© 2005 Oxford University Press
REVIEW |
Epigenetic Changes in Prostate Cancer: Implication for Diagnosis and Treatment
Affiliations of authors: Department of Urology, Veterans Affairs Medical Center, and University of California San Francisco, San Francisco
Correspondence to: Rajvir Dahiya, PhD, Urology Research Center (112F), Veterans Affairs Medical Center and UCSF, 4150 Clement St., San Francisco, CA 94121 (e-mail: rdahiya{at}urol.ucsf.edu)
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ABSTRACT |
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 TopNotes Abstract IntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer death among men in the United States. DNA methylation and histone modifications are important epigenetic mechanisms of gene regulation and play essential roles both independently and cooperatively in tumor initiation and progression. Aberrant epigenetic events such as DNA hypo- and hypermethylation and altered histone acetylation have both been observed in prostate cancer, in which they affect a large number of genes. Although the list of aberrantly epigenetically regulated genes continues to grow, only a few genes have, so far, given promising results as potential tumor biomarkers for early diagnosis and risk assessment of prostate cancer. Thus, large-scale screening of aberrant epigenetic events such as DNA hypermethylation is needed to identify prostate cancer–specific epigenetic fingerprints. The reversibility of epigenetic aberrations has made them attractive targets for cancer treatment with modulators that demethylate DNA and inhibit histone deacetylases, leading to reactivation of silenced genes. More studies into the mechanism and consequence of demethylation are required before the cancer epigenome can be safely manipulated with therapeutics as a treatment modality. In this review, we examine the current literature on epigenetic changes in prostate cancer and discuss the clinical potential of cancer epigenetics for the diagnosis and treatment of this disease.
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INTRODUCTION |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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Prostate cancer is a common malignancy and a leading cause of cancer death among men in the United States. The molecular mechanisms underlying its development and progression remain poorly understood. Genetic alterations, such as mutations, and epigenetic changes, defined as heritable changes in gene expression that occur without changes in DNA sequence (1), appear to contribute to the malignant transformation and progression of prostate cancer.
One type of epigenetic aberration is DNA methylation—the addition of a methyl group to the 5'-carbon of cytosine in CpG sequences—catalyzed by DNA methyltransferases (DNMTs). Methylcytosine residues are often found in short stretches of CpG-rich regions (i.e., CpG islands) that are 0.5–2 kb long and found in the 5' region of approximately 60% of genes (2). Most CpG islands are unmethylated, with the exception of certain imprinted genes and genes on the inactive X chromosomes of females (3). DNA methylation aberrations can occur as either hypo- or hypermethylation. Both forms can lead to chromosomal instability and transcriptional gene silencing, and both have been implicated in a variety of human malignancies, including prostate cancer (4).
A second type of epigenetic aberration involves chromatin structure. The basic chromatin unit is the nucleosome (5). The N-terminal tails of histones, positioned peripheral to the nucleosome core, are subject to various covalent modifications, such as acetylation, methylation, phosphorylation, and ubiquitination by specific chromatin-modifying enzymes (6). The pattern of these modifications has been referred to as "the histone code," and it acts as a second layer of epigenetic regulation of gene expression affecting chromatin structure and remodeling (7). Acetylation and deacetylation of histone tails are catalyzed by histone acetyltransferases and deacetylases (HDACs), respectively (8). Histone acetyltransferases have been shown to increase the activity of several transcription factors, including nuclear hormone receptors, by inducing histone acetylation, which facilitates promoter access to the transcriptional machinery (9). Conversely, HDACs reduce levels of histone acetylation and are associated with transcriptional repression.
These two epigenetic regulatory mechanisms, DNA methylation and histone modifications, are closely related. Successful epigenetic control of gene expression often requires the cooperation and interaction of both mechanisms, and disruption of either of those processes leads to aberrant gene expression seen in almost all human cancers. Here, we review current knowledge of the epigenetic changes in prostate cancer regarding DNA methylation and histone modifications and discuss the implication for understanding the molecular basis, clinical diagnosis, and treatment of this disease.
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DNA METHYLATION IN PROSTATE CANCER |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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Hypermethylation
DNA hypermethylation is the most common and best characterized epigenetic abnormality in human malignancies, including prostate cancer. By searching the Medline database using the query "prostate AND methylation" (10), 242 records were returned as of June 20, 2004, of which 52 papers reported more than 30 genes that undergo aberrant hypermethylation in prostate cancer (Table 1). These genes include classic and putative tumor-suppressor genes and genes involved in a number of cellular pathways such as hormonal responses, tumor-cell invasion and/or tumor architecture, cell cycle control, and DNA damage repair. For many of these genes, promoter hypermethylation is often the main mechanism underlying their functional loss in prostate cancer. Inappropriate silencing of these genes can contribute to cancer initiation, progression, invasion, and metastasis. Hypermethylation of these genes in prostate cancer can correlate with pathologic grade or clinical stage and with androgen independence. To assess the overall prevalence of hypermethylation of these genes in prostate cancer, we reviewed available data from the 52 publications and summarized the clinical results in Figure 1. Some frequently hypermethylated genes, such as those involved in hormone responses, cell cycle regulation, cell invasion and architecture, and tumor suppression constitute a potential prostate cancer–specific methylation signature and are discussed below.
