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Background: In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of TERT gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating TERT gene expression in cancer cells is as yet not fully understood., Methods: Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines, including primary, immortalized and cancer cell types, as well as in control and reference samples., Results: In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally found that hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells., Conclusions: Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to attenuation of TERT activation in cancer cells. This type of fine tuning of TERT expression may account for the modest activation of TERT expression in most cancers.

A level of epigenetic programming, encoded by complex sets of chemical marks on DNA and histones, and by context-specific DNA, RNA, protein interactions, that all regulate the structure, organization, and function of the genome, is critical to establish both normal and neoplastic cell identities and functions. This structure-function relationship of the genome encoded by the epigenetic programming can be thought of as an epigenetic cityscape that is built on the underlying genetic landscape. Alterations in the epigenetic cityscape of prostate cancer cells compared with normal prostate tissues have a complex interplay with genetic alterations to drive prostate cancer initiation and progression. Indeed, mutations in genes encoding epigenetic enzymes are often observed in human cancers including prostate cancer. Interestingly, alterations in the prostate cancer epigenetic cityscape can be highly recurrent, a facet that can be exploited for development of biomarkers and potentially as therapeutic targets., (Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved.)

The epigenetic and anti-cancer activities of the nucleoside analog DNA methyltransferase (DNMT) inhibitors decitabine (5-aza-2'-deoxycytidine, DAC), azacitidine, and guadecitabine are thought to require cellular uptake, metabolism to 5-aza-2'-deoxycytidine triphosphate, and incorporation into DNA. This genomic incorporation can then lead to trapping and degradation of DNMT enzymes, and ultimately, passive loss of DNA methylation. To facilitate measurement of critical exposure-response relationships of nucleoside analog DNMT inhibitors, a sensitive and reliable method was developed to simultaneously quantitate 5-aza-2'-deoxycytidine genomic incorporation and genomic 5-methylcytosine content using LC-MS/MS. Genomic DNA was extracted and digested into single nucleosides. Chromatographic separation was achieved with a Thermo Hyperpcarb porous graphite column (100mm×2.1mm, 5μm) and isocratic elution with a 10mM ammonium acetate:acetonitrile with 0.1% formic acid (70:30, v/v) mobile phase over a 5min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5-aza-2'-deoxycytidine, 2'-deoxycytidine, and 5-methyl-2'-deoxycytidine. The assay range was 2-400ng/mL for 5-aza-2'-deoxycytidine, 50-10,000ng/mL for 2'-deoxycytidine, and was 5-1000ng/mL for 5-methyl-2'-deoxycytidine. The assay proved to be accurate (93.0-102.2%) and precise (CV≤6.3%) across all analytes. All analytes exhibited long-term frozen digest matrix stability at -70°C for at least 117 days. The method was applied for the measurement of genomic 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine content following exposure of in vitro cell culture and in vivo animal models to decitabine., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Solid tumors are characterized by a plethora of epigenetic changes. In particular, patterns methylation of cytosines at the 5-position (5mC) in the context of CpGs are frequently altered in tumors. Recent evidence suggests that 5mC can get converted to 5-hydroxylmethylcytosine (5hmC) in an enzymatic process involving ten eleven translocation (TET) protein family members, and this process appears to be important in facilitating plasticity of cytosine methylation. Here we evaluated the global levels of 5hmC using a validated immunohistochemical staining method in a large series of clear cell renal cell carcinoma (n = 111), urothelial cell carcinoma (n = 55) and testicular germ cell tumors (n = 84) and matched adjacent benign tissues. Whereas tumor-adjacent benign tissues were mostly characterized by high levels of 5hmC, renal cell carcinoma and urothelial cell carcinoma showed dramatically reduced staining for 5hmC. 5hmC levels were low in both primary tumors and metastases of clear cell renal cell carcinoma and showed no association with disease outcomes. In normal testis, robust 5hmC staining was only observed in stroma and Sertoli cells. Seminoma showed greatly reduced 5hmC immunolabeling, whereas differentiated teratoma, embryonal and yolk sack tumors exhibited high 5hmC levels. The substantial tumor specific loss of 5hmC, particularly in clear cell renal cell carcinoma and urothelial cell carcinoma, suggests that alterations in pathways involved in establishing and maintaining 5hmC levels might be very common in cancer and could potentially be exploited for diagnosis and treatment.

