Identification of epigenetically silenced genes in tumor endothelial cells

Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDAC are involved in regulation of endothelial cell gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes down-regulated in tumor-conditioned versus quiescent endothelial cells, and reexpressed by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA). Among the 81 genes identified, 77% harbored a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant down-regulation in tumor-conditioned endothelial cells and reactivation by treatment with a combination of DAC and TSA, as well as by both compounds separately. Silencing of these genes in tumor-conditioned endothelial cells correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, but did not involve DNA methylation of promoter CpG islands. For six genes, down-regulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1, and quiescin Q6 are negative regulators of endothelial cell growth and angiogenesis. In summary, our data identify novel angiogenesis-suppressing genes that become silenced in tumor-conditioned endothelial cells in association with promoter histone modifications and reactivated by DNMT and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis.     

MUC2 expression is regulated by histone H3 modification and DNA methylation in pancreatic cancer

Mucins are highly glycosylated proteins that play important roles in carcinogenesis. In pancreatic neoplasia, MUC2 mucin has been demonstrated as a tumor suppressor and we have reported that MUC2 is a favorable prognostic factor. Regulation of MUC2 gene expression is known to be controlled by DNA methylation, but the role of histone modification for MUC2 gene expression has yet to be clarified. Herein, we provide the first report that the histone H3 modification of the MUC2 promoter region regulates MUC2 gene expression. To investigate the histone modification and DNA methylation of the promoter region of the MUC2 gene, we treated 2 human pancreatic cancer cell lines, PANC1 (MUC2-negative) and BxPC3 (MUC2-positive) with the DNA methyltransferase inhibitor 5-azacytidine (5-aza), the histone deacetylase inhibitor trichostatin A (TSA), and a combination of these agents. The DNA methylation level of PANC1 cells was decreased by all 3 treatments, whereas histone H3-K4/K9 methylation and H3-K9/K27 acetylation in PANC1 cells was changed to the level in BxPC3 cells by treatment with TSA alone and with the 5-aza/TSA combination. The expression level of MUC2 mRNA in PANC1 cells exhibited a definite increase when treated with TSA and 5-aza/TSA, whereas 5-aza alone induced only a slight increase. Our results suggest that histone H3 modification in the 5' flanking region play an important role in MUC2 gene expression, possibly affecting DNA methylation. An understanding of these intimately correlated epigenetic changes may be of importance for predicting the outcome of patients with pancreatic neoplasms.     

组蛋白修饰对喉癌细胞系中RASSF1A基因表达的影响

目的：探讨喉癌细胞中RASSF1A基因表达水平与基因组蛋白修饰的关系。方法：应用染色质免疫沉淀技术（ChIP）和实时定量逆转录聚合酶链反应（Realtime-PCR）分析喉癌Hep-2细胞系在以下药物：5-氮杂-2＇-脱氧胞苷（5-Aza-2＇-deoxyeytidine,5-Aza-dC,DNA甲基转移酶抑制剂,5-AZa-dC）;曲古抑菌素A（TriChostatinA,TSA,组蛋白去乙酰化酶抑制剂）作用前后RASSF1A基因启动子区域组蛋白H3-K9甲基化、H3-K4甲基化、H3-K9乙酰化和RASSF1A基因表达情况。结果：TSA能轻度降低RASSF1A基因启动子区域组蛋白H3-K9甲基化水平、轻度提高H3-K4甲基化水平、明显提高H3-K9乙酰化水平。5-Aza-dC明显降低启动子区域H3-K9甲基化程度、明显提高H3-K4甲基化水平、提高H3-K9乙酰化水平,联合给予5-Aza-dC和TSA起协同增效作用。H3-K9甲基化水平降低、H3-K4甲基化和H3-K9乙酰化水平提高可使RASSF1A基因表达上调。结论：喉癌细胞中RASSF1A肿瘤抑制基因表达下调与其启动子区H3-K9甲基化、H3-K4甲基化和H3-K9乙酰化相关。甲基化抑制剂及乙酰化制剂均可使表达下调的基因表达上调。     

DNA demethylating agents and histone deacetylase inhibitors in hematologic malignancies

The pivotal role of aberrant promoter methylation in gene silencing and cancer development has fueled the interest in DNA methyltransferase inhibitors as novel anticancer drugs. Modulation of gene expression through targeting of epigenetic marks is one of the emerging and promising strategies that has demonstrated successful clinical outcome in hematologic malignancies. Epigenetic modifiers, including DNA methyltransferase inhibitors and histone deacetylase inhibitors, have demonstrated significant clinical activity; several are or are likely to soon be approved by the U.S. Food and Drug Administration. However, the exact mechanism of the clinical response achieved is not fully understood. This review focuses on the pharmacology of the known DNA methyltransferase and histone deacetylase inhibitors and their potential as promising anticancer drugs.     

