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1.  Explanatory Chapter: Next Generation Sequencing 
Methods in enzymology  2013;529:201-208.
Technological breakthroughs in sequencing technologies have driven the advancement of molecular biology and molecular genetics research. The advent of high-throughput Sanger sequencing (for information on the method, see Sanger Dideoxy Sequencing of DNA) in the mid- to late-1990s made possible the accelerated completion of the human genome project, which has since revolutionized the pace of discovery in biomedical research. Similarly, the advent of next generation sequencing is poised to revolutionize biomedical research and usher a new era of individualized, rational medicine.
The term next generation sequencing refers to technologies that have enabled the massively parallel analysis of DNA sequence facilitated through the convergence of advancements in molecular biology, nucleic acid chemistry and biochemistry, computational biology, and electrical and mechanical engineering. The current next generation sequencing technologies are capable of sequencing tens to hundreds of millions of DNA templates simultaneously and generate >4 gigabases of sequence in a single day. These technologies have largely started to replace high-throughput Sanger sequencing for large-scale genomic projects, and have created significant enthusiasm for the advent of a new era of individualized medicine.
doi:10.1016/B978-0-12-418687-3.00016-1
PMCID: PMC3978176  PMID: 24011047
2.  Correction: Dietary Chemoprevention of PhIP Induced Carcinogenesis in Male Fischer 344 Rats with Tomato and Broccoli 
PLoS ONE  2014;9(1):10.1371/annotation/1ac6412e-619f-4575-8b63-f5e16bd3252b.
doi:10.1371/annotation/1ac6412e-619f-4575-8b63-f5e16bd3252b
PMCID: PMC3883743
3.  Correction: Dietary Chemoprevention of PhIP Induced Carcinogenesis in Male Fischer 344 Rats with Tomato and Broccoli 
PLoS ONE  2014;9(1):10.1371/annotation/3fd9703c-a6d9-4715-9c93-cf5279ea4fa6.
doi:10.1371/annotation/3fd9703c-a6d9-4715-9c93-cf5279ea4fa6
PMCID: PMC3883746
4.  Dietary Chemoprevention of PhIP Induced Carcinogenesis in Male Fischer 344 Rats with Tomato and Broccoli 
PLoS ONE  2013;8(11):e79842.
The heterocyclic amine, 2-amino-1-methyl-6-phenylimidazo[4,5-B]pyridine (PhIP), found in meats cooked at high temperatures, has been implicated in epidemiological and rodent studies for causing breast, prostate, and colorectal cancers. A previous animal study using a xenograft model has shown that whole tomato and broccoli, when eaten in combination, exhibit a marked effect on tumor reduction compared to when eaten alone. Our aim was to determine if PhIP-induced carcinogenesis can be prevented by dietary consumption of whole tomato + broccoli powders. Male Fischer 344 rats (n = 45) were randomized into the following treatment groups: control (AIN93G diet), PhIP (200 ppm in AIN93G diet for the first 20 weeks of the study), or tomato + broccoli + PhIP (mixed in AIN93G diet at 10% each and fed with PhIP for 20 weeks, and then without PhIP for 32 weeks). Study animals were monitored for 52 weeks and were euthanized as necessary based on a set of criteria for health status and tumor burden. Although there appeared to be some hepatic and intestinal toxicity due to the combination of PhIP and tomato + broccoli, these rodents had improved survival and reduced incidence and/or severity of PhIP-induced neoplastic lesions compared to the PhIP-alone treated group. Rats eating tomato + broccoli exhibited a marked decrease in the number and size of cribiform prostatic intraepitheilial neoplasia/carcinoma in situ (cribiform PIN/CIS) lesions and in the incidence of invasive intestinal adenocarcinomas and skin carcinomas. Although the apparent toxic effects of combined PhIP and tomato + broccoli need additional study, the results of this study support the hypothesis that a diet rich in tomato and broccoli can reduce or prevent dietary carcinogen-induced cancers.
doi:10.1371/journal.pone.0079842
PMCID: PMC3842290  PMID: 24312188
5.  Prospective study of genomic hypomethylation of leukocyte DNA and colorectal cancer risk 
Background
Systematic genome-wide reductions of methylated cytosine (5-mC) levels have been observed in colorectal cancer tissue and are suspected to play a role in carcinogenesis, possibly as a consequence of inadequate folate intake. Reduced 5-mC levels in peripheral blood leukocytes have been associated with increased risk of colorectal cancer and adenoma in cross-sectional studies.