Epigenetic control of NRF2, a master regulator of many critical antioxidative stress defense genes in human prostate cancer (CaP), is unknown. Our previous animal study found decreased Nrf2 expression through promoter CpG methylation/histone modifications during prostate cancer progression in TRAMP mice. In this study, we evaluated CpG methylation of human NRF2 promoter in 27 clinical prostate cancer samples and in LNCaP cells using MAQMA analysis and bisulfite genomic DNA sequencing. Prostate cancer tissue microarray (TMA) containing normal and prostate cancer tissues was studied by immunohistochemistry. Luciferase reporter assay using specific human NRF2 DNA promoter segments and chromatin immunoprecipitation (ChIP) assay against histone modifying proteins were performed in LNCaP cells. Three specific CpG sites in the NRF2 promoter were found to be hypermethylated in clinical prostate cancer samples (BPH

Human cancers almost ubiquitously harbor epigenetic alterations. Although such alterations in epigenetic marks, including DNA methylation, are potentially heritable, they can also be dynamically altered. Given this potential for plasticity, the degree to which epigenetic changes can be subject to selection and act as drivers of neoplasia has been questioned. We carried out genome-scale analyses of DNA methylation alterations in lethal metastatic prostate cancer and created DNA methylation "cityscape" plots to visualize these complex data. We show that somatic DNA methylation alterations, despite showing marked interindividual heterogeneity among men with lethal metastatic prostate cancer, were maintained across all metastases within the same individual. The overall extent of maintenance in DNA methylation changes was comparable to that of genetic copy number alterations. Regions that were frequently hypermethylated across individuals were markedly enriched for cancer- and development/differentiation-related genes. Additionally, regions exhibiting high consistency of hypermethylation across metastases within individuals, even if variably hypermethylated across individuals, showed enrichment for cancer-related genes. Whereas some regions showed intraindividual metastatic tumor heterogeneity in promoter methylation, such methylation alterations were generally not correlated with gene expression. This was despite a general tendency for promoter methylation patterns to be strongly correlated with gene expression, particularly at regions that were variably methylated across individuals. These findings suggest that DNA methylation alterations have the potential for producing selectable driver events in carcinogenesis and disease progression and highlight the possibility of targeting such epigenome alterations for development of longitudinal markers and therapeutic strategies.

Context: Prostate cancer (PCa) is one of the most common human malignancies and arises through genetic and epigenetic alterations. Epigenetic modifications include DNA methylation, histone modifications, and microRNAs (miRNA) and produce heritable changes in gene expression without altering the DNA coding sequence., Objective: To review progress in the understanding of PCa epigenetics and to focus upon translational applications of this knowledge., Evidence Acquisition: PubMed was searched for publications regarding PCa and DNA methylation, histone modifications, and miRNAs. Reports were selected based on the detail of analysis, mechanistic support of data, novelty, and potential clinical applications., Evidence Synthesis: Aberrant DNA methylation (hypo- and hypermethylation) is the best-characterized alteration in PCa and leads to genomic instability and inappropriate gene expression. Global and locus-specific changes in chromatin remodeling are implicated in PCa, with evidence suggesting a causative dysfunction of histone-modifying enzymes. MicroRNA deregulation also contributes to prostate carcinogenesis, including interference with androgen receptor signaling and apoptosis. There are important connections between common genetic alterations (eg, E twenty-six fusion genes) and the altered epigenetic landscape. Owing to the ubiquitous nature of epigenetic alterations, they provide potential biomarkers for PCa detection, diagnosis, assessment of prognosis, and post-treatment surveillance., Conclusions: Altered epigenetic gene regulation is involved in the genesis and progression of PCa. Epigenetic alterations may provide valuable tools for the management of PCa patients and be targeted by pharmacologic compounds that reverse their nature. The potential for epigenetic changes in PCa requires further exploration and validation to enable translation to the clinic., (Copyright © 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

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.

