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
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
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.
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.
DNA methylation; Epigenetics; Microarray
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
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.
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.
By recapitulating human π-class GST expression, hGSTP1+mGstp1/2−/− mice may better model human drug and xenobiotic toxicology.
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.
Myc; EZH2; prostate cancer
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
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.
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.
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.
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.
DNA methylation; prostate cancer; tiling microarray; epigenetics; methylated DNA binding domain; MBD-chip; ADAMTS1; SCARF2; DSCR9; HLCS
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
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.
Nrf2; Keap1; Prostate cancer; mutation; chemo-resistance; radio-resistance; RNAi
Androgen Receptor (AR) mediated oncogenic pathways have not been fully elucidated. In this study we utilized high throughput microarray analysis on two AR-positive Prostate Cancer (CaP) cell lines to identify 16 AR-responsive microRNAs (miRNAs). We focused on miR-21 because of its previously reported oncogenic activity in other cancers. We demonstrate androgen-induced AR binding to the defined miR-21 promoter, miPPR-21, suggesting direct transcriptional regulation. Moreover, inhibition of miR-21 diminished androgen-induced CaP cell proliferation, providing new evidence that microRNAs can contribute to androgen-driven cell growth. Elevated expression of miR-21 enhanced CaP tumor growth in vivo and, surprisingly, was sufficient for androgen-dependent tumors to overcome castration-mediated growth arrest. Thus, elevated miR-21 expression alone is sufficient to impart castration resistance. Moreover, quantitative RT-PCR analysis revealed elevated miR-21 expression in CaP when compared to adjacent normal tissue. These results suggest that miR-21 may contribute to CaP pathogenesis.
Prostate cancer; microRNA; Androgen Receptor
Prostate cancer, the majority of which is adenocarcinoma, is the most common epithelial cancer affecting a majority of elderly men in Western nations. Its manifestation, however, varies from clinically asymptomatic insidious neoplasms that progress slowly and do not threaten life to one that is highly aggressive with a propensity for metastatic spread and lethality if not treated in time. A number of somatic genetic and epigenetic alterations occur in prostate cancer cells. Some of these changes, such as loss of the tumor suppressors PTEN and p53, are linked to disease progression. Others, such as ETS gene fusions, appear to be linked more with early phases of the disease, such as invasion. Alterations in chromosome 8q24 in the region of MYC have also been linked to disease aggressiveness for many years. However, a number of recent studies in human tissues have indicated that MYC appears to be activated at the earliest phases of prostate cancer (e.g., in tumor-initiating cells) in prostatic intraepithelial neoplasia, a key precursor lesion to invasive prostatic adenocarcinoma. The initiation and early progression of prostate cancer can be recapitulated in genetically engineered mouse models, permitting a richer understanding of the cause and effects of loss of tumor suppressors and activation of MYC. The combination of studies using human tissues and mouse models paints an emerging molecular picture of prostate cancer development and early progression. This picture reveals that MYC contributes to disease initiation and progression by stimulating an embryonic stem cell–like signature characterized by an enrichment of genes involved in ribosome biogenesis and by repressing differentiation. These insights pave the way to potential novel therapeutic concepts based on MYC biology.
prostate cancer; tumor initiating cells; prostatic intraepithelial neoplasia
prostatic; neoplasia; chromosome; aberration; clonal
Lo-MYC and Hi-MYC mice develop prostatic intraepithelial neoplasia (PIN) and prostatic adenocarcinoma as a result of MYC overexpression in the mouse prostate. However, prior studies have not determined precisely when, and in which cell types, MYC is induced. Using immunohistochemistry (IHC) to localize MYC expression in Lo-MYC transgenic mice, we show that morphological and molecular alterations characteristic of high grade PIN arise in luminal epithelial cells as soon as MYC overexpression is detected. These changes include increased nuclear and nucleolar size and large scale chromatin remodeling. Mouse PIN cells retained a columnar architecture and abundant cytoplasm and appeared as either a single layer of neoplastic cells or as pseudo-stratified/multilayered structures with open glandular lumina—features highly analogous to human high grade PIN. Also using IHC, we show that the onset of MYC overexpression and PIN development coincided precisely with decreased expression of the homeodomain transcription factor and tumor suppressor, Nkx3.1. Virtually all normal appearing prostate luminal cells expressed high levels of Nkx3.1, but all cells expressing MYC in PIN lesions showed marked reductions in Nkx3.1, implicating MYC as a key factor that represses Nkx3.1 in PIN lesions. To determine the effects of less pronounced overexpression of MYC we generated a new line of mice expressing MYC in the prostate under the transcriptional control of the mouse Nkx3.1 control region. These “Super-Lo-MYC” mice also developed PIN, albeit a less aggressive form. We also identified a histologically defined intermediate step in the progression of mouse PIN into invasive adenocarcinoma. These lesions are characterized by a loss of cell polarity, multi-layering, and cribriform formation, and by a “paradoxical” increase in Nkx3.1 protein. Similar histopathological changes occurred in Hi-MYC mice, albeit with accelerated kinetics. Our results using IHC provide novel insights that support the contention that MYC overexpression is sufficient to transform prostate luminal epithelial cells into PIN cells in vivo. We also identified a novel histopathologically identifiable intermediate step prior to invasion that should facilitate studies of molecular pathway alterations occurring during early progression of prostatic adenocarcinomas.