Methods
To minimize disease- and/or treatment-related effects, we studied leukocyte 5-mC levels in prospectively collected blood specimens of 370 cases and 493 controls who were cancer-free at blood collection from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial., Leukocyte 5-mC level was determined by an HPLC/Tandem Mass Spectrometry method and expressed as the relative amount of methyl- to total cytosine residues, or %5-mC. We estimated the association between colorectal cancer risk and %5-mC categories by computing odds ratios (ORs) and 95% confidence intervals (CIs) through logistic regression modeling.
Results
We observed no dose-dependent association between colorectal cancer and %5-mC categories (lowest tertile vs. highest: OR=1.14, 95% CI=0.80–1.63; P trend=0.51). However, among subjects whose 5-mC levels were at the highest tertile, we observed an inverse association between natural folate intake and colorectal cancer (highest tertile of natural folate vs. lowest: OR=0.35, 95% CI=0.17–0.71; P trend=0.003; P interaction=0.003).
Conclusions
This prospective investigation show no clear association between leukocyte 5-mC level and subsequent colorectal cancer risk, but a suggestive risk modification between 5-mC level and natural folate intake.
Impact
Adequate folate status may protect against colorectal carcinogenesis through mechanisms involving adequate DNA methylation in the genome.
doi:10.1158/1055-9965.EPI-12-0700-T
PMCID: PMC3493855  PMID: 23001241
5-mC; PLCO; folate; colorectal
6.  Tracking the clonal origin of lethal prostate cancer 
The Journal of Clinical Investigation  2013;123(11):4918-4922.
Recent controversies surrounding prostate cancer overtreatment emphasize the critical need to delineate the molecular features associated with progression to lethal metastatic disease. Here, we have used whole-genome sequencing and molecular pathological analyses to characterize the lethal cell clone in a patient who died of prostate cancer. We tracked the evolution of the lethal cell clone from the primary cancer to metastases through samples collected during disease progression and at the time of death. Surprisingly, these analyses revealed that the lethal clone arose from a small, relatively low-grade cancer focus in the primary tumor, and not from the bulk, higher-grade primary cancer or from a lymph node metastasis resected at prostatectomy. Despite being limited to one case, these findings highlight the potential importance of developing and implementing molecular prognostic and predictive markers, such as alterations of tumor suppressor proteins PTEN or p53, to augment current pathological evaluation and delineate clonal heterogeneity. Furthermore, this case illustrates the potential need in precision medicine to longitudinally sample metastatic lesions to capture the evolving constellation of alterations during progression. Similar comprehensive studies of additional prostate cancer cases are warranted to understand the extent to which these issues may challenge prostate cancer clinical management.
doi:10.1172/JCI70354
PMCID: PMC3809798  PMID: 24135135
7.  DNA methylation alterations exhibit intra-individual stability and inter-individual heterogeneity in prostate cancer metastases 
Science translational medicine  2013;5(169):169ra10.
Human cancers nearly ubiquitously harbor epigenetic alterations. While 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. Here, 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 inter-individual 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 of cancer-related genes. Interestingly, whereas some regions showed intra-individual 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.
doi:10.1126/scitranslmed.3005211
PMCID: PMC3577373  PMID: 23345608
8.  Distinct transcriptional programs mediated by the ligand-dependent full-length androgen receptor and its splice variants in castration-resistant prostate cancer 
Cancer research  2012;72(14):3457-3462.