Background: The clinical success of the nucleoside analogs 5-aza-cytidine (5-azaC) and 5-aza-2'deoxycytidine (5-aza-dC) as DNA methyltransferase (DNMT) inhibitors has spurred interest in the development of non-nucleoside inhibitors with improved pharmacologic and safety profiles. Because DNMT catalysis features attack of cytosine bases by an enzyme thiol group, we tested whether disulfiram (DSF), a thiol-reactive compound with known clinical safety, demonstrated DNMT inhibitory activity., Methods: Inhibition of DNMT1 activity by DSF was assessed using methyltransferase activity assays with recombinant DNMT1. Next, prostate cancer cell lines were exposed to DSF and assessed for: i) reduction of global 5-methyl cytosine ((5me)C) content using liquid chromatography/tandem mass spectrometry (LC-MS/MS); ii) gene-specific promoter demethylation by methylation-specific PCR (MSP); and iii) gene-reactivation by real-time RT-PCR. DSF was also tested for growth inhibition using prostate cancer cell lines propagated in vitro in cell culture and in vivo as xenografts in nude mice., Results: Disulfiram showed a dose-dependent inhibition of DNMT1 activity on a hemimethylated DNA substrate. In prostate cancer cells in culture, DSF exposure led to reduction of global genomic (5me)C content, increase in unmethylated APC and RARB gene promoters, and associated re-expression of these genes, but did not significantly alter prostate-specific antigen (PSA) expression. DSF significantly inhibited growth and clonogenic survival of prostate cancer cell lines in culture and showed a trend for reduced growth of prostate cancer xenografts., Conclusions: Disulfiram is a non-nucleoside DNMT1 inhibitor that can reduce global (5me)C content, reactivate epigenetically silenced genes, and significantly inhibit growth in prostate cancer cell lines., (Copyright © 2010 Wiley-Liss, Inc.)

Hypomethylation of CpG dinucleotides in genomic DNA was one of the first somatic epigenetic alterations discovered in human cancers. DNA hypomethylation is postulated to occur very early in almost all human cancers, perhaps facilitating genetic instability and cancer initiation and progression. We therefore examined the nature, extent, and timing of DNA hypomethylation changes in human prostate cancer. Contrary to the prevailing view that global DNA hypomethylation changes occur extremely early in all human cancers, we show that reductions in (5me)C content in the genome occur very late in prostate cancer progression, appearing at a significant extent only at the stage of metastatic disease. Furthermore, we found that, whereas some LINE1 promoter hypomethylation does occur in primary prostate cancers compared with normal tissues, this LINE1 hypomethylation is significantly more pronounced in metastatic prostate cancer. Next, we carried out a tiered gene expression microarray and bisulfite genomic sequencing-based approach to identify genes that are silenced by CpG island methylation in normal prostate cells but become overexpressed in prostate cancer cells as a result of CpG island hypomethylation. Through this analysis, we show that a class of cancer testis antigen genes undergoes CpG island hypomethylation and overexpression in primary prostate cancers, but more so in metastatic prostate cancers. Finally, we show that DNA hypomethylation patterns are quite heterogeneous across different metastatic sites within the same patients. These findings provide evidence that DNA hypomethylation changes occur later in prostate carcinogenesis than the CpG island hypermethylation changes and occur heterogeneously during prostate cancer progression and metastatic dissemination.

Alterations in methylation of CpG dinucleotides at the 5 position of deoxycytidine residues (5(m)C) are a hallmark of cancer cells, including testicular germ cell tumors. Virtually all testicular germ cell tumors are believed to be derived from intratubular germ cell neoplasia unclassified (IGCNU), which is thought to arise from primordial germ cells. Prior studies revealed that seminomas contain reduced levels of global DNA methylation as compared with nonseminomatous germ cell tumors. Smiraglia et al have proposed a model whereby seminomas arise from IGCNU cells derived from primordial germ cells that have undergone 5(m)C erasure, and nonseminomas arise from IGCNU cells derived from primordial germ cells that have already undergone de novo methylation after the original erasure of methylation and contain normal 5(m)C levels. Yet the methylation status of IGCNU has not been determined previously. We used immunohistochemical staining against 5(m)C to evaluate global methylation in IGCNU and associated invasive testicular germ cell tumors. Strikingly, staining for 5(m)C was undetectable (or markedly reduced) in the majority of IGCNU and seminomas, yet there was robust staining in nonseminomatous germ cell tumors. The lack of staining for 5(m)C in IGCNU and seminomas was also found in mixed germ cell tumors containing both seminomatous and nonseminomatous components. Lack of 5(m)C staining was not related to a lack of the maintenance methyltransferase (DNA methyltransferase 1) protein. We conclude that testicular germ cell tumors are derived in most cases from IGCNU cells that have undergone developmentally programmed 5(m)C erasure and that the degree of subsequent de novo methylation is most closely related to the differentiation state of the neoplastic cells. That is, IGCNU cells and seminoma cells remain unmethylated, whereas all other histological types appear to arise after de novo methylation.

