The Translational Research Working Group (TRWG) was created as a national initiative to evaluate the current status of the National Cancer Institute’s investment in translational research and envision its future. The TRWG conceptualized translational research as a set of six developmental processes or pathways focused on various clinical goals. One of those pathways describes the development of agents—both small molecules and biologics—for the treatment and prevention of cancer. The Agents Developmental Pathway was conceived not as a comprehensive description of the corresponding real-world processes, but rather as a tool designed to facilitate movement of an agent through the translational process to the point where it can begin definitive clinical testing. This article presents the Agents Developmental Pathway and discusses key challenges associated with the processes described.
Contrary to the hypothesis that the racial/ethnic disparity in prostate cancer has a hormonal basis, we did not observe a difference in serum testosterone concentration between non-Hispanic black and white men in the Third National Health and Nutrition Examination Survey (NHANES III), although non-Hispanic black men had a higher estradiol level. Unexpectedly, Mexican-American men had the highest testosterone level. Next, we evaluated whether the same patterns are observed during adolescence, the time of prostate maturation.
We measured serum testosterone, estradiol, and sex hormone binding globulin (SHBG) by immunoassay in 134 males aged 12–19 in NHANES III. Mean concentrations were compared by race/ethnicity adjusting for age, Tanner stage, percent body fat, waist, physical activity, tobacco smoke, and the other hormones.
After multivariable adjustment, in the 12–15 year-old males, testosterone concentration was lower in non-Hispanic blacks than whites (P=0.043), SHBG concentration did not significantly differ between the two groups. Mexican-Americans had the highest testosterone (versus non-Hispanic black: P=0.002) and lowest SHBG (versus non-Hispanic white: P=0.010; versus non-Hispanic black: P=0.047) concentrations. Estradiol concentration was lower in non-Hispanic blacks (P=0.11) and Mexican-Americans (P=0.033) compared with non-Hispanic whites. After multivariable adjustment, in the 16–19 year-old males, testosterone, estradiol, and SHBG concentrations did not differ between non-Hispanic blacks and whites. Mexican-Americans had the highest testosterone concentration (versus non-Hispanic white: P=0.08), but did not differ from the other groups on estradiol and SHBG concentrations. In both age groups, these patterns were generally present, but less pronounced after adjusting for age and Tanner stage only.
In adolescent males, non-Hispanic blacks did not have a higher testosterone concentration than non-Hispanic whites, and Mexican-Americans had the highest testosterone concentration, patterns similar to adult males.
testosterone; adolescence; race and ethnicity
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.
Obesity is associated with a variety of chronic diseases, including cancer, which may partly be explained by its influence on sex steroid hormone concentrations. Whether different measures of obesity, i.e., body mass index (BMI), waist circumference, and percent body fat were differentially associated with circulating levels of sex steroid hormones was examined in 1,265 men, aged 20 to 90+ years, attending the morning examination session of the Third National Health and Nutrition Examination Survey (NHANES III).
Methods and Methods
Serum hormones were measured by immunoassay. Weight, height, and waist circumference were measured by trained staff. Percent body fat was estimated from bioelectrical impedance. Multivariate linear regression was used to estimate associations between body fatness measures and hormone levels.
Total and free testosterone and sex hormone binding globulin concentrations decreased, whereas total and free estradiol increased with increasing BMI, waist circumference, and percent body fat (all P-trend <0.05). The magnitude of change in these hormones was similar for a one quartile increase in each body fatness measure.
Measured BMI, waist circumference, and percent body fat led to similar inferences about their association with hormone levels in men.
NHANES III; testosterone; estradiol; obesity
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.
Low or high hematocrit levels are associated with increased morbidity and mortality, mediated via anemia or thromboembolic events, respectively. It is therefore important to identify factors that influence hematocrit. Although androgens are known to stimulate hematopoietic cells, it is unknown whether circulating sex steroid hormones affect hematocrit. The association between serum sex steroid hormone concentrations and hematocrit in men aged ≥20 years was evaluated in a cross-sectional study of 1273 men in the Third National Health and Nutrition Examination Survey (1988–1991). Outcomes were low (<10th percentile), high (>90th percentile), and mean hematocrit. Men with low free testosterone levels had a lower hematocrit than men with normal free testosterone levels (P = .03), although no relationship was found between total testosterone level and hematocrit. The relationship between sex hormone–binding globulin (SHBG) and hematocrit was complex, with both low (P < .001) and high (P = .01) SHBG levels associated with lower hematocrit in men aged ≥20 years and only high (P = .01) SHBG levels in men aged ≥50 years. The odds ratio (OR) of high vs normal hematocrit increased as total estradiol (OR, 2.84; P trend = .04) and free estradiol (OR, 2.23; P trend = .09) levels increased. In this nationally representative study of men, sex steroid hormone levels, particularly low free testosterone and high SHBG levels, were associated with lower hematocrit, and high total and free estradiol levels were associated with high hematocrit. Thus, changes in sex hormone levels with aging may contribute to the increased prevalence of anemia and thromboembolic stroke in men as they age.
