. Therefore, it seems that decitabine can be used as a potential drug for the first line treatment of patients with B-ALL, but further in vivo investigation is necessary.     

Comparison of Aberrant Methylation of CpG Islands in the p16/CDKN2A and p14/ARF Promoters in Non-Small Cell Lung Cancer and Acute Lymphoblastic Leukemia

Multiplex methylation-sensitive (MSe-PCR) and methylation-specific (MSp-PCR) PCRs were used to detect aberrant methylation of CpG islands in the promoter regions and first exons of p16/CDKN2A and p14/ARF in non-small cell lung cancer (NSCLC, 54 specimens) and B-cell acute lymphoblastic leukemia (B-ALL, 61 specimens). A difference in CpG methylation was observed for individual specimens and for the two malignancies. A high methylation frequency of the first exon of p16/CDKN2A was detected both in NSCLC (68%) and in B-ALL (55%). The CpG island of the p14/ARF first exon proved to be nonmethylated in both malignancies. Particular CpG-rich fragments were examined in the p16/CDKN2A and p14/ARF promoters. It was shown that methylation frequency can differ between the 5 regions of one promoter. The sensitivity was compared for MSe-PCR and MSp-PCR, which are commonly employed in methylation analysis.     

p16基因甲基化状态与散发性大肠癌的相关性研究

为探讨p1 6基因甲基化状态与散发性大肠癌发生发展的关系 ,用甲基化特异性的聚合酶链反应 (methylati omspecificPCR ,MSP)结合测序检测散发性大肠癌及相应癌旁组织p1 6基因甲基化状态。研究发现p1 6基因在散发性大肠癌中甲基化率为 2 8 9% (1 3 4 5 ) ,有 8例癌及癌旁组织都发生了甲基化 ;有淋巴结及远处转移的甲基化率为5 0 % (8 1 6 ) ,高于无转移的甲基化率 2 0 8(5 2 4 ) (P <0 0 5 )。p1 6基因高甲基化是散发性大肠癌中常见的分子改变之一 ,大肠癌中p1 6基因高甲基化可能发生在癌变早期并与大肠癌的恶性进展有相关性     

Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis

Aberrant DNA methylation patterns have been reported in inflamed tissues and may play a role in disease. We studied DNA methylation and gene expression profiles of purified intestinal epithelial cells from ulcerative colitis patients, comparing inflamed and non-inflamed areas of the colon. We identified 577 differentially methylated sites (false discovery rate <0.2) mapping to 210 genes. From gene expression data from the same epithelial cells, we identified 62 differentially expressed genes with increased expression in the presence of inflammation at prostate cancer susceptibility genes PRAC1 and PRAC2. Four genes showed inverse correlation between methylation and gene expression; ROR1, GXYLT2, FOXA2, and, notably, RARB, a gene previously identified as a tumor suppressor in colorectal adenocarcinoma as well as breast, lung and prostate cancer. We highlight targeted and specific patterns of DNA methylation and gene expression in epithelial cells from inflamed colon, while challenging the importance of epithelial cells in the pathogenesis of chronic inflammation.     

Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation.Key words: MS-HRM, pyrosequencing, digital PCR, heterogeneous DNA methylation, DAPK1, chronic lymphocytic leukemia     

Epigenetic inactivation of the miR-124-1 in haematological malignancies

miR-124-1 is a tumour suppressor microRNA (miR). Epigenetic deregulation of miRs is implicated in carcinogenesis. Promoter DNA methylation and histone modification of miR-124-1 was studied in 5 normal marrow controls, 4 lymphoma, 8 multiple myeloma (MM) cell lines, 230 diagnostic primary samples of acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), MM, and non-Hodgkin's lymphoma (NHL), and 53 MM samples at stable disease or relapse. Promoter of miR-124-1 was unmethylated in normal controls but homozygously methylated in 4 of 4 lymphoma and 4 of 8 myeloma cell lines. Treatment of 5-Aza-2'-deoxycytidine led to miR-124-1 demethylation and re-expression of mature miR-124, which also associated with emergence of euchromatic trimethyl H3K4 and consequent downregulation of CDK6 in myeloma cells harboring homozygous miR-124-1 methylation. In primary samples at diagnosis, miR-124-1 methylation was absent in CML but detected in 2% each of MM at diagnosis and relapse/progression, 5% ALL, 15% AML, 14% CLL and 58.1% of NHL (p<0.001). Amongst lymphoid malignancies, miR-124-1 was preferentially methylated in NHL than MM, CLL or ALL. In primary lymphoma samples, miR-124-1 was preferentially hypermethylated in B- or NK/T-cell lymphomas and associated with reduced miR-124 expression. In conclusion, miR-124-1 was hypermethylated in a tumour-specific manner, with a heterochromatic histone configuration. Hypomethylation led to partial restoration of euchromatic histone code and miR re-expression. Infrequent miR-124-1 methylation detected in diagnostic and relapse MM samples showed an unimportant role in MM pathogenesis, despite frequent methylation found in cell lines. Amongst haematological cancers, miR-124-1 was more frequently hypermethylated in NHL, and hence warrants further study.     

DNA methylation analyses of the connexin gene family reveal silencing of GJC1 (Connexin45) by promoter hypermethylation in colorectal cancer

Gap junctions are specialized plasma membrane domains consisting of channels formed by members of the connexin protein family. Gap junctional intercellular communication is often lost in cancers due to aberrant localization or downregulation of connexins, and connexins are therefore suggested to act as tumor suppressor genes in various tissues. The aim of this study was to investigate the expression pattern and DNA promoter methylation status of connexins in colorectal cancer. Expression of six (GJA1, GJA9, GJB1, GJB2, GJC1 and GJD3) connexin genes was detected in normal colonic tissue samples. GJC1 expression was reduced in colorectal carcinomas compared to normal tissue samples. All analyzed connexins were hypermethylated in colon cancer cell lines, although at various frequencies. GJA4, GJB6 and GJD2 were hypermethylated in 60% (29/48), 25% (12/48) and 96% (46/48) of primary colorectal carcinomas, respectively. However, the methylation status was not associated with gene expression. GJC1 has two alternative promoters, which were methylated in 42% (32/76) and 38% (25/65) of colorectal tumors, and in none of the normal mucosa samples. Expression of GJC1 was significantly lower in methylated compared with unmethylated samples (p < 0.01) and was restored in cell lines treated with the demethylating drug 5-aza-2'deoxycytidine. Taken together, DNA hypermethylation of the promoter region of GJC1, encoding connexin45, is an important mechanism in silencing gene expression in colorectal cancer.