Purpose: We have noted that hypermethylation at GSTP1 in the preoperative serum of men with localized prostate cancer predicts early prostate specific antigen failure following surgical treatment. In this study we investigated the hypermethylation profile of several genes in the serum of men with localized and hormone refractory prostate cancer., Materials and Methods: We assayed the serum of 192 men with clinically localized prostate cancer and 18 with hormone refractory metastatic disease. A total of 35 serum samples from patients with negative prostate biopsy served as a negative control. CpG Island hypermethylation status of certain genes was assessed, including MDR1, EDNRB, CD44, NEP, PTGS2, RASSF1A, RAR-beta and ESR1. The results of hypermethylation at GSTP1 were included from a previous study., Results: CpG island hypermethylation at MDR1 was positive in 38.2% of cases without PSA recurrence and in 16.1% of those with biochemical recurrence after radical prostatectomy. DNA hypermethylation at the remaining 7 gene loci was not detected in the serum of patients with localized prostate cancer. In serum from metastatic prostate cancer cases CpG island hypermethylation was detected at MDR1 in 15 (83.3%), EDNRB in 9 (50%), RAR-beta in 7 (38.9%), GTSP1 in 5 (27.8%) and NEP or RASSF1A in 3 (16.7%). CpG island hypermethylation at CD44, PTGS2 or ESR was not detected in any samples. All histologically normal cases were negative for CpG island hypermethylation., Conclusions: DNA hypermethylation at MDR1 was detected in cases of localized prostate cancer. CpG island hypermethylation at several gene loci was detected in men with advanced disease. No single gene was consistently observed to be hypermethylated in men with hormone refractory disease. These results suggest that the CpG island hypermethylation status of a defined panel of genes may be a useful biomarker in men with hormone refractory prostate cancer.

Human prostatic carcinogenesis is characterized by the accumulation of both genetic and epigenetic alterations. The epigenetic changes appear earlier and more consistently, and because the DNA sequence remains intact, may be therapeutically reversible. The mechanism(s) by which epigenetic changes appear during the pathogenesis of prostate cancer have not been established. Nonetheless, new methods for the detection of abnormal DNA methylation, a molecular biomarker of epigenetic alterations, are poised to provide clinical tests potentially useful for prostate cancer detection and diagnosis. In addition, new drugs targeting DNA methyltransferases and other enzymes involved in the maintenance of chromatic structure have been introduced into clinical trials for the treatment of advanced prostate cancers. If sufficiently safe strategies for chromatin modulation can be discovered and developed, epigenetic alterations may become rational targets for both prostate cancer prevention and prostate cancer treatment.

Purpose: We evaluated the concordance between post-digital rectal examination and post-prostate biopsy urine samples using conventional methylation specific polymerase chain reaction analysis of 3 gene promoters in patients with suspected or confirmed prostate cancer., Materials and Methods: Voided urine specimens were collected from 17 men after 15-second digital rectal examination and again after transrectal ultrasound guided biopsy of the prostate for suspected malignancy or for followup biopsy as part of an expectant management protocol. Urine sediment DNA was isolated and subjected to bisulfite modification. Methylation of GSTP1, EDNRB and APC promoters was determined by conventional methylation specific polymerase chain reaction analysis in post-digital rectal examination and post-biopsy samples, and correlated with clinical information., Results: Prostate cancer was detected on prostate biopsy in 12 of 17 patients (71%). Promoter methylation was detected in post-digital rectal examination urine specimens for GSTP1 (24%), APC (12%) and EDNRB (66%). Promoter methylation was detected in post-biopsy urine specimens for GSTP1 (18%), APC (18%) and EDNRB (77%). The concordance between post-digital rectal examination and post-biopsy urine samples was 94% for GSTP1 and APC, and 82% for EDNRB. Overall 100% of patients with biopsy proven prostate cancer had at least 1 gene methylated in urine vs 60% of those without evidence of prostate cancer on biopsy., Conclusions: Gene analysis using conventional methylation specific polymerase chain reaction is a reliable method for detecting abnormal DNA methylation in voided urine samples obtained following digital rectal examination or prostate needle biopsy. The concordance between post-digital rectal examination and post-biopsy urinary samples for promoter methylation is high (82% to 94%), suggesting that urine collected after digital rectal examination may be used for genetic analysis with results similar to those in post-biopsy urine samples.