Testosterone; estradiol; sex hormone–binding globulin; NHANES III
To investigate serologic evidence of infection by cytomegalovirus (CMV), a herpesvirus with known oncogenic potential that has been detected in malignant prostate tissue, in relation to prostate cancer (PCa) risk in a large case-control study nested in the Prostate Cancer Prevention Trial (PCPT).
Cases were men with a confirmed diagnosis of PCa after visit 2 (n=614) and controls were men not diagnosed with PCa during the trial who also had a negative end-of-study biopsy (n=616). Controls were frequency-matched to cases by age, treatment arm, and family history of PCa. Sera from visit 2 were tested for CMV IgG antibodies.
No association was observed between CMV serostatus and PCa risk (adjusted CMV seroprevalence=67.9% for cases and 65.2% for controls, odds ratio=1.13, 95% confidence interval: 0.89–1.45).
Considering our null findings in the context of the full CMV literature, CMV infection, as measured by serostatus, does not appear to increase PCa risk.
Cytomegalovirus; infection; prostate cancer; epidemiology
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.
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.
chronic myeloid leukemia; CML; leukemic stem cell; LSC; normal hematopoietic stem cell; HSC; myeloid progenitor cells; CD34; CD38; ALDH; IL2RA; CD25; DPP4; CD26; GAS2
About 20% of all human cancers are caused by chronic infection or chronic inflammatory states. Recently, a new hypothesis has been proposed for prostate carcinogenesis. It proposes that exposure to environmental factors such as infectious agents and dietary carcinogens, and hormonal imbalances lead to injury of the prostate and to the development of chronic inflammation and regenerative ‘risk factor’ lesions, referred to as proliferative inflammatory atrophy (PIA). By developing new experimental animal models coupled with classical epidemiological studies, genetic epidemiological studies and molecular pathological approaches, we should be able to determine whether prostate cancer is driven by inflammation, and if so, to develop new strategies to prevent the disease.
Vitamin E may protect against prostate cancer, possibly only in smokers and, we hypothesize, through altered sex steroid hormones. A controlled trial in smokers showed that sex hormone levels were inversely associated with baseline serum α-tocopherol and decreased in response to vitamin E supplementation. The vitamin E-hormone relation is understudied in non-smokers.
Serum sex steroid hormones and α-tocopherol were measured for 1,457 men in NHANES III. Multivariable-adjusted geometric mean hormone concentrations by α-tocopherol quintile were estimated.
We observed lower mean testosterone, estradiol, and SHBG concentrations with increasing serum α-tocopherol (Q1=5.5 and Q5=4.6 ng/mL, p-trend=0.0007; Q1=37.8 and Q5=33.1 pg/mL, p-trend=0.02; Q1=38.8 and Q5=30.6 pg/mL, p-trend=0.05, respectively). Interactions between serum α-tocopherol and exposure to cigarette smoke for total testosterone, total estradiol, and SHBG were found with the inverse relation observed only among smokers.
Results from this nationally representative, cross-sectional study indicate an inverse association between serum α-tocopherol and circulating testosterone, estradiol, and SHBG, but only in men who smoked. Our findings support vitamin E selectively influencing sex hormones in smokers, and afford possible mechanisms through which vitamin E may impact prostate cancer risk.
Gonadal Steroid Hormones; alpha-Tocopherol; Smoking; Prostatic Neoplasms; Cross-Sectional Studies
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.
epigenetics; rDNA; rRNA; DNA methylation; prostate cancer
Physiologic processes during aging leading to multi-morbidity and diseases that increase risk of premature death may be influenced by aging-associated changes in endogenous hormone production.
To evaluate the decline in sex steroid hormone levels across age and estimate the number of US men 40+ years old who may have low hormone levels.
We measured serum testosterone, estradiol, and sex hormone binding globulin by immunoassay in 1,351 men 20+ years old in NHANES III. We estimated free hormones by mass action.