Continued androgen receptor (AR) signaling is an established mechanism underlying castration-resistant prostate cancer (CRPC), and suppression of AR signaling remains a therapeutic goal of CRPC therapy. Constitutively active androgen receptor splice variants (AR-Vs) lack the AR ligand-binding domain (AR-LBD), the intended target of androgen deprivation therapies (ADT) including CRPC therapies such as abiraterone and MDV3100. While the canonical full-length AR (AR-FL) and AR-Vs are both increased in CRPC, their expression regulation, associated transcriptional programs, and functional relationships have not been dissected. In this study, we show that suppression of ligand-mediated AR-FL signaling by targeting AR-LBD leads to increased AR-V expression in two cell line models of CRPC. Importantly, treatment-induced AR-Vs activated a distinct expression signature enriched for cell cycle genes without requiring the presence of AR-FL. Conversely, activation of AR-FL signaling suppressed the AR-V signature and activated expression programs mainly associated with macromolecular synthesis, metabolism, and differentiation. In prostate cancer cells and CRPC xenografts treated with MDV3100 or abiraterone, increased expression of two constitutively active AR-Vs, AR-V7 and ARV567ES, but not AR-FL, paralleled increased expression of the AR-driven cell cycle gene UBE2C. Expression of AR-V7, but not AR-FL, was positively correlated with UBE2C in clinical CRPC specimens. Together, our findings support an adaptive shift toward AR-V-mediated signaling in a subset of CRPC tumors as the AR-LBD is rendered inactive, suggesting an important mechanism contributing to drug resistance to CRPC therapy.
doi:10.1158/0008-5472.CAN-11-3892
PMCID: PMC3415705  PMID: 22710436
androgen receptor; castration-resistant prostate cancer; androgen receptor splice variants; MDV3100; abiraterone
9.  Genetic and epigenetic inactivation of LPL gene in human prostate cancer 
Lipoprotein lipase (LPL) is in chromosome 8p22, site of one of the most common somatic deletions in prostate tumors. Additionally, a CpG island (CGI) was identified in the LPL promoter region. To test the hypothesis that LPL is a tumor suppressor gene, which is inactivated by somatic deletion and hypermethylation in prostate cancer, we evaluated somatic DNA deletion and methylation status at LPL in 56 pairs of DNA samples isolated from prostate cancer tissues and matching normal controls and 11 prostate cell lines. We found that the DNA in 21 of 56 primary cancers (38%) was methylated in the LPL promoter CGI, whereas no methylation was detected in any normal samples. In addition, we found a hemizygous deletion at LPL in 38 of the 56 tumors (68%). When the results of deletion and methylation were considered together, we found LPL promoter CGI methylation occurred in 45% of LPL deleted tumors and in 22% of LPL retained tumors. Within several clinical characteristics tested, the preoperative PSA levels were found to be significantly higher in subjects with LPL promoter CGI methylation compared with subjects without LPL promoter methylation (p = 0.0012). Additionally, demethylation of the LPL promoter CGI was accompanied by transcriptional reactivation of LPL in the prostate cancer cell lines DU145 and PC3. In summary, we report a novel finding that the LPL gene is commonly methylated in prostate tumors, and our results suggest that biallelic inactivation of LPL by chromosomal deletion and promoter hypermethylation may play a role in human prostate cancer.
doi:10.1002/ijc.23972
PMCID: PMC3667349  PMID: 19004026
LPL; promoter methylation; prostate cancer; somatic deletion; biallelic inactivation
10.  Genome-wide comparison of the transcriptomes of highly enriched normal and chronic myeloid leukemia stem and progenitor cell populations 
Oncotarget  2013;4(5):715-728.