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

Purpose: Hypermethylation of the CpG island at the promoter region of the pi-class glutathione S-transferase gene (GSTP1) is the most common somatic genome abnormality in human prostate cancer. We evaluated circulating cell-free DNA GSTP1 CpG island hypermethylation as a prognostic biomarker in the serum of men with prostate cancer., Experimental Design: Prostate cancer DNA GSTP1 CpG island hypermethylation was detected using a restriction endonuclease quantitative PCR technique. We analyzed preoperative serum from 85 men with clinically localized prostate cancer treated with radical prostatectomy and from 35 men with a negative prostate biopsy. We then assayed preoperative serum from a data set of 55 pairs of men with clinically localized prostate cancer treated with radical prostatectomy, matched for Gleason score, comprising 55 men suffering prostate-specific antigen (PSA) recurrence (median, 2 years) and 55 men who were free of disease at last follow-up (median, 3 years). The association of serum GSTP1 CpG island hypermethylation and PSA recurrence was determined., Results: Circulating cell-free DNA with GSTP1 CpG island hypermethylation was not detected in the serum of men with a negative prostate biopsy but was detected in 12% of men with clinically localized disease and 28% of men with metastatic cancer (P = 0.003). In the matched data set, eight men (15%) who developed PSA recurrence were positive for DNA with GSTP1 CpG hypermethylation, whereas no patient who was free of disease was positive for GSTP1 CpG island hypermethylation (McNemar test, chi(2) = 6.1, P = 0.01). In a multivariable analysis that accounted for recognized prognostic factors, the presence of serum DNA with GTSP1 CpG island hypermethylation was the most significant predictor of PSA recurrence (hazard ratio, 4.4; 95% confidence interval, 2.2, 8.8; P < 0.001)., Conclusion: Our study suggests that GSTP1 CpG island hypermethylation may be an important DNA-based prognostic serum biomarker for prostate cancer.

CpG island hypermethylation may be one of the earliest somatic genome alterations to occur during the development of multiple cancers. Recently, aberrant methylation patterns for different tumors have been reported. We present a comprehensive review of the literature describing the role of CpG island hypermethylation of DNA from prostatic tissue and bodily fluids from men with prostate cancer. We reviewed the literature to evaluate CpG island hypermethylation in tissue and bodily fluids of men with primary and metastatic prostate cancer. Additionally, we reviewed the literature with respect to CpG island hypermethylation patterns in other urological malignancies. Using modern analytic methods, CpG island hypermethylation detection can be achieved. In men with prostate cancer, correlations between specific gene regulatory region hypermethylation analyses and Gleason score, pathologic stage and tumor recurrence have been demonstrated. CpG island hypermethylation may serve as a useful molecular biomarker for the detection and diagnosis of patients with prostate cancer.