Epigenetic regulation of tumor endothelial cell anergy: silencing of intercellular adhesion molecule-1 by histone modifications

Tumors can escape from immunity by repressing leukocyte adhesion molecule expression on tumor endothelial cells and by rendering endothelial cells unresponsive to inflammatory activation. This endothelial cell anergy is induced by angiogenic growth factors and results in reduced leukocyte-vessel wall interactions, thereby attenuating infiltration of leukocytes into the tumor. This report describes a novel mechanism of endothelial cell anergy regulation. We recently reported that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors have angiostatic activity. Here, we studied whether epigenetic mechanisms regulate this angiogenesis-mediated escape from immunity. We found that DNMT inhibitors 5-aza-2'-deoxycytidine and zebularine, as well as HDAC inhibitor trichostatin A, reexpressed intercellular adhesion molecule-1 (ICAM-1) on tumor-conditioned endothelial cells in vitro, resulting in restored leukocyte-endothelial cell adhesion. In addition, treatment with DNMT or HDAC inhibitors in vivo also restored ICAM-1 expression on tumor endothelial cells from two different mouse tumor models. Furthermore, leukocyte-vessel wall interactions in mouse tumors were increased by these compounds, as measured by intravital microscopy, resulting in enhanced leukocyte infiltration. We show that ICAM-1 down-regulation in tumor endothelial cells is associated with ICAM-1 promoter histone H3 deacetylation and loss of histone H3 Lys(4) methylation but not with DNA hypermethylation. In conclusion, our data show that ICAM-1 is epigenetically silenced in tumor endothelial cells by promoter histone modifications, which can be overcome by DNMT and HDAC inhibitors, suggesting a new molecular mechanism based on which novel therapeutic approaches for cancer can be pursued.     

Selenite reactivates silenced genes by modifying DNA methylation and histones in prostate cancer cells

DNA hypermethylation is a common epigenetic alteration in human prostate cancer and is considered to contribute to development of this disease. Accumulating data suggest that dietary factors may alter cancer risk by modifications of epigenetic processes in the cell. The present study was designed to investigate whether selenium (Se) would alter epigenetic events to regulate methylation-silenced genes in human prostate cancer cells. DNA methylation, histone modifications and gene expression were studied in LNCaP cells after selenite treatment using polymerase chain reaction, western blot analysis, chromatin immunoprecipitation assay and enzymatic activity assay. Our study shows that selenite treatment caused partial promoter DNA demethylation and reexpression of the pi-class glutathione-S-transferase (GSTP1) in LNCaP cells in a dose- and time-dependent manner. Selenite treatment decreased messenger RNA levels of DNA methyltransferases (DNMTs) 1 and 3A and protein levels of DNMT1. Selenite also decreased histone deacetylase activity and increased levels of acetylated lysine 9 on histone H3 (H3-Lys 9), but decreased levels of methylated H3-Lys 9. Selenite treatment reduced levels of DNMT1 and methylated H3-Lys 9 associated with the GSTP1 promoter, but increased levels of acetylated H3-Lys 9 associated with this promoter. Additionally, selenite treatment decreased general DNA methylation and caused partial promoter demethylation and reexpression of the tumor suppressor adenomatous polyposis coli and cellular stress response 1, a gene involving tumor growth and metastasis. Our study demonstrates that Se can epigenetically modulate DNA and histones to activate methylation-silenced genes. These epigenetic modifications may contribute to cancer prevention by Se.     