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Hormonal Response Genes
The prostate is an endocrine gland that responds to sex hormones such as androgens, estrogens, and progesterones through their specific receptors. Epigenetic modifications such as DNA methylation and histone acetylation participate in the transcriptional regulation of steroid and thyroid nuclear receptors (11,12).
The androgen receptor (AR) mediates androgen activity, which is essential for the development of both the normal prostate and prostate cancer. Prostate cancer is initially androgen dependent, but can eventually become androgen independent after androgen deprivation therapy. Androgen-independent prostate cancers are characterized by a heterogeneous loss of AR expression (13–15). Jarrard et al. (13) first reported aberrant promoter methylation in AR-negative prostate cancer cell lines. Consistent with these results, Izbicka et al. (16) showed that 5,6-dihydro-5'-azacytidine, an inhibitor of cytosine DNA methyltransferase, could restore androgen sensitivity in androgen-insensitive human prostate carcinoma cell lines, which then become sensitive to growth inhibition by antiandrogens. Furthermore, the incidence of methylation-mediated AR inactivation in prostate cancer tissue ranged from 0% to 20% in primary prostate cancers (11,17–19) and from 13% to 28% in androgen-independent cancers (17,18).
Genetic alterations, including AR gene mutation (20) and amplification (21) without loss of AR expression, that alter the sensitivity of the AR to androgen are thought to play a key role in the development of androgen-independent advanced prostate cancer. However, as many as 20%–30% of androgen-independent cancers do not express AR (22). The loss of AR expression in this subgroup is not associated with AR amplification. AR promoter methylation is more prevalent in androgen-independent prostate cancer than in primary androgen-dependent prostate cancer (17,18), suggesting that epigenetic silencing of AR by DNA hypermethylation could be an alternative mechanism leading to androgen independence in a subset of patients with advanced prostate cancer.
Although estrogens have been historically used for the treatment of prostate cancer, their function in the prostate remains unclear (23). The action of estrogens was thought to be mediated via a blockade of the pituitary-testicular axis (24). However, estrogens have been shown to exert direct effects on prostatic cancer cells via their own receptors (25,26).
The prostate expresses two types of estrogen receptors (ERs): ER
(ESR1) and ER
(ESR2) (27). Lost or decreased expression of ESR1 and ESR2 in prostate cancer has been documented (28–31). Low ESR1 expression is also associated with poor prognosis for effective endocrine therapy using estrogens (32). The ESR1 gene is frequently methylated in prostate cancer, and the methylation status is associated with tumor progression (33). The ESR2 promoter contains a typical CpG island (34). Subsequent studies from our laboratory (11,35) and others (36–38) support the concept that hypermethylation is the main known mechanism responsible for inactivation of ER expression in prostate cancer. It is interesting to note that metastatic prostate cancer cells may regain ESR2 expression (27), which is accompanied by loss of ESR2 promoter methylation (37). This observation provides further evidence that ESR2 inactivation in primary prostate cancer is epigenetic by nature and thus reversible.
Cell Cycle Control Genes
An important characteristic of tumor cells is their increased proliferative ability, which is often associated with impaired regulation of the cell cycle. The cell cycle has multiple checkpoints that are controlled by a number of complex modulation systems, including the retinoblastoma (RB1) protein, cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CDKIs) (39). RB1 was the first tumor suppressor gene to be identified, and its alteration has been observed in many tumor types (40). RB1 inactivation resulting from promoter methylation is a common event in retinoblastomas (41,42) but a rare event in prostate cancer (43). RB1 inactivation in prostate cancer is apparently the result of loss of heterozygosity and mutation (43,44).
CDKIs are negative regulators of cell cycle progression and thus are considered to be potential tumor suppressor genes. Currently, CDKIs are grouped into two families: the INK4 family, which includes CDKN2A (p16), CDKN2B (p15), CDKN2C (p18), and CDKN2D (p19), specifically inhibits cyclin D–associated kinases (CDKs 4 and 6); and the CIP/KIP (kinase inhibitor protein) family, which includes CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57), inhibits most CDKs (45). Failure of cell cycle arrest secondary to alterations in CDKI expression has been implicated in prostate cancer (46,47). CDKN2A is inactivated by a variety of mechanisms, including deletion, point mutation, and silencing by hypermethylation in a number of cancers, including the prostate (48–50). Methylation-mediated inactivation of the CDKN2A gene has been reported in prostate cancer cell lines (51,52) and prostate cancer tissue, with incidence of inactivation ranging from 0% to 16% (52–56). Methylation at exon 2 of the CDKN2A gene is more frequent in prostate cancer tissue (66%) than methylation of the promoter region (56); however, exon 2 methylation does not affect gene expression (55,56) making the functional relevance of this epigenetic event unclear. However, because CDKN2A exon 2 is hypermethylated only in cancer tissues, it may serve as a biomarker to detect or confirm a prostate neoplasm. Inactivation of other cell cycle genes such as CDKN2B, CDKN1A, and CDKN1B by hypermethylation is rare in prostate cancer (56,57).