Purpose: Preoperative histologic classification of solid renal masses remains limited with current technology. We determine the utility of molecular profiling based on quantitative methylation analysis for characterization of sporadic renal cell carcinoma., Experimental Design: Primary renal cell carcinomas representing three different histologic subtypes were obtained from a total of 38 patients who underwent radical nephrectomy for suspected malignant disease. Genomic DNA was isolated from tumors and was subjected to sodium bisulfite modification. The normalized index of methylation (NIM) for each sample was determined by quantitative real-time methylation-specific PCR at 17 different gene promoters. Hierarchical cluster analysis was performed by using an unsupervised neural network with binary tree topology., Results: The majority of gene promoters that were analyzed in this study demonstrated very low levels of methylation (NIM <1.0). The RASSF1A gene promoter, however, was methylated in 30 of 38 (79%) cases. The frequency of RASSF1A methylation in papillary, clear-cell, and oncocytoma subtypes was 100, 90, and 25%, respectively. The highest levels of RASSF1A methylation were observed in the papillary (mean NIM = 78.9) and clear-cell (mean NIM = 13.4) subtypes. The vast majority of oncocytomas were completely unmethylated, and none demonstrated >1% methylation (mean NIM = 0.11). Hierarchical cluster analysis based on quantitative methylation levels resulted in stratification of sporadic renal cell carcinomas into their discrete histologic subtypes., Conclusions: Classification of sporadic renal cell carcinomas into histologic subtypes can be accomplished via multigene quantitative methylation profiling. Validation of this approach and selection of appropriate methylation markers may ultimately lead to use of this technology in the preoperative assessment of suspicious renal masses.

Purpose: We assessed the feasibility of a novel urinary test for prostate cancer based on the presence of alpha methylacyl coenzyme A racemase (AMACR) protein in voided urine specimens obtained after prostate biopsy., Materials and Methods: Clean catch voided urine specimens were prospectively collected from 26 consecutive men immediately after transrectal ultrasound guided prostate biopsy for suspected malignancy. The presence of AMACR was evaluated in a blinded manner by Western blot analysis and correlated with biopsy results and patient clinical information., Results: AMACR was detected in the urine in 18 of 26 patients (69%). AMACR was detected in all 12 patients with biopsy confirmed adenocarcinoma of the prostate (100% sensitivity, 95% CI 75 to 100), in 5 of 12 with no evidence of cancer on biopsy (58% specificity, 95% CI 29 to 78) and in 1 of 2 (50%, 95% CI 3 to 80) with atypia on biopsy. Overall AMACR detection was associated with cancer status by prostate biopsy in 21 of 26 patients (86%)., Conclusions: We report the feasibility of a novel, noninvasive, nonprostate specific antigen based molecular approach to detect prostate cancer in voided urine. To our knowledge this is the first report of AMACR protein detection in the urine of patients with prostate cancer. A screening test based on urinary AMACR may develop into a useful adjunct to serum prostate specific antigen and digital rectal examination for identifying men at increased risk for harboring prostate cancer despite negative biopsy. Such a test has potential application for stratifying patients into low and high risk groups for surveillance vs repeat biopsy.

Resistance to the cytotoxic actions of antineoplastic drugs, whether intrinsic or acquired, remains a barrier to the establishment of curative chemotherapy regimens for advanced breast cancer. Over-expression of P-glycoprotein (P-gp), encoded by the MDR1 gene and known to mediate resistance to many antineoplastic drugs, may contribute to poor breast cancer treatment outcome. Nonetheless, the precise molecular mechanisms responsible for high or low level P-gp expression in breast cancer cells have not been established. We assessed the role of DNA hypermethylation near the MDR1 transcriptional regulatory region in MDR1 expression in MCF-7 breast cancer cells, which fail to express MDR1 mRNA, and MCF-7/ADR cells, known to express high MDR1 mRNA levels. When compared to MCF-7/ADR cells, MCF-7 cells manifested markedly diminished MDR1 transcription rates by nuclear run-off assay, but equivalent MDR1 promoter trans-activation activity in transient transfection experiments, indicating that cis factors were most likely responsible for the differences in MDR1 transcription between MCF-7/ADR cells and MCF-7 cells. Bisulfite genomic sequencing analyses revealed substantially less extensive MDR1 promoter methylation in MCF-7/ADR cells than in MCF-7 cells, suggesting that CpG dinucleotide methylation might contribute to the observed MDR1 transcription differences. Chromatin immunoprecipitation analyses indicated an inactive MDR1 chromatin conformation in MCF-7 cells, with a paucity of acetylated histones and the presence of 5-mC-binding proteins MeCP2 and MBD2, and an active MDR1 chromatin conformation in MCF-7/ADR cells, with an abundance of acetylated histones and the presence of the transcriptional trans-activator YB-1. Stable MCF-7 sublines which had been treated with the DNA methyltransferase inhibitor 5-azacytidine, exhibited a reduction in MDR1 promoter methylation and a complex MDR1 chromatin configuration, characterized by the simultaneous presence of transcriptional activators and repressors. In this state, MDR1 expression was markedly sensitive to treatment with the histone deacetylase inhibitor trichostatin A.