Decitabine and its role in the treatment of hematopoietic malignancies

DNA methylation is responsible for abnormal silencing of many genes, including tumor suppressor genes, in cancer. Decitabine, an S-phase specific inhibitor of DNA methyltransferase, has been shown to decrease levels of abnormal methylation in neoplasia. Though initially investigated at high doses as a cytotoxic agent, recent studies show that when administered at low doses, the hypomethylating activity of decitabine is increased with a demonstrated increase in activity in hematopoietic malignancies. Multiple clinical trials, both in the United States and in Europe, have demonstrated the efficacy of decitabine in acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome (MDS). Recently approved by the United States Food and Drug Administration for the treatment of (MDS), decitabine represents an effective and well-tolerated therapeutic option in this disease, for which treatment options were previously scarce. While the activity in MDS is promising, primary and secondary resistance remain a problem. Investigations of combinations of decitabine with other agents, including histone deacetylase inhibitors, are currently ongoing in the hope of substantially prolonging survival in patients with hematologic malignancies.     

Resetting the histone code at CDKN2A in HNSCC by inhibition of DNA methylation

Head and neck squamous cell carcinoma (HNSCC) is the fifth most frequent cancer in the US. Several genetic and epigenetic alterations are associated with HNSCC tumorigenesis, including inactivation of CDKN2A, which encodes the p16 tumor suppressor, in cell lines and primary tumors by DNA methylation. Reactivation of tumor suppressor genes by DNA-demethylating agents and histone deacetylase (HDAC) inhibitors shows therapeutic promise for other cancers. Therefore, we investigated the ability of these agents to reactivate p16 in Tu159 HNSCC cells. Treatment of cells with 5-aza-2'deoxycytidine (5-aza-dC) increases CDKN2A expression and slightly increases histone H3 acetylation at this gene. No reactivation of CDKN2A is observed upon treatment with the HDAC inhibitor trichostatin A (TSA), but synergistic reactivation of CDKN2A is observed upon sequential treatment of Tu159 cells with both 5-aza-dC and TSA. Silencing of CDKN2A in Tu159 cells is correlated with increased methylation of histone H3 at lysine 9 and decreased methylation at lysine 4 relative to the upstream p15 gene promoter. Interestingly, global levels of H3-K9 methylation are decreased upon treatment with 5-aza-dC. Together these data indicate that DNA methylation is a dominant epigenetic mark for silencing of CDKN2A in Tu159 tumor cells. Moreover, changes in DNA methylation can reset the histone code by impacting multiple H3 modifications.     

Secreted frizzled‐related protein‐5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer

Secreted frizzled‐related protein‐5 (sFRP‐5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP‐5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP‐5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real‐time RT‐PCR, we found that sFRP‐5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP‐5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP‐5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP‐5 promoter region were significantly increased after treatment with demethylation agent (5‐Aza‐dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP‐5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP‐5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP‐5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.     

Azacitidine induces demethylation of the Epstein-Barr virus genome in tumors.

PURPOSE: To determine whether therapy with a DNA methyltransferase inhibitor is effective in achieving demethylation and gene re-expression in tumor DNA in patients. METHODS: Biopsy specimens were obtained from patients with Epstein-Barr virus-associated tumors, enrolled on a clinical trial of 5-azacitidine, within 72 hours of the conclusion of the last infusion of the first cycle of therapy, and compared to pretreatment specimens. Methylation-specific polymerase chain reaction, bisulfite genomic sequencing, and immunohistochemistry were used to assess demethylation and gene re-expression. RESULTS: Substantial degrees of demethylation were detected in all latent and lytic Epstein-Barr virus promoters examined. Immunohistochemistry suggested activation of a previously silent viral antigen expression in one instance. CONCLUSION: Pharmacologic reversal of dense CpG methylation in tumor tissue can be achieved in patients.     

Effects of specific DNMT gene depletion on cancer cell transformation and breast cancer cell invasion; toward selective DNMT inhibitors

A hallmark of cancer is aberrant DNA methylation, consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes. DNA methyltransferase inhibitors have been recognized as promising candidate anticancer drugs. Drug development has focused on DNA methylation inhibitors with the goal of activating tumor suppressor genes silenced by DNA methylation. 5-azacytidine (5-AC; Vidaza), a global DNA methyltransferase inhibitor, was Food and Drug Administration approved to treat myelodysplastic syndromes and is clinically tested for solid tumors. In this paper, it was demonstrated that 5'-aza-2'-deoxycytidine (5-azaCdR) activated both silenced tumor suppressor genes and pro-metastatic genes by demethylation, raising the concern that it would promote metastasis. 5-AzaCdR treatment increased the invasiveness of non-invasive breast cancer cell lines MCF-7 cells and ZR-75-1 and dramatically induced pro-metastatic genes; Urokinase plasminogen activator (uPA), matrix metalloproteinase 2 (MMP2), metastasis-associated gene (H-MTS1; S100A4) and C-X-C chemokine receptor 4 (CXCR4). The hypothesis that the blocking of cellular transformation activity of DNA methyltransferase inhibitor could be separated from the pro-metastatic activity was tested using short interfering RNA (siRNA) targeted to the different DNA methyltransferase (DNMT) genes. Although depletion of DNMT1 had the strongest effect on colony growth suppression in cellular transformation assays, it did not result in demethylation and activation of uPA, S100A4, MMP2 and CXCR4 in MCF-7 cells. Depletion of DNMT1 did not induce cellular invasion in MCF-7 and ZR-75-1 non-invasive breast cancer cell lines. These data have implications on the design of new DNA methyltransferase inhibitor and on the proper utilization of current inhibitors.     