Cell Invasion and Cell Architecture Genes
The cadherin–catenin adhesion system is critical to the preservation of normal tissue architecture and is regulated by a family of proteins collectively termed cell adhesion molecules. Decreased expression of E-cadherin (CDH1) and other cell adhesion molecules has been reported to have prognostic significance in various human cancers, including prostate cancer (58,59).
In human prostate tumors, expression of CDH1 is strongly reduced and its promoter is methylated to varying degrees (54,60–62). The 5' CpG island of CDH1 is densely methylated in prostate cancer cell lines (63). A study of prostate cancer tissue samples from our own laboratory showed that methylation of the CDH1 promoter, as detected by bisulfite genomic sequencing of 29 CpG sites within the promoter and first exon, occurs in 30% of low-grade and 70% of high-grade prostate cancer samples and is associated with absent or reduced CDH1 protein expression, as detected by immunohistochemical analysis (60). Consistent with our data, Kallakury et al. (61) reported an 80% prevalence of CDH1 methylation in prostate cancer samples analyzed by methylation-specific polymerase chain reaction (PCR). In addition, methylation of the CDH1 promoter is increased in advanced prostate tumors, suggesting that it might be a useful biomarker to assess tumor progression (60).
There are, however, some discrepancies regarding the prevalence of CDH1 methylation in prostate cancer. Woodson et al. (64) reported that methylation of the CDH1 promoter (-159 to -51 region) could not be detected in any of 101 prostate cancer samples using real-time PCR. However, the same group (62) later reported a 22.4% prevalence of CDH1 methylation in prostate cancer using the same assay method but covering a different region (+59 to +140). Thus, different methodologies (methylation-specific polymerase chain reaction versus bisulfite genomic sequencing) and different genomic regions (promoter versus exon) examined may contribute to the observed discrepancies.
CD44 is an integral membrane protein that is involved in matrix adhesion and signal transduction. In prostate cancer, loss of CD44 protein expression is associated with methylation of its gene promoter (64–69). CD44 expression and promoter methylation are associated with prostate cancer stage and patient prognosis (68,70). Other genes involved in the cadherin–catenin adhesion system have also shown methylation-mediated inactivation in prostate cancer, including H-cadherin (CDH13) (71), adenomatous polyposis coli (APC) (71), caveolin-1 (CAV1) (72), laminin 3 (LAMA3), laminin 3 (LAMB3), and laminin 
2 (LAMC2) (73) (Table 1).
DNA Damage Repair Genes
DNA repair is a correcting mechanism that maintains genome integrity during replication or after DNA damage. Cells defective in components of DNA repair pathways exhibit higher rates of spontaneous DNA mutations, which can lead to cancer (74). Hypermethylation of two genes involved in DNA damage repair, the detoxifier gene glutathione S-transferase Pi (GSTP1) and the DNA alkyl-repair gene O6-methylguanine DNA methyltransferase (MGMT), has been reported in prostate cancer. The glutathione S-transferase Pi gene belongs to a supergene family of glutathione S-transferases (GSTs) that play an important role in the detoxification of electrophilic compounds (such as carcinogens and cytotoxic drugs) by glutathione conjugation (75). GSTP1 inactivation may lead to increased cell vulnerability to oxidative DNA damage and to the accumulation of DNA base adducts, which can precede other relevant genetic alterations in carcinogenesis (76).
In prostate cancer, methylation of the GSTP1 gene promoter is the most frequently detected epigenetic alteration, with a frequency ranging from 70% to 100% in prostate cancer DNA specimens (77–79). Notably, GSTP1 methylation is also detected in 50%–70% of prostatic intraepithelial neoplasia (80,81), a precursor lesion of prostate cancer (82). Hypermethylation of the GSTP1 gene has also been detected in nonmalignant prostate tissue, but at a much lower level and frequency than in malignant tissue (19,81,83). The cancer–specific hypermethylation of the GSTP1 gene in prostate cancer cell lines and tissues provides a good model for the study of molecular mechanisms underlying methylation-mediated gene silencing and may also serve as a potential tumor biomarker for clinical detection of prostate cancer.
MGMT is a DNA repair protein that removes mutagenic and cytotoxic alkyl adducts from genomic DNA. Tumors that lack MGMT expression have a higher incidence of point mutations in the genes encoding p53 and K-ras, which may influence cancer progression (84). In addition, MGMT-deficient tumors exhibit high sensitivity to the effects of chemotherapeutic alkylating agents. Results from studies evaluating MGMT promoter methylation in prostate cancer have been equivocal, with moderate to high levels of methylation detected in some studies (54,56) but not others (19,71). Further work will be necessary to resolve this discrepancy.
Putative Tumor Suppressor Genes
Functional losses of classic tumor suppressor genes, such as the retinoblastoma-1 gene (85), the mismatch repair gene MLH1 (86), and the von Hippel–Lindau gene (87), through DNA hypermethylation are rare in prostate cancer, although common in other types of cancer. However, some putative tumor suppressor genes have been reported to be silenced by DNA hypermethylation in prostate cancer, most notably, the Ras association domain family 1 gene (RASSF1A).