Aberrant DNA methylation patterns may be the earliest somatic genome changes in prostate cancer. Using real-time methylation-specific PCR, we assessed the extent of hypermethylation at 16 CpG islands in DNA from seven prostate cancer cell lines (LNCaP, PC-3, DU-145, LAPC-4, CWR22Rv1, VCaP, and C42B), normal prostate epithelial cells, normal prostate stromal cells, 73 primary prostate cancers, 91 metastatic prostate cancers, and 25 noncancerous prostate tissues. We found that CpG islands at GSTP1, APC, RASSF1a, PTGS2, and MDR1 were hypermethylated in >85% of prostate cancers and cancer cell lines but not in normal prostate cells and tissues; CpG islands at EDNRB, ESR1, CDKN2a, and hMLH1 exhibited low to moderate rates of hypermethylation in prostate cancer tissues and cancer cell lines but were entirely unmethylated in normal tissues; and CpG islands at DAPK1, TIMP3, MGMT, CDKN2b, p14/ARF, and CDH1 were not abnormally hypermethylated in prostate cancers. Receiver operator characteristic curve analyses suggested that CpG island hypermethylation changes at GSTP1, APC, RASSF1a, PTGS2, and MDR1 in various combinations can distinguish primary prostate cancer from benign prostate tissues with sensitivities of 97.3-100% and specificities of 92-100%. Hypermethylation of the CpG island at EDNRB was correlated with the grade and stage of the primary prostate cancers. PTGS2 CpG island hypermethylation portended an increased risk of recurrence. Furthermore, CpG island hypermethylation patterns in prostate cancer metastases were very similar to the primary prostate cancers and tended to show greater differences between cases than between anatomical sites of metastasis.

Somatic hypermethylation of CpG island sequences at GSTP1, the gene encoding the pi-class glutathione S-transferase, appears to be characteristic of human prostatic carcinogenesis. To consider the potential utility of this epigenetic alteration as a biomarker for prostate cancer, we present here a comprehensive review of the literature describing somatic GSTP1 changes in DNA from prostate cells and tissues. GSTP1 CpG island hypermethylation has been detected in prostate cancer DNA using a variety of assay techniques, including (i) Southern blot analysis (SB), after treatment with (5-m)C-sensitive restriction endonucleases, (ii) the polymerase chain reaction, following treatment with (5-m)C-sensitive restriction endonucleases (RE-PCR), (iii) bisulfite genomic sequencing (BGS), and (iv) bisulfite modification followed by the polymerase chain reaction, using primers selective for target sequences containing (5-m)C (MSP). In the majority of the case series so far reported, GSTP1 CpG island hypermethylation was present in DNA from at least 90% of prostate cancer cases. When analyses have been carefully conducted, GSTP1 CpG island hypermethylation has not been found in DNA from normal prostate tissues, or from benign prostatic hyperplasia (BPH) tissues, though GSTP1 CpG island hypermethylation changes have been detected in DNA from candidate prostate cancer precursor lesions proliferative inflammatory atrophy (PIA) and prostatic intraepithelial neoplasia (PIN). Using PCR methods, GSTP1 CpG island hypermethylation has also been detected in urine, ejaculate, and plasma from men with prostate cancer. GSTP1 CpG island hypermethylation, a somatic epigenetic alteration, appears poised to serve as a molecular biomarker useful for prostate cancer screening, detection, and diagnosis., (Copyright 2003 Wiley-Liss, Inc.)