The persistence leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) despite tyrosine kinase inhibition (TKI) may explain relapse after TKI withdrawal. Here we performed genome-wide transcriptome analysis of highly refined CML and normal stem and progenitor cell populations to identify novel targets for the eradication of CML LSCs using exon microarrays. We identified 97 genes that were differentially expressed in CML versus normal stem and progenitor cells. These included cell surface genes significantly upregulated in CML LSCs: DPP4 (CD26), IL2RA (CD25), PTPRD, CACNA1D, IL1RAP, SLC4A4, and KCNK5. Further analyses of the LSCs revealed dysregulation of normal cellular processes, evidenced by alternative splicing of genes in key cancer signaling pathways such as p53 signaling (e.g. PERP, CDKN1A), kinase binding (e.g. DUSP12, MARCKS), and cell proliferation (MYCN, TIMELESS); downregulation of pro-differentiation and TGF-β/BMP signaling pathways; upregulation of oxidative metabolism and DNA repair pathways; and activation of inflammatory cytokines, including CCL2, and multiple oncogenes (e.g., CCND1). These data represent an important resource for understanding the molecular changes in CML LSCs, which may be exploited to develop novel therapies for eradication these cells and achieve cure.
PMCID: PMC3742832  PMID: 23651669
chronic myeloid leukemia; CML; leukemic stem cell; LSC; normal hematopoietic stem cell; HSC; myeloid progenitor cells; CD34; CD38; ALDH; IL2RA; CD25; DPP4; CD26; GAS2
11.  Overexpression of Ribosomal RNA in Prostate Cancer is Common but not Linked to rDNA Promoter Hypomethylation 
Oncogene  2011;31(10):1254-1263.
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 rRNA precursor that is then processed to yield the mature 18S, 5.8S and 28S RNA species. While 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 to 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 to 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.
doi:10.1038/onc.2011.319
PMCID: PMC3298623  PMID: 21822302
epigenetics; rDNA; rRNA; DNA methylation; prostate cancer
12.  Decreased 5-Hydroxymethylcytosine Is Associated with Neural Progenitor Phenotype in Normal Brain and Shorter Survival in Malignant Glioma 
PLoS ONE  2012;7(7):e41036.
Epigenetic modification of DNA by cytosine methylation to produce 5-methylcytosine (5mC) has become well-recognized as an important epigenetic process in human health and disease. Recently, further modification of 5mC by the ten eleven translocated (TET) family of enzymes to produce 5-hydroxymethylcytosine (5hmC) has been described. In the present study, we used immunohistochemistry to evaluate the distribution of 5hmC in human brain during different periods of development and in a large series of gliomas (n = 225). We found that during development, 5hmC levels are high in more differentiated compartments like the fetal cortex, but low in the periventricular progenitor cell regions. In adults, we found 5hmC levels to be highest in the cortex, but present in all intrinsic cell types in the brain including stromal elements. In brain tumors, 5hmC levels were high in low grade tumors and reduced in malignant glioma, but did not exhibit any correlation with IDH1 mutation status. Additionally, we identified a significant relationship between low levels of 5hmC and reduced survival in malignant glioma. This observation was further supported by in silico analysis showing differential expression of genes involved in 5hmC homeostasis in aggressive subsets of glioblastoma. Finally, we show that several genes involved in regulating the levels of 5hmC are also prognostic in malignant glioma. These findings suggest that 5hmC regulation in malignant glioma may represent an important determinant of tumor differentiation and aggressive behavior, as well as a potential therapeutic target.