RASSF1A, located at 3p21.3, encodes a protein similar to the RAS effector proteins. The biologic activity of RASSF1 is largely unknown. Both in vitro and in vivo studies show that overexpression of RASSF1 in cancer cells leads to cell cycle arrest (88), reduced colony formation, and inhibition of tumor growth in nude mice (89). Thus, a tumor suppressor role has been proposed for RASSF1 (90,91). In prostate cancer, RASSF1 promoter methylation is a common event, occurring in 54%–96% of tumor samples (54,57,62,71,90,91). RASSF1 promoter methylation is detected in some nonmalignant prostate tissue samples (71) but not in many others (54,57,62). A large percentage (64%) of prostatic intraepithelial neoplasia samples exhibit RASSF1 promoter hypermethylation (54). Increased RASSFI promoter methylation is also associated with advanced tumors (i.e., those with high Gleason scores) (54,71,90). These findings indicate that RASSF1 promoter methylation occurs in early prostate cancer development, increases as the cancer progresses, and is a potential tumor biomarker for prostate cancer diagnosis and risk assessment.
Additional genes with putative tumor suppressor function undergoing epigenetic inactivation by hypermethylation in prostate cancer include KAI1 (a prostate-specific tumor metastasis suppressor gene) (92), inhibin- (a member of the transforming growth factor– family of growth and differentiation factors) (93), and DAB2IP, a novel GTPase-activating protein for modulating the Ras-mediated signal pathway (94). It is unknown, however, whether hypermethylation of these genes plays a role in prostate carcinogenesis or has a role as a biomarker for prostate cancer diagnosis.
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HYPOMETHYLATION |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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DNA methylation in mammalian genomes is a defense mechanism by which repetitive DNA (which accounts for at least 50% of genome’s content) is transcriptionally silenced to prevent it from propagating (95). Demethylation of normally methylated DNA, also known as hypomethylation, can disrupt such a defense mechanism, leading to structural and functional alterations of the genome.
There are two types of hypomethylation: global or genomic hypomethylation, which refers to an overall decrease of 5-methylcytosine content in the genome; and localized or gene-specific hypomethylation, which refers to a decrease in cytosine methylation relative to the "normal" methylation level. The latter process affects specific regions of the genome, such as the promoter regions of proto-oncogenes or normally highly methylated sequences such as repetitive sequences and oncogenes (96). Both global hypomethylation and gene-specific hypomethylation have been implicated in human cancer.
Global Hypomethylation
Net decreases in the content of methylcytosines in cancer often exceed the localized increases in DNA methylation associated with carcinogenesis (97,98). For example, in colon adenocarcinomas, the genomic 5-methylcytosine content is reduced by an average of 10% (99). Global DNA hypomethylation has also been found in the premalignant or early stages of some neoplasms (99,100) and is implicated as an important factor for tumor progression (77,101). It is unclear whether this epigenetic alteration is a cause or consequence of tumorigenesis. To add to the complexity, hypomethylation induced by disrupting DNMT1 has been found to either inhibit (102) or promote (103) tumor growth. In a murine model of intestinal neoplasia, mice carrying a germ-line mutation in the APC gene (APCMin/+) crossed with mice heterozygous for the DNMT1 mutation had substantially fewer tumors than Min mice with wild-type DNMT1 (102,104). By contrast, genomic hypomethylation has been associated with the induction of T-cell lymphomas in mice carrying a hypomorphic DNMT1 allele, which reduces DNMT1 expression to 10% of wild-type levels and results in substantial genome-wide hypomethylation in all tissues. Whether hypomethylation promotes or inhibits tumor progression might be related to differences in model systems or tissue specificity (105).
The initial findings regarding DNA methylation in the prostate came from studies by Bedford and van Helden (106) more than a decade ago. They observed that the overall 5-methylcytosine content in DNA from prostates with benign prostatic hyperplasia and metastatic tumors was significantly lower than that in DNA from nonmetastatic prostate tumors. Further studies found that global hypomethylation is associated with clinical stage (77) and metastatic state (107) of prostate cancer.
Gene-Specific Hypomethylation
Genes from cancer cells but not from normal cells are substantially hypomethylated (108). Moreover, compared with adjacent normal tissues, cancer tissues contain two hypomethylated ras oncogenes, c-Ha-ras and c-Ki-ras (109). Other examples of hypomethylated genes include the c-jun and c-MYC proto-oncogenes in liver cancer (110,111) and the pS2 gene in breast cancer (112).
Hypomethylation of a locus transcriptionally controlled by methylation may enhance transcription of associated genes (110). In the prostate, the PLAU gene is highly expressed in most prostate cancer tissues (113) and invasive prostate cancer cell lines (114). The PLAU gene encodes urokinase plasminogen activator, a multifunctional protein that can promote tumor invasion and metastasis in several malignancies including prostate cancer. Helenius et al. (114) have attributed PLAU overexpression to gene amplification, as evidenced by the two- to threefold increase in the number of copies of the gene in PC3 cells. DNA methylation may also play a role in the regulation of the PLAU gene in prostate cancer (115), with hypomethylation of the PLAU promoter being associated with its increased expression in hormone-independent prostate cancer cells, higher invasive capacity in vitro, and increased tumorigenesis in vivo (115). However, in normal prostate epithelial cells and in hormone-dependent LNCaP cells, the PLAU promoter is methylated, resulting in lower expression of the gene (115).