Hypermethylation,but not LOH,is associated with the low expression of MT1G and CRABP1 in papillary thyroid carcinoma

We previously obtained gene expression profiles of 8 matched papillary thyroid carcinoma (PTC) and normal tissues using DNA microarrays. To identify novel tumor suppressor genes involved in thyroid carcinogenesis, we here analyze genes showing lower expression in PTC tumors than in normal thyroid tissues. A search for loss of heterozygosity (LOH) in 49 regions that harbor consistently down-regulated genes revealed LOH in only 4 regions and in just a very small number of tumors. To determine whether the underexpression might be due to promoter methylation, we used combined bisulfite restriction analysis and bisulfite sequencing to study 7 underexpressed genes. Loss of expression of MT1G and CRABP1 is accompanied by hypermethylation in the 5' regions of these genes, but methylation was not seen in other genes tested. Combined treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) and the histone deacetylase inhibitor trichostatin A (TSA) resulted in demethylation and re-expression of the MT1G gene in the cell line K2. Treatment with 5-Aza-dC alone restored CRABP1 expression in a colorectal cancer cell line, SW48. In conclusion, LOH is a remarkably rare mechanism of loss of gene function in PTC. In contrast, hypermethylation of promoter CpG islands seems to occur at higher frequency. MT1G and CRABP1 are novel genes that are likely involved in the pathogenesis of sporadic PTC.     

CpG methylation-dependent repression of the human O6-methylguanine-DNA methyltransferase gene linked to chromatin structure alteration

The mechanism of inactivation of the O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic and cytotoxic O6-alkylguanine, in Mex- tumor cells, is not completely understood. We have examined the role of CpG methylation in the human MGMT promoter in a luciferase (luc) reporter plasmid and associated alteration in chromatin structure. Methylation of 16% CpG sequences in promoter and flanking sequences in the plasmid with HpaII methylase reduced luciferase activity by 10-12-fold, while methylation of all CpG sites, including those in the luc coding sequence, as well as the promoter sequence blocked expression completely. Repression of luc expression due to partial but not complete CpG methylation could be reversed by histone deacetylase inhibitor trichostatin A (TSA). However, 5-azacytidine, which reverses CpG methylation, but not TSA, could reactivate silent MGMT gene in Mex- HeLa MR cells. Furthermore, chromatin immunoprecipitation (ChIP) assay showed reduced level of acetylation of H4 histone bound to the methylated promoter compared with the non-methylated promoter. These results suggest that complete repression of the MGMT gene in Mex- cells requires methylation of CpG sequences in both promoter and neighboring regions of the gene, resulting in inactive, condensed chromatin state of the gene.     

Epigenetic reactivation of tumor suppressor genes by a novel small-molecule inhibitor of human DNA methyltransferases

DNA methylation regulates gene expression in normal and malignant cells. The possibility to reactivate epigenetically silenced genes has generated considerable interest in the development of DNA methyltransferase inhibitors. Here, we provide a detailed characterization of RG108, a novel small molecule that effectively blocked DNA methyltransferases in vitro and did not cause covalent enzyme trapping in human cell lines. Incubation of cells with low micromolar concentrations of the compound resulted in significant demethylation of genomic DNA without any detectable toxicity. Intriguingly, RG108 caused demethylation and reactivation of tumor suppressor genes, but it did not affect the methylation of centromeric satellite sequences. These results establish RG108 as a DNA methyltransferase inhibitor with fundamentally novel characteristics that will be particularly useful for the experimental modulation of epigenetic gene regulation.