doi:10.1371/journal.pone.0041036
PMCID: PMC3400598  PMID: 22829908
13.  Transcription-induced DNA double strand breaks: both oncogenic force and potential therapeutic target? 
An emerging model of transcriptional activation suggests that induction of transcriptional programs, for instance by stimulating prostate or breast cells with androgens or estrogens, respectively, involves the formation of DNA damage, including double strand breaks (DSB), recruitment of DSB repair proteins, and movement of newly activated genes to transcription hubs. The DSB can be mediated by the class II topoisomerase TOP2B, which is recruited with the androgen receptor (AR) and estrogen receptor (ER) to regulatory sites on target genes and is apparently required for efficient transcriptional activation of these genes. These DSB are recognized by the DNA repair machinery triggering the recruitment of repair proteins such as PARP1, ATM and DNA-PK. If illegitimately repaired, such DSB can seed the formation of genomic rearrangements like the TMPRSS2-ERG fusion oncogene in prostate cancer. Here we hypothesize that these transcription induced TOP2B mediated DSB can also be exploited therapeutically and propose that, in hormone dependent tumors like breast and prostate cancers, a hormone cycling therapy, in combination with topoisomerase II poisons or inhibitors of the DNA repair components PARP1 and DNA-PK, could overwhelm cancer cells with transcription-associated double strand breaks. Such strategies may find particular utility in cancers, like prostate cancer, that show low proliferation rates, where other chemotherapeutic strategies that target rapidly proliferating cells have had limited success.
doi:10.1158/1078-0432.CCR-10-2044
PMCID: PMC3117909  PMID: 21385925
14.  A Novel Two-Stage, Transdisciplinary Study Identifies Digoxin as a Possible Drug for Prostate Cancer Treatment 
Cancer discovery  2011;2011(1):68-77.
Identification of novel indications for commonly prescribed drugs could accelerate translation of therapies. We investigated whether any clinically-used drugs might have utility for treating prostate cancer by coupling an efficient, high-throughput laboratory-based screen and a large, prospective cohort study. In stage 1, we conducted an in vitro prostate cancer cell cytotoxicity screen of 3,187 compounds. Digoxin emerged as the leading candidate given its potency in inhibiting proliferation in vitro (mean IC50=163 nM) and common use. In stage 2, we evaluated the association between the leading candidate drug from stage 1 and prostate cancer risk in 47,884 men followed 1986–2006. Regular digoxin users (versus nonusers: RR=0.76, 95% CI 0.61–0.95), especially users for ≥10 years (RR=0.54, 95% CI 0.37–0.79, P-trend<0.001), had a lower prostate cancer risk. Digoxin was highly potent in inhibiting prostate cancer cell growth in vitro and its use was associated with a 25% lower prostate cancer risk.
doi:10.1158/2159-8274.CD-10-0020
PMCID: PMC3227223  PMID: 22140654
Digoxin; prostate cancer; risk; cohort; transdisciplinary; cytotoxicity
15.  A novel source for miR-21 expression through the alternative polyadenylation of VMP1 gene transcripts 
Nucleic Acids Research  2012;40(14):6821-6833.
miR-21 is the most commonly over-expressed microRNA (miRNA) in cancer and a proven oncogene. Hsa-miR-21 is located on chromosome 17q23.2, immediately downstream of the vacuole membrane protein-1 (VMP1) gene, also known as TMEM49. VMP1 transcripts initiate ∼130 kb upstream of miR-21, are spliced, and polyadenylated only a few hundred base pairs upstream of the miR-21 hairpin. On the other hand, primary miR-21 transcripts (pri-miR-21) originate within the last introns of VMP1, but bypass VMP1 polyadenylation signals to include the miR-21 hairpin. Here, we report that VMP1 transcripts can also bypass these polyadenylation signals to include miR-21, thus providing a novel and independently regulated source of miR-21, termed VMP1–miR-21. Northern blotting, gene-specific RT-PCR, RNA pull-down and DNA branching assays support that VMP1–miR-21 is expressed at significant levels in a number of cancer cell lines and that it is processed by the Microprocessor complex to produce mature miR-21. VMP1 and pri-miR-21 are induced by common stimuli, such as phorbol-12-myristate-13-acetate (PMA) and androgens, but show differential responses to some stimuli such as epigenetic modifying agents. Collectively, these results indicate that miR-21 is a unique miRNA capable of being regulated by alternative polyadenylation and two independent gene promoters.
doi:10.1093/nar/gks308
PMCID: PMC3413119  PMID: 22505577
16.  Accurate genome-scale percentage DNA methylation estimates from microarray data 
Biostatistics (Oxford, England)  2010;12(2):197-210.
