Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.Published versio

Overexpression of ribosomal RNA in prostate cancer is common but not linked to rDNA promoter hypomethylation

Alterations in nucleoli, including increased numbers, increased size, altered architecture and increased function are hallmarks of prostate cancer cells. The mechanisms that result in increased nucleolar size, number and function in prostate cancer have not been fully elucidated. The nucleolus is formed around repeats of a transcriptional unit encoding a 45S ribosomal RNA (rRNA) precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. Although it has been generally accepted that tumor cells overexpress rRNA species, this has not been examined in clinical prostate cancer. We find that indeed levels of the 45S rRNA, 28S, 18S and 5.8S are overexpressed in the majority of human primary prostate cancer specimens as compared with matched benign tissues. One mechanism that can alter nucleolar function and structure in cancer cells is hypomethylation of CpG dinucleotides of the upstream rDNA promoter region. However, this mechanism has not been examined in prostate cancer. To determine whether rRNA overexpression could be explained by hypomethylation of these CpG sites, we also evaluated the DNA methylation status of the rDNA promoter in prostate cancer cell lines and the clinical specimens. Bisulfite sequencing of genomic DNA revealed two roughly equal populations of loci in cell lines consisting of those that contained densely methylated deoxycytidine residues within CpGs and those that were largely unmethylated. All clinical specimens also contained two populations with no marked changes in methylation of this region in cancer as compared with normal. We recently reported that MYC can regulate rRNA levels in human prostate cancer; here we show that MYC mRNA levels are correlated with 45S, 18S and 5.8S rRNA levels. Further, as a surrogate for nucleolar size and number, we examined the expression of fibrillarin, which did not correlate with rRNA levels. We conclude that rRNA levels are increased in human prostate cancer, but that hypomethylation of the rDNA promoter does not explain this increase, nor does hypomethylation explain alterations in nucleolar number and structure in prostate cancer cells. Rather, rRNA levels and nucleolar size and number relate more closely to MYC overexpression

Regulatory T cell DNA methyltransferase inhibition accelerates resolution of lung inflammation

Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4+CD25hi Foxp3+ Tregs from D AC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1-null mice and Treg-depleted (diphtheria toxin-treated Foxp3DTR) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 ×105 DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS

Control of prostate cancer associated with withdrawal of a supplement containing folic acid, L-methyltetrahydrofolate and vitamin B12: a case report

<p>Abstract</p> <p>Introduction</p> <p>This is the first report of possible direct stimulation of hormone-resistant prostate cancer or interference of docetaxel cytotoxicity of prostate cancer in a patient with biochemical relapse of prostatic-specific antigen. This observation is of clinical and metabolic importance, especially at a time when more than 80 countries have fortified food supplies with folic acid and some contemplate further fortification with vitamin B₁₂.</p> <p>Case presentation</p> <p>Our patient is a 71-year-old Caucasian man who had been diagnosed in 1997 with prostate cancer, stage T1c, and Gleason score 3+4 = 7. His primary treatment included intermittent androgen deprivation therapy including leuprolide + bicalutamide + deutasteride, ketoconazole + hydrocortisone, nilandrone and flutamide to resistance defined as biochemical relapse of PSA. While undergoing docetaxel therapy to treat a continually increasing prostate-specific antigen level, withdrawal of 10 daily doses of a supplement containing 500 μg of vitamin B₁₂as cyanocobalamin, as well as 400 μg of folic acid as pteroylglutamic acid and 400 μg of L-5-methyltetrahydrofolate for a combined total of 800 μg of mixed folates, was associated with a return to a normal serum prostatic-specific antigen level.</p> <p>Conclusion</p> <p>This case report illustrates the importance of the effects of supplements containing large amounts of folic acid, L-5-methyltetrahydrofolate, and cyanocobalamin on the metabolism of prostate cancer cells directly and/or B vitamin interference with docetaxel efficacy. Physicians caring for patients with prostate cancer undergoing watchful waiting, hormone therapy, and/or chemotherapy should consider the possible acceleration of tumor growth and/or metastasis and the development of drug resistance associated with supplement ingestion. We describe several pathways of metabolic and epigenetic interactions that could affect the observed changes in serum levels of prostate-specific antigen.</p