Other hypomethylated genes in prostate cancer include CAGE, a novel cancer/testis antigen gene (116), and heparanase. Hypomethylation of CAGE, which occurs at a frequency of approximately 40% in prostate cancers, is responsible for its exclusive expression in cancer tissues (117). Heparanase, an endo--D-glucuronidase, is highly expressed in prostate cancers. Data from our laboratory (Ogishima T, Shiina H, Igawa M, Breault JE, Terashima M, Honda S, et al., unpublished data) shows substantial hypomethylation of the heparanase gene in prostate cancer compared with benign prostatic hyperplasia samples.
There is little information regarding the paradoxical coexistence of global and regional hypo- and hypermethylation in cancer, in which DNA methyltransferase activity is generally high (33,118,119). DNA methylation has been considered as a mechanism by which tissue-specific expression of genes is regulated (120). Therefore, the gene-specific hypomethylation observed in some cancers may result from disrupted transcriptional inhibition of normally silenced tumor-promoting genes. Additionally, gene-specific hypomethylation may be associated with global hypomethylation (121) but not with gene-specific hypermethylation (122). Thus, hypo- and hypermethylation may contribute individually to the process of carcinogenesis (122).
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HISTONE MODIFICATIONS |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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In prostate cancer, the expression of several genes may be potentially regulated by histone acetylation. Although at least one study has directly demonstrated changes in histone acetylation associated with a particular gene locus by using the chromatin immunoprecipitation assay (94), other studies reported that treatment of prostate cancer cells with HDAC inhibitors increased expression of specific genes such as insulin-like growth factor–binding protein 3 (123) and carboxypeptidase A3 (CPA3) (124), and thus inferred a role for histone acetylation in gene regulation.
Several genes of biologic significance to prostate cancer are potentially regulated by histone modification. One such gene, coxsackie and adenovirus receptor (CAR), is the primary receptor for group C adenoviruses and is important for adenovirus attachment to the cell membrane. Efficient adenovirus infection in gene therapy requires adequate expression of CAR in target cells. Decreased CAR gene expression has been detected in prostate cancer and is associated with an increased Gleason score (125). In urogenital cancer cells, including the prostate cancer cell line PC-3, activation of the CAR gene is modulated by histone acetylation and can be induced by depsipeptide, an HDAC inhibitor (126). Exposing cancer cells to low concentrations of depsipeptide has the functional consequence of preferentially increasing the efficiency of adenoviral transgene expression (127).
Another gene regulated by histone modification is the vitamin D receptor, via which 1,25-(OH)2-vitamin D3 acts to exert cell cycle regulatory antiproliferative effects in a variety of tumor cells, including those of the prostate (128–131). However, prostate cancer cells display a range of sensitivities to the antiproliferative effects of 1,25-(OH)2-vitamin D3 (132,133) for reasons that are unclear. Prostate cancer cells that are insensitive to 1,25-(OH)2-vitamin D3 have increased levels of nuclear receptor corepressor SMRT (silencing mediator of retinoid and thyroid), which could result in increased deacetylase activity and decreased transcriptional activity of the vitamin D receptor (134). In addition, combined treatment of prostate cancer cell lines with the HDAC inhibitor trichostatin A (TSA) and 1,25-(OH)2-vitamin D3 synergistically inhibits cell proliferation (134). This finding may be useful in the clinical setting, in which use of 1,25-(OH)2-vitamin D3 and its analogs in combination with HDAC inhibitors could activate the vitamin D receptor while minimizing unwanted side effects associated with 1,25-(OH)2-vitamin D3, such as hypocalcemia.
Histones can also be modified through methylation of lysine and arginine residues (135). Methylation of H3 at lysine 4 is associated with inactive transcription of the prostate-specific antigen (PSA) gene in the prostate cancer cell line LNCaP, and AR-mediated transcription of the PSA gene was accompanied by rapid decreases in di- and trimethylated H3 at lysine 4 (136).
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INTERACTION BETWEEN DNA METHYLATION AND HISTONE MODIFICATION |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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Although DNA methylation and histone acetylation can each modulate gene expression separately, they also interact to form a transcriptionally inactive chromatin state through the binding of methylated DNA binding proteins such as MeCP2. This interaction then recruits HDAC activity to methylated promoters, resulting in gene silencing (137). In addition, DNMTs can directly recruit HDAC activity to silence gene expression (138,139). In vitro studies have demonstrated that DNA methylation and histone acetylation cooperate in regulating expression of several genes in prostate-derived cell lines. For example, TSA and the demethylating agent 5-aza-deoxycytidine cooperatively activate DAB2IP mRNA expression in PC-3 cells, which have low basal levels of acetylated H3 (94). Another gene that is regulated by both DNA methylation and histone acetylation is the retinoic acid receptor
gene (RARB) gene, which is methylated in a majority of prostate cancer tissues and cell lines (140). All RARB-negative cells (LNCaP, PC3, and DU145) are hypoacetylated at both H3 and H4. Combined TSA and all-trans retinoic acid treatment after 5-azacytidine treatment increases the accumulation of acetylated histones, leading to reactivation of the methylated RARB promoter and subsequently the expression of RARB (140). These studies provide evidence that promoter hypermethylation and histone deacetylation cooperate in ensuring inactive chromatin status and provide a rationale for a combined treatment with DNA methylation and HDAC inhibitors in reversing the epigenetic silencing of key tumor suppressor genes. DNA methylation may interact with histone methylation to modulate chromatin structure and regulate gene transcription (141,142). MeCP2, which binds to methylated CpG dinucleotides, not only recruits HDAC activity, which results in chromatin remodeling, but also facilitates methylation of H3 lysine 9 and thereby reinforces an inactive chromatin state (142). In Neurospora, histone methylation directs DNA methylation mediated by the adaptor protein, heterochromatin protein HP1 (143). This mechanism could also be operational in mammals, in which a direct interaction between DNMTs and histone methyltransferase has been observed (141,144). It is unclear which methylation event happens first; the available data support both possibilities (145,146). However, in LNCaP prostate cancer cells, transcriptional shutdown of the GSTP1 gene requires several sequential changes involving DNA methylation as the initial event, followed by histone deacetylation, and then histone methylation (147).
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EPIGENETIC CHANGES AS PROSTATE CANCER BIOMARKERS |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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To be clinically applicable, an ideal tumor biomarker must be readily detectable in clinical specimens obtained through minimally invasive procedures. DNA hypermethylation seems to fulfill this requirement and has been considered to be a promising biomarker for several reasons (148,149). First, unlike mutations, methylation always occurs in defined regions (i.e., CpG islands) and can be detected using techniques with high sensitivity, such as methylation-specific PCR (150), and high resolution, such as bisulfite genomic sequencing (151). Second, hypermethylated DNA is associated with virtually every type of tumor (152), with each type of tumor apparently having its own signature of methylated genes, such as the methylation of GSTP1 in prostate cancer (77,78) (Fig. 1), von Hippel–Lindau gene in renal cancer (87,153), the mismatch repair gene MLH1 in colon cancer (154), and APC in esophageal cancer (155). In addition, some methylation occurs early in cancer development (149). These features make DNA hypermethylation an excellent tumor biomarker candidate.
Because GSTP1 is the most frequently methylated gene in prostate cancer, attempts have been made to detect prostate cancer by identifying methylated GSTP1 CpG islands in clinical samples, including plasma and serum (156,157), prostate secretions (158,159), voided urine (156,160–162), and prostate biopsy specimens (163,164). Goessl et al. (161) examined DNA methylation of the GSTP1 gene in urine after prostatic massage and detected prostate cancer with a specificity of 98% and an overall sensitivity of 73%. In a similar study using urine samples collected after biopsy (162), the specificity was only 67% and the sensitivity only 58%. Specificity and sensitivity were even lower if simple voided urine was used as the DNA source (160).
In a recent study, Harden et al. (165) compared the results of a blinded histologic review of sextant biopsy samples from 72 excised prostates with the relative methylation levels of GSTP1 (i.e., a relative methylation level is the ratio of GSTP1 to the level a reference gene, MYOD1). They found that histologic analysis alone detected prostate carcinoma with a sensitivity of 64% and a specificity of 100%, whereas the combination of histologic analysis and GSTP1 methylation at an assay threshold (i.e., a cut-off level for the GSTP1/MYOD1 ratio) of greater than 10 detected prostate carcinoma with a sensitivity of 75% and a specificity of 100%.
GSTP1 methylation can also be used to detect occult prostate cancer cells in lymph nodes. Kollermann et al. (166) found evidence of GSTP1 hypermethylation in 90% of lymph nodes from prostate cancer patients but in only 11.1% of lymph nodes from a noncancer cohort, suggesting that detection of GSTP1 methylation could have a role in molecular staging of prostate cancer. CD44 may have a similar role. However, although CD44 hypermethylation is readily detectable in the serum of prostate cancer patients, there is a lack of specificity for the disease because CD44 is also found in normal epithelial specimens (67).
Using the methylation status of a single gene as a biomarker of prostate cancer has limitations, including insufficient sensitivity, lack of specificity in differentiating prostate cancer from nonmalignant disease and from cancers originating from other organs, and poor risk assessment. An examination of the methylation pattern of multiple genes may overcome these limitations and offer better diagnostic and prognostic possibilities than that of a single gene. By profiling the methylation pattern of multiple genes in prostate tissue, several recent studies (54,57) have shown improved sensitivity and specificity in detecting prostate cancer. For example, examining the methylation of GSTP1, APC, RASSF1, and MDR1 can distinguish primary prostate cancer from benign prostate tissues, with sensitivities of 97%–100% and specificities of 92%–100% (57). Similar methylation patterns were observed in studies conducted with Korean prostate cancer patients (54) and Western patients (57). The prevalence of hypermethylation for three of four genes (GSTP1, RASSF1A, APC, and MGMT) studied in both populations was similar (54,57), indicating the existence of a unique prostate cancer-specific methylation fingerprint that is not defined by race/ethnicity.
For methylation profiling of multiple genes to be a clinically practical tumor marker, these results need to be validated in clinical specimens of prostate cancer patients. In addition, although most current studies focus on candidate gene approaches, there is an urgent need to perform genome-wide screening of unknown methylated loci in prostate cancer and add these loci to the pool of known methylated genes for methylation profiling. Microchips spotted with five to 10 genes representing the best prostate cancer methylation fingerprints may make the rapid, accurate diagnosis and risk assessment of patients with prostate cancer possible.
Another potentially useful application of methylation profiles is in the molecular classification of prostate cancer. The current prostate cancer classification system depends largely on histopathologic observations that are unable to predict whether a latent tumor will progress to a clinically relevant tumor or whether it will respond to androgen ablation treatment. Classification based on methylation profiles alone or in combination with pathologic diagnosis could be useful in predicting the behavior of a tumor. Attempts have been made to use both quantitative methylation analysis of the GSTP1 gene and a histologic review in the diagnosis of prostatic sextant biopsies. Using these techniques, improved sensitivity and specificity were noted (164).
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EPIGENETIC MODULATORS FOR PROSTATE CANCER TREATMENT |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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Reversal of Hypermethylation by DNA Methyltransferase Inhibitors
Unlike genetic alterations such as mutations, epigenetic changes such as DNA methylation are potentially reversible. This property makes epigenetic changes an attractive target for cancer therapy (97). 5-Azacytidine and 5-aza-deoxycytidine (decitabine), nucleoside analog inhibitors of DNMTs, have been widely used in in vitro cell culture systems to reverse abnormal DNA hypermethylation and restore silenced gene expression. However, only limited success has been achieved in clinical trials with these drugs (167,168). In a phase II study conducted by Thibault et al. (168), 14 men with progressive, metastatic prostate cancer that recurred after total androgen blockade and flutamide withdrawal were treated with decitabine, but only 2 of 12 patients evaluated for response had stable disease, with delayed time to progression. The authors concluded that decitabine is a well-tolerated regimen with modest clinical activity against hormone-independent prostate cancer.
Because nucleoside analog inhibitors of DNMTs have many potential side effects, such as myelotoxicity (169), mutagenesis (170), and tumorigenesis (171), non-nucleoside analog DNA methyltransferase inhibitors are an attractive alternative for possible clinical use. Lin et al. (172) reported that procainamide, a widely used antiarrhythmia drug and a known non-nucleoside inhibitor of DNMTs, reversed GSTP1 CpG island hypermethylation and restored GSTP1 expression in LNCaP cells grown in vitro or in vivo as xenograft tumors in athymic nude mice. Procainamide also restored expression of several other genes silenced by promoter methylation (173,174). The demethylating effect of procainamide is thought to occur through inhibition of DNMT-catalyzed transfer of methyl groups from S-adenosylmethionine to DNA (175). It is likely that additional compounds with a weak demethylating effect such as those existing in various dietary plants will be identified (176).
Although demethylating agents may protect against some cancers (102), they may also promote genomic instability and increase the risk of cancer in other tissues (103). Indeed, hypomethylation can have hazardous effects such as promoting carcinogenesis as demonstrated in certain model systems (103). Therefore, caution should be used in selecting the type of cancer patient for clinical trials involving DNA methyltransferase inhibitors. It is also important to note that the effects of DNMT inhibitors and even the effects of knocking down DNMT1 per se may be independent of the mechanisms of epigenetic reactivation such as DNA demethylation and histone hypermethylation (177). Therefore, it is possible that many of the antiproliferative effects seen in either normal or cancer cells may be attributed to methylation-independent roles of DNMT1 rather than the loss of DNA methylation.
Reversal of Hypomethylation for the Suppression of Overexpressed Genes
Gene transcription from a transfected plasmid DNA can be suppressed by in vitro DNA methylation of the upstream promoter by using SssI methylase (178,179). Recently, a new class of oligonucleotide termed methylated sense oligonucleotide has been used to manipulate sequence-specific DNA methylation in vitro and in vivo (37,180). This technique uses a synthetic oligonucleotide in which the cytosine residues are replaced by 5'-methylcytosine. Binding of the methylated sense oligonucleotide to one strand of the DNA forms a hemimethylated DNA intermediate that has a "replication fork"–like structure and is a preferred substrate of DNMTs. The latter DNMTs would methylate the second strand and spread the methylation around the targeted site (180).
Unlike DNMT inhibitors, this innovative approach changes only the epigenetics in a gene-specific manner and could be potentially powerful in suppressing hypomethylated and thus overexpressed tumor-promoting genes by introducing de novo methylation in their promoter. In this regard, introduction of a methylated sense oligonucleotide that targets the ESR2 gene promoter into PC-3 prostate cancer cells results in sequence-specific methylation and the suppression of ESR2 gene expression, which is overexpressed in metastatic prostate cancer because of a hypomethylated promoter (36). In an in vivo study of mice with implanted hepatocellular carcinoma, injection of a methylated sense oligonucleotide targeting the insulin-like growth factor 2 gene led to improved survival (180). However, before this technique can be tested as a human therapy, its efficacy in achieving sustained inhibition of gene expression needs to be compared with that of other posttranscriptional and posttranslation gene silencing approaches such as RNA interference.
HDAC Inhibitors for Activating Transcriptionally Silenced Genes
HDAC complexes enzymatically remove the acetyl group from lysine residues of the amino-terminal tails of histones maintaining chromatin in a transcriptionally inactive state (181). This transcriptional blockade can be overcome by agents that inhibit HDACs (8). A variety of agents, many of which are natural products, exhibit HDAC inhibitory activity and therefore may have antitumor activity. Commonly used HDAC inhibitors include TSA (182), sodium butyrate (182), depsipeptide (FR901228 and FK228) (183), valproic acid (184), MS-275 (185), suberoylanilide hydroxamic acid (SAHA) (186), pyroxamide (186), and phenylbutyrate (187). Some of these agents such as depsipeptide are in clinical trials. A comprehensive review of various HDAC inhibitors in cancer treatment has been published recently (188).
A number of in vitro studies have used the antiproliferative effects of various HDAC inhibitors in cultured human prostate cancer cells. All of the agents tested have inhibitory activity against prostate cancer cell growth (186,187), but the underlying mechanism varies widely. For example, sodium butyrate and TSA synergize with 1,25-(OH)2-vitamin D3 to inhibit the growth of LNCaP, PC-3, and DU-145 prostate cancer cells by inducing apoptosis (12). Valproic acid induces prostate cancer cell apoptosis by increasing the expression of several pro-apoptotic genes (184). Although the study did not address whether there were locus-specific histone acetylation changes, a marked global decrease in nuclear HDAC activity was noticed in valproic acid–treated cells (184). Other growth inhibitory mechanisms have also been identified, such as increasing expression of the cell cycle regulator CDKN1A (186,189), decreasing telomerase activity (182), and suppressing angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (183). The observed decrease in histone deacetylating activity induced by HDAC inhibitors is mainly related to the induction of gene transcription. In several instances, however, HDAC inhibitors may actually decrease expression of hyperacetylated genes (182,183). In particular, depsipeptide inhibits PC-3 cell growth by suppressing the expression of VEGF mRNA even though it induces accumulation of acetylated histones in chromatin associated with the VEGF gene promoter (183). It is unclear why accumulation of acetylated histones in the VEGF gene promoter causes transcriptional inhibition of the associated gene.
Several HDAC inhibitors have also been tested in animal models of prostate cancer and have had promising antitumor activity (183,186,190,191). SAHA, at a dose without detectable toxicity, reduced tumor growth by 97% in mice transplanted with CWR222 human prostate tumors (190). Similarly, depsipeptide, sodium butyrate, and tributyrin slowed prostate cancer tumor growth by 50%–98%, depending on the cell line used for establishing xenografts in mice (189,191).
Combined Action of DNA Methyltransferase Inhibitors and HDAC Inhibitors
It is unlikely that a single agent has the potential to reverse epigenetic silencing of genes once and for all without inducing adverse effects related to cytotoxicity or undesired epigenetic effects. Emerging evidence supports the concept that epigenetic silencing of genes involves multiple mechanisms including DNA methylation, histone methylation and acetylation, and chromatin remodeling and requires their sequential action (147). Several studies have shown that the combination of HDAC and DNMT inhibitors can generate additive or synergistic effects on apoptosis, differentiation, and/or cell growth arrest in cancer cells (192–194). HDAC inhibitors such as TSA can potentiate the induction of tumor suppressor genes by DNA-demethylating agents in cancer cells (194). To maximally achieve gene reactivation, it may be necessary to simultaneously block the processes essential to both the formation and maintenance of the transcriptionally repressive chromatin associated with such genes (94).
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CONCLUSIONS AND FUTURE DIRECTIONS |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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Although the number of genes that undergo aberrant epigenetic inactivation associated with prostate cancer is growing, some specific questions need to be answered before we fully understand the biologic significance and consequence of this process. For example, what is the mechanism controlling selective methylation of genes in prostate cancer cells that have increased expression and activity of DNMTs, and why do hypo- and hypermethylation both occur in prostate cancer cells? Is there an active demethylation process to account for hypomethylation or it is caused by decreased hypermethylation?
Ample evidence suggests that DNA methylation detection can serve as a promising tumor biomarker. However in the prostate, only the GSTP1 gene shows sufficient specificity and sensitivity in detecting prostate cancer to warrant further testing as a tumor marker in patients’ body fluids such as serum, urine, and ejaculate. Further work should also focus on identifying new aberrantly methylated genes using high-throughput screening such as CpG island microarray assays as the first step toward tumor marker validation. Furthermore, a prostate cancer methylation fingerprint comprising multiple genes may be more accurate than that of individual genes in the early detection and risk assessment of prostate cancer and molecular diagnosis of resected specimens.
Before we fully understand the cause and consequence of global hypomethylation in prostate cancer, therapeutics targeting DNMTs in cancer should be used with caution. Ideal treatments are those that can selectively activate a group of methylated genes without inducing undesired demethylation to the rest of the genome. Given the close relationship between DNA methylation and histone deacetylation in epigenetic inactivation, a combination of DNMT inhibitors and histone deacetylation inhibitors may be an attractive strategy for the treatment of prostate cancer patients.
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NOTES |
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TopNotesAbstractIntroductionDNA Methylation in Prostate...HypomethylationHistone ModificationsInteraction Between DNA...Epigenetic Changes as Prostate...Epigenetic Modulators for...Conclusions and Future...References |
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Supported by grants RO1AG21418 and RO1CA1018447 from the National Institutes of Health, a VA REAP award, and Merit Review grants.
We thank Dr. Roger Erickson for his editorial assistance.
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REFERENCES |
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