DNA methylation is a key regulator of gene function in a multitude of both normal and abnormal biological processes, but tools to elucidate its roles on a genome-wide scale are still in their infancy. Methylation sensitive restriction enzymes and microarrays provide a potential high-throughput, low-cost platform to allow methylation profiling. However, accurate absolute methylation estimates have been elusive due to systematic errors and unwanted variability. Previous microarray preprocessing procedures, mostly developed for expression arrays, fail to adequately normalize methylation-related data since they rely on key assumptions that are violated in the case of DNA methylation. We develop a normalization strategy tailored to DNA methylation data and an empirical Bayes percentage methylation estimator that together yield accurate absolute methylation estimates that can be compared across samples. We illustrate the method on data generated to detect methylation differences between tissues and between normal and tumor colon samples.
doi:10.1093/biostatistics/kxq055
PMCID: PMC3062148  PMID: 20858772
DNA methylation; Epigenetics; Microarray
17.  Molecular Alterations in Prostate Cancer as Diagnostic, Prognostic and Therapeutic Targets 
Advances in anatomic pathology  2008;15(6):319-331.
Prostatic adenocarcinoma is extremely common in Western nations, representing the second leading cause of cancer death in American men. The recent application of increasingly sophisticated molecular approaches to the study of prostate cancer in this “post-genomic” era has resulted in a rapid increase in the identification of somatic genome alterations as well as germline heritable risk factors in this disease. These findings are leading to a new understanding of the pathogenesis of prostate cancer and to the generation of new targets for diagnosis, prognosis, and prediction of therapeutic response. Although we are still in the very early phase of clinical development, some of the molecular alterations identified in prostate cancer are being translated into clinical practice.
The purpose of this review is to update the practicing surgical pathologist, and residents-in-training in pathology, regarding recent findings in the molecular pathobiology of prostate cancer. We will highlight some of the somatic molecular alterations associated with prostate cancer development and progression, with a focus on newer discoveries. In addition, recent studies in which new molecular diagnostic approaches have been applied in the clinic will be discussed.
doi:10.1097/PAP.0b013e31818a5c19
PMCID: PMC3214657  PMID: 18948763
Prostatic adenocarcinoma; molecular pathology; hypermethylation; GSTP1; PCA3; urine
18.  Humanizing π-Class Glutathione S-Transferase Regulation in a Mouse Model Alters Liver Toxicity in Response to Acetaminophen Overdose 
PLoS ONE  2011;6(10):e25707.
Background
Glutathione S-transferases (GSTs) metabolize drugs and xenobiotics. Yet despite high protein sequence homology, expression of π-class GSTs, the most abundant of the enzymes, varies significantly between species. In mouse liver, hepatocytes exhibit high mGstp expression, while in human liver, hepatocytes contain little or no hGSTP1 mRNA or hGSTP1 protein. π-class GSTs are known to be critical determinants of liver responses to drugs and toxins: when treated with high doses of acetaminophen, mGstp1/2+/+ mice suffer marked liver damage, while mGstp1/2−/− mice escape liver injury.
Methodology/Principal Findings
To more faithfully model the contribution of π-class GSTs to human liver toxicology, we introduced hGSTP1, with its exons, introns, and flanking sequences, into the germline of mice carrying disrupted mGstp genes. In the resultant hGSTP1+mGstp1/2−/− strain, π-class GSTs were regulated differently than in wild-type mice. In the liver, enzyme expression was restricted to bile duct cells, Kupffer cells, macrophages, and endothelial cells, reminiscent of human liver, while in the prostate, enzyme production was limited to basal epithelial cells, reminiscent of human prostate. The human patterns of hGSTP1 transgene regulation were accompanied by human patterns of DNA methylation, with bisulfite genomic sequencing revealing establishment of an unmethylated CpG island sequence encompassing the gene promoter. Unlike wild-type or mGstp1/2−/− mice, when hGSTP1+mGstp1/2−/− mice were overdosed with acetaminophen, liver tissues showed limited centrilobular necrosis, suggesting that π-class GSTs may be critical determinants of toxin-induced hepatocyte injury even when not expressed by hepatocytes.