Particle Swarm Optimization with Reinforcement Learning for the Prediction of CpG Islands in the Human Genome

BACKGROUND: Regions with abundant GC nucleotides, a high CpG number, and a length greater than 200 bp in a genome are often referred to as CpG islands. These islands are usually located in the 5' end of genes. Recently, several algorithms for the prediction of CpG islands have been proposed. METHODOLOGY/PRINCIPAL FINDINGS: We propose here a new method called CPSORL to predict CpG islands, which consists of a complement particle swarm optimization algorithm combined with reinforcement learning to predict CpG islands more reliably. Several CpG island prediction tools equipped with the sliding window technique have been developed previously. However, the quality of the results seems to rely too much on the choices that are made for the window sizes, and thus these methods leave room for improvement. CONCLUSIONS/SIGNIFICANCE: Experimental results indicate that CPSORL provides results of a higher sensitivity and a higher correlation coefficient in all selected experimental contigs than the other methods it was compared to (CpGIS, CpGcluster, CpGProd and CpGPlot). A higher number of CpG islands were identified in chromosomes 21 and 22 of the human genome than with the other methods from the literature. CPSORL also achieved the highest coverage rate (3.4%). CPSORL is an application for identifying promoter and TSS regions associated with CpG islands in entire human genomic. When compared to CpGcluster, the islands predicted by CPSORL covered a larger region in the TSS (12.2%) and promoter (26.1%) region. If Alu sequences are considered, the islands predicted by CPSORL (Alu) covered a larger TSS (40.5%) and promoter (67.8%) region than CpGIS. Furthermore, CPSORL was used to verify that the average methylation density was 5.33% for CpG islands in the entire human genome

Changes in cortical cytoskeletal and extracellular matrix gene expression in prostate cancer are related to oncogenic ERG deregulation

Abstract Background The cortical cytoskeleton network connects the actin cytoskeleton to various membrane proteins, influencing cell adhesion, polarity, migration and response to extracellular signals. Previous studies have suggested changes in the expression of specific components in prostate cancer, especially of 4.1 proteins (encoded by EPB41 genes) which form nodes in this network. Methods Expression of EPB41L1, EPB41L2, EPB41L3 (protein: 4.1B), EPB41L4B (EHM2), EPB41L5, EPB49 (dematin), VIL2 (ezrin), and DLG1 (summarized as „cortical cytoskeleton" genes) as well as ERG was measured by quantitative RT-PCR in a well-characterized set of 45 M0 prostate adenocarcinoma and 13 benign tissues. Hypermethylation of EPB41L3 and GSTP1 was compared in 93 cancer tissues by methylation-specific PCR. Expression of 4.1B was further studied by immunohistochemistry. Results EPB41L1 and EPB41L3 were significantly downregulated and EPB41L4B was upregulated in cancer tissues. Low EPB41L1 or high EPB41L4B expression were associated with earlier biochemical recurrence. None of the other cortical cytoskeleton genes displayed expression changes, in particular EPB49 and VIL2, despite hints from previous studies. EPB41L3 downregulation was significantly associated with hypermethylation of its promoter and strongly correlated with GSTP1 hypermethylation. Protein 4.1B was detected most strongly in the basal cells of normal prostate epithelia. Its expression in carcinoma cells was similar to the weaker one in normal luminal cells. EPB41L3 downregulation and EPB41L4B upregulation were essentially restricted to the 22 cases with ERG overexpression. Expression changes in EPB41L3 and EPB41L4B closely paralleled those previously observed for the extracellular matrix genes FBLN1 and SPOCK1, respectively. Conclusions Specific changes in the cortical cytoskeleton were observed during prostate cancer progression. They parallel changes in the expression of extracellular matrix components and all together appear to be associated with oncogenic ERG overexpression. We hypothesize that these alterations may contribute to the increased invasivity conferred to prostate cancer cells by ERG deregulation.</p

Androgen-Regulated Transcriptional Control of Sialyltransferases in Prostate Cancer Cells