Free testosterone declined most rapidly with age (a 2% decline in geometric mean concentration occurred after aging 1.3 years), followed by total testosterone (2.4 years), free estradiol (4.1 years), and total estradiol (8.1 years). These hormone changes with age translated into 25.0% and 30.2% of men 70+ years old having low total (which we defined as <10.4 nmol/L) and free (<0.17 nmol/L) testosterone, respectively, and 8.3% and 23.9% having low total (<73.4 pmol/L) and free (<2.2 pmol/L) estradiol. Using population size projections between the 2000 and 2010 Censuses, we estimated that 8.4 (95% CI 4.7-12.2), 6.2 (3.1-9.2), and 6.0 (3.1-9.0) million 40+ year old men may have low total testosterone, free testosterone, and free estradiol, respectively. The prevalences were only modestly lower in men without prevalent chronic diseases.
Although no consensus exists for defining low hormone levels in aging men, a substantial number of US men may have low sex steroid hormone levels, possibly putting them at risk for adverse health consequences and pre-mature death.
NHANES III; testosterone; men; aging
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.
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.
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.
Digoxin; prostate cancer; risk; cohort; transdisciplinary; cytotoxicity
Histone deacetylases (HDAC) play a critical role in chromatin modification and gene expression. Recent evidence indicates that HDACs can also regulate functions of nonhistone proteins by catalyzing the removal of acetylated lysine residues. Here, we show that the HDAC inhibitor LBH589 down-regulates DNA methyltransferase 1 (DNMT1) protein expression in the nucleus of human breast cancer cells. Cotreatment with the proteasomal inhibitor MG-132 abolishes the ability of LBH589 to reduce DNMT1, suggesting that the proteasomal pathway mediates DNMT1 degradation on HDAC inhibition. Deletion of the NH2-terminal 120 amino acids of DNMT1 diminishes LBH589-induced ubiquitination, indicating that this domain is essential for its proteasomal degradation. DNMT1 recruits the molecular chaperone heat shock protein 90 (Hsp90) to form a chaperone complex. Treatment with LBH589 induces hyperacetylation of Hsp90, thereby inhibiting the association of DNMT1 with Hsp90 and promoting ubiquitination of DNMT1. In addition, inactivation of HDAC1 activity by small interfering RNA and MS-275 is associated with Hsp90 acetylation in conjunction with reduction of DNMT1 protein expression. We conclude that the stability of DNMT1 is maintained in part through its association with Hsp90. Disruption of Hsp90 function by HDAC inhibition is a unique mechanism that mediates the ubiquitin-proteasome pathway for DNMT1 degradation. Our studies suggest a new role for HDAC1 and identify a novel mechanism of action for the HDAC inhibitors as down-regulators of DNMT1.
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.
Prostatic adenocarcinoma; molecular pathology; hypermethylation; GSTP1; PCA3; urine
Small cell carcinoma of the prostate is an uncommon neoplasm, the origin of which has been controversial. To address this, we performed transcriptome profiling and TP53 sequencing of concurrent small cell and prostatic adenocarcinoma to determine the relationship between these entities.
We identified an unusual case of primary prostate cancer that contained adjacent acinar adenocarcinoma (Gleason score 4+3=7) and small cell carcinoma. We performed laser capture microdissection to isolate tumor components and performed gene expression and TP53 gene sequence analysis on each component, with results validated by immunohistochemistry for PSA, PSAP, PSMA, androgen receptor, NKX 3.1 and neuroendocrine markers.
Transcriptome profiling of the carcinoma components identified 99 genes with a greater than 10-fold differential expression between prostatic adenocarcinoma and small cell carcinoma, many of which have not been previously reported in prostate cancer. The small cell carcinoma component demonstrated upregulation of proliferative and neuroendocrine markers and tyrosine kinase receptors, and downregulation of cell adhesion molecules, supporting the aggressive nature of this form of carcinoma. Sequencing of the TP53 gene suggested a common clonal origin for both components.
This is the first report of a primary small cell carcinoma of the prostate subjected to extensive molecular analysis and the first to show a clonal relation between two morphologically distinct prostate cancer types. The evidence of progression to small cell carcinoma may yield important insights into the pathogenesis of this entity and provide a novel spectrum of molecular markers to further dissect cellular pathways important in tumor progression.
carcinoma; small cell; prostate; genes; p53; DNA sequence
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.
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.
5-hydroxymethylcytosine; 5hmC; DNA methylation; differentiation; cancer; tissue stem/progenitor cells
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.
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.
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.
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.
DNA methyltransferase inhibitor; DNA methylation; Prostate cancer; Disulfiram