Conclusions
By recapitulating human π-class GST expression, hGSTP1+mGstp1/2−/− mice may better model human drug and xenobiotic toxicology.
doi:10.1371/journal.pone.0025707
PMCID: PMC3191143  PMID: 22022436
19.  Myc Enforces Overexpression of EZH2 in Early Prostatic Neoplasia via Transcriptional and Post-transcriptional Mechanisms 
Oncotarget  2011;2(9):669-683.
EZH2 is part of the PRC2 polycomb repressive complex that is overexpressed in multiple cancer types and has been implicated in prostate cancer initiation and progression. Here, we identify EZH2 as a target of the MYC oncogene in prostate cancer and show that MYC coordinately regulates EZH2 through transcriptional and post-transcriptional means. Although prior studies in prostate cancer have revealed a number of possible mechanisms of EZH2 upregulation, these changes cannot account for the overexpression EZH2 in many primary prostate cancers, nor in most cases of high grade PIN. We report that upregulation of Myc in the mouse prostate results in overexpression of EZH2 mRNA and protein which coincides with reductions in miR-26a and miR-26b, known regulators of EZH2 in some non-prostate cell types, albeit not in others. Further, in human prostate cancer cells, Myc negatively regulates miR-26a and miR-26b via direct binding to their parental Pol II gene promoters, and forced overexpression of miR-26a and miR-26b in prostate cancer cells results in decreased EZH2 levels and suppressed proliferation. In human clinical samples, miR-26a and miR-26b are downregulated in most primary prostate cancers. As a separate mechanism of EZH2 mRNA upregulation, we find that Myc binds directly to and activates the transcription of the EZH2 promoter. These results link two major pathways in prostate cancer by providing two additional and complementary Myc-regulated mechanisms by which EZH2 upregulation occurs and is enforced during prostatic carcinogenesis. Further, the results implicate EZH2-driven mechanisms by which Myc may stimulate prostate tumor initiation and disease progression.
PMCID: PMC3248223  PMID: 21941025
Myc; EZH2; prostate cancer
20.  Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements 
Nature genetics  2010;42(8):668-675.
DNA double strand breaks (DSB) can lead to development of genomic rearrangements, which are hallmarks of cancer. TMPRSS2-ERG gene fusions in prostate cancer (PCa) are among the most common genomic rearrangements observed in human cancer. We show that androgen signaling promotes co-recruitment of androgen receptor (AR) and topoisomerase II beta (TOP2B) to sites of TMPRSS2-ERG genomic breakpoints, triggering recombinogenic TOP2B-mediated DSB. Furthermore, androgen stimulation resulted in de novo production of TMPRSS2-ERG fusion transcripts in a process requiring TOP2B and components of DSB repair machinery. Finally, unlike normal prostate epithelium, prostatic intraepithelial neoplasia (PIN) cells showed strong co-expression of AR and TOP2B. These findings implicate androgen-induced TOP2B-mediated DSB in generating TMPRSS2-ERG rearrangements.
doi:10.1038/ng.613
PMCID: PMC3157086  PMID: 20601956
21.  Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers 
Oncotarget  2011;2(8):627-637.