The expression of gangliosides is often associated with cancer progression. Sialyltransferases have received much attention in terms of their relationship with cancer because they modulate the expression of gangliosides. We previously demonstrated that GD1a production was high in castration-resistant prostate cancer cell lines, PC3 and DU145, mainly due to their high expression of β-galactoside α2,3-sialyltransferase (ST3Gal) II (not ST3Gal I), and the expression of both ST3Gals was regulated by NF-κB, mainly by RelB. We herein demonstrate that GD1a was produced in abundance in cancerous tissue samples from human patients with hormone-sensitive prostate cancers as well as castration-resistant prostate cancers. The expression of ST3Gal II was constitutively activated in castration-resistant prostate cancer cell lines, PC3 and DU145, because of the hypomethylation of CpG island in its promoter. However, in androgen-depleted LNCap cells, a hormone-sensitive prostate cancer cell line, the expression of ST3Gal II was silenced because of the hypermethylation of the promoter region. The expression of ST3Gal II in LNCap cells increased with testosterone treatment because of the demethylation of the CpG sites. This testosterone-dependent ST3Gal II expression was suppressed by RelB siRNA, indicating that RelB activated ST3Gal II transcription in the testosterone-induced demethylated promoter. Therefore, in hormone-sensitive prostate cancers, the production of GD1a may be regulated by androgen. This is the first report indicating that the expression of a sialyltransferase is transcriptionally regulated by androgen-dependent demethylation of the CpG sites in its gene promoter

Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines

A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral “contamination”, much like routine mycoplasma testing

Downregulation of miR-205 and miR-31 confers resistance to chemotherapy-induced apoptosis in prostate cancer cells

Advanced prostate cancers are known to acquire not only invasive capabilities but also significant resistance to chemotherapy-induced apoptosis. To understand how microRNAs (miRNAs) may contribute to prostate cancer resistance to apoptosis, we compared microRNA expression profiles of a benign prostate cancer cell line WPE1-NA22 and a highly malignant WPE1-NB26 cell line (derived from a common lineage). We found that miR-205 and miR-31 are significantly downregulated in WPE1-NB26 cells, as well as in other cell lines representing advanced-stage prostate cancers. Antiapoptotic genes BCL2L2 (encoding Bcl-w) and E2F6 are identified as the targets of miR-205 and miR-31, respectively. By downregulating Bcl-w and E2F6, miR-205 and miR-31 promote chemotherapeutic agents-induced apoptosis in prostate cancer cells. The promoter region of the miR-205 gene was cloned and was found to be hypermethylated in cell lines derived from advanced prostate cancers, contributing to the downregulation of the gene. Treatment with DNA methylation inhibitor 5-aza-2′-deoxycytidine induced miR-205 expression, downregulated Bcl-w, and sensitized prostate cancer cells to chemotherapy-induced apoptosis. Thus, downregulation of miR-205 and miR-31 has an important role in apoptosis resistance in advanced prostate cancer

Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

<p>Abstract</p> <p>Background</p> <p>Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors.</p> <p>Methods</p> <p>A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters.</p> <p>Results</p> <p>Significant differences in methylation levels among the four subtypes of renal tumors were found for <it>CDH1 </it>(<it>p </it>= 0.0007), <it>PTGS2 </it>(<it>p </it>= 0.002), and <it>RASSF1A </it>(<it>p </it>= 0.0001). <it>CDH1 </it>hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (<it>p </it>= 0.00016 and <it>p </it>= 0.0034, respectively), whereas <it>PTGS2 </it>methylation levels were significantly higher in ccRCC compared to pRCC (<it>p </it>= 0.004). <it>RASSF1A </it>methylation levels were significantly higher in pRCC than in normal tissue (<it>p </it>= 0.035). In pRCC, <it>CDH1 </it>and <it>RASSF1A </it>methylation levels were inversely correlated with tumor stage (<it>p </it>= 0.031) and nuclear grade (<it>p </it>= 0.022), respectively.</p> <p>Conclusion</p> <p>The major subtypes of renal epithelial neoplasms display differential aberrant <it>CDH1</it>, <it>PTGS2</it>, and <it>RASSF1A </it>promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.</p