Objectives: To evaluate the utility of methylation-specific polymerase chain reaction analysis of the pi-class glutathione-S-transferase (GSTP1) gene promoter in prostatic secretions for cancer detection and prognostication., Methods: Prostatic secretions were obtained from a total of 100 radical prostatectomy specimens immediately after surgical extirpation. GSTP1 promoter methylation was assessed by methylation-specific polymerase chain reaction analysis using two different primer sets. Correlations between GSTP1 promoter methylation and clinical and pathologic variables were examined., Results: The sensitivity for detection of GSTP1 methylation in prostatic secretions from men with clinically localized prostate cancer using two different primer sets was 76% and 54%. Methylation of the GSTP1 promoter was detected by both primer sets in 44% and by at least one primer set in 86% of the prostatic secretion specimens. The degree of methylation detected in the prostatic secretions was associated with the extent of cancer (predominant involvement of one or both sides of the gland; P = 0.02) and increasing age (P = 0.009)., Conclusions: Genomic DNA with GSTP1 promoter methylation can be detected in prostatic secretion specimens from the great majority of men with localized prostate cancer. Assays of GSTP1 promoter methylation in prostatic massage fluid or ejaculate may therefore serve as useful adjuncts to existing methods for prostate cancer screening and prognostication.

Somatic inactivation of the glutathione S-transferase-pi gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in approximately 70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-alpha (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA lesions. These data support the hypothesis that atrophic epithelium in a subset of PIA lesions may lead to high-grade PIN and/or adenocarcinoma. Because these atrophic lesions are so prevalent and extensive, even though only a small subset contains this somatic DNA alteration, the clinical impact may be substantial.

Purpose: We assess the feasibility of a urinary test for prostate cancer detection in a high-risk patient cohort based on methylation-specific PCR analysis of the pi class glutathione S-transferase (GSTP1) gene promoter., Experimental Design: A total of 45 men underwent transrectal ultrasound-guided biopsy of the prostate for suspected malignancy. Clean-catch voided urine specimens were prospectively collected from each patient immediately after biopsy. Genomic DNA was isolated from urine specimens and subjected to sodium bisulfite modification. Methylation of the GSTP1 promoter was examined in a blinded manner by methylation-specific PCR analysis and correlated with pathology results, and clinical information was obtained from the patient record., Results: Methylation of GSTP1 in the urine was detected in a total of 18 of 36 (50%) informative cases. A total of 7 of 18 (39%) patients with prostate adenocarcinoma identified on their initial biopsy had detectable urinary GSTP1 methylation (58% sensitivity among informative cases). Abnormal urinary GSTP1 methylation was also detected in 7 of 21 (33%) patients without evidence of cancer on biopsy and in 4 of 6 (67%) patients diagnosed with atypia or high-grade prostatic intraepithelial neoplasia., Conclusions: We have demonstrated the feasibility of a novel, noninvasive molecular approach for the detection of epigenetic changes associated with prostate cancer. A screening test based on GSTP1 methylation in the urine specimens of patients with suspected prostate malignancy may be a useful adjunct to serum screening tests and digital rectal examination findings for identification of men at increased risk of harboring cancer despite a negative biopsy. This molecular assay has potential application for stratification of patients into low- and high-risk groups for surveillance versus repeat biopsy.

GSTP1, encoding the pi-class glutathione S-transferase, is commonly inactivated by somatic CpGisland hypermethylation in cancers of the prostate, liver, and breast. We report here thathypermethylation of CpG dinucleotides at the 5' transcriptional regulatory region was sufficient to inhibit GSTP1 transcription in MCF-7 breast cancer cells and that repression of GSTP1 transcription was mediated in part by the methyl-CpG-binding domain (MBD) protein MBD2. MCF-7 breast cancer cells contained only hypermethylated GSTP1 CpG island alleles and failed to express GSTP1 mRNA or GSTP1 polypeptides. In contrast, MCF-7/ADR cells contained only unmethylated GSTP1 CpG island alleles and exhibited abundant GSTP1 expression. Chromatin immunoprecipitation analysis detected the presence of MBD2 and DNMT1 at the GSTP1 promoter in MCF-7 breast cancer cells but not in MCF-7/ADR breast cancer cells. In a test of the contribution of MBD2 to GSTP1 repression in MCF-7 breast cancer cells, transfection of small interference RNA complementary to MBD2 mRNA into MCF-7 cells both reduced MBD2 polypeptide levels and stimulated GSTP1 mRNA expression. These findings implicate MBD2 in GSTP1 silencing associated with somatic GSTP1 CpG island hypermethylation in breast cancer cells.

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.