DNA methylation at the 5-position of cytosines (5mC) represents an important epigenetic modification involved in tissue differentiation and is frequently altered in cancer. Recent evidence suggests that 5mC can be converted to 5-hydroxymethylcytosine (5hmC) in an enzymatic process involving members of the TET protein family. Such 5hmC modifications are known to be prevalent in DNA of embryonic stem cells and in the brain, but the distribution of 5hmC in the majority of embryonic and adult tissues has not been rigorously explored. Here, we describe an immunohistochemical detection method for 5hmC and the application of this technique to study the distribution of 5hmC in a large set of mouse and human tissues. We found that 5hmC was abundant in the majority of embryonic and adult tissues. Additionally, the level of 5hmC closely tracked with the differentiation state of cells in hierarchically organized tissues. The highest 5hmC levels were observed in terminally differentiated cells, while less differentiated tissue stem/progenitor cell compartments had very low 5hmC levels. Furthermore, 5hmC levels were profoundly reduced in carcinoma of the prostate, breast and colon compared to normal tissues. Our findings suggest a distinct role for 5hmC in tissue differentiation, and provide evidence for its large-scale loss in cancers.
PMCID: PMC3248214  PMID: 21896958
5-hydroxymethylcytosine; 5hmC; DNA methylation; differentiation; cancer; tissue stem/progenitor cells
22.  Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines 
PLoS ONE  2011;6(6):e20874.
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.
doi:10.1371/journal.pone.0020874
PMCID: PMC3117837  PMID: 21698104
23.  Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences 
BMC Genomics  2011;12:313.
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.
doi:10.1186/1471-2164-12-313
PMCID: PMC3124442  PMID: 21669002
DNA methylation; prostate cancer; tiling microarray; epigenetics; methylated DNA binding domain; MBD-chip; ADAMTS1; SCARF2; DSCR9; HLCS
24.  Disulfiram Is a DNA Demethylating Agent and Inhibits Prostate Cancer Cell Growth 
The Prostate  2010;71(4):333-343.
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 (5meC) 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 5meC 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 5meC content, reactivate epigenetically silenced genes, and significantly inhibit growth in prostate cancer cell lines.
doi:10.1002/pros.21247
PMCID: PMC3043358  PMID: 20809552
DNA methyltransferase inhibitor; DNA methylation; Prostate cancer; Disulfiram
25.  Loss of Keap1 Function in Prostate Cancer Cells Causes Chemo- and Radio-resistance and Promotes Tumor Growth 
Loss-of-function mutations in the nuclear factor erythroid-2 related factor-2 (Nrf2) inhibitor, Kelch-like-ECH-associated protein (Keap1), result in increased Nrf2 activity in non–small-cell lung cancer (NSCLC) and confer therapeutic resistance. We detected point mutations in Keap1 gene leading to non-conservative amino acid substitutions in prostate cancer cells. We found novel transcriptional and post-transcriptional mechanisms of Keap1 inactivation such as promoter CpG island hypermethylation and aberrant splicing of Keap1 in DU-145 cells. Very low levels of Keap1 mRNA were detected in DU-145 cells, which significantly increased by treatment with DNA methyltransferase inhibitor 5-aza-cytidine. The loss of Keap1 function led to an enhanced activity of Nrf2 and its downstream electrophile/drug detoxification pathway. Inhibition of Nrf2 expression in DU-145 cells by RNAi attenuated the expression of glutathione, thioredoxin, and the drug efflux pathways involved in counteracting electrophiles, oxidative stress, and detoxification of a broad spectrum of drugs. DU-145 cells expressing Nrf2-shRNA had lower levels of total glutathione and higher levels of intracellular reactive oxygen species. Attenuation of Nrf2 function in DU-145 cells enhanced sensitivity to chemotherapeutic drugs and radiation-induced cell death. In addition, Inhibition of Nrf2 greatly suppressed in vitro and in vivo tumor growth of DU-145 prostate cancer cells. Thus, targeting Nrf2 pathway in prostate cancer cells may provide a novel strategy to enhance chemo- and radio-therapy responsiveness and ameliorate the growth and tumorigenecity leading to improved clinical outcomes.
doi:10.1158/1535-7163.MCT-09-0589
PMCID: PMC2821808  PMID: 20124447
Nrf2; Keap1; Prostate cancer; mutation; chemo-resistance; radio-resistance; RNAi

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