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 5meC 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 while some LINE1 promoter hypomethylation does occur in primary prostate cancers compared to 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 over-expressed 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 over-expression 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 , and occur heterogeneously during prostate cancer progression and metastatic dissemination.
DNA methylation; hypomethylation; prostate cancer; metastasis; epigenetics; LINE1; tumor heterogeneity; cancer testis antigens
Demethylation of 5-methylcytosine in genomic DNA is believed to be one of the mechanisms underlying replicative life-span of mammalian cells. Both proliferation associated SNF2-like gene (PASG, also termed Lsh) and DNA methyltransferase 3B (Dnmt3b) knockout mice result in embryonic genomic hypomethylation and a replicative senescent phenotype. However, it is unclear whether gradual demethylation of DNA during somatic cell division is directly involved in senescence. In this study, we retrovirally transduced TIG-7 human fibroblasts with a shRNA against PASG and compared the rate of change in DNA methylation as well as the replicative life-span to control cells under low (3%) and ambient (20%) oxygen. Expression of PASG protein was decreased by approximately 80% compared to control cells following transduction of PASG shRNA gene. The rate of cell growth was the same in both control and PASG-suppressed cells. The rate of demethylation of DNA was significantly increased in PASG-suppressed cells as compared control cells. However, decreased PASG expression did not shorten the replicative life-span of TIG-7 cells. Culture under low oxygen extended the life-span of TIG-7 cells but did not alter the rate of DNA demethylation. While knockout of PASG during development results in genomic hypomethylation and premature senescence, our results show that while downregulation of PASG expression in a somatic cell also leads to DNA hypomethylation, there is no associated senescent phenotype. These results suggest differences in cellular consequences of hypomethylation mediated by PASG during development compared to that in somatic cells.
epigenetics; DNA methylation; PASG; LSH; senescence
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-β and ESR1. The results of hypermethylation at GSTP1 were included from a previous study.
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-β 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.
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.
prostate; prostatic neoplasms; tumor markers; biological; glutathione S-transferase pi; neoplasm recurrence; local
CpG island hypermethylation occurs in most cases of cancer, typically resulting in the transcriptional silencing of critical cancer genes. Procainamide has been shown to inhibit DNA methyltransferase activity and reactivate silenced gene expression in cancer cells by reversing CpG island hypermethylation. We report here that procainamide specifically inhibits the hemimethylase activity of DNA methyltransferase 1 (DNMT1), the mammalian enzyme thought to be responsible for maintaining DNA methylation patterns during replication. At micromolar concentrations, procainamide was found to be a partial competitive inhibitor of DNMT1, reducing the affinity of the enzyme for its two substrates, hemimethylated DNA and S-adenosyl-l-methionine. By doing so, procainamide significantly decreased the processivity of DNMT1 on hemimethylated DNA. Procainamide was not a potent inhibitor of the de novo methyltransferases DNMT3a and DNMT3b2. As further evidence of the specificity of procainamide for DNMT1, procainamide failed to lower genomic 5-methyl-2′-deoxycytidine levels in HCT116 colorectal cancer cells when DNMT1 was genetically deleted but significantly reduced genomic 5-methyl-2′-deoxycyti-dine content in parental HCT116 cells and in HCT116 cells where DNMT3b was genetically deleted. Because many reports have strongly linked DNMT1 with epigenetic alterations in carcinogenesis, procainamide may be a useful drug in the prevention of